c

 


  
 V

cV

A. Domains - Bacteria, Archaea, Eukarya

B. Kingdoms - Eubacteria, Archaebacteria, Archaezoa, Protista, Plantae, Chromista, Fungi,
Animal
C. Cladogram - ?
V

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A. Definition - by most definitions, a plant:V

÷V is multicellular;V
÷V is non-motileV
÷V has eukaryotic cellsV
÷V has cell walls comprised of celluloseV
÷V is autotrophic; andV
÷V exhibits alternation of generations - has a distinctive diploid (sporophyte) and
haploid (gametophyte) phase.V

B. Examples - the Plant Kingdom includes the angiosperms (flowering plants), gymnosperms
(cone-bearing plants), ferns, and bryophytes (mosses & liverworts). Recent classification systems
suggest that these organisms, in addition to the red algae and green algae, should be classified
in the Plant Kingdom (Plantae).V

ccc


  
 V

A. Definitions (numerous) - Plant physiology is the study of:V

÷V the functions and processes occurring in plantsV

÷V the vital processes occurring in plantsV
÷V how plants workV

B. In essence, plant physiology is a study of the plant way of life, which include various aspects
of the plant lifestyle and survival including: metabolism, water relations, mineral nutrition,
development, movement, irritability (response to the environment), organization, growth, and
transport processes.V

C. Plant physiology is a
 .V

D. Plant physiology is an 


 .V

E. Plant physiology relies heavily on 
   .V

c 
 

 
 V

V Food - plants are the route by which solar energy enters ecosystemsV
 è
V Economically important products - plants produce countless products from fibers
to medicines to wood. For example, you can check out the web notes for
my Plants and Human Affairs course or The Society for Economic Botany V

V Applications to other disciplines (i.e., agriculture, forestry, horticulture)V

V Theoretical importance (like a mountain - it·s there!)V

V ‰obs! - see career pamphlets in file box. Also, check out the web sites for
the American Society of Plant Biologists, Botanical Society of America, American
Phytopathology Society and others.V

V It's fun & exciting (but, I guess not everyone necessarily agrees!)V

V Botany Without Borders is a good online film that highlights the importance of
plants. It was created by Dr. K Niklas (Cornell).V






  

There is an incredible wealth of information about plant physiology. Never say, "nothing is
known about...." unless you are positive. Chances are someone, somewhere, sometime, has
studied the phenomena. I think it was Bertrand Russell who said something like, "it·s easier to
make a scientific discovery than to discover if it·s already been discovered." Since, there·s so
much literature available, it pays to learn a little about the types of resources that are
available. V

One reason why there is so much information is that plant physiology material is found in both
the biological and chemical literature.V

A. Types of literatureV

V Primary - original reports of research; journals. Two excellent journals that are
published by the American Society of Plant Biology arem 
m
?
and m 


, both of which are available in the Clemens Library. We·ll check out a copy
of m 
m
?
and point out volume, number, date of publication, publisher,
organization, format, etc. V

è
V Non-primary - revisions, summaries, compilations, texts. One particularly good

example is  

?


m 
?
 You can also find helpful information
in other volumes including the  

?


m
 
,  

?





 


?, and  

?


?
?
 
?



. A new volume is published every year by Annual Reviews, Inc. For a
listing of some good textbooks and general books in plant physiology, click here. V

B. Keeping track of the literature - note cards, duplicate copies of articles, reprints, computer
tracking programs.V

C. Citation Format - various formats, varies with the journal/discipline. We·ll use the format
adopted by m 
m
?
:V

÷V Format for citing a journal article: Author AB, Author BC (1977) Title of article. Plant
Physiology 59: 121-125V
÷V Format for citing an article in a book: Author AB, Author BC, Author CD (1974) Title
of article. In A Smith, B ‰ones, ed., Title of Book, Ed 2 Vol. 3. Publisher, City, pp. 14-
19V
÷V Format for citing a book: Author AB (1998) Title of Book. Publisher, City.V
 ÷V Citations in text - again, many formats. We will cite references by author.
Examples: Smith (1998) said that .... or, Apples grow on trees (Smith, 1998).V

c
 


Do you remember the proper form for citing a scientific name? For more information about
scientific nomenclature click here. Here is the general format for scientific names: 



?
 Author citation (Family)V

Notes:V

÷V Author citation should appear the first time the name appearsV
÷V Family should be included for each species the first time it appearsV
÷V Family names almost always end in 'aceae'. There are a few exceptions (
, Cruciferae,
Compositae, Labiatae, Graminae).V
÷V Abbreviate the name after the first usage; i.e., 

?
V

As an example: ´

 L. (Fagaceae)V

cc  c

  

V


V Ëndergraduate Training - you should consider taking as many courses in botany and
chemistry as possible, including biochemistry and physical chemistry. You can obtain
entry level positions with a bachelor's degree, other positions may require graduate
training. There are many excellent graduate schools - all of the Big 10 schools have good
programs and there are many others (?
, ËC-Davis, Cornell, Washington Ëniversity).V

V Professional Societies - join a society. The American Society of Plant Biologists is the main
organization for plant physiology. The Botanical Society of America is another good
society to consider joining. Most societies have student rates and are worth every penny.
Attend their annual meetings.V

V Read m 
m
?
, the  

?


m 
?
, and other journals and books
regularly to stay abreast of recent developments in the field.V


V Careers - there are lots of career opportunities available in government and industry at
both the entry and upper level. Visit our Career Resourcesoffices for more information.
Read the pamphlets in the "Careers" file in the filing cabinet. Check the course "Links"
page for links to good websites with career information. Especially check the web pages
for the American Society of Plant Biologists, Botanical Society of America, and American
Phytopathology Society.V

ccc
!
V

÷V Study Guide for Plant PhysiologyV

V
 

 "# $ %

V

G




 V


c



Recall - by most definitions, a plant:V

÷V is multicellular;V
÷V is non-motileV
÷V has eukaryotic cellsV
÷V has cell walls composed of celluloseV
÷V is a photosynthetic autotrophic; andV
÷V exhibits alternation of generations - has a distinctive diploid (sporophyte) and
haploid (gametophyte) phase.V

Examples include the angiosperms (flowering plants), gymnosperms (cone-bearing plants),

ferns, and bryophytes (mosses & liverworts). Recent classification systems suggest that these
organisms, in addition to the red algae and green algae, should be classified in the Plant
Kingdom (Plantae).V

cc

 









 



 
Autotrophism! Yup, that's my guess, too. We should recognize that a systematist (someone
who studies classification systems) familiar with the most recent notions of classification might
disagree since members of a "new" kingdom, Chromista, are also photosynthetic autotrophs.
Nevertheless, both of these groups are closely related so we can still safely agree that
autotrophism is important to the plant way of life. V

A. Take-Home-Lesson 1: An autotroph makes its own food (energy-rich organic compounds)

from simple, inorganic materials in the environment. Plants use light as their energy source,
hence they are photosynthetic (vs. chemo-synthetic for certain bacteria). The general equation
for photosynthesis is:V

CO2 + H2O + light ń (CH2O)n +O2V

In contrast, animals are heterotrophic, meaning that they must obtain their food (pre-
fabricated organic compounds) from the environment. They cannot manufacture their own
food. Examples of heterotrophs include mycotrophs (plants that obtain their nutrient source from
a fungus like Indian pipes ( ), decomposers (fungi, bacteria), carnivores, and
herbivores. Some parasitic plants (holoparasites like dodder ( ) and dwarf mistletoe that
lack chlorophyll are obligate heterotrophs that can only obtain their nutrients from another
plant. Others parasites, like mistletoe (m 
 ) and Indian paintbrush are green and can
make their own organic compounds but obtain water and minerals from a host plant (Hershey).
Finally, some plants, like the carnivorous species, feed both autotrophically and
heterotrophically.V

B. Take-Home-Lesson 2: The autotrophic mode of nutrition evolved early in the evolution of

life,  3 billion years ago. This event set in motion the evolutionary events that culminated in
modern plants. Therefore,  



 
 
 

 & 


 

 . Plants colonized land about 440 million years
ago. The transition from water to land required the evolution of (in approximate sequence):
cuticle (to resist drying out), stomata (gas exchange), and vascular tissue (for water/nutrient
transport). V

ccc  & 

 



Plants required specialized structures adapted for the autotrophic mode of nutrition.
Specialization occurs at all levels of biological organization (
, organ, tissue, cell, organelle).
Specific problems, and their solutions, related to autotrophic nutrition are:V



   
  

 
In order for photosynthesis to function properly and efficiently, it was necessary to separate it
from other reactions that occur in the cell. Thus, the evolution of a specialized organelle for this
process - the chloroplasts. Even within the chloroplast specialization was required. Recall from
intro bio that there are three major areas in a chloroplast - the stroma, inner membrane, and
inter-membrane space. Each of these three regions is important for the functioning of
photosynthesis. Electron transfer reactions require the highly ordered environment provided by
the inner membrane. The Calvin cycle (light-independent reactions) are aqueous biochemical
reactions which occur in the stroma and the inter-membrane space is needed to generate the
pH gradient across the membrane that is important for photophosphorylation (ATP production).V



   
 
  
 
Leaves are perfect solar collectors. These organs are broad and flat to allow for efficient light
harvest. The leaves are broad to maximize surface area for light harvest and they are thin since
light cannot penetrate too deeply into the leaf (the amount of light decreases exponentially
with distance). As an aside, although the majority of light is absorbed near the leaf surface, in
some situations plant tissues act like fiber optic cables that can funnel some light deeply into the
plant body (Briggs

). The window plant in the Namib desert funnels light through translucent
cell into the photosynthetic tissue that is buried in the soil (Attenborough)V

Even within the thin leaf, most chloroplasts are found in the upper layer of cells, the palisade
layer, which is the tissue layer just beneath the upper epidermis. This makes "sense" since these
cells will be receiving the greatest amount of light of any region in the leaf. Thus, this is an
example of specialization at the tissue level.V



   
 

 
  
Leaves double as a means to exchange photosynthetic gases (take up carbon dioxide and
get rid of oxygen) with the environment. Leaves have pores in the surface (stomata) that
regulate the entry/exit of gases and prevent the loss of excessive water.V

The spongy layer of the leaf acts like a "lung" increasing the internal surface area and provides
for more rapid diffusion within the leaf. Note again that leaves are thin - this avoids the need for
lungs or other type of pump to move gases. Since diffusion rates are inversely related to
distance, diffusion can account for gas movements into/out of a leaf. As a consequence, no
cell if more than 2 or 3 cells from the air. An added advantage of having large leaves for light
harvest is that they provide lots of surface area for absorption of carbon dioxide.V

Again, note the specialization of the leaf at the organ, tissue, and cellular levels for gas
exchange.V



  
 


     
.
This problem was solved by the evolution of the cell wall which provided for the support of thin
structures without the need (or potential) for significant numbers of internal support structures.
Leaves also have some internal "struts" (in other words, veins).V



   
 

 
With the exception of the algae and aquatic plants, plants obtain their water through the
roots from soil. Essentially the roots "mine" the soil for water. Thus, photosynthesis and the transition
to a terrestrial environment necessitated the evolution of a root system to obtain water
(specialization at the organ level). And, it required the evolution of specialized transport tissue
(xylem) to move the water from the roots to the leaves.V



   
 


 

 
 
  
Once carbohydrate is produced during photosynthesis there must be a mechanism to
transport it to other locations throughout the plant. The evolution of vascular tissue, specifically
phloem, permitted movement of photosynthate from leaves to roots, fruits and other tissues
where required.V

c  & 

 

 '(



 
 &)#

One of the main reasons for motility is to obtain food. Since the nutrients required by plants are
"omnipotent" there was never an evolutionary pressure for "motility." Let·s quickly compare the
nutrients used by plants and animals:V

Table 1: Comparison of Plant & Animal NutritionV

NutrientV PlantV AnimalV

form of uptakeV inorganic (CO2, water, ions)V organic (proteins, carbohydrates, fats)V

concentrationV dilute (i.e., CO2 = 0.03%)V concentratedV

distributionV omnipotentV localizedV

Conclusion: plants must be adapted for harvesting dilute nutrients that occur everywhere,
whereas animals are adapted for searching out and trapping widely dispersed, concentrated
packets of food.V

Supportive Evidence: if this is true, then we hypothesize that animals with a nutrient source like a
plant should have similar features to a plant. Check out corals, sea fans, and hydra. These are all
non-motile animals that occur in aquatic environments which enables them to "feed like a plant"
- food is essentially brought to them via water currents. Thus, they never had any pressure for
motility and they have very similar lifestyles/forms as plants.V

In addition, note that motility is really not possible for terrestrial plants. Once plants evolved
roots it precluded movement. These evolutionary "choices" are closely connected.V

However, being stationary has its own problems/consequences.V

  & 

  

' 




*


 * 
.

: a fixed (stationary) organism must be able to continually obtain nutrients without
using them up. Plants face the additional problem that their nutrients are "dilute." Thus, plants
must be designed for collecting dilute nutrients in the environment. Plants have several solutions
to this "problem":V

A. Plants are dendritic.

In other words, the basic shape of the plant body is dendritic - which means "tree-like" or
"filamentous". The advantage of this shape is that it provides a large surface-to-volume (s/v) ratio
which enables a plant to exploit a large area of the environment. In contrast, animals are more
compact (spherical) to minimize their s/v ratio. Among other things, this is an advantage for
motility. Surface-to-volume ratios are very important in many areas of biology. In class we
will investigate surface/volume ratios in more detail.V

B. Plants have indeterminate growth.

Process by which a plant continues to grow and get larger throughout its life cycle. The
advantage of this is that it allows the plant, especially roots, to grow into new areas. In contrast,
determinate growth is where an organism or part reaches a certain size and then stops growing.
This is characteristic of animals and some plant parts (
, leaves, fruits).V

C. Plants have an architectural design.

In other words, the plant body is constructed like a building - modular (Silverton & Gordon). It is
built of a limited number of units, each of which is relatively independent of the others and that
are united into a single structure. Thus, just like a building is made of rooms, the leaves, stems and
roots of a plant are analogous to a rooms in the building. Each room is somewhat independent,
yet they all function together to make an integrated whole. You can seal off a room in a
building, or remove a leaf or fruit, with little harm to the overall integrity of the structure. This is
critical for plants to be able to add or remove parts (leaves, stems, flowers, fruits) as necessary.
One conclusion is that because of their indeterminate growth and architectural design, 

 
  

This gives plants the ability to colonize and exploit new areas for
resources.V

In contrast, an animal has a mechanical design. In other words, animals are built more like a
machine, made of numerous, different parts that function together. The parts are highly
integrated. Parts cannot be added or removed without reducing the efficiency of the operation
of the whole. Animals are limited by size.V

As a consequence, 
  - plants constantly change shape by

adding/loosing parts - by accumulating modular units. Animals don·t change shape - they
remain the same general shape throughout their life. Thus, growth in plants occurs by the
addition of new "units" not enlargement.V

D. Plants have a well developed ability to reproduce asexually.

This can be viewed as a quick and energetically inexpensive way to expand the influence of
the parent into a new location. One testable prediction from this hypothesis is that plants under
nutrient stress should increase their rate of asexual reproduction (see foraging data below).V

E. Plants (may) exhibit heterophylly.

Heterophylly refers to leaves with different shapes. For example, the aerial leaves of aquatic
plants are entire but the submerged leaves are dissected. Sun leaves tend to be smaller and
thicker than shade leaves. Dandelions are toothier when grown in a carbon dioxide enriched
(700 vs. 350 ppm) environment. The leaves of the vine  
 are more or less heart-shaped
and pressed to the trunk as the vine climbs into the tropical forest canopy. Once in the canopy,
the leaves take on the mature form with slits and holes. V
F. Leaves are arranged to minimize overlapping.
Phyllotaxy is the fancy term for leaf arrangement. Interestingly, phyllotaxy patterns have
always been shown to be related to Fibonacci number series (1, 1, 2, 3, 5, 8, 13, 21....etc). For
more on phyllaxy, visit this web site.V

G. Plants can forage.

The growth patterns of plants, especially vines and plants with stolons (runners), are similar to
the foraging tactics of animals. As an example, rhizomes of ÿ


veer from patches of
grass to avoid competition. A brief overview of the anatomy of a clonal plant like
 




 (ground ivy): parent plant, stolon (internode), ramet (individual of a clone).V

For a Case Study on Foraging, click here.V

Thus, plant growth is essentially analogous to animal behavior. One of the first to express this
idea was Arber (1950ë



m ?


m 
. Cambridge). She said, "Among
plants, form may be held to include something corresponding to behavior in the zoological
field...for most though but not for all plants the only available forms of action are either growth,
or ascending of parts, both of which involve a change in the size and form of the organism."V

c  & 

  

' 
   
 * 
.
 
: a non-motile organism is unable to move to a more favorable location to carry out
its vital functions. Thus, plants have at least three major problems to contend with:V

A. Environmental Positioning/Location - or, Getting Started in the Right Spot.

Obviously a motile organism can move to a favorable location, but a plant is stuck once the
seed germinates. For most plants getting started in the right place is a matter of luck. Thus, it is no
surprise that plants exhibit a Type III survivorship curve (produce lots of offspring, few survive, no
parental care of offspring - think oak tree and acorns). However, there are a few "tricks" that
plants use to help increase the odds that seeds will germinate in a favorable environment:V

V Light - some seeds, like certain varieties of lettuce, require light for germination. This is a
mechanism to insure that they germinate on the soil surface. It's no surprise that a garden
develops a healthy crop of weeds after the soil is turned - it brings light-sensitive seeds to
the surface. Light sensitive seeds are usually small and without much stored food. Thus, it
is important that they begin to photosynthesize soon after germination.V
è
V Ethylene - some seeds require ethylene to germinate. This naturally occurring plant
hormone is produced by plants and soil microbes. Once the ethylene concentration
reaches a critical level it induces the seeds to germinate. This happens if the seed is
buried. These seeds are usually larger than light sensitive ones. One advantage of being
buried is that the seeds will be more likely to be in a moist, humid
environment.
witchweed (?)V

V Specialized Dispersal Mechanisms - some plants have specialized mechanisms for
dispersal that will increase the odds of the seed getting into the proper place. For
example, mistletoes have sticky seeds that often stick to the beak of a hungry bird. The
bird will try to rub it off on a branch where the seed will adhere and germinate.V

B. Axis orientation.
Once a seed germinates in a favorable environment it must determine which way is up/down
to insure that the roots grow down and shoots up. Thus, gravitropism is a very important
physiological response characteristic of all plants. V
C. Fine Tuning.
Even non-motile organisms need to "fine-tune" their position in the environment. Thus plants
have a variety of mechanisms that enable them to optimize their position in the environment
including:V

V   

- grow toward light, maximize light reception. Although plants are typically
positively phototropic, some show negative phototropism. For example, the tendrils of a
tropical vine (?  ?

) grow away from the light and ivy stems bend away
from the light (hence the reason they tend to come into a window) but the leaves grow
towards the light. V
è
V º  

- growth of vines (e.g.,  
) toward a darkened region of the
environment. Mechanism by which some tropical vines find a support to grow up (Ray,
1975). Video clip from m? 

?


m  series;V

V 


    - response to touch in which the plant is shorter with thicker stems
- prevents plants from getting too spindly and reduces risk of breaking in wind;V

V º 

 (i.e., flowers follow the movement of the sun) - keeps pollen dry, maximize
photosynthesis;V

V ` 
 - pattern of leaves which minimizes overlapping such as ivy on a building
(Oxlade, 1998);V

V ©   - the response of plants to growth in the dark or with reduce light. Etiolated
plants are typically yellow, have elongated internodes (stems) with unfolded leaves and
the stems are thinner. These features can be considered ways of conserving energy until
conditions improve (i.e., light).V

V ÿ    - one example of habitat selection is shown by rhizomatous plants like
western ragweed (?
?
) that preferentially colonize non-saline soil
(Salzman, 1985). In this case, the growth rate is higher in saline soil than non-saline -
"moving away from the salt" increasing the likelihood of finding new habitat. Stilt palms
grow to avoid competition and the roots of many plants grow to avoid one another.
Dodder, a parasite, actually "chooses" its host when presented with several potential
species. Roots track humidity and mineral gradients and can change branching
patterns in response.V

V Climbing plants hold their leaves at right angles from the support stem to better intercept
light (Dicks 2000)
V
cc  & 

  

'
 
 + 

 * 

 
: plants, like other organisms, must be able to respond to changes in their
environment. Plants respond to their environment in a variety of ways. V

V 
 
 



 to unpredictable, usually short-
term environmental fluctuations, like changes in temperature or light. For example, some
flowers like crocus are temperature sensitive and open when it is warm and close when it
is cooler. The flowers of an alpine species (
? 
? close up before a
thunderstorm. The flowers actually respond to the decreased temperature associated
with the storm front and close to avoid pollen being washed out of the flower by the
rain. V
è
V 


    

. Light is one of the most important environmental
cues for plant development. This phenomenon is called photomorphogenesis and one
classic example is etiolation (discussed above). Another example: increased levels of
carbon dioxide in the air has lead to a decrease in the number of stomata on leaf
 surfaces and have similarly been shown to increase the toothiness of dandelions. The
leaves of plants in the rainforest usually have an elongated tip (drip tip) to help funnel
water off the leaf surface to minimize the growth of fungi and other leaf epiphytes that
might cause disease or block sunlight. Leaf margins are also a response to the
environment. For example, entire leaf margins are correlated with warmer temperatures
in tropical forests. We also discussed sun and shade leaves earlier.V

V   
  
    
!

 

  
   
   
       
 
 

 
 . Since these changes take time, a plant must "know" or
"predict" when the environment will change and prepare for the change. Indeterminate
growth is important here since it provides plants with the ability to change
developmentally through the life cycle. Some examples of this phenomenon include:
preparing for winter (by forming buds in summer) and photoperiodism (timing flowering
so the appropriate pollinator is available and that the seeds have enough time to
develop before winter); circadian rhythms (various types in response to day/night), and
nyctinasty (sleep movements). In contrast, 

   



 
G

. Since they are motile, they can "move" to a
favorable position. In fact, because they are motile they need a nervous system to
respond to the environment. Plants don·t need a nervous system since they are
constrained to respond to the environment by growth/developmental changes; hence
they never had a pressure to evolve a nervous system.V

ccc  & 

  

' 




* 
 


  
 * 

Problem: a non-motile organism cannot flee when conditions get tough. It must "fight" it out.
Both the physical environment and biological environment threatens the well being of plants.V

A. Physical dangers - wind, water (flood), drought, cold (winter) are among the physical dangers
that a plant faces. In general, plants cope with these (at least the predictable ones like winter
and drought during summer) by dormancy, senescence, and even death. The evergreen and
deciduous lifestyle are in part a response to adverse conditions. Evergreens are much better
able to tolerate cold, dry conditions. They also do better in poor soil because they don't loose as
many leaves. Plants also respond to enviromental challenges morphologically - for example,
xeric plants reduce their S/V to minimize water loss. Arctic or montane herbs are small and hug
the ground.V

B. Biological dangers - predators (=herbivores) and competitors (=other plants). Plants have
evolved:V

V  ?

  (thorns, hairs, thick cuticle). Some plants are even "smart"
enough to stop producing defenses when they are out of range of a herbivore.
For example, the upper parts of Acacia trees above giraffe height produce few
thorns. Similarly, holly leaves have few prickles above herbivore (
, deer)
height; V
è
V
?

  - produce toxic, unpalatable chemicals. These can be
inducible (produced in response to attack) or constitutive (always present)
(Karban & Myers. 1989. Ann Rev Systemat. Ecol 20:331); 

is chemical
warfare between plants. m

? are chemicals produced by plants to resist
microbial infection. V
 
V ??
- "tricking" predators. For example, lithops in S. African deserts look like
pebbles - are stone mimics. NZ mistletoe leaves look just like the host tree leaves
to avoid being eaten which is important because they contain even higher
amounts of nitrogen. 
? is a non-toxic New Zealand plant that looks very
similar to ? 


 (a toxic species). When young, NZ lancewood
looks very unappetizing - much like a woody umbrella that is folded up. However,
when it gets about 15 feet tall, above predator (
, moa) height, it branches
out and has a more "traditional" appearance.V

V 



 some plants, like wild tobacco, when attacked by herbivores
release volatile chemicals that summon predatory insects to the damaged
plants. These insects in turn, kill the herbivores.V

Overall, volatile chemicals play a very important role in plant defense. These chemicals,
released when the plant is attacked by an herbivore serve as signals that: (a) warn other plants
that danger is imminent. For example, tobacco eaten by herbivores produces salicylate (similar
to aspirin) that stimulates its own defense response and is converted into methylsalicyate which
is volatile. This compound travels to other plants to induce their defense response; (b) tell other
herbivores that it is being attacked and that they should look for another food source unless they
want to battle it out (compete) with another herbivore; (c) alert predator insects that their are
tasty herbivores in the area (see #4); (d) tell herbivores that the chemical defense system of the
plant is ready for them and that should go pick on someone else; and (e) the volatiles
themselves help to repel the attack.V

Plants are more sensitive to being handling than perhaps we give them credit. Recent studies
have shown that simply stroking the leaf of a plant just once will affect herbivory - some plants
suffer greater damage while others less damage (Sci News 159: 119, 2001).
V
c,  & 

  

'
 
 
: A non-motile organism cannot seek a mate (for gamete transfer) or easily disperse
offspring. Plants solve the gamete transfer problem by relying on various pollination vectors.
Fruits/seed dispersal mechanisms help disperse offspring. Check out the m? 

?



m  video, Volume 1 (dispersal) and Volume 3 (pollination) for some great examples. These
document some absolutely fascinating stories about plants and their pollination and dispersal
vectors. To add a recent story, the fragrance of a particular orchid changes after pollination.
Prior to pollination the orchid released a fragrance that was an "aphrodisiac" for the male but
once the flower was pollinated it produced a fragrance to repel him. And, when a few fruits
ofÿ
?

, a neo-tropical tree, are removed it stimulates the rest of the fruit to ripen - it
essentially tells them that a dispersal agent is in the area.
V
,%
 
  & - & 





 
: As we mentioned, plants evolved cell walls: (a) as a means of support; (b) to prevent
the protoplast from bursting in a hypotonic medium; and (c) some speculate that walls may
even be a way to dispose of excess carbon. In any event, by surrounding their cells with a rigid
box, this imposed certain limitations. These include:V

V 


  



 (primary meristems - responsible for growth in
length such as apical meristems at root and shoot tips; and secondary meristems -
responsible for growth in girth, like vascular cambium). In contrast growth in animals
occurs throughout the body.V
 è
V Since plants are built from rigid structures,

    
   ,
not cell movement (as is found in development of animals)V

V 
   
 

   
 rather than behaviorallyV

V Plants exhibit  

 
 (vs. determinate for animals)V

V Plants 
 

 
  

 (?
., architectural design);
whereas animals have a fixed shape that enlarges (from Adrian Bell, 1986)V

V Since each cell is walled off from neighboring cells,     
 
 ! ! 

 . Plants accomplish this through:

 
 -
cytoplasmic connections; hormonal regulation and some electrical signals.V

,c



   ./
For many years, I have joked that "plants are smarter than you think." Hopefully, our discussion
of the plant way of life has led you to agree. Although my comment was somewhat tongue-in-
cheek, there have been recent discussions about the intelligence of plants. Historically, plants
have not been considered to be "intelligent" because this concept has been associated with
movement. Thus, animals are smart, plants are not. But, if you agree with Anthony Trewavas
(2002) who first argued in 
that if "intelligence is defined as adaptively variable behaviour
during the life of the individual, then, in plants, behavioural plasticity is where intelligence should
be apparent." The examples cited in this document and lots of others from Trewavas, convince
me that Trewavas is correct - plants are intelligent. For more reading, Trewavas has expanded
his 
article into a longer review which is excellent and available on-line. David Hershey
agrees, and has also addressed this issue in an eloquent essay, "Plants are indeed,
intelligent," that was submitted to the 
?
?



. Both of these articles contain
lots of examples why plants are so "cool." V

One caveat, don't associate the concept of intelligence with the notion of intelligent design,
which is an idea that suggests God must have created the earth and its inhabitants because
everything is too perfect to have happened via the somewhat random or chancy process of
evolution.
V
,cc

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  0% 
 * 0



0

 1
Hopefully, I've made a good case for why it's important to study plants and why are they are
"cool." Which brings up a question - why don't more people like or want to study plants? Why is
plant physiology normally a small class? Evidence shows that even grade school kids prefer
zoological topics to botanical ones. But why?V

Wandersee and Schussler (1999) argue that the main problem is that by human nature we are
"blind" to plants due to limitations in our visual perception. Others, like Hoekstra (2000) and
Hershey (2002) argue that a more critical concern is that plants are neglected in the curriculum
and that botanical concepts are often either not taught, or not presented well. Whatever the
reason, this semester we are going to be Plant Chauvinists practicing Animal Neglect, seeing
clearly the beauty and excitement in the study of plant physiology.
V
Π-V

÷V Alexander, R (1975) Size and Shape. Study in Biology 29. Edward Arnold. London.V
÷V Barker, M (1995) 'A plant is an animal standing on its head' ‰ourn. Biol. Educ. 29: 201 - 208.V
÷V Barrett, S. Mimicry in Plants. Scientific American.V
÷V Becker, Wayne. "What it Means to Be a Plant. Some Musings on the Comparative Biology
of Plants and Animals". In Ënpublished Class Lecture Notes, Ëniv. of Wisconsin, Madison.V
÷V Bradshaw, A.D. 1972. Some of the Evolutionary Consequences of Being a Plant. Evol. Biol.
5:25-47.V
÷V Burnham K‰ (2001) Habitat related error in estimating temperature through leaf margins
in a humid tropical forest. Amer. ‰ourn. Botany 88: 1096 - 1102.V
÷V Bynum MR, SMith WK (2001) Floral movements in response to thunderstorms improve
reproductive efforts in the alpine species 
? 
?(Gentianaceae) Amer. ‰ourn.
of Botany 88: 1088 - 1095. V
÷V Cain, ML, DA Dudle, ‰P Evans. 1996. Spatial models of foraging in clonal plant species.
American ‰ournal of Botany 83: 76 -85.V
÷V Cook, R.B. 1981. Plant Parenthood. Natural History. ‰uly.V
÷V Darley, W.M. 1990. The essence of "plantness" Amer. Biol. Teacher 52:354-356.V
÷V Dicks, L. 1999/2000. The holly and the ivy. New Scientist pp 27 - 29. ‰an 25, 1999 - 1 ‰an,
2000.V
÷V Dirzo, R. and ‰. Sarukhan, eds. 1984. Perspectives on Plant Population Ecology. Sinauer,
MS.V
÷V Flannercy, MC (1999) Seeing plants a little more clearly. Amer. Biol Teach 61: 303 -307.V
÷V Galen, C (1999) Sun stalkers. Natural History May. pp 49 - 51.V
÷V Hershey, David (2002) Plants are indeed intelligent. Available on-line.V
÷V Hershey, David (2002) Plant blindness: "We have met the enemy and he is us." Plant
Science Bulletin 48: 78 - 85.V
÷V Hoekstra, B (2000) Plant blindness - The ultimate challenge to botanists. American Biology
Teacher 62: 82-83.V
÷V Horn, H.S. 1970. Adaptive Geometry of Trees. Princeton Ëniv. Press, N‰.V
÷V Hunter AF, Aarssen LW (1988) Plants helping plants. BioScience 38: 34-40.V
÷V Hutchings, M. & A. Slade. 1988. Foraging for resources and the structure of plants. Plants
Today 1 (1): 28.V
÷V Niklas, K. 1989. The cellular mechanics of plants. Amer. Sci. 77:344.V
÷V Niklas, K. (1994) One giant step for life. Natural History. ‰une, pp 22 - 25.V
÷V Niklas, K. 1996. How to build a tree. Natural History. February. 49-52.V
÷V Norman, A.G. 1963. The Ëniqueness of Plants. BioScience.V
÷V McMahon, T.A. and ‰.T. Bonner. 1983. On Size and Life. Scientific American Books, Inc.,
NY.V
÷V Milius, S (2001) Chemical SOS not just for farm, lab plants. Science News 159: 166.V
÷V Milius, S (2001) Phew! Orchid perfume turns revolting. Science News 159: 174.V
÷V Oxlade, EL 1998. An Investigation of Leaf mosaics. ‰. Biol. Educ. 32: 34 - 40.V
÷V Pate ‰S (1993) Plants stand on stilt's above Australia's scorching sand. Natural History
August pp 36 -37.V
÷V Patrusky, B. 1991. Drosophila Botanica. Mosaic 22: 32-43.V
÷V Patrusky, B. 1983. Plants in their own behalf. Mosaic. March/April.V
÷V Ray, ‰r., T.S. 1975. Slow-motion world of plant 'behavior' visible in rain forest. Smithsonian 9
(12): 121.V
÷V Sabelis MW, ‰anssen A, Kant MR (2001) The enemy of my enemy is my ally. Science 291:
2104 - 2105.V
÷V Salzman, Amy. 1985. Habitat selection in a clonal plant. Science 228: 603-604.V
÷V Saunders, F (1997) Keep the aspirin flying. DiscoverV
÷V Schultz, ‰.C. 1983. Tree tactics. Natural History. May.V
÷V Silvertown, ‰. and D. M. Gordon. 1989. A framework for plant behavior. Ann. Rev. Ecol.
Syst. 20: 349-366.V
÷V Sutherland, W.‰. & RA Stillman. 1988. The foraging tactics of plants. Oikos 52:239-244.V
 ÷V Thomas, SC and FA Bazzaz (1996) Elevated CO2 and leaf shape: Are dandelions getting
toothier? American ‰ournal of Botany 83: 106 - 111.V
÷V Tooley, Chris. 1989. Investigating morphological changes induced by light in the clonal
herb 
 



. ‰ournal Biol. Educ. 23:263-264.V
÷V Trewavas, A (2002) Mindless mastery Nature 415: 841.V
÷V Trewavas, A (2003) Aspects of Plant Intelligence. Annals of Botany 92: 1 - 20.V
÷V VanValen, L. 1978. Arborescent animals and other colonoids. 276 : 318.V
÷V Wandersee ‰H, Schussler EE (1999) Preventing plant blindness Amer. Biol. Teach 61: 82 -
86.V
÷V White, ‰. 1979. The plant as a metapopulation. Ann Rev. Ecol. Syst. 10: 109-45.V
÷V Wiebe, H. 1978. The significance of plant vacuoles. BioScience 28:327.V
÷V Wijesinghe DK, Whigham DF (2001) Nutrient foraging in woodlands herbs: a comparison
of 3 species of Ë ? (Liliaceae) with contrasting belowground strategies Amer ‰.
Botany 88: 1071 - 1079.V
÷V Wolkomir, R. 1984. It's a jungle out there. National Wildlife.V
÷V Waisel, Y., A. Eshel, V. Kafkofs. 1991. Plant Roots. The Hidden Half. Marcel Dekker, Inc., NY.V

 
( c-


 V

c

 : Concept mapping is an important learning tool that can be used in a variety of
situations. A concept map is a visual representation of how ideas (=concepts or propositions)
are interconnected. To make a concept map, prepare a list of the main
ideas/concepts/propositions you are interested in. Then, select the most inclusive (largest) of
these and write it in a circle at the top of your paper. Then, begin connecting the remaining
ideas by writing them below and connecting the ideas with a line or arrow. Label the line to
represent the interconnection. In class, I will show you an example making a concept map to
the following ideas in the Plant Way of Life notes. V

÷V Motility is not required by an autotrophV

÷V Plants are photosynthetic autotrophsV
÷V Sedentary organisms need to be able to position themselvesV
÷V Sedentary organisms need to be able to reproduceV
÷V Sedentary organisms need to exploit the environmentV
÷V Sedentary organisms need to protect themselvesV
÷V Sedentary organisms need to respond to the environmentV
÷V Specialized structures required for autotrophismV
V

A. Plasma or Cell membrane

Cell boundary; selectively permeable; bilayer of phospholipids with inserted protein.
Phospholipids are unique molecules - they are amphipathic, meaning that they have both
hydrophilic and hydrophobic regions. They have a glycerol backbone; one of the hydroxyls is
bonded to a phosphate and another charged group, the other two hydroxyls are esterified to
fatty acids. The fatty acids range in length from C14 - C24. One fatty acid is usually unsaturated
and the other is saturated. The unsaturated fatty acid is kinked which helps to keep plant cell
membranes fluid at cool temperatures. As a result plant phospholipids usually have a higher
degree of unsaturation than animals. Hydrophobic interactions between the tail regions of the
phospholipids hold the membrane together. Some proteins are found: (1) just on the outside or
inside surfaces of the membrane (peripheral proteins - non-covalent interactions and anchored
proteins - covalently bound to lipids, etc); or (2) embedded in the membrane (integral protein),
many of which span the membrane (trans-membrane proteins). Hydrophilic regions of the
integral proteins are oriented to the outside of the membrane whereas hydrophobic regions are
embedded within the phospholipid bilayer. Lipid soluble materials can readily pass through but
charged or ionized substances (hydrophilic) pass through very slowly, if at all. The function of the
membrane is to: (1) regulate traffic; (2) separate the internal from external environment; (3)
serve as a platform on which some reactions can occur; (4) participate in some reactions (?
,
the membrane components are important intermediates or enzymes); and (5) provide some
structural integrity for the cell.V

B. Nucleus
The cell "brain". Surrounded by a double membrane (two phospholipid bilayers) - the nuclear
membrane. Has pores. The structure of the pores is complex comprised of more than 100
proteins. The pore opening is surrounding by a series of proteins and these are attached to a
series radial spokes. Nucleoplasm - matrix within nucleus. DNA, which is found in the nucleus,
may be condensed into chromosomes or not (chromatin). There may be one or more nucleolus
(site of ribosome production). The nucleus is 5-20 mm in diameter. There is a layer of
intermediate filaments (see below) just inside the nuclear envelope; called the nuclear lamina.V

C. Cytoplasm/cytosol
The cytosol is the gel-like matrix within the cell in which the other structures are embedded.
The cytoplasm refers to the cell contents inside the membrane.V

D. Mitochondria
These organelles, like the nucleus and plastids, are double-membrane bound. They vary in
shape from tubular (like sausages) to spherical. They reproduce by fission, have their own
ribosomes and DNA (a circular loop like prokaryotic cells). The inner membrane has a larger
surface area so it must be folded into finger-like projections (called cristae) to fit inside the outer
membrane. Mitochondria are found in all eukaryotic cells. They are the sites of cellular
respiration - process by which energy is released from fuels such as sugar. The mitochondria are
the power plant of the cell. They are small (1-5mm) and generally numerous (500-2000 per cell).
A popular misconception is that "plants have chloroplasts, animals have mitochondria." Plant
cells, at least green plant cells (?
, leaf cells), have both. Root cells only have mitochondria.
Mitochondrial DNA which comprises about 200 kbases, codes for some of the genes required for
cellular respiration including the 70S ribosomes and components of the electron transport
system. The inner membrane differs from the plasma membrane in that it has a higher protein
content (70%) and unique phospholipids (?
., cardiolipin).V

E. Ribosome
Sites of protein synthesis (translation). Two subunits; one large and the other small. Made in the
nucleus from rRNA and protein. Ribosomes are tiny (0.25mm) and numerous (5 - 50 X 1010 per
cell). Since ribosomes are not surrounded by a membrane, they are not considered to be "true"
organelles. Some ribosomes are 'free' (produce proteins that remain in the cell) while others are
attached to the ER (produce proteins for export). To export a protein, the mRNA and subunits of
the ribosome bind together. A signal recognition particle (SRP) binds to specific amino acids in
the newly forming protein. The SRP, which is bound to the protein/mRNA/ribosome, then binds
to a receptor in the ER membrane. As the protein is made it is released into the lumen of the ER
and the SRP sequence of the protein is snipped off.V

F. Endoplasmic reticulum
A series of membranous tubes and sacs (cisternae) that run throughout the cell. Rough ER has
ribosomes associated with it and is laminar while smooth ER lacks ribosomes and is tubular.
Totally man. The ER has several functions including: (1) synthesis of lipids and membranes
(smooth ER); (2) serving as a site for the synthesis of proteins by the ribosomes (rough ER); (3)
transport (a type of cell 'highway' system); and (4) support. V

G. Peroxisomes
Membrane sac containing enzymes for metabolizing waste products from photosynthesis, fats
and amino acids. Hydrogen peroxide is a product of metabolism in peroxisomes. Catalase,
which breaks down the peroxide is also present and serves as a marker enzyme for these
organelles.V

H. Glyoxisomes
Membrane sac containing enzymes for fat metabolism. Especially common in seeds. Also
contain catalase.V

I. Golgi apparatus
Pancake- or pita bread-like stack of membranes. Particularly important in cells that produce
materials for export (secretion). They have a polarity (cis - imports vesicles from ER; trans - exports
vesicles). The Golgi is the site of processing and packaging cellular components. Vesicles
containing proteins, lipids and other materials, fuse with the Golgi (cis side), release contents,
which then get processed, sorted, packaged and re-released from the other side (trans face).
The Golgi also is active in synthesizing many cell components, especially carbohydrates and is
involved in tagging proteins with carbohydrates and other side chains for sorting them to their
final destination. There are two models for the movement of materials thru the Golgi: (1) Vesicle
Migration Model - in this case a vesicle fuses with the cis side, then ultimately a new vesicle pinch
off this stack and fuses with teh next one, and so on, until the vesicle reaches the trans side; and
(2) Escalator Model - a vesicle fuses with the cis side and never leaves this stack. Rather, the
stack on the trans side releases vesicles and then disintegrates while a new stack forms on the cis
side. The original vesicle is now in the "second" stack, and so on until it reaches the trans side.
Vesicles are tagged with various proteins to direct them to the appropriate locations.V

‰. Microtubules
Hollow tubes made of a mix of alpha and beta tubulin, which are globular proteins. There are
essentially 13 columns of proteins. The tubes are about 25 mm in diameter. Microtubules are
involved in the cell cytoskeleton (for support), cell movements (cilia, flagella) and cell division
(spindle). Assembly of microtubules is prevented by colchicine, an inhibitor derived
from  bulbs. Low calcium concentration favors the formation of microtubulesV

K. Microfilaments
Protein strands. Solid. Made from G-actin. Involved with the cell cytoskeleton. Main function is
support. They are about 7 nm in diameter.V

L. Intermediate filaments
These are similar to microfilaments. They are also made of protein in the keratin family; about
10 nm diameter.V

M. Cilia/flagella
For cellular movements. Cilia = many, short; flagella = few, long. Have a 9+2 arrangement of
microtubules. Prongs on the tubules are ATPases (dynein) to hydrolyze ATP to provide energy for
movement. These are not particularly common in plants.V
N. The Cytomembrane system
The membranous organelles (ER, vesicles, golgi, cell membrane) comprise a group of
organelles that cooperate and function together. For example, imagine the synthesis of
cellulose in the cell wall of a plant. Cellulose synthesis requires the enzyme cellulose synthase.
Ribosomes (rough ER)  makes enzyme  passes through RER to smooth ER  packaged into a
vesicle  pinches off  to golgi (cis face)  processed  repackaged into vesicle  pinches
off (trans face)  cell membrane  fuses  releases contents  cellulose synthase makes
cellulose.V

O. Others V

÷V Microbodies - a general term for any single membrane bound organelle typically derived
from the ER that contain catalase and/or hydrogen peroxide producing enzymes. This
includes the peroxisomes and glyoxisomes;V
÷V Microsomes - a "biochemical" term for the fraction that is obtained from high speed
centrifugation of cell homogenates. It includes membrane fragments and ribosomes.V
÷V Oleosome (spherosomes) - these are lipid bodies. The coolest thing about them is that
they are encased by one-half of a cell membrane; in other words, just a single
phospholipid layer.V


c# 

2 &


'


Plastids are double membrane-bound organelles in plants. They contain their own DNA (in
nucleoid region) and ribosomes. They are semi-autonomous and reproduce by fission similar to
the division process in prokaryotes. If plastids only arise from other plastids and can·t be built
"from scratch", then where do they come from? The egg. Plastids are inherited cytoplasmically,
primarily through the female - however, there are examples of paternal inheritance of plastids.
The plastid DNA carries several genes including the large subunit of rubisco and those for
resistance to some herbicides. The chemistry of the membranes differs from the plasma
membrane - plastid membranes are comprised of glycosylglycerides rather than phospholipids
(the phosphate in the polar head group in glycosylglycerides is replaced with galactose or a
related sugar).V

There are several types of plastids including:V

V Proplastids - small, precursors to the other plastid types, found in young cells, actively
growing tissues;V
è
V Chloroplasts - sites of photosynthesis (energy capture). They contain photosynthetic
pigments including chlorophyll, carotenes and xanthophylls. The chloroplast is packed
with membranes, called thylakoids. The thylakoids may be stacked into pancake- like
piles called grana (granum, singular). The "liquidy" material in the chloroplast is the
stroma. A chloroplast is from 5-20 Jm in diameter and there are usually 50-200 per cell.
The chloroplast genome has about 145 Kbase pairs, it is smaller than that of the
mitochondria (200 kbases). About 1/3 of the total cell DNA is extranuclear (in the
chloroplasts and mitochondria);V

V Chromoplasts - non-photosynthetic, colored plastids; give some fruits (tomatoes, carrots)
and flowers their color;V

V Amyloplasts - colorless, starch-storing plastids;V

V Leucoplast - another term for amyloplast;V
 
V Etioplast - plastid whose development into a chloroplast has been arrested (stopped).
These contain a dark crystalline body, prolamellar body, which is essentially a cluster of
thylakoids in a somewhat tubular form.V

Plastids can dedifferentiate and convert from one form into another. For example, think about
the ripening processing in tomato. Initially, green tomatoes have oodles of chloroplasts which
then begin to accumulate lycopene (red) and become chromoplasts. Ësually you find only
chromoplasts or chloroplasts in a cell, but not both.V

# 

2 &


'

This is the large, central cavity containing fluid, called cell sap, found in plant cells. The
vacuole is surrounded by a membrane (tonoplast). Back to the water balloon in the box model -
imagine the vacuole to be analogous to another water balloon inside our protoplast balloon.
This water balloon is a separate entity that can be physically removed from the cell. The vacuole
is penetrated by strands of cytoplasm - transvacuolar strands.V

The tonoplast and plasma membrane have different properties such as thickness (tonoplast
thicker).V

Virtually every plant cell has a large, well-developed vacuole that makes up to 90% or more of
the cell volume. Wow! Meristematic and embryonic cells are exceptions. Young tissues have
many small vacuoles. As the cell grows the vacuoles expand and eventually coalesce. These
small vacuoles appear to be derived from the Golgi.V

The central vacuole contains water, ions, organic acids, sugars, enzymes, and a variety of
secondary metabolites. Among the hydrolytic enzymes are proteases (digest protein),
ribonucleases (digest RNA) and glycosidases (break links between monosaccharides). These
enzymes are typically not used for recycling cellular components but rather leak out on cell
senescence. There are smaller lytic vacuoles, which contain digestive enzymes, that are used
for this purpose. Another type of vacuole, protein bodies, are vacuoles that store proteins. V

Why do plants cells have a large central vacuole? Important roles of the vacuoles are:V

A. Energetically efficient means to increase surface to volume ratio in the dendritic growth form
Since 90% of the cell volume is vacuole, therefore 90% of the cell is water, which is relatively
cheap in metabolic terms. Thus, it allows plants to get big with a minimal energy investment.
Plants are particularly 'smart,' since, the cell wall, which comprises much of the remaining 10% or
so of the cell is a polymer of glucose. Cellulose is a great bargain! It is stronger and cheaper than
comparable polymers. Let·s compare:V

compound/production tensile
value (weight strength
polymerV
product/weight (newton
glucose to make)V m2 x 109)V

celluloseV 0.83V 30V

collagenV 0.40V 2V

silkV 0.40V 10V

B. Water storage
Probably a minor role; mostly in succulentsV

C. Waste disposal
The vacuole can be considered the cell cesspool. It contains many secondary metabolites
including a variety of hydrolytic enzymes like the "marker enzyme" alpha mannosidase. In this
regard, the vacuole is analogous to the lysosome.V

Let·s consider differences between plants and animals in terms of wastes. Plants have little
waste. Their nutrients are in dilute form, they use them efficiently and hence, there is little left
over. Plants remind me of my Dad who was a Marine during WWII. He said that the Marine
philosophy at dinner was to "eat all you want, but all that you take." Plants do just that - most
everything they "ingest" they use. The minimal wastes plants produce can be stored in the cells in
the vacuoles (or disposed of in other ways - released as a gas into the air, leached out of roots
or leaves).V

In contrast, animals rely on nutrients in a concentrated form. They are rarely selective about
what they eat; much un-usable material is ingested along with the "good stuff". Thus, they have
a large quantity of waste and needed to evolve specialized digestive/excretory systems to get
rid of it. Further, animals don·t have the luxury of having bulky vacuoles and internally storing
wastes because these adaptations would limit motility.V

Some related ideas:V

V Heterotrophic plants (like carnivorous plants) have excessive wastes. The plant
strategy is usually to discard the structure with the wastes (?
., insect remains) and
produce a new one. For example, pitcher plant leaves fill up with insect parts and
the plant produces a new batch of leaves the next season. Plants are able to do
this because of their architectural design; V
è
V Humans start out life very much like a plant. Newborns are non-motile and breast-
fed. Breast milk is dilute and nutritious. And, it is highly digestible leaving relatively
few leftovers. Hence, newborn diapers are relatively innocuous. However, as
babies become more animal-like and begin solid food, diapers are best left to
the strong of stomach; V

V Cell walls - have been suggested that the cell wall first evolved as a site for waste
disposal for excess carbon. Whatdayathink?V

D. pH regulation
The vacuole is a pool to dump excess protons. There is an active proton pump in the
tonoplast. The cell sap has a pH of 2-5.7, whereas the cytosol is ca. 7.0.V

E. Storage of essential ions

Ions are pumped into the vacuole for water balance. Potassium and calcium. in particular,
are stored in the vacuole.V

F. Cell enlargement
Cell growth requires some force to allow for the cell to increase in size. Water pressure provides
the force and it moves into the vacuole. For example, root hair enlargement is due entirely to
vacuolar enlargement.V
G. Facilitates diffusion
The cytosol essentially forms a thin coating around the large vacuole which in effect,
increases the surface-to-volume ratio of the cytoplasm. It provides easier access and shorter
diffusion distances between any part of the cytoplasm and the external environment of the cell.
This can be particularly important for chloroplasts.V

H. Structural support
The vacuole helps to maintain turgor pressure in plant cells due to the opposing forces of
tensile strength of the wall vs. compression strength of water.V

c
 3: click hereV

ccΠ:V

÷V Wiebe, H.H. 1978. The Significance of Plant Vacuoles. ??


28: 327-331.V
÷V The Annual Review of Plant Physiology and Plant Molecular Biology is an excellent source
of references.V
÷V The Nuclear Family - article about the origin of mitochondria and chloroplasts. Scientific
American. November 19, 2001. V



'

+4 
 V

c4 
 




:









?





? ?  V

V maintaining/determining cell shape (analogous to an external skeleton for every cell).

Since protoplasts are invariably round, this is good evidence that the wall ultimately
determines the shape of plant cells.
V
è
V Support and mechanical strength (allows plants to get tall, hold out thin leaves to obtain
light)
V

V prevents the cell membrane from bursting in a hypotonic medium (?
 resists water
pressure)
V

V controls the rate and direction of cell growth and regulates cell volume
V

V ultimately responsible for the plant architectural design and controlling plant
morphogenesis since the wall dictates that plants develop by cell addition (not cell
migration)
V

V has a metabolic role (?

some of the proteins in the wall are enzymes for transport,
secretion)
V

V physical barrier to: (a) pathogens; and (b) water in suberized cells. However, remember
that the wall is very porous and allows the free passage of small molecules, including
proteins up to 60,000 MW. The pores are about 4 nm (Tepfert & Taylor 1987)
V
 
V carbohydrate storage - the components of the wall can be reused in other metabolic
processes (especially in seeds). Thus, in one sense the wall serves as a storage repository
for carbohydrates
V

V signaling - fragments of wall, called oligosaccharins, act as hormones. Oligosaccharins,
which can result from normal development or pathogen attack, serve a variety of
functions including: (a) stimulate ethylene synthesis; (b) induce phytoalexin (defense
chemicals produced in response to a fungal/bacterial infection) synthesis; (c) induce
chitinase and other enzymes; (d) increase cytoplasmic calcium levels and (d) cause an
"oxidative burst". This burst produces hydrogen peroxide, superoxide and other active
oxygen species that attack the pathogen directly or cause increased cross-links in the
wall making the wall harder to penetrate.

Let's look at how this system works. Consider a pathogenic fungus like m
  .
In contact with the host plant the fungus releases enzymes such as pectinase that break
down plant wall components into oligosaccharins. The oligosaccharins stimulate the
oxidative burst and phytoalexin synthesis, both which will deter the advance of the
fungus. In addition, the oligosaccharins stimulate chitinase and glucanase production in
the plant. These are released and begin to digest the fungal wall. Fragments of fungal
wall also act as oligosaccharins in the plant to further induce phytoalexin synthesis. Cool!
V

Vrecognition responses - for example: (a) the wall of roots of legumes is important in the
nitrogen-fixing bacteria colonizing the root to form nodules; and (b) pollen-style
interactions are mediated by wall chemistry.
V

Veconomic products - cell walls are important for products such as paper, wood, fiber,
energy, shelter, and even roughage in our diet.V

cc

  
' 


The main ingredient in cell walls are polysaccharides (or complex carbohydrates or complex
sugars) which are built from monosaccharides (or simple sugars). Eleven different
monosaccharides are common in these polysaccharides including glucose and galactose.
Carbohydrates are good building blocks because they can produce a nearly infinite variety of
structures. There are a variety of other components in the wall including protein, and lignin. Let's
look at these wall components in more detail:V

A. Cellulose
Ô1,4-glucan (

 ?

?
. Made of as many as 25,000 individual glucose
molecules. Every other molecule (called residues) is "upside down".     (glucose-glucose
disaccharide) is the basic building block. Cellulose readily forms hydrogen bonds with itself
(
!
  
ÿ! ) and with other cellulose chains (
!
  
ÿ! ). A cellulose
chain will form hydrogen bonds with about 36 other chains to yield a


. This is somewhat
analogous to the formation of a thick rope from thin fibers. Microfibrils are 5-12 nm wide and give
the wall strength - they have a tensile strength equivalent to steel. Some regions of the
microfibrils are highly crystalline while others are more "amorphous". V

B. Cross-linking glycans (=Hemicellulose)

Diverse group of carbohydrates that used to be called hemicellulose. Characterized by
being soluble in strong alkali. They are linear (straight), flat, with a Ô-1,4 backbone and relatively
short side chains. Two common types include xyloglucans and glucuronarabinoxylans. Other less
common ones include glucomannans, galactoglucomannans, and galactomannans. The main
feature of this group is that they don·t aggregate with themselves - in other words, they don·t
form microfibrils. However, they form hydrogen bonds with cellulose and hence the reason they
are called "? !? 

 ". There may be a fucose sugar at the end of the side chains
which may help keep the molecules planar by interacting with other regions of the chain.V

C. Pectic polysaccharides
These are extracted from the wall with hot water or dilute acid or calcium chelators (like
EDTA). They are the easiest constituents to remove from the wall. They form gels (?
 used in jelly
making). They are also a diverse group of polysaccharides and are particularly rich in
galacturonic acid (galacturonans = pectic acids). They are polymers of primarily Ô 1,4
galacturonans (=polygalacturonans) are called homogalacturons (HGA) and are particularly
common. These are helical in shape. Divalent cations, like calcium, also form cross-linkages to
join adjacent polymers creating a gel. Pectic polysaccharides can also be cross-linked by
dihydrocinnamic or diferulic acids. The HGA's (galacturonans) are initially secreted from the
golgi as methylated polymers; the methyl groups are removed by pectin methylesterase to
initiate calcium binding.

Other pectic acids include Rhamnogalacturonan II (RGII) which features rhamnose and
galacturonic acid in combination with a large diversity of other sugars in varying linkages.
Dimers of RGII can be cross-linked by boron atoms linked to apiose sugars in a side chain.

Although most pectic polysaccharides are acidic, others are composed of neutral sugars
including arabinans and galactans. The pectic polysaccharides serve a variety of functions
including determining wall porosity, providing a charged wall surface for cell-cell adhesion - or in
other words gluing cells together (i.e,. middle lamella), cell-cell recognition, pathogen
recognition and others.V

D. Protein
Wall proteins are typically glycoproteins (polypeptide backbone with carbohydrate side
chains). The proteins are particularly rich in the amino acids hydroxyproline (hydroxyproline-rich
glycoprotein, HPRG), proline (proline-rich protein, PRP), and glycine (glycine-rich protein, GRP).
These proteins form rods (HRGP, PRP) or beta-pleated sheets (GRP). ©  is a well-studied
HRGP. HRGP is induced by wounding and pathogen attack. The wall proteins also have a
structural role since: (1) the amino acids are characteristic of other structural proteins such as
collagen; and (2) to extract the protein from the wall requires destructive conditions. Protein
appears to be cross-linked to pectic substances and may have sites for lignification. The proteins
may serve as the scaffolding used to construct the other wall components.

Another group of wall proteins are heavily glycosylated with arabinose and galactose. These
arabinogalactan proteins, or AGP's, seem to be tissue specific and may function in cell signaling.
They may be important in embryogenesis and growth and guidance of the pollen tube.V

E. Lignin
Polymer of phenolics, especially phenylpropanoids. Lignin is primarily a strengthening agent in
the wall. It also resists fungal/pathogen attack.V

F. Suberin, wax, cutin

A variety of lipids are associated with the wall for strength and waterproofing.V

G. Water
The wall is largely hydrated and comprised of between 75-80% water. This is responsible for
some of the wall properties. For example, hydrated walls have greater flexibility and extensibility
than non-hydrated walls.V

ccc(
 
 



- 




 

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 V

V Middle lamella - outermost layer, glue that binds adjacent cells, composed primarily of
pectic polysaccharides.V
è
V Primary wall - wall deposited by cells before and during active growth. The primary wall
of cultured sycamore cells is comprised of pectic polysaccharides (ca. 30%), cross-linking
glycans (hemicellulose; ca 25%), cellulose (15-30%) and protein (ca. 20%) (see Darvill et
al, 1980). The actual content of the wall components varies with species and age. All
plant cells have a middle lamella and primary wall.V

V Secondary Wall - some cells deposit additional layers inside the primary wall. This occurs
after growth stops or when the cells begins to differentiate (specialize). The secondary
wall is mainly for support and is comprised primarily of cellulose and lignin. Often can
distinguish distinct layers, S1, S2 and S3 - which differ in the orientation, or direction, of the
cellulose microfibrils.V

c 
 




The wall is similar to a tire that has a series of steel belts or cords embedded in an amorphous
matrix of rubber. In the plant cell wall, the "cords" are analogous to the cellulose microfibrils and
they provide the structural strength of the wall. The matrix of the wall is analogous to the rubber
in the tire and is comprised of non-cellulosic wall components. How are the various wall polymers
arranged? It appears that:V

V cross-linking glycans (hemicellulosic polysaccharides) are hydrogen bonded to the

cellulose microfibrilsV
è
V cross-linking glycans may also be entrapped inside cellulose microfibrils as they formV

V the different types of pectic polysaccharides are covalently bonded to one anotherV

V calcium bridges link pectic acidsV

V connections between the protein and other wall polymers are still not clearV

V pectic polysaccharides and cross-linking glycans interact V

V cross-linking glycans are linked by ferulic acid bridges or boronV



4

The cell wall is made during cell division when the cell plate is formed between daughter cell
nuclei. The cell plate forms from a series of vesicles produced by the golgi apparatus. The
vesicles migrate along the cytoskeleton and move to the cell equator. The vesicles coalesce
and dump their contents. The membranes of the vesicle become the new cell membrane. The
golgi synthesizes the non-cellulosic polysaccharides. At first, the golgi vesicles contain mostly
pectic polysaccharides that are used to build the middle lamella. As the wall is deposited, other
non-cellulosic polysaccharides are made in the golgi and transported to the growing wall.V

Cellulose is made at the cell surface. The process is catalyzed by the enzyme cellulose
synthase that occurs in a rosette complex in the membrane. Cellulose synthase, which is initially
made in by the ribosomes (rough ER) and move from the ER ń vesicles ń
golgi ń vesicle ń cell membrane. The enzyme apparently has two catalytic sites that transfer
two glucoses at a time (?
, cellobiose) from ËDP-glucose to the growing cellulose chain.
Sucrose may supply the glucose that binds to the ËDP. Wall protein is presumably incorporated
into the wall in a similar fashion.V

Remember that the wall is made from the outside in. Thus, as the wall gets thicker the lumen
(space within the wall) gets smaller.V

Exactly how the wall components join together to form the wall once they are in place is not
completely understood. Two methods seem likely: V

V self assembly. This means that the wall components spontaneously aggregate; and
V
è
V enzymatic assembly ² various enzymatic reactions (XET) are designed for wall assembly.
For example, one group of enzymes "stitches" xylans together in the wall to form long
chains. Oxidases may catalyze additional cross-linking between wall components and
pectin methyl esterase may play an important role (see below).V

c
 

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How can the wall be strong (it must withstand pressures of 100 MPa!), yet still allow for
expansion? Good question, eh? The answer requires that the wall:V

A. Be capable of expansion
In other words, only cells with primary walls are capable of growth since the formation of the
secondary wall precludes further expansion of the cell. The sequence of microfibril orientation
changes during development. Initially the microfibrils are laid down somewhat randomly
(isotropically). Such a cell can expand in any direction. As the cell matures, most microfibrils are
laid down laterally, like the hoops of a barrel, which restricts lateral growth but permits growth in
length. As the cell elongates the microfibrils take on an overlapping cross-hatched pattern,
similar to fiberglass. This occurs because the cell expands like a slinky - the width of the cell
doesn't change by the microfibrils become aligned in the direction of growth just like the spring.
This overlapping of microfibrils, which is strong and lightweight, prohibits further expansion. V

But, what determines the orientation of the microfibrils? They are correlated with the direction
of the microtubules in the cell. Evidence: treating a cell with colchicine or oryzalin (which inhibit
microtubule formation) destroys the orientation of the microfibrils. The microtubules apparently
direct the cellulose synthesizing enzymes to the plasma membrane.V

In addition to cellulose microfibril orientation, mature walls apparently loose their ability to
expand because the wall components become resistant to loosening-activities. This would
occur if there were increased cross-linking between wall components during maturation. This
would result from:V

V producing wall polysaccharides in a form that makes tighter complexes with cellulose or
other materialsV
è
V increasing the lignin in the wall would increase cross-links between polymersV

V de-esterifying the pectic acids would increase calcium bridges;V

B. Loosening (or Stress relaxation) the wall at the appropriate time

Even though the microfibrils may be in the proper position to permit loosening, the wall is still
rather strong. Recall that our wall model proposed strong (covalent) and weak (hydrogen
bonds) links between the wall components. When the wall is loosened, weak bonds are
temporarily broken to allow the wall components to slide or creep past one another. So, how is
the wall temporarily loosened?V

1. Protons are the primary wall loosening factor (%3
 
). This idea was first
proposed by David Rayle and R. Cleland in 1970. Some evidence:V

÷V acid buffers stimulate elongation and rapid responses 5-15 min even in non-living
tissues (Evans,1974);V
÷V acid secretion is associated with sites of cell elongation (see Evans & Mulkey,
1981)V
÷V Fusicoccin, a diterpene glycoside extracted from a fungus, stimulates proton
secretion (activates a H+/K+ pump) and stimulates elongation.V

2. Mechanism of proton action: Protons stimulate wall loosening by:V

÷V disrupting acid-labile bonds such as H-bonds and calcium bridges; andV

÷V enhancing the activity of enzymes that break wall cross-links including H-bonds
and calcium bridges. Evidence for the enzyme involvement includes: (1) when
primary walls are heated or treated with protein denaturing agents they can't be
"loosened" by acid; and (2) adding proteins extracted from growing walls to
heat-treated walls restores the acid response.

Expansins ² appear to be the primary wall-loosening enzymes. This class of

proteins are activated by low pH and break the hydrogen bonds between
cellulose and the cross-linking glycans. Other candidates for enzymes involved
include: (1) pectin methyl esterase which would break the calcium bridges
between pectins by esterifying the carboxyl groups; and (2) hydrolases ² which
would hydrolyze the cross-linking glycans (hemicelluloses). For example,
xyloglucan endotransglycosylase (XET) has been shown to cleave cross-linking
glycans that could allow slippage of the wall componentsV

3. The acid effect is induced by indole-3-acetic acid (IAA, auxin), one of the major plant
hormones. IAA stimulates proton excretion and cell growth/elongation. Evidence:V

÷V peeled coleoptiles + IAA  medium acidic; peeled coleoptiles + water  not

acidic; andV
÷V flooding auxin-treated tissue with neutral buffers prevents the growth response.V

4. Mechanism of Auxin Action ² How does auxin stimulate proton excretion and wall
elongation? There are two ideas:V

 
6: Auxin activates pre-existing H+-ATPase pump proteins in the cell membrane. These
proteins transport protons from the protoplast into the wall. Auxin probably first binds to a
receptor molecule and this complex then actives the pump. This process is active - thus the
pump requires ATP. Evidence: ATP stimulated acidification is observed soon after auxin
treatment.V

 
7- Auxin stimulates transcription and translation. Transcription/translation (protein

synthesis) would be required to produce proton pump proteins (a wonderful alliteration),
respiratory enzymes to provide ATP to power the process; and even enzymes for the synthesis of
wall components and cell solutes (see C. & D. below). Evidence for the involvement of
transcription/translation:V

÷V Nooden (1968) found that artichoke disks increased in size when

incubated with IAA but that the addition of antimycin (a protein synthesis
inhibitor) prevented this response;V
÷V soybean hypocotyls incubated with 2,4-D (an analog of IAA) produce at
least 3 new polypeptides within three hours (Zurfluh & Guilfoyle, 1980);V
÷V ?
? translation of mRNA occurs within 15 minutes of IAA treatmentV
÷V The proton effect is short-lived. Cell elongation stops 30-60 minutes after
acidification. Continuous elongation requires longer term metabolic
changes such as protein synthesis.V

C. Wall synthesis occursV

As the cell grows, wall synthesis needs to occur. Think about the color of a balloon as it is
blown up - it gets lighter in color as the balloon gets larger because the thickness of the balloon
decreases as it expands and stretches. Ësing this logic, we expect that plant cells should
become thinner as they expand. Right? Wrong - cell walls remain a relatively uniform thickness
throughout cell growth. Thus, we can conclude that new wall material must be made during cell
elongation.V

D. Enhanced solute synthesisV

The solute concentration of the cell remains constant during cell enlargement. This suggests
that solutes are being synthesized since the volume of the cell is increasing. Maintaining a high
solute concentration is necessary to allow for water uptake.V

E. Lock wall in place after expansion is complete

Once wall elongation is completed, the cell needs to "lock it" in place. This likely happens as
the temporary bonds that were broken reform, and due to increased interactions (including
enzymatic) between wall molecules.V

F. Water Ëptake/PressureV


-V

÷V Carpita, N, McCann (2000) Cell Walls. Chapter 2. In ?

?

%


?



m 

Buchanan, BB, Gruissem W, ‰ones, RL. eds. American Society of Plant Biology,
Beltsville, MD. This is a great article!V

ΠV

÷V Albersheim, P. 1975. The Walls of Growing Plant Cells. Sci. Amer. 232: 81-95.V
÷V Albersheim, P. 1985. Oligosaccharins. Sci. Amer. 253: 58.V
÷V Brett, C. T. and ‰.R.Hillman. 1985. Biochemistry of Plant Cell Walls. Cambridge Ëniversity
Press, NY.V
÷V Brett, C. & K. Waldron. 1996. Physiology and Biochemistry of Plant Cell Walls. 2nd edn.
Chapman & Hall, NY.V
÷V Cosgrove, D. 1986. Cell growth. ARPP 37: 377V
÷V Delmer, D. 1987. Cellulose biosynthesis. ARPP 38: 259V
÷V Fry, S.C. 1989. Dissecting the complexity of the plant cell wall. Plants Today 2: 126-132.V
 ÷V Mulkey, T.‰., K.M. Kuzmanoff and M.L. Evans. 1981. The agar-dye method for visualizing
acid efflux paterns during tropistic curvatures. What's New in Plant Physiol. 12:9-12.V
÷V Preston, R.D. 1979. Polysaccharide conformation and cell wall formation. ARPP 30:50.V
÷V Taiz, L. 1984. Plant cell expansion. ARPP 35: 585-647V


"8  #V



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L. ‰. Henderson
 "   ©

, 1913V

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*  V

The evidence:V

V Most organisms are comprised of at least 70% or more water. Some plants, like a head of
lettuce, are made up of nearly 95% water;V
è
V When organisms go dormant, they loose most of their water. For example, seeds and
buds are typically less than 10% water, as are desiccated rotifers, nematodes and yeast
cells;V

V Earth is the water planet (that's why astronomers get so excited about finding water in
space).V

V Water is the limiting resource for crop productivity in most agricultural systems (





? 
)V

Tom Robbins, author of 

?





, has eloquently stated the importance of
water:V

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Now, let's change our perspective for a minute and put ourselves in the place of water.
According to Robbins, water is so important that "[i]t has even been suggested that life evolved
as a means to transport water." Well, this is certainly good fodder for a late night discussion, but I
doubt that anyone would question Frank Salisbury·s and Cleon Ross·s (1992) statement that
"Plant physiology is ... the study of water."V

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   V

A. Water is Polar
In other words, the water molecule has a positively-charged (hydrogen side) and negatively-
charged side (oxygen). This occurs because:V

V the hydrogen atoms are arranged at an angle of about 105 degrees;V
è
V the covalent bond between O-H is polarized. This is caused by an unequal sharing of
electrons between these atoms which, in turn, results in a slight negative charge on the
oxygen atom (electronegative) and slight positive charge on the hydrogen;V

V oxygen has an unshared pair of electrons (the molecule is tetrahedral-shaped).V

B. Hydrogen Bonds
The 'fancy' definition of a hydrogen bond is that it is a weak bond that forms between a
hydrogen atom that is covalently bonded to an electronegative atom (like oxygen) and
another electronegative atom. In other words, a positively-charged hydrogen atom is attracted
to a negatively-charged oxygen.V

The end result is that water readily forms hydrogen bonds with itself and other polar molecules.
When likes attract it is termed  (i.e., hydrogen bonds between water molecules). When
unlikes attract, it is called  (i.e., when a paper towel absorbs water, water and cellulose
adhere to one another). Cohesion and adhesion are responsible for 

 
 , the
movement of water up a thin tube.V

In liquid water, hydrogen bonds between water molecules are continuously made and
broken. The molecules can even form temporary "quasi-crystalline" areas. Individually, each
hydrogen bond is weak (20 k‰ mol-1), but collectively they give water many unique properties (a
Marxist molecule!).V

ccc 

V

#
   



 (i.e., between 0-100 C).
In other words, water has a 
 
and a 

 
when compared to
other similar-sized molecules such as ammonia, carbon dioxide, hydrogen sulfide. These other
molecules are gases at room temperature. This is important because if life exists anywhere, we
predict that it occurs between approx. 0 and 100 C. Temperatures much below 0 are too cold
to permit significant chemistry for metabolism; temperatures above 100 tend to disrupt bonds.V

{#
  
  

In other words, it takes a lot of energy (ca. 44 k‰ mol-1) to convert water from a liquid to a gas;
or stated another way



?

?  This property is responsible for water's use
in *
*
  systems, hence the reason dog's pant, people perspire, and leaves
transpire.V

#
  G .

It takes a lot of energy (4.184 ‰ g-1 C-1, or the non-SI unit is the calorie where 1 cal = 4.184 ‰) to
raise the temperature of water (because it requires a lot of energy to break/make hydrogen
bonds). Thus, water is slow to heat up and cool down, or stated another way



?





  
 This is why you can swim reasonably comfortably in the Sag in late fall but
not the spring. In contrast, a sidewalk has low specific heat - it heats up quickly (try walking
barefoot on a sidewalk in summer on a sunny day), but cools down quickly. This property is
important in water's role as a

. It's not surprising that desert plants are succulent - to
help resist temperature fluctuations.V

6#
    .

It takes a lot of energy to convert water from a solid to a liquid, or put another way




?


'? . Energy is required to break the collective hydrogen bonds holding water in its
solid configuration. Conversely, a lot of energy (6 k‰ mol-1) must be released by water to freeze.
This property is used by citrus growers - prior to a light freeze they spray fruit with water; ice forms
releasing the heat of fusion which will help protect the crop from serious damage.V

©#

   .

It takes a lot of energy to break through the surface of water, because water molecules at the
surface are attracted (cohesion) to others within the liquid much more than they are to air.
Thus, 



 
?


!?  This phenomenon is important at air/water interfaces
and explains why: (1) water rises up a thin tube (capillary action); (2) raindrops are round (the
molecules at the surface attract one another); (3) water striders and other bugs can "walk on
water"; (4) a meniscus forms; and (5) a belly-whopper into a pool of ammonia would not hurt
nearly as much as one into water.V

"   
 
  
  .

Good thing too, or else ice fishing would be a moist business. This occurs because when ice
forms each water molecule is hydrogen bonded to exactly four others. At four degrees, water is
it's densest, and each water molecule is attracted to slightly more than four others. Thus, as
water cools it gets denser and denser until it reaches 4 C, then, it gets less dense. And ice floats.V

#
 
  .

Water dissolves more different kinds of molecules than any other solvent. Hydrophilic (water-
loving) molecules dissolve readily in water (likes dissolve likes), hydrophobic (water-fearing) ones
do not.V

ÿ#
  



.
In other words, if you put water in a tube and put a piston on either end, you won·t be able to
push the pistons together. Thus, water is good for hydraulic systems because when it is squeezed
it doesn't compress and produces positive pressures (hydrostatic pressures). This pressure
provides the driving force for cell growth and other plant movements. The pressure is measured
in units of Pascals (or actually MegaPascals, MPa). One MPa is approximately equal to ten
atmospheres or 10 bars.V

In a similar fashion, if you fill the tube with water, remove any air bubbles, and then pull the
pistons away from one another, the water column resists breaking. This will result in a suction on
the water column - just like putting your finger on the end of a syringe and pulling back the
plunger. Negative pressures (
  ) can develop in the water column. Very sizable tensions
can be generated in a thin water column. However, *

 " when air comes out of solution
at negative pressures, can be a problem.V

c#


 .

This is important because chloroplasts (inside a cell) are obviously surrounded by water. If
water were opaque, plants couldn't photosynthesize. From an ecological perspective, the
penetration of in water determines the distribution of aquatic plants.V

‰#


.
It doesn't react unless it is enzymatically designed to do so.V

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  .

This serves as the basis for the pH system (see below).V
`#
   $

    
   .
For example, many ions (such as sodium) and molecules (such as DNA and wall components)
are normally hydrated. This means that water is hydrogen bonded to them and in some cases
(i.e., sodium) forms a hydration shell around them. V


c4 
 
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?




 V

V is a major component of cellsV

è
V is a solvent for the uptake and transport of materialsV

V is a good medium for biochemical reactionsV

V is a reactant in many biochemical reactions (?
 photosynthesis)V

V provides structural support via turgor pressure (?

leaves)V

V is the medium for the transfer of plant gametes (sperms swim to eggs in water, some
aquatic plants shed pollen underwater)V

V offspring (propagule) dispersal (think "coconut")V

V plant movements are the result of water moving into and out of those parts (?
 diurnal
movements, stomatal opening, flower opening)V

V cell elongation and growthV

Vthermal bufferV

Vperhaps most importantly, water has directed the evolution of all organisms. You can
think of morphological features of organisms as a consequence of water availability. For
example, consider organisms growing in xeric (dry), mesic (moderate) and hydric
(aquatic) environments.V

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Water ionizes to a small degree to form a hydrogen ion (or proton) and hydroxide ion (OH-). In
reality, two water molecules form a hydronium ion (H30+) and a hydroxide ion (OH-).V

In pure
water,V V
[H+]V = V [OH-] This solution is neutralV
[H+]V >V [OH-] Then, the solution is an acid (acidic)V
[H+]V < V [OH-] Then the solution is a base (alkaline)V

Thus:V

V An acid is a substance that increases the [H+], or as the chemists say, is a proton donor.

. HCl ń H+ + Cl-V

è
V A base is a substance that increases the [OH-]; or from the perspective of a proton, a
base is a substance that decreases the proton concentration; it is a proton acceptor.
e.g. NaOH ń Na+ + OH- (accepts protons to make water)
e.g. NH3 (ammonia) + H+ ń NH4+ (ammonium ion)V
The pH Scale:
pH is the scale to express the degree of acidity (or alkalinity) of a solution. The scale ranges
from 0 to 14 where 1 is highly acidic, 7 is neutral, and 14 is highly alkaline.V

As the pH increases, the [H+] decreases and the [OH-] increases

As the pH decreases, the [H+] increases and the [OH-] decreaseV

pH = - log[H+]V

m? 


:V

V the pH scale is based on proton concentration; andV

è
V the pH scale is logarithmic, there is a 10-fold difference in concentration between each
pH unit.V


9-V
V V
[H+]V =V [OH-]V
0.0000001 mol H+ liter-1 = 10-
7 H+V
=V 0.0000001 OH- liter-1 V

pHV =V log [H+]V

=V -log[10-7]V
V
=V - (-7)V
V
=V 7V
V
The products of [H+] x [OH-] always equals 10-14. Thus, you can always determine
concentration of one if you know the other. For example, if the [H+] = 10-2, then the [OH-] is 10-12.V

c*  
*  
*


Organisms must maintain pH within tolerable ranges. This is a good example of homeostasis.V

A buffer is a solution that resists fluctuations in pH when additional OH- or H+ are added. They
maintain a constant pH and usually consist of a proton donor and a proton acceptor. [e.g.,
blood pH must be between 7.36 (venous) and 7.41 (arterial). The carbonic acid/bicarbonate
buffer helps to maintain pH:V

H2CO3 (carbonic acid; proton donor) ń H+ + HCO3- (bicarbonate ion; proton acceptor)V

cc
(*
- There are two major ways to move molecules:V

A. Bulk (or Mass) Flow.

This is the mass movement of molecules in response to a pressure gradient. The molecules
move from hi ń low pressure, following a pressure gradient. A good example would be a faucet.
When you turn a faucet on, water comes out. This occurs because the water in the tap is under
pressure relative to the air outside the faucet. A toilet is another example; high pressure in the
tank/bowl but lower pressure in the sewer system. Some molecular movements rely on bulk flow
which requires a mechanism to generate the pressure gradient. For example, animals have
evolved a pump (?
., heart) that is designed for the bulk flow of molecules through the
circulatory system.V
B. Diffusion
The net, random movement of individual molecules from one area to another. The molecules
move from [hi] ń [low], following a concentration gradient. Another way of stating this is that
the molecules move from an area of high free energy (higher concentration) to one of low free
energy (lower concentration). The net movement stops when a  &
 is
achieved.V

Imagine opening a bottle of perfume containing volatile essential oils in a very, very still room.
Initially, the essential oils are concentrated in a corner of the room. As the molecules move
randomly, in every different direction, over time they will eventually appear throughout the
room. Ëltimately the essential oils will reach a point, dynamic equilibrium, at which they are
evenly distributed throughout the room. At this point the molecules are still moving. They
continue to move randomly in every direction. The only difference is that there is no net change
in the overall distribution of the perfume in the room.V

Now imagine that the room is divided by a partition with holes (which is analogous to a
membrane). If we place a drop of perfume on one side of the partition and then count at
intervals the number of essential oil molecules on either side of the partition and graph the
results:V

insert: plot # molecules vs. time on both sides of the partition. G 
 
:V

We will observe that the number of molecules on one side will decrease while the other will
increase until they reach dynamic equilibrium. At equilibrium the molecules continue to move
randomly, back and forth from one side of the partition to the other. Hence the number of
molecules on either side of the partition at any given time is simply chance. The number
oscillates about the midpoint.V

%*

Although this theoretical example can help us to better understand the nature of diffusion, ?
?


 ?

 . The molecular movement attributed to diffusion in this example is really due
to air movements in the room, or convection. True examples of diffusion are hard to come by
(see Vogel, 1994; Wheatley, 1993). Nevertheless, it serves our purpose to illustrate the general
concept of diffusion.V

C. Osmosis
This is a specialized case of diffusion; it represents the diffusion of a solvent (typically water)
across a membrane.V

D. Dialysis
Another specialized case of diffusion; it is the diffusion of solute across a semi-permeable
membrane. Example ² consider a cell containing a sugar dissolved in water. If water (the
solvent) moves out of the cell into the surroundings it moves osmotically; if the sugar (solute)
moves into the surroundings, it is an example of dialysis.V

cc4
 
  

 - 



? 







?? 




? 
V

  
 
 .

As previously stated, solutes move from an area of high concentration to one of lower
concentration; in other words, in response to a concentration gradient (ƦC). Although this is true
for most solutes, it is NOT important for water. The concentration of water (55.2 - 55.5 mol L-1) is
nearly constant under all conditions (?
., MW = 18 g/mol, and 1000 g/liter; thus, 1000/18 = 55.5
mol/L).V

Fick·s Law - is an equation that relates the rate of diffusion to the concentration gradient (C1 ²
C2) and resistance (r). Diffusion rate, also called flux density (‰s, in units of mol m-2 s-1) can be
expressed in the simplified version of Fick's equation as:V

‰s = (C1 - C2) / rV

The take-home-lessons from this equation are that:V

V the rate of diffusion is directly proportional to the concentration gradient. The

greater the difference in concentration between two areas, the greater the rate
of diffusion. Thus, when the gradient is zero, there will be no net diffusion; diffusion
will only occur so long as a concentration gradient exists;V
è
V the rate of diffusion is indirectly proportional to resistance. In other words, the
greater the resistance to diffusion, the lower the rate of diffusion. Resistance refers
to anything that reduces the rate of diffusion such as the partition in our perfume
example. The width of the partitions is a resistance; the wider the partitions, the
lower the resistance. And, the membrane is a resistance to the movement of ions
and other charged substances in or out of cells; andV

V the rate of diffusion is inversely proportional to distance traveled (also a function
of resistance). For example, some typical diffusion rates for water are 10 µm - 0.1
sec; 100 µm -1 sec; and 1 mm - 100 sec. As the text demonstrates nicely, diffusion
is effective over short distances, but is pathetically slow over long distances.V

{%   
º . According to kinetic theory, particles like atoms and molecules are in
always in motion at temperatures above absolute zero (0 K = -273 C). The take-home-lesson is
that molecular movement is:V

V directly proportional to temperature; andV

è
V indirectly related to molecular weight (heavier particles move more slowly than
lighter, smaller ones). At room temperature, the average velocity of a molecule is
fast - about 2 km/sec (=3997 mph!).V




- increases the rate of molecular movement, therefore, increases the rate of
diffusionV

6

- increases speed of molecules, therefore, increase the rate of diffusionV

©º       

      . Solute particles decrease the free
energy of a solvent. The critical factor is the number of particles, not charge or particle size.
Essentially solvent molecules, such as water in a biological system, move from a region of greater
mole fraction to a region where it has a lower mole fraction. The mole fraction of solvent = #
solvent molecules/ total (# solvent molecules + # solute molecules). This is particularly important
in the movement of water. Water moves from an area of higher mole fraction or higher energy
to an area of lower mole fraction or lower energy.V
ccc
 



Water potential is a measure of the energy state of water. This is a particularly important
concept in plant physiology because it determines the direction and movement of water.V

"

  V

V Free energy of water - energy available to do work (‰ = n m)V

è
V Chemical potential (µ) - free energy/unit quantity (usually per mole) (‰ mol-1)V

V Water potential (ƹw ) - chemical potential of water, compared to pure water at
the same temperature and pressure. The units are in pressure because: (a) plant
cells are under pressure (remember the wall?); and (b) it is easier to measure
pressure. V

V Derivation of units - Water potential is official defined as the chemical potential of
water (‰ mol-1). When divided by the partial molar volume (L mol-1):

‰ mol-1/ L mol-1 = n x m mol-1/ m3 mol-1 = n m-2 = MPa


!? 

?
 



‰ mol-1/ L mol-1 = ‰ / liter = energy / volume = (weight x distance)/area x distance

= force/area = pressure units V

V Pressure is measured in MPa (megapascals). 1 MPa = 10 bars = 10 atm. (as an
aside, 1 atm = 760 mm Hg = 14. 7 lbs sq in-1)V

{©  

   (
 




 
? 




??



):V

ƹw = ƹp + ƹs + ƹgV

where ƹw = water potential; ƹp = pressure potential; ƹs = solute or osmotic potential; and ƹg =

gravity potential.V

1. Solute (or osmotic) potential (ƹs)

This is the contribution due to dissolved solutes. Solutes always decrease the free energy of
water, thus there contribution is always negative. The solute potential of a solution can be
calculated with the van·t Hoff equation: ƹs = - miRT where m = molality (moles/1000 g); i =
ionization constant (often 1.0); R = gas constant (0.0083 liter x MPa/mol deg); and T =
temperature (K).V

2. Pressure (or Pressure Potential; ƹp)

Due to the pressure build up in cells thanks to the wall. It is usually positive, although may be
negative (tension) as in the xylem. Pressure can be measured with an osmometer.V

3. Matric potential
This is the contribution to water potential due to the force of attraction of water for colloidal,
charged surfaces. It is negative because it reduces the ability of water to move. In large
volumes of water it is very small and usually ignored. However, it can be very important in the
soil, especially when referring to the root/soil interface.V
4. Gravity (ƹg)
Contributions due to gravity which is usually ignored unless referring to the tops of tall trees.V

 
   


 Water potentials in intact plant tissue are usually
negative (because of the large quantities of dissolved solutes in cells).V

6#
   

   
    
 
  

  
where: ƹw = ƹp + ƹs + etc.V

©% 
#
   - we will discuss the following techniques in class/lab:V

1. Pressure bomb - a steel chamber that can be pressurized, usually with nitrogen. The sample is
placed in the chamber with the petiole or surface exposed through a hole in the lid. The sample
is pressurized and the pressure that is required to force water to appear on the cut surface is
assumed to be equivalent to the water potential of the tissue.V

2. Chardakov Method - Dye Drop methodV

3. Gravimetric methodV

"% 
º     

Solute potential can be measured by:V

V Freezing Point Depression - dissolved solutes lower the freezing point of a liquid
(think salt and MN roads in the winter). A 1 molal solution with an osmotic
potential of -2.27 MPa lowers the freezing point (fp) by 1.86 degrees. We can use
this relatinoship  Vset up a ratio: -2.27 MPa/1.86 degree = unknown ƹs / fp.
Rearranging we get the equation: ƹs (MPa) = -1.22 x fpV
è
V Incipient Plasmolysis - a tissue is incubated in a series of solutions of known water
potential. The point at which membrane just pulls away from tissue is "incipient
plasmolysis" and considered to the equivalent to the osmotic potential of the
tissue.V

V Vapor pressure osmometer - dissolved solutes increase the boiling point or
decrease the vapor pressure of a liquid. A thermocouple hooked to a recorder is
placed in an airtight chamber with the tissue sample or standard. The
thermocouple is also linked to a reference junction. Thermocouples are made of
two different metals (constantan and chrome) and a current will flow if there is a
difference in temperature between the reference junction and thermocouple. A
water droplet or KCl (aq) solutions of known osmolarity are placed on the
thermocouple. Depending on the osmotic potential of the solution, water will
either evaporate from or condense on the droplet. This is turn causes a current
change in the thermocouple which can be detected by a meter. The cooling
rate is plotted vs. ƹs or [KCl] to yield a standard curve from which the ƹs of the
tissue is determined.V

c,(*

  
   
.

Individual water molecules diffuse across the membrane. In addition, there are integral
proteins in the membrane that form a channel or pore through which water moves. These pores
are important and water molecules essentially move through these pores by bulk flow. The
proteins are called aquaporins and are essentially water transport channels. Water is moving
passively (following a gradient of free energy).V

Π-V

÷V Boyer, ‰S (1969) Measurement of the water status of plants. Ann. Rev Plant Physiol. 20: 351
- 364.V
÷V Hebrank, MR. 1997. Reduce confusion about diffusion. American Biology Teacher 59: 160.V
÷V Odom, AL. 1995. Secondary & College Biology Students· misconceptions about diffusion
& osmosis. American Biology Teacher 57: 409 - 415.V
÷V Vogel, Steven. 1994. Dealing Honestly with diffusion. American Biology Teacher 56: 405-
407.V
÷V Wheatley, D. 1993. Diffusion theory in biology: its validity and relevance. ‰ournal of
Biological Education 27: 181-187.V
÷V Zuckerman, ‰T. 1994. Problem solvers· conceptions about osmosis. American Biology
Teacher 56: 22-25.V


 
V

c
'

'
 . The movement of water follows the pathway:V

soil  uptake  root  stem  leaf  transpiration  airV

The driving force for water movement is the water potential gradient that exists from soil to air. Or
in other words:V

ƹsoil > ƹroot > ƹstem > ƹleaf > ƹairV

Some typical values water potential values (in MPa) for a tree are: trunk -0.7; twig -2.3, leaf -2.5.
In class, we may also look at some data for ivy.V

cc„V 


A. What is soil?
Soil is a mixture of organic (dung, decayed organic materials, decomposers) and inorganic
(weathered rock) materials, gases (oxygen, carbon dioxide, ethylene), and liquid.V

B. Soil type - determined by: (1) composition; (2) texture or particle size ( sand > silt > clay. A
loam is a soil with 10-25% clay and equal parts of sand and silt); and (3) structure (?
.,
compaction)V

C. Water and soilV

V Saturated - soil before drained. Gravitational water - water that drains and is not tightly
bound; ƹ = 0 MPaV
è
V Field capacity - soil that holds all the water it can against gravity. Capillary water -water
held by capillary action, water at field capacity; ƹ = -0.015 MPaV
 
V Permanent wilting percentage - soil moisture content at which plants can't get enough
water. For most, ƹ = -1.5 MPaV

V Graphic relationship of soil water potential vs. water content (%). Take-home lessonsV

V between PWP and FC is the water available for a plant to use;V

V clay holds more water than sand at any ńƹ ; andV

V clay holds water more tightly (?
., @10% water Ysand > ƹclay). This is essentially a
s/v problem, since smaller particles in clay they have a larger total surface and
hence, has more charged surfaces that will bind water tightly.V

V Soil water potential is a function of osmotic potential (which is usually near zero except in
saline soils) and mostly pressure (used to call it matrix potential; this refers to the tension
generated because of the attraction of colloidal particles; ?
., adhesion). The pressure in
the soil can be calculated from the equation: ƹp = -2T/r 

T = surface tension (7.28 x
10-8 MPa) and r = radius of curvature of the meniscus). Water movement through soil -
mostly due to bulk flow as a result of pressure gradients, with some diffusion.
V

V Spuds McSaupe plays with spongesV

ccc

%G  
 :
Or more simply stated, the movement of water from plant to air occurs via transpiration. Air
has a very high capacity for holding water. For example at 20 C, the water potential of water in
air at 100% RH = 0 MPa; 98% RH -2.7 MPa; 50% RH = -93.5 MPa. Conclusion - there is a very steep
water potential gradient from soil to air. Essentially, the plant just inserts itself between the two
and takes advantage of passive transport.V


c
Œ
V


V Root anatomy

We will go over structure of the root including epidermis, cortex, endodermis,
Casparian strip, stele, phloem, xylem, pericycle.
V

V Root Formation
Roots develop from the pericycle; film loop
V

V Apoplast vs. symplast
Recall that the apoplast refers to the "non-living" regions of the plant and the symplast
is the "living" areas.
V


V Region of Water Absorption
Most of the water is absorbed near the tip of the root. The further from the tip, the less
water that is taken up by the root. This roughly correlates to the region of the root that is
suberized.
V

V Route of Water Movement
There are three routes water can follow: (a) Apoplastic ² water follows an apoplastic
route from soil through cortex. However, it must enter the stele symplast because of
the  
. Once inside, it leaks back out and enters the apoplast (xylem) where
it is transported to the apex of the plant. This appears to be the major route of transport;
(b) Symplastic: Transmembrane ² in other words, the water moves from cell-to-cell
 crossing membranes as it goes; and (c) Symplastic: Plasmodesmata ² the water moves
from cell-to-cell via the plasmodesmata.
V

V Root as an osmometer
Analogy - allows for the development of root pressures in the stem. These can be
measured and are about 0.2 - 0.3 MPa.
V

V Guttation and hydathodesV
Œ
*V

A. What is the transport tissue for water? Xylem.

Evidence comes from various tracer studies where xylem is loaded with dyes. I·ll bet you·ve
done the classic "celery stalk in food coloring" experiment.V

We·ll see a film loop and maybe play with some celeryV

B. In which cells does water move? Vessels & Tracheids

There are four major types of cells in the xylem: (a) tracheids - long, tapered ends, thick
secondary wall; (b) vessel elements, - shorter, ends attached; (c) fibers - long and skinny with
thick secondary wall, mostly for support; and (d) parenchyma - alive, thin, store starch and other
materials, lateral transport. The primary water transport cells are tracheids and vessels. Note that
gymnosperms only have tracheids whereas angiosperms have both and primarily rely on vessels
for water transport. Both tracheids and vessels have pits, thin circular regions, in the walls.V

C. How much pressure is required to move water to the top of a tall tree, that is say, 100 meters
tall?
Let's calculate. We can measure the velocity of flow in the xylem to be 4 - 13 mm s-1 in vessels
with a diameter of 100 - 200 mm. For our calculations, let's use a flow rate of 4 mm s-1 (= 4 x 10-3 m
s-1) and a vessel radius of 40 Jm (= 0.00004 m).V

According to Poiseuille's Law - flow rate is directly proportional to the pressure gradient and
the cross sectional area of the pipe but inversely proportional to the viscosity of the fluid. Thus,
this is mathematically expressed as the Poiseuille equation:V

‰v = ((Ǒ)(r4)( P))/8 (Lj) where Lj = viscosity of water (assume it is the same as in a cell, 10-3 Pa s)V

Now, divide the equation by the cross-sectional area of a vessel (Ǒ r2). Thus, the equation
simplifies to:V

‰v = (( r2 )( P ))/8 (Lj)V

Substituting the values for flow and vessel diameter:V

4 x 10-3 m/sec = (0.00004 m )2(P)/ 8 (10-3 Pa s)V

P = 20,000 Pa m-1V

P = 0.02 MPa m-1V

If the tree is 100 meters, then: 0.02 MPa m-1 x 100 m = 2 MPaV

However, we must also take into account the effects of gravity (0.01 MPa m-1). Thus, for a 100 m
tree: 0.01 MPa m-1 x 100 m = 1 MPa for gravityV

Finally, the total pressure required to move water to the top of a 100 meter tree equals:V

2.0 + 1.0 = 3 MPaV

D. How is Water Moved to the top of trees?V

1. Is water moved to the tops of trees by a "push from the bottom" pump? - #
Several lines of evidence show that this type of pump doesn't exist: (a) dissections showed
there is no anatomical area in the stem or root that could serve as a pump; (b) when German
physiologists cut off a tree in a vat of picric acid it continued to transport water. This suggested
that a stem pump was not involved since the picric acid should have killed living cells stopping
the pump; (c) a root pump isn't involved or else when a plant is decapitated, the stump should
continue to gush water; and (d) recall that root pressures only generate 0.2-0.3 MPA but that a
pressure of at least 3 MPa is required to move water to the tops of tall trees. To summarize, root
pressure doesn't have nearly enough power. V

2. Is water moved to the tops of trees by "capillary action" - #

Capillary action is the movement of water up a thin tube due to surface tension and the
cohesive and adhesive properties of water. Essentially the meniscus "pulls" the water up the
tube. Without worrying about the derivation of the equation, the height to which a column of
water can move is inversely related to the radius of the pipe and is mathematically expressed
as: h = 14.87/r (where r = radius in Jm; and h = height in meters). Let's look at some actual dataV

Table 1: Capillary Heights of Water MovementV

Tube Radius (Jm)V Column Height (m)V

10V 1.4877V

40 (tracheid)V 0.37V

100V 0.148V

0.005 (size of pores in 2975 (. 3

wall)V kilometers)V

Vessels are too wide to support movement very high and obviously capillarity cannot be
responsible for water movement. Further, even it could, it would only move to the top of the
plant once; capillary action can't continually pull the water up.V

3. Cohesion-Tension Theory - YES!

This idea was first proposed by HH Dixon ( ??
 






?
m , 1914).
According to this hypothesis, water is drawn up and out of the plant by the force of transpiration.
Because of the cohesive/adhesive properties of water, as one water molecule evaporates at
the opening it pulls the other molecules and sends this pull all the way down the column. If this is
true them, water transport in plants must meet the following criteria:V

÷V 







?


? 
. The vessels and tracheids are hollow at
maturity. Imagine how difficult it would be to move water through a clogged
pipe. Let's calculate how much pressure would be necessary if the water
transport cells were "alive." We'll use Fick's Law:

‰v = Lp ƹ (Lp = hydraulic conductance which is the inverse of resistance)

If we assume that water movement occurs at the rate we used earlier (‰v = 4 x 10-
3 m s-1) and we use a typical value of 4 x 10-7 m s-1MPa-1 for Lp, then:V

4 x 10-3 m s-1 = (4 x 10-7 m s-1 MPa-1) (ƹ)

ƹ = 4 x 10-3 m s-1/4 x 10-7 m s-1 MPa-1

ƹ = 104 MPa (this is the pressure required to move the water across just one membrane!
Compare to the value we calculated above)V

if, the cell length is 100 Jm (10-4 m), then we can calculate the force required per meter:V

104 MPa/10-4 m = 108 MPa m-1V

÷V 

 






 ? 









?.
If not, it would be analogous to having a chain with a single broken link ² it
would be impossible to pull anything attached to the other end. The tracheids
and vessels form a continuous water column. If there are gaps - or air bubbles -
water must be routed around these bubbles. Cavitation, or vacuum boiling, is
the fancy term for air coming out of solution when the water columns break.

By the way, this is one reason why you don't want to go outside and beat on
the trunk of a tree on a hot sunny day...it could cause many of the columns to
break so that a plant may have a difficult time transporting water. Check out the
Per Scholander stories written by Dr. V Berg.

If cavitation does occurs, the plant responds by: (a) transporting water around
the blocked cell; or (b) redissolving the air bubble, which usually occurs at night;
and/or (3) forming new xylem cells; in other words, xylem is disposable. Only the
most recent cells in the latest seasons growth are actually functional. The
remainder of wood in a tree is non-functional because it has cavitated and/or
filled with other waste materials. In addition, it is thought that one function of
bordered pits is to stop the movement of air bubbles from a cavitated cell to
another thereby isolating the impact of cavitation.
V
÷V 





??

? 

.
Even though . 3 MPa are required to move water to the top of a tall tree, the
water potential gradient from soil to air is considerably steeper (on the order of -
100 MPa.)
V
÷V 




 


 



? .
Several lines of evidence support this prediction: (a) Cut a stem and the water
 will snap up into the top and accumulate at the cut surface on the bottom; (b)
Dendrometer studies - this device is essentially a band wrapped around a tree
that is hooked to a pressure transducer. As the tree transpires the diameter of the
tree is measured. These experiments show that the diameter of the stem is
smallest during the day when transpiration is occurring and largest at night, as we
would. Imagine putting your finger on the end of a straw and then sucking on the
other end. The straw will get thinner (collapse) as you apply tension to the air in
the straw - just like a plant stem; (c) Puncturing the xylem of an actively transpiring
tree with an ice pick may result in a hissing sound as air is sucked into the stem
(see Dr. Berg's water stories); and (d) Dye solutions are rapidly sucked into a tree
trunk when punctured with a knife and then transported in both upward and
downward directions. Since the pressure in the stem is lower than atmospheric
the dye solution is quickly sucked in. We will see a demonstration of this in a video
that a previous class made (BIOL327- Spring 2001) made.
V
÷V 


?












?  


.
In other words, the columns of water must not snap as they are being pulled.
As the water is pulled up the tree the water column puts up a resistance, much
like stretching a rubber band. ‰ust like a rubber band will snap if pulled too hard,
so too will a water column break or cavitate. This occurs because the reduced
pressures in the water column cause gases to come out of solution and form a
vapor lock in that cell. Cavitation can be heard by placing a sensitive
microphone on the plant. Tiny popping noises can be heard, a little like a bowl of
rice krispies.

The fact that water has a very high tensile strength, more than sufficient to
withstand the pulling forces necessary to move water to the top of tall trees, was
demonstrated by an elegant experiment in which water was centrifuged in Z-
shaped tubes.

As an aside, the tensile strength of water may be one of the factors that limits
the height of trees - the tensions in the stems of taller trees would be too great
and the water columns would snap. It's perhaps not a surprise that the tallest
trees, California redwoods, grow along the fog enshrouded coast. This helps to
minimize the rate of water loss and ultimately reduce the tensions in the xylem
(Zimmer, 2000).
V
÷V 
?
 









?  

? 
? 

??  Hence the reason that they have thick cell walls with circular
thickenings. It's no surprise that wood is hard.V

4. Pressure Compensation Theory - Controversial.

Recent work by Martin Canny and others have challenged the validity of the Cohesion-
Tension Theory (see Canny, 1995). V

cc4
0;G:
Don·t you just love a paradox?....recall that flow rate is directly related to the radius of the
pipe (Poiseuille·s law). Thus, flow rates in vessels are greater than those in tracheids. But, why
aren·t vessels (and tracheids for that matter) larger, especially since it means that they could
transport more water? The answer - cavitation. As the pipes get larger, the chance of cavitation
increases.
cccΠV

÷V Dr. Berg's Favorite Water Relations Stories (Northern Iowa)V

÷V Zimmer, C (2000) High and dry. Natural History. October. pp36-37.V
÷V Canny, M (1998) Transporting water in plants. Amer. Scientist 86: 152 - 159.V

35 0 
 V

c8 
 V

÷V Transpiration - evaporation of water from a plant surfaceV

÷V Evapotranspiration - evaporation of water from a plant surface and soil (including abiotic
surroundings).V
÷V Take-home-lesson: Plants loose a lot of water by transpiration.V

cc 

0 
  G


"< <
<8
<:
Recall the equation for photosynthesis where:V

CO2 + H2O ń (CH2O) n + O2V

This equation suggests that:V

V Gases, such as carbon dioxide and oxygen, are important in the overall energy
metabolism of plants;V
è
V Plants must exchange gases with the environment; andV

V In order to obtain carbon dioxide plants will necessarily loose water (transpire). In other
words, transpiration is a necessary evil of photosynthesis.V

ccc

 
 V


V 






?

&?



??



  


Thus, animals have lungs or gills whereas plants have leaves with extensive spongy
mesophyll (it may be time to review your leaf anatomy).V

V 








  ? 













?? 


Animals solve this problem by placing the absorptive surface inside a humid cavity
(lung) opened with a small exit pore(s). Plants put the absorptive surface (spongy
mesophyll) inside the leaf and cover it with a water impermeable layer (cuticle)
peppered with a series of pores (stomata). The cuticle is comprised of waxes, which are
an assortment of long chain hydrocarbons and, in particular, cutin (C16-C18
hydroxylated fatty acids). Note that even though these strategies minimize desiccation
from the absorptive surfaces of plants and animals, they don·t completely eliminate it.V

V m? 


?



? ?



 ?





??









? 







?




Animals use an active pumping mechanism (
, lungs/diaphragm) to move gases
inside the organism by bulk flow. The gases are circulated by another pumping system
 (heart). The distances needed to move the gases are too great to be accounted for by
simple diffusion. Plants do not have a pumping mechanism for moving gases; they rely
on diffusion (and to a lesser degree, bulk flow). In either case, plants do not actively
move gases. This is one reason why leaves are so thin (recall our previous discussion) -
diffusion is not efficient over long distances (?
, diffusion is inversely related to the square
of distance). For example, it would take about 2.5 seconds for glucose to diffuse the
distance across a cell membrane (0.5 Ǎm) but approximately 32 years to go one meter!V


V m
 
?


In order to obtain carbon dioxide for photosynthesis plants needed to evolve a large,
thin absorptive surface (leaves with spongy layer) and then protect it from desiccation.
Thus we can consider this the photosynthesis/transpiration "paradox". Actually, it might
better be considered a "compromise" since that is what a plant needs to do - strike a
compromise or balance between the amount of carbon dioxide absorbed and the
amount of water lost by transpiration.V

c4


 
Not only is water loss a "necessary evil" of photosynthesis, but to make matters worse, the
tendency to loose water is greater than the tendency of carbon dioxide to diffuse into the plant.
As evidence, let's calculate the transpiration ratio, which is a measure of the the amount of
water loss relative to the amount of carbon fixation. V

transpiration ratio = mol water transpired / mol CO2 fixedV

If carbon dioxide uptake (or fixation) and water loss are equal, this ratio should be one. In reality,
experiments show that this ratio is closer to 200! Thus, for every 200 kg of water transpired, 1 kg of
dry matter is fixed by a plant. Let·s see why:V


(??
 
?



Recall that during diffusion molecules move from an area of higher chemical potential (or
concentration or chemical energy) to an area of lower chemical potential (or concentration or
free energy) and that the driving force for diffusion is the gradient from one area to another. We
can express this relationship mathematically using Fick·s Law: ‰v = (c1 - c2)/r 




‰v = flux
density, (mol m-2s-1); c1 - c2 = concentration gradient, and r = resistance (a function of distance,
medium viscosity, membrane permeability, etc.). We can simplify this equation to:V

diffusion = gradient/resistanceV

Now let·s compare the rates of diffusion for both water and carbon dioxide. Since resistance, or
the distance that either carbon dioxide or water must diffuse into/out of the leaf is the same,
then diffusion is directly proportional to the gradient.V

V Carbon dioxide - has a very shallow, or small, gradient from inside to outside of the leaf.
Ambient carbon dioxide concentrations are approximately 0.03% (= 0.36 mmol mol-1).
The internal concentration cannot be less than zero. Thus, the gradient is no larger than
0.36 mmol mol-1(0.36 - 0).V
è
V Water - has a very steep gradient from inside to outside. At a relative humidity of 50%
and 25 C the water potential of water in air is . -100 MPa (= 32 mmol mol-1). The air in
the substomatal cavity of a leaf is typically fully saturated, with a RH near 100%, and has
a water potential near zero. Thus, the water potential gradient is 100 MPa (or 32 mmol
mol-1).V
Conclusion: based on gradient alone, the water has approximately 100x greater tendency to
diffuse out of the leaf than carbon dioxide to diffuse into the leaf.V


(??
 




? 


Recall that diffusion is inversely related to molecular weight. Simply put, the heavier the
molecule the more slowly it will diffuse. No big surprise. Or, to express this mathematically:
rate = 1 / sq rt MWV

The relationship between the rate of diffusion of two molecules can be summarized by the
following relationship:
Rate A / Rate B = sq rt B / sq rt AV

Thus to calculate the ratio of water loss to carbon dioxide uptake:

H20 loss/CO2 uptake = sq rt 44 / sq rt 18 = 1.56V

Conclusion: Based on molecular weight alone, the tendency for water to diffuse out of the leaf
is 1.56 times greater than the tendency for carbon dioxide to diffuse into the leaf.V



0
 
 *


Although it seems as though water loss is a serious, intractable problem for a plant, it is NOT.
The reason - plants continually compromise between the amount of carbon dioxide absorbed
and the amount of water loss. This compromise is mediated by the stomata, whose function is to
regulate gas exchange.V

c




V


V  




G

- guard cells, subsidiary cells, substomatal cavity,

cuticle, ledge (or lip), stomatal apparatus. The subsidiary cells are epidermal cells that
may be specialized and different from the other epidermal cells. The function of the
ledge is to prevent liquid water from seeping into the pore. Interestingly, cutin covers
most of the cells in the substomatal cavity; only regions near the actual opening are free
of cuticle and most water is lost from this area. For images of various guard cells/stoma
click here.V

V 





 (1) elliptical or kidney-shaped. These are characteristic of eudicots
and other non-grasses; and (2) dumb-bell or dog-bone shaped - characteristic of grasses
(called graminaceous type)V

V 


 - (1) thickened inner walls; and (2) radial micellation - the cellulose
microfibrils radiate out around the circumference of the pore; (3) chloroplasts - these are
the only epidermal cells with chloroplasts; and (4) connected end-to-end.V

cc




The evolution of a water-impermeable covering of the absorptive surface that was peppered
with oodles of pores was a great idea. The stomata are ideal structures for regulating gas
exchange because:V


V 











 

. In fact, there can be as many as 1000 mm-2.
Obviously, the larger the number of pores, the greater the amount of total diffusion that
can occur (see accompanying data.
m

??
G 

)







 

??
G 
)



&
). A large number of pores is necessary so that
plants are able to absorb enough carbon dioxide for photosynthesis.V
 
V T



?













  In fact, stomata occupy as
much as 2-3% of the total leaf surface area. As expected, the greater the pore area the
greater the rate of diffusion [

??






G*
see
accompanying data] which is advantageous for maximizing carbon dioxide uptake.V

V 





 On average, stomata are about 14 Ǎm in diameter. Although more
total diffusion occurs through large pores [
??
G 
)



?
G ], small
pores are more efficient than larger ones [d??


G 
)
*



?

G 
see accompanying data]. This is due to the edge effect (related to surface-to-
volume ratios). Smaller pores have a greater proportion of edge. As molecules reach
the edge they "spill over" and in effect have a shorter diffusion distance to get away from
the pore.V


V 





?


 A significant boundary layer of humid air forms around
leaf surfaces. This humid area reduces the rate of further transpiration. It occurs because
diffusion shells from adjacent pores fuse. If the pores were much farther separated, they
wouldn't form a nice boundary layer. The thickness of the boundary layer is further
affected by: (1) wind speed; (2) presence of hairs; and (3) sunken chambers.V

V 





?





In grasses, the stomata are distributed approximately
equally on both sides of the leave whereas in herbaceous eudicots there are generally
more on the underside (abaxial) than the upper (adaxial) side. In woody eudicots there
are usually few stomata in the upper surface, whereas aquatic plants with floating leaves
have most stomata in the upper surface. These modifications are important to
minimize/control water loss. Conifers and xeric plants often put the stoma in sunken
chambers.V

V 






? +? 





?










 
?

 






? 
 In effect, any factor that can impact the rate of photosynthesis or
overall water status of the plant will influence the action of the guard cells (see below). I
like our textbook description that stomata are "multisensory hydraulic valves."V

V Test these ideas by studying the data supplied (click here for Diffusion from a standard
leaf)V

ccc( 3 

%

Guard cells open because of the osmotic entry of water into the GC. In turn, this increases the
turgidity (water pressure) in the GC and causes them to elongate. The radial orientation of
cellulose microfibrils prevents increase in girth. Since GC are attached at the ends and because
the inner wall is thicker, the guard cells belly out with the outer wall moving more pulling open
the guard cell. Guard cell closure essential involves reversing this process. We can summarize
the mechanics of GC action as follows:V

stoma closed G
? ń water uptake (osmosis) ń increase pressure ń stoma open G

?V

c,  
 3 

%
  
c
Since water is the driving force for GC action, this means that there must be a gradient in
water potential between the GC and the surrounding cells (subsidiary cells). Thus, to open a
stoma, there must be a mechanism to generate a water potential gradient.V



ÿ










?
?

?

?

? 
V

V There is a rapid decrease in pressure in surrounding cells (i.e., subsidiary cells shrink which
would result in the GC expanding and taking up water). Ësing a fine needle transducer,
 Mary Edwards and Hans Meidner showed that there is some decrease in surrounding cell
P, but this is not a major factor.V
è
V There is an increase in the stretchability of the GC cell wall. This would result in an
expansion of the GC with a concomitant uptake of water. Little evidence exists for this
idea.V

V There is a decrease in the osmotic potential in the GC

Much evidence supports this
hypothesis. For example, Humble and Raschke (1971) showed that the solute potential of
turgid GC (open stoma) of broad bean is -3.5 MPA but when the guard cells are flaccid
(stoma closed), the osmotic potential is -1.9 MPa. Thus, the solute potential of the GC
decreases (becomes more negative) when open. Since the GC volume increases, this
must mean that there is an accumulation or synthesis of solute. V

Thus, we can modify our schematic diagram:V

stoma closed (GC flaccid) ń add solute ń lower ƹs ń decrease ƹw ń water uptake
(osmosis) ń increase pressure ń stoma open (GC turgid)V



 
?




 





?


$V

V Carbohydrates, such as sucrose

Since guard cells are the only epidermal cells with chloroplasts, plant physiologists
have long hypothesized that sucrose and related carbohydrates are osmotic regulators
of guard cells. For example, the starch content of guard cell chloroplasts decreases as
the stomata open. This idea, the "starch-sugar hypothesis", was the first postulated
mechanism for guard cell activity. It lost popularity after the role of potassium ions was
discovered, but most now agree that both sugar and potassium ions play a role in guard
cell regulation. Sucrose seems to be especially important in closing guard cells.  

 &V

Where does the sucrose come from? (a) hydrolysis of starch in the GC chloroplasts. In
other words, an indirect product of photosynthesis (evidence: starch grains disappear
during opening); or (b) a direct product of carbon fixation (photosynthesis).
V

è
V Malate
Malate is an organic acid (C4). You may already be familiar with its role in the Kreb's
cycle in the mitochondria. In plants, malate is also derived from the hydrolysis of
starches. The enzyme phosophoenolpyruvate carboxylase (PEPase) binds carbon dioxide
(actually bicarbonate ions) to phosphoenolpyruvate (PEP; 3-carbon atoms; an
intermediate in glycolysis) to produce oxaloacetate (C4) which is then reduced to
malate and stored in the vacuole.V

V Chloride ions
Chloride ions are transported into the cell from the apoplast via a Cl-/H+ symport in
which a proton gradient is used to "drag" the chloride into the cell.V

V Potassium ions
This appears to be the primary osmotic agent, especially for opening the GC in the
morning. The potassium comes comes from surrounding cells. Evidence: (a) if you strip
epidermis from a leaf, which breaks many epidermal cells but not the more resistant GC,
the GC will only open if K+ is provided in the medium; (b) potassium concentrations
increase in the guard cells upon opening (see Table 1)V
 Table 1: Potassium in the stomatal aperture of 
? 

 ?V

VV OpenV ClosedV

K+ (mol) in GCV 0.45V 0.10V

K+ (mol) in epidermal cellsV 0.07V 0.45V

Thus, we can modify our original scheme:V

stoma closed (GC flaccid) ń sucrose/potassium/malate/chloride ions ń lower ƹs ń decrease

ƹw ń water uptake (osmosis) ń increase pressure ń stoma open (GC turgid)V

To close the stoma, the reverse process occurs. *, time course studies indicate that
potassium uptake is associated with opening of the stomata in the morning, but sucrose loss is
more closely associated with closure in the afternoon. Thus, the final modification to our scheme:V

stoma closed (GC flaccid) ń potassium and chloride ion uptake, malate synthesis ń lower ƹs
ń decrease ƹw ń water uptake (osmosis) from subsidiary cells ń pressure increases ń stoma
open (GC turgid) ń ||||| ń sucrose (potassium, chloride, malate) decreases ń ƹs increases
ń ƹw increases ń water loss ń pressure decreases ń stoma closed (GC flaccid)V

,5 * 

 

3 %

Whatever physiological mechanism we finally postulate for the GC, it must also be compatible
with the action of various environmental factors that are known to regulate stomatal activity.
Since guard cells respond to their environment, especially any factors that impact the
photosynthesis/transpiration compromise. We expect any factor important in photosynthesis to
exert regulatory control on GC. And, we expect water to have the "final word" on control since if
a plant dries out too much it's as good as dead!V

A. Light - exerts strong control. In general: light = open; dark = closed G




 V

What kind of light is important?V

V Red & blue light

The action spectrum for the process suggests that both red and blue light are
important regulators of guard cell activity and that their action is, at least partially,
mediated by photosynthesis (recall red and blue light are used in photosynthesis).
Further evidence that photosynthesis is important - DCMË (an inhibitor of PS) prevents
stomatal action.V

So, what is photosynthesis doing? (a) provides sugars (sucrose and glucose) for
osmotic regulation; (b) provides ATP (via photophosphorylation) to power ion pumps
(see below); (c) reduces internal CO2 levels which stimulates opening (see below); and
(d) reduces the pH in the lumen of the thylakoid that stimulates the synthesis of the blue
 light receptor pigment (see below).
V

è
V Blue light

There is an additional effect of blue light on stomatal activity that is irrespective of its
role in photosynthesis. The evidence: (a) blue light is 10x more effective than red light; (b)
in saturating levels of red light, treatment with blue light will cause additional GC
opening; (c) the action spectrum for blue light is similar to other "blue light responses", a
"three-finger pattern"; (d) photosynthesis inhibitors block the red light effect but not the
blue light effect.V

What is blue light doing?V

÷V Blue light activates a H+-ATPase in the membrane (recall the proton pump for cell
elongation?). Evidence: (a) potassium accumulates in isolated GC protoplasts
treated with blue light and causes them to swell; (b) blue light causes the
acidification of the medium of GC protoplasts under saturating red light
conditions; (c) fusicoccin, which stimulates proton pumping, also stimulates
stomatal action; (d) vanadate (VO3-, blocks the proton pump) and CCCP
(carbonyl cyanide m-chlorophenylhydrazone, an ionophore that makes the
membrane leaky to protons) both inhibit stomatal opening.V

÷V Blue light stimulates starch breakdown and malate synthesis.V

÷V Blue light stimulates cellular respiration (which among other things may be
required to produce ATP for the proton pump).V

What is the receptor for the blue light? V

The action spectrum for the blue light response shows a "three finger pattern," which is
characteristic of other blue light responses (i.e., phototropism). Absorption spectra of potential
receptor pigments show a good match between zeaxanthin, a carotenoid pigment (C40) that
occurs in the chloroplast thylakoid, and the action spectrum. Further ² zeaxanthin levels are
directly correlated with stomatal aperture.V

How does blue light cause stomatal closure?V

Photosynthetically active radiation (red and blue light) cause an acidification of the
chloroplast lumen. This activates the synthesis of zeaxanthin, which in turn, zeaxanthin activates
a calcium-ATP pump in the chloroplast membrane that decreases calcium concentrations in
the cytosol. This, in turn activates the proton pump in the cell membrane.V

B. Low oxygen levels ń GC openV

C. Carbon dioxide - intracellular level is an important regulatory control.V

÷V lo CO2 (?
., during the day, used by photosynthesis) = openV
÷V hi CO2 (?
, at night, produced during respiration) = closedV

D. pH effectV
 ÷V hi pH ń openV
÷V lo pH ń closedV

This effect seems mediated by:V

1. Carbon dioxide Concentration.

Recall the interaction of carbon dioxide and water:V

CO2 + H2O ń CO2 (aq) ń H2CO3 (aq) ń H+ + HCO3- ńH+ + CO3-V

therefore: V

low CO2 = hi pH = open

hi CO2 = lo pH = closedV

2. H+/ATPase proton pump.

The pump is required for stomatal opening (see above). Protons are transferred from the
cytosol into the apoplast. As protons are removed from the cytosol, the pH increases. V

E. Water - protects against excessive water loss.

This is the prevailing and overriding control mechanism. There are two mechanisms by which
water loss regulates stomatal closure, one of which is active and the other passive.V

V Hydropassive Control - simply put, as the plant looses water, the turgidity of the leaf cells,
including guard cells, decreases and this results in stomatal closure. The plant is not
"intentionally" closing the stoma, it is simply a consequence of drying out.V
è
V Hydroactive Control - this mechanism is one in which the plant actually seems to monitor
its water status. When the water potential drops below some critical level, it engages a
cascade of events that close the stomata. Presumably the plant is measuring pressure
(turgor) and then synthesizes or releases an anti-transpirant that is translocated (moved)
to the GC to cause closure.V

The anti-transpirant is abscisic acid (ABA), one of the major plant growth regulators. It is active
in very low concentration (10-6M) and appears very rapidly after water stress (within 7 minutes).
After 4-8 hours, the [ABA] increases nearly 50x. ABA comes from two sources: (a) root ² in
response to water stress, the xylem sap pH increases which in turn stimulates the release of ABA
into the xylem sap for transport to the leaves. This seems to be a root signal to the leaves that
"water stress is coming"; and (b) leaves ² water stress stimulates a synthesis of new ABA and
redistribution of existing ABA.V

Mechanism of Action:
Treatment with ABA results in decrease of potassium, chloride and malate levels in the guard
cells which in turn increase the water potential resulting in water efflux. Evidence suggests that
there is an ABA receptor in the cell membrane. The receptor: (a) activates calcium channels in
the membrane causing calcium uptake from the apoplast; (b) activates calcium channels in
the tonoplast causing calcium release from the vacuole into the cytosol; (c) activates chloride
(and malate)
 channels; (d) inactivates potassium ion ü? ü channels; (e) inactivates the cell
membrane proton pump; and (f) causes an increase in pH that activates
potassium
 channels. Thus, in short, ABA treatment causes an increase in cellular calcium
levels which in turn results in decreases potassium and chloride levels and turns off the proton
pump.V
F. Temperature
Increased temperatures usually increase stomatal action, presumably to open them for
evaporative cooling. If the temperature becomes too high the stomata close due to water stress
and increased CO2 that results from respiration.V

G. Wind
Often causes closure because it: (a) brings CO2 enriched air; and (b) increases the rate of
transpiration that causes water stress which causes the stomata to close. In some cases, wind
causing stomatal opening to increase transpiration for cooling.V

,c  
 3 

%
 ' 
cc

Now, let's pull everything we've learned together to hypothesize a mechanism of action. First,
there are a couple of other observations that we also need to reconcile with our mechanism:V


V Starch disappears in open stomata.

Alkaline conditions favors the starch hydrolysis and acidic conditions favors starch
synthesis. Starch hydrolysis is activated by blue light; andV

V PEPcase activity is high in GC.
Phosphoenolpyruvate carboxylase catalyzes the reaction: phosphoenolpyruvate (PEP)
+ HCO 3- ń oxaloacetate (OAA).V



 
- 


+4 
 V

c8 
 
Solute transport in plants, translocation, primarily occurs in the phloem, but it can occur in the
xylem. V

cc

 
 
,
V

÷V Some solutes are transported in the xylemV

÷V Water and dissolved ions are the main substances in vessels/tracheidsV
÷V These materials are transported via transpiration streamV
÷V Xylem sap may also contain organic materials, usually in relatively low concentration
(with a notable exception being maple sap in the spring which is comprised of 2% or
more sucrose).







.V
÷V Substances move at different rates depending on matrix effects, metabolic needs, etc.V

ccc

 
 
 
V


V Phloem is difficult to study in plants because: (1) the transport cells/tissue in plants are
small (microscopic) in comparison to the transport structures in animals; (2) there is a very
rapid response of the phloem to wounding (contents under pressure); (3) transport in
plants is intracellular (vs. extracellular in animals); and (4) the transport cells are alive.V

V Phloem is the primary transport tissue for photosynthates (photoassimilates, or simply
stated - organic materials).
Radiotracer studies in which leaves are briefly exposed to 14C-labeled carbon dioxide
show that radioactive photosynthates are localized in the phloem.V

V Aphids Don't Suck
Kennedy & Mittler (1953) first noted that aphids could be used as a direct pipeline to
the phloem. Phloem-feeding aphids stick their hollow, syringe-like stylet directly into
phloem cells. Surprisingly, the phloem doesn·t seal itself in response. Aphids don't suck;
rather, the phloem contents are forced into the aphid (thus the phloem is under pressure)
and the excess oozes out the anus (honeydew). Thus, aphid studies demonstrate that the
phloem is under pressure. Further, the honeydew can be collected and we can identify
its composition. Better yet, after anaesthetizing the aphid with CO2 the body is severed
from the stylet leaving a miniature spile tapped directly into the phloem.V

V Phloem Content
G







Analysis - early studies to determine the content of the phloem involved cutting into
the plant and analyzing the contents of the sap that was recovered. The problem is that
you couldn't be sure that your sample wasn't contaminated by xylem exudates or other
materials. Aphid studies described above helped to solve this problem. Phloem is rich in:

1. Carbohydrates - make up 16-25% of sap. The major organic transport materials are
sucrose, stachyose (sucrose-gal), raffinose (stachyose-gal). These are excellent choices
for transport materials for two reasons: (a) they are non-reducing sugars (the hydroxyl
group on the anomeric carbon, the number one carbon, is tied up) which means that
they are less reactive and more chemically stable; and (b) the linkage between sucrose
and fructose is a "high-energy" linkage similar to that of ATP. Thus, sucrose is a good
transport form that provides a high energy, yet stable packet of energy;

2. Amines/amides (0.04-4%) such as asparagine, glutamine, aspartic acid, ureides like

ureas, citrulline, allantoin and allantoic acid. These compounds serve to transport
"nitrogen";

3. ATP, hormones, sugar alcohols like sorbitol (apple, pear, prune) and mannitol
(mangrove, olive), and an assortment of other organic materials; and

4. Inorganic substances including magnesium and potassium.V


V Direction of phloem transport
Information derived from several experiments; check out the Phloem Case Studies.

(1) Classic girdling experiments (removing the bark of a woody plant) by Malphigi (1675)
and Hales (1725) provided some of the earliest evidence. These experiments showed the
accumulation of material above the girdle, and that carbohydrates were not
translocated below the girdle. Thus, plants transport substances in the phloem downward
toward the roots.

(2) Sophisticated girdling experiments, using tracers like 32P, 13C, and 14C demonstrate
that substances in the phloem are transported downward towards the roots  upwards
toward the shoot meristem. 





.

(3) Aphids and tracers G





Conclusions - phloem transports organic materials from sites of production (called

a ) to a site of need (called a  .). Thus, the typical direction of transport is
downward from the primary source (leaves) to the major sink (roots).
V

V Rate of phloem transport
Aphid experiments once again provide an answer...translocation rates average about
 30 cm hour-1 or even faster.
V

V The phloem is under pressure
Studies with aphids showed that the sap was "pushed" out of the plant suggesting the
phloem is under pressure. More recent studies with sophisticated pressure probes have
shown a pressure gradient from source to sink.V


c 
% 
 V

A. Cell types.V

V Sieve tube members or sieve elements.

These cells are joined end to end to make a sieve tube. Theye are called sieve cells in
gymnosperms. At maturity, these cells: (a) are alive, (b) have a functional plasma
membrane and therefore are osmotically active/responsive; (c) no tonoplast or vacuole;
(d) no nucleus, thus no DNA-directed protein synthesis, (e) few mitochondria or plastids;
(f) the ER is primarily beneath plasma membrane and it is mostly smooth.V

Sieve elements are joined by sieve plates. These have numerous pores lined with
callose (ǃ 1-3 glucan). Callose forms rings around the pore, like a grommet. The wall
region in the middle of the grommet hollows out and the membranes from the two
adjacent cells are connected. Callose can plug the pore if the cell is damaged. The
amount of callose observed varies with season, age, metabolism. Callose synthase is in
the cell membrane.V

è
V Companion cells (angiosperms; albuminous cells - gymnosperms)

These cells have a dense cytoplasm, mitochondria, nucleus, golgi, ER, chloroplasts -
the standard goodies. Although their function is not well understood, they can be
considered "nurse cells" to the sieve tube members. These cells are derived from the
same cambial initial cell as the sieve tube members. There are three types of companion
cells:

(a) "ordinary" ² with chloroplasts, few plasmodesmata connections to other cells except
sieve elements, smooth inner walls, normal chloroplasts;

(b) transfer ² more plasmodesmata, ingrowths in the wall to increase the S/V ratio; and

(c) intermediary ² many plasmodesmata, vacuoles, undeveloped chloroplasts. The

transfer and "ordinary" companion cells likely function to remove solutes from the
apoplastV

V Parenchyma cells
These are vacuolated, storage cells. They help in lateral conduction and may help in
transferring material to/from sieve cells. Transfer cells are specialized parenchyma cells.
V

V Fibers - primarily for support.
V

V Side note: Mesophyll cells in a leaf are close (perhaps 1-3 cells away) to a minor vein.V

 
 V
 ÷V MW 14,000-158,000V
÷V Originally thought to be a carbohydrate and was called slime because it gelled when
exposed to the airV
÷V Various forms, bundles of fibers or amorphous areas or even crystallineV
÷V Appear early in development of sieve elementsV
÷V Only in angiospermsV
÷V at least two proteins, PP1 and PP2V
÷V Once the sieve pores form, the P-protein disperses through the pore.V
÷V The protein is fibrousV
÷V P protein plugs the pore when the cell is damaged.V
÷V synthesized in companion cellsV

c( 

 
V


V Requirements
The model must account for: (1) speed of transport. The process is much faster than
simple diffusion. For example, a conservative estimate of the mass transfer rate in phloem
is 15 g cm-2 hr-1. If the rate was based solely on diffusion is would be predicted to be
200 Ǎg cm-2 hr-1; (2) bidirectional flow - recall that substances can be transported down
or up in the phloem; and (3) pressures in the phloem
V

V Pressure flow (or Bulk Flow) hypothesis of Munch. This is the best model that fits the data.
(
- Phloem transport is analogous to the operation of a double
osmometer G

?. If solute is added to bulb A ń osmotic potential decreases ń

osmotic uptake of water ń pressure increases ń bulk flow of water and solute to bulb B
ń pressures increases in bulb ń water potential in B greater than in beaker ń osmotic
flow of water into the beaker ń water returns to side A via the connection. This system
could be maintained indefinitely if there is a mechanism to remove solute (sucrose) at
the end (sink) and a mechanism to add solute (source).V

Sinks include young leaves, roots, developing fruits. Sources include mature leaves,
cotyledons, endosperm, and bulbs and storage roots in spring. Sinks and sources can
change depending upon the nutritional need of the plant. Thus, roots can be a source
in the spring but are sinks for the majority of the growing season.
V

V Plants as osmometers. If this model is valid then.....V

V Sieve tubes should be continuous pipes...they are.V

è
V Sieve tubes should provide minimal resistance to flow.
In other words, the sieve tubes shouldn·t be clogged by P-protein. This is true in
specimens that are rapidly prepared. However, this was a major concern in early
experiments because the phloem always appeared clogged up in TEM pictures.
Further, this explains why sieve tube members have few "typical" cellular structures
- they would "get in the way."V

V The phloem should be under pressure. As the aphid experiments suggest....it is.
In fact, mini-pressure gauges can be attached to a severed aphid stylet and
the pressure can be measured. It varies from 0.1-2.5 MPa. Further, there should
 be a pressure gradient from source to sink (driving force for movement). There
is...see overhead.V

V Sieve elements must have a membrane (for development of pressure gradients) -
they do.V

V There should be an osmotic potential gradient from source to sink (there is...see
overhead).
The source region of the phloem has a considerably lower osmotic potential
than the sink regions.V

V There must be a mechanism to load solutes from the source into sieve cells.
This process must be active since the solutes (usually sucrose) are being loaded
against a concentration gradient. Evidence - respiratory inhibitors block the
process. The loading mechanism should be:V

÷V Selective - it should only load the materials that are transported. This is
supported by radiotracer studies; abraded leaves have been shown to
only load materials that are normally transported;V

÷V Allow for apoplastic (from protoplast to wall to protoplast) or symplastic

(from protoplasts to protoplast via plasmodesmata) transport. In some
species, sucrose transport is symplastic - from mesophyll protoplast to cc-
se protoplast via plasmodesmata. In others, sucrose loading into the cc-se
complex involves an apoplastic step (mesophyll protoplasts to apoplast to
cc-se protoplast.V

÷V Provide a mechanism to transport sucrose across the membrane - the

sucrose/proton cotransport system. According to this model, protons are
pumped out of the sieve cells into the apoplast by a membrane-bound
H+-ATPase Vthe proton concentration increases in the apoplast  pH
decreases  K+ is brought into the sieve cell to balance the charge  the
proton gradient provides the driving force for transporting sucrose against
a gradient  the sucrose and protons bind to a carrier protein in the
membrane and are released in the sieve tube member. Evidence: the pH
is high in sieve tubes; if the pH of the apoplast is increased there will be no
sucrose uptake; there is a hi potassium conc. in sieve tube members. A
membrane carrier is likely involved since PCMBS (p-chloromercuribenzene
sulfonic acid), an inhibitor of membrane proteins, interferes with sucrose
uptake.V


V There must be a mechanism to unload solute at the sink. Sucrose is unloaded into
the apoplast in some tissues (?
, ovules) and into the symplast of others
(growing/respiring tissues like young leaves, meristems).V

V Apoplastic transport and unloading can occur via two methods: (a) sucrose is
hydrolyzed by acid invertase to glucose and fructose upon reaching the sink. This
maintains the gradient for transport. The glucose and fructose are taken up by
the sink cells and stored or further metabolized as in maize; or (b) sucrose is
unloaded into the sink by a carrier co-transport system like in sucrose loading.V

V The empty ovule technique has been useful in these studies.V

VSome metabolism is required (for loading/unloading) and to maintain sucrose
against a concentration gradient. This explains the response to respiratory
 inhibitors. Phloem transport is also inhibited by anoxia and cold temperatures -
both thought to exert their effect through energy metabolism.V


3

 0 V

c

 '
 

You betcha. In class we will provide some case studies, including early experiments by Darwin
(1880), Boysen-‰ensen (1913), Paal (1918) and Went (1923) involving phototropic curvature in
canary grass coleoptiles and more recent work concerning fruit set in soybean.V

Conclusion: from these studies, and countless others, we conclude that plants possess a well-
developed system of chemical messengers that induce (inhibit or promote) growth and
developmental responses. These chemical messengers are termed "hormones".V

cc

  -  




$






?

 V

V smallV
è
V organic compounds;V

V synthesized by the plant;V

V active in low concentration (<10-6);V

V promote or inhibit growth and developmental responses;V

V often show a separation of the site of production and the site of action (although
this isn·t as clear a distinction as in animals).V

Based on these criteria, would the following substances be considered hormones? Ca2+, sucrose,
2 4 - D, glycine, or K+? V

ccc

* 

There are a few significant differences in the nature of hormones found in plants and animals.
These are summarized in the following table:V

VV PlantsV AnimalsV
number of
fewerV manyV
hormonesV
specificity of actionV non-specificV specificV
work togetherV yesV noV
site noV yesV
action/production
separationV

Thus, there are comparatively few plant hormones, each elicits a variety of responses and
often works together with other hormones. In contrast, animals have numerous different kinds of
hormones, each with a specific function, and it works alone to induce a response.V
 Why the differences? Good question. It may be partly a function of our ignorance; in other
words, there are likely to be many more plant hormones that simply haven·t yet been identified.
Nevertheless, I suspect that at least part of the reason for the differences is related to body
design. Recall that plants have an architectural design with a limited number of parts that are
repeated. It follows that only a limited number of hormones would be necessary to induce
growth/developmental responses. With a mechanical design and numerous separate parts,
animals required unique chemical messengers to interact with each one.V

c

 
 There are five major groups, based on chemical structure. With the exception of the latter
two, each group represents a family of related compounds. These groups are: (1) auxins; (2)
gibberellins; (3) cytokinins; (4) abscisic acid; and (5) ethylene. In addition, there are a variety of
other plant "hormones" including the brassinosteroids, oligosaccharides, polyamines, jasmonic
acid, salicylic acid, systemin, and putative hormones like those involved in flowering (florigen). V

. 
  

In the past, physiologists simply applied the substance to a plant or excised part to see if it
caused a response. If there was a response, then we assumed that the substance was a
hormone involved in the response. However, responding to an exogenous application of a
hormone doesn·t necessarily mean that the substance has any endogenous (?
? ) action.
Thus, we·ve become a little more picky and only accept that a substance is a hormone if:V

V Exogenous application causes the response;V

è
V Lowering endogenous levels prevents the response;V

V Lowering endogenous levels followed by exogenous application restores the
response;V

V Endogenous levels should be related to the response (i.e., increase 

the
response occurs).V

Check out the case study concerning bolting in 


?  (‰ones and Zeevart, 1980,
Planta 149:269).V

c(  
 

Hormones act on target tissues to activate a receptor. The general mechanism is: V

hormone à target tissue/cell à receptor à signal amplification à responseV

Thus, for a response to occur:V

V the hormone must be present in sufficient quantity;V

è
V the target tissue must be sensitive (sensitized) to the hormone;V

V the target tissue recognizes the hormone (i.e., there must be a receptor to which
the hormone can bind);V

V the binding of the hormone/receptor should initiate a change in the receptor
(amplification). Calcium is often involved and its interaction is mediated by the
protein calmodulin; andV

V the activated receptor initiates a physiological responseV

cc &

  V

A. Bioassays
A bioassay examines the effect of a test substance on a plant tissue. To perform a hormone
bioassay, a test plant is chosen that lacks the hormone for a response. Known amounts of
hormone are added to the plant, the response is measured, and a "standard curve" is
produced. To determine if a sample contains the hormone, the test plant is treated in a similar
fashion. If present the hormone can be quantified by comparing its response to the samples of
known concentration. For example, a variety of rice (Tauginbozu) lacks GA and is often used in
bioassay studies. After treatment with various concentrations of GA, leaf length vs. [GA] is
plotted.V

Table: Comparison of the advantages and

disadvantages of bioassaysV
AdvantagesV DisadvantagesV
simple & easyV sensitive to impuritiesV

inexpensiveV false positive tests can resultV

lower sensitivity than other

methodsV

B. Immunological studies
Antibodies are made against the plant hormones and then used as specific probes to localize
and quantify. The antibodies are usually coupled to radioisotopes or fluorescent dyes to make it
easier to trace. This technique is very sensitivity and specific.V

C. Instrumental Methods: GC-MS; HPLC; high specificity and sensitivityV

ccc(


   
*

The internal levels of plant hormones must be tightly controlled so that the response occurs
only at the appropriate time. Regulation of hormonal action is achieved by: (1) controlling the
rate of hormone synthesis; (2) forming conjugates, which are inactive storage forms where the
hormone is covalently bonded to a sugar, amino acid or other molecule; (3) enzyme
degradation; (4) transporting the hormone away/toward the site; and (5) compartmentalizing
the substance in an organelle such as the chloroplast.V


 '% V

cc

 
 Auxin is a general name for a group of hormones that are involved with growth responses (i.e.,
elongate cells, stimulate cell division in callus). Not surprisingly, the term "auxin" is derived from
the Greek word "to increase or grow". This was the first group of plant hormones discovered.V

cc 
 0

V

A. Naturally Occurring Auxins

The most important auxin found in plants is indole-3-acetic acid (IAA). IAA is comprised of an
indole ring linked to acetic acid (  
 ). Other auxins that have been isolated from
plants include indole ethanol, indole acetaldehyde, indole acetonitrile, phenylacetic acid
(PAA), and 4-chloro-indoleacetic acid. These are probably converted to IAA ?
? .V

B. Synthetics with Auxin Activity

There are a variety of substances that are not known to occur in plants that have auxin
activity. These include indolebutyric acid (IBA); naphthalene acetic acid (NAA); 2,4- dichloro-
phenoxyacetic acid (2,4-D), and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). The exact
mechanism of action of these compounds is not known, but they may inhibit nucleic acid
synthesis.V

C. Chemistry of action
Although a variety of molecular structures have auxin activity (i.e., derivatives of indole,
benzene and naphthalene), these molecules seem to share a few features that appear to be
required for activity. Auxin activity seems to be correlated with a flat, hydrophobic ring system
that separates negatively-charged (acidic, carboxyl group) side chain and positively charged
region. There is a charge separation of about 0.5 nm. Note that the indole unit isn't required for
activity, but a planar ring system is.V

D. Conjugated forms
Auxins, as do other hormones, occur in a free or conjugated (bound to sugars, alcohols or
other molecules) form. In fact, up to 98% of the auxin may be bound. Auxins may be conjugated
with inositol, coenzyme A or glucosides (sugars).V

E. Anti-auxins
PCIB (p-chlorophenoxyisobutryic acid) is a compound that competes with auxin for binding
sites. However, it doesn't cause any growth response. V

ccc$
V

A. Site
Auxin is made in actively growing tissue which includes young leaves, fruits, and especially the
shoot apex. Made in cytosol of cellsV

B. Routes
There are two major routes to the production of IAA. V

V Tryptophan-dependent Pathways. The similarity of chemical structure of IAA and

tryptophan suggested a connection between these. Considerable research has shown
that tryptophan, one of the protein amino acids, is a precursor of auxin biosynthesis.
Overall, the conversion of tryptophan to IAA can occur by: (1) a transamination followed
by a decarboxylation; (2) a decarboxylation followed by a transamination; or (3)
formation of IAA via an oxime (C=NOH) and nitrile (CN).V
è
V Tryptophan-independent Pathway - this route doesn't involve tryptophan directly as an
intermediate to the formation of auxin. V

c 
V

A. Basipetal (or Polar) Transport

Auxin is transported in a basipetal (towards the base, base-seeking) direction. In other words,
auxin moves from the shoot tip towards the roots and from the root tip towards the shoot.V
Evidence - (1) Seedling vs. [auxin]; and (2) 14C labeled IAA applied to the top of a stem section is
recovered only from the bottom of the stem section. When auxin is applied to the end of stem
segments, it is only transported from the "top" of the section to the "bottom" as demonstrated in
these data:V

V ËprightV Ëpside downV

14C-IAAin donor block (dpm)C-IAA in 10,000V 10,000V

donor block (dpm)V

14C-IAA in receiver block (dpm)C-IAA 7,500V 300V

in receiver block (dpm)V

B. Mechanism of polar transportV

V Transport Rate ² IAA is not transported through the transpiration stream or phloem
because the rate of movement is too slow. The rate of transport is consistent with
diffusion.V
è
V Tissue of transport - appears to occur in parenchyma cells associated with the
vascular tissue.V

V Model for polar transport: (a) Protons are moved out of the cell by a proton
pump that requires ATP; (b) IAA is protonated at low pH; (c) IAA-H passes through
the lipid membrane. It can enter or leave anywhere; (d) once inside the cell, the
IAA-H ionizes in response to the higher pH; (e) IAA- requires a permease to pass
through the membrane; (f) histochemical studies have shown that a permease is
only located at the bottom of the cell - resulting in a net movement of auxin out
of the bottom of the cells.V

V Evidence for polar transport model: (a) transport is blocked by respiratory poisons
(i.e., demonstrates the need for ATP and a proton pump); (b) unlabeled IAA
competes with C14 labeled IAA for uptake in to the cells - this suggests that a
carrier of some sort is required ; (c) fluorescein-labeled antibodies show that the
permease is localized at bottom of cells; and (d) the transport of auxin is blocked
by NPA (napthylthalamic acid), TIBA (tri-iodobenzoic acid) and flavanoids. These
inhibitors appear to block the permease.V

V Proton-Auxin CoTransport Mechanism - In addition to the passive pH-dependent
mechanism described, there a membrane transport protein seems involved that
cotransports protons and IAA into the cell. This tranport protein is localized in the
upper side of the cells.V

$ 

There are four classic bioassays for auxin. These tests, which are all based on the ability of
auxin to stimulate shoot growth (or inhibit root growth) are: V

A. 
 coleoptile curvature test
Pioneered by F. Went. The angle of curvature of a decapitated oat coleoptile is measured
after placing an agar block containing auxin on one side. Then, angle of curvature vs. [IAA] is
plotted.V
B. 
 coleoptile elongation
The ratio of final length/original length for oat coleoptiles or pea stem sections is measured
after the tissues are floated in solutions containing different concentrations of IAA. Elongation vs.
[IAA] is plotted.V

C. Split pea curvature test

A section of pea hypocotyl is obtained and split halfway down the middle. After incubating
the sections in solutions of known IAA concentration, the angle of curvature is measured. Note
that only the epidermal cells are responding to the treatment.V

D. Cress root inhibition

This bioassay is based on the ability of auxin to stop root growth. A ratio of treatment/control
growth is plotted vs. log [IAA].V

c%  

IAA is involved in the following responses:V

A. Cell elongation and wall relaxation

Discussed earlier in the semester. Basis of several bioassays and the discovery of auxin. NOTE:
Normally exogenous application of IAA has little effect on plants.

B. Cell differentiation
Promotes differentiation of vascular tissue (i.e., xylem & phloem).

C. Ethylene production
IAA apparently stimulates the production of ethylene.

D. Inhibition of root growth

[IAA] > 10-6 M inhibit root elongation. However, very low (>10-8 M) favor root elongation.

E. Stimulate root initiation (lateral roots, adventitious roots)

Roots always form at the basal end of cutting

F. Flowering
Although most plants don·t initiate the production of flowers after auxin treatment, pineapple
and its relatives (Bromeliaceae) do. Once flowers are initiated, in many species, IAA promotes
the formation of female flowers, especially in cucurbits (gourd family).

G. Parthenocarpic fruit development - as per Π$

H. Apical dominance
The apical meristem (apex) controls or dominates the development of the lateral buds. (PS - a
bud is an embryonic shoot with immature leaves and stem). Apical dominance also occurs in
roots. It is responsible for the Xmas tree shape of many trees and prevents an individual from
becoming too top-heavy.V

V Function: (a) maintain upward growth; (b) maximize light absorption; (c) prevent
top-heaviness; and (d) provide mechanism to replace the apical bud if it is
removed by grazing or damage.
V
è
V Thimann & Skoog (1934) - first suggested a correlation between [IAA] and apical
dominance. This idea was further developed by Cholodny-Went who proposed
that plant tissues responded to different concentrations of IAA (see figure). For
example, hi [IAA] stimulates shoot growth but inhibits bud and roots. Thus, IAA is
produced at the tip of the plant and is transported downward. The high
concentrations near the apex inhibit lateral buds. As the concentration
decreases it frees the buds from the inhibition and they develop.V

Evidence:V


V every gardener knows that removing the apical meristem results in bushier
plants. This is consistent with the Cholodny-Went hypothesis. If IAA is
applied to a de-tipped plant it prevents lateral bud developmentV

V IAA stimulates the formation of ethylene which is known to inhibit lateral
bud formation.V

V branching mutants of tomato don't export 14C-IAAV

But:V


V [IAA] in buds after the tip is removed should decrease, and they don't V

V [IAA] that act to replace the tip are much higher than levels in the intact
plantV

V
V 14C-IAA doesn·t enter the lateral buds when applied at tip

V
V Some plants don·t respond to exogenous IAA


V Nutrient diversion hypothesis

There is little development of vascular tissue to the lateral buds. This has lead
some to propose that the lateral buds fail to develop because they get no
nutrients because the nutritional traffic is being diverted to the apex.

Evidence: (a) application of cytokinins stimulates lateral bud development;

cytokinins known to direct nutritional traffic, especially during senescence; (b)
tomato mutants that exhibit strong apical dominance have lower levels of
cytokinin; (c) there is a good correlation between cytokinin level and bud
development.V

V Bud Inhibition
Another possible reason why buds fail to develop is because they have an
inhibitor. It has been shown that ABA inhibits bud and seed development in many
species. The buds of decapitated plants have a lower [ABA] than intact plants.
High [ABA] in the bud are maintained by applying IAA to decapitated apex.V

I. Tropic responses - such as gravitropism and phototropismV

‰. Abscission
Formation of the abscission layer is correlated with the [IAA] in leaf. As long as the [IAA] in the
leaf is high relative to the stem, then the abscission layer doesn·t form. When the [IAA] in the leaf
drops, which occurs normally during the growing season, it stimulates the formation of the
abscission layer forms and the leaf falls. The IAA presumably works by decreasing the sensitivity
of the cells to ethylene which is the primary hormone involved in initiating leaf drop.V

ccŒ

 5  *

All of the general methods (i.e., degradation, conjugates, regulate rate of synthesis,
sequestering in different regions) of regulation are probably operative. In particular, IAA is
probably shuffled back and forth between the two main areas, or pools, where it is stored ² the
cytosol and chloroplast. IAA oxidase, a type of peroxidase, converts IAA to inactive metabolites.
There are several degradative pathways.V

%#Œ(#5'3c$$5Œ5cV

c3 
V

÷V "foolish seedling" disease (bakanae) - rice plants tall, weak and spindly with little grain -
was observed early this century by ‰apanese farmers.V
÷V associated with the fungus, ?


"?!?.V
÷V liquid from the culture medium applied to plants caused symptoms (Kurosawa, 1926).V
÷V three gibberellins were isolated and identified from the medium & fungusV
÷V independent confirmation in labs in Europe and ËS.V
÷V gibberellins known from angiosperms, gymnosperms, ferns, mosses, algae, fungi and
even a few bacteria.V

cc 
 V

÷V diterpenes (C20, though some have lost a carbon and are C19) ² based on isoprene
skeletonV
÷V characterized by having a complex system of 4-5 rings (ent-kaurene ring system) and a
carboxyl (acidic) side chainV
÷V more than 110 different gibberellins are known - abbreviated GA1...GAn.V
÷V No more than 12 or so different gibberellins occur in any one speciesV
÷V only a few (about 15) of the GA's have biological activity; the rest are likely breakdown
products of, or precursors to, the active onesV
÷V GA1 is the most active GAV
÷V GA3 was the first one discovered and is readily available commercially (produced by 

"?!? cultures).V
÷V C19 GA's are more active than C20V
÷V GA's can occur in a free or conjugated form (i.e., glucosides)V

ccc$
V

A. Site - young leaves, roots, and developing seeds (developing endosperm) and fruits.V

B. PathwayV

÷V terpene pathwayV
÷V basic terpene building block is isoprene (isopentenylpryophosphate, IPP), a five carbon
unitV
 ÷V five major steps: (1) synthesis of IPP; (2) condensation of 4 isoprene units to form
geranylgeranylpyrophosphate (GGPP); (3) GGPP cyclizes to form the kaurene ring
system; (4) a methyl group of kaurene is oxidized to a carboxyl group to form GA12-
aldehyde; (5) GA12-aldehyde is the precursor to the other GA'sV
÷V two routes to the production of IPP (isoprene): (1) Mevalonate-dependent pathway -
occurs in cytosol; mevalonate is especially important for sterol biosynthesis; mevalonate
is derived from acetyl CoA; and (2) Mevalonate independent pathway ² especially in
plastids, important for carotenoid biosynthesisV

C. GA synthesis inhibitorsV

÷V Kaurene synthesis inhibitors - Phosphon D, CCC (cycocel), and Amo1618.V

÷V Kaurene oxidation inhibitors - Ancymidol (A-rest) and paclobuturalV
÷V Another inhibitor is B-Nine (alar).V


c$ 0% 
V

A. Three common bioassays used for gibberellin are:V

÷V Lettuce hypocotyl elongationV

÷V Dwarf rice (var. Tanginbozu rice) leaf sheath elongationV
÷V alpha-amylase production in barleyV

B. Instrumental methods ² now the method of choice, especially GC-MSV

 
V

÷V made in the tissue in which it is usedV

÷V transport occurs through xylem, phloem, or cell-to-cell.V
÷V phloem seems to be most important transport routeV
÷V transport is not polar, as it is for auxin.V
÷V not purely diffusion.V

c8
0Œ

   
*
V

÷V Formation of conjugatesV
÷V Slow hydrolysisV

cc%
 V

A. Promotes stem elongation

When applied to intact plants, GA usually causes an increase, unlike auxin. It overcomes
dwarfism in mutants that have a mutation in the GA synthesis pathway. dwarf = short; wild type =
tall; dwarf + GA = tall. Thus, GA application: (1) stimulates elongation; and (2) acts on intact
plants.V

GA stimulates stem elongation by:V


V stimulating cell division. Specifically, GA increases the transition from G1 à S phase of the
cell cycle;V
è
V increasing amylase (and other hydrolytic enzymes) production à increases hydrolysis of
starch à provides glucose and other sugars that: (a) lower water potential which
provides driving force for water uptake; (b) provide energy through cell respiration; and
(c) provide materials for building cell walls; andV

V increase cell wall plasticity (by a mechanism other than how auxin works).V

B. Overcomes dormancy in seeds and buds

Treating dormant seeds with GA stimulates germination (see below)V

C. Involved in parthenocarpic fruit development - remember

V

D. Flowering
Recall the   we did? To summarize: LD = tall (bolts); SD = short; SD + GA = tall.
Thus, GA stimulates bolting in Long Day plants and can substitute for long days or cold
treatments that are necessary for flowering.V

E. Mobilization of food reserves in grass seed germination

GA is produced by the scutellum (cotyledon) of the embryo ń stimulates the production of
amylase by the aleurone layer à amylase hydrolyzes starch to simple sugars ń absorbed by
scutellum and translocated to embryo for growth.V

The production of amylase occurs 

 . That is, gibberellin stimulates transcription. In short:
GA ń binds to membrane receptor ń interacts with a protein complex (heterotrimeric G
protein) that ń activates a GA signaling intermediate ń turns off a repressor ń transcription of
GA-MYB mRNA ńtranslated in cytosol to make GA-MYB protein ń returns to nucleus to bind to
alpha-amylase gene promoter region ń activates transcription of alpha-amylase
mRNA ń translated in ribosomes on RER ń transported to golgi ń secretory vesicles release
alpha-amylase. This last step is apparently regulated by a calcium dependent mechanism that
was also activated by the heterotrimeric G protein complex.V

Brewers take advantage of GA's ability to stimulate germination and enzymes which are
important in the brewing process.V

F. ‰uvenility
Plants exist in a juvenile and adult form. As in humans, the main difference is whether the
plants are able to flower (reproduce). In some plants there is little morphological difference
between juvenile and adult forms, whereas in others, the two forms are very distinct. For
example, in beans, the first (juvenile) leaves are entire (heart-shaped) while the adult leaves are
trifoliate. Lancewood is a New Zealand plant that has very distinctive juvenile and adult forms. In
fact, they are so different that botanists originally mistook the two forms for different species. The
juvenile form is unbranched with long (. 12 in) linear, drooping leaves that look a little like the
ribs of a folded umbrella. When the plant reaches about 15 foot it switches to the adult form
which is branched and has smaller, ovate leaves. It has been suggested that this is a
modification to prevent predation by moa, a large, formerly common but now extinct bird.V

Gibberellin stimulates the transition between the juvenile and adult forms. In ivy, the adult form
(unlobed leaves, shorter internodes) is converted to the juvenile form (lobed leaves, longer
internodes) by GA treatment.V
G. Sex expression
In plants with separate male and female flowers, GA application can determine sex. For
example, in cucumber, hemp and spinach, GA treatment increases the proportion of male
flowers. In maize, GA treatment causes female flower development.V

c, 
%

 V

÷V increase size of grapes (spray at time of blooming and fruit set stage)V
÷V increase distance between grapes in a cluster to minimize fungi/diseaseV
÷V Breweries - increase starch digestion for malting processV
÷V Delay senescence - spray on fruit like naval orangesV
÷V celery stalk elongation (but must use carefully because of increased storage problems)V
÷V sugar cane ² increased growth and yieldsV
÷V Minimize lodgingV


 '
.  V

c3 
V

÷V Called "cytokinins" because they stimulate cell division (?

, cytokinesis)V
÷V Haberlandt (1913) noted that non-dividing potato parenchyma cells would revert to
actively dividing ones in the presence of phloem sap. This observation suggested a
soluble material was responsible for cell division. V
÷V Folke Skoog (1940's) and colleagues at Ëniv. of Wisconsin found that cultured tobacco
pith tissue explants would proliferate only if they were supplemented with various
substances such as autoclaved herring sperm or coconut milk.V
÷V Miller (1956) identified the first cytokinin, called kinetin, in the herring sperm.V
÷V Cytokinins occur in most plants including mosses, ferns, conifers, algae and diatomsV

cc 
 V

A. GeneralV

÷V adenine derivatives (amino purines)V

÷V occur as: (a) the free nitrogenous base; (b) a nucleoside (base + ribose); (c) a
nucleotide (base + ribose + phosphate); or (d) glycosidesV
÷V The free base is the active form.V
÷V approximately 40 different structures known.V
÷V Zeatin (Z), which was first isolated from maize (§


) is the most common cytokinin.V
÷V Other naturally occurring cytokinins include, dihydrozeatin (DHZ) and
isopentenyladenosine (IPA).V

B. Synthetic cytokininsV

÷V kinetin (

) ² probably byproduct of zeatin degradationV
÷V there are several other substances with cytokinin activity such as benzyl adenine
(benzylaminopurine; BA).V

C. Cytokinins and nucleic acidsV

 ÷V can occur as a modified base in tRNA, but the bases exist in the ? form, rather than the
typical   form. These modified bases that are found in all organisms from bacteria to
plants to humans.V
÷V The function of the tRNA cytokinins is not clear, but after hydrolysis of the tRNA the
products can act as a cytokinin. The importance of the tRNA derived cytokinins in overall
growth and development is not clear, either.V
÷V Interestingly plants have different sets of tRNA·s with different cytokinins that participate
in protein synthesis in the cytoplasm and the plastids.V


ccc
V

÷V Site: synthesized primarily in the meristematic region of the roots. This is known in part
because roots can be cultured (grown in artificial medium in a flask) without added
cytokinin, but stem cells cannot. V
÷V Cytokinins are also produced in developing embryos and crown gall tissuesV
÷V first major precursor to the cytokinin is AMP (adenosine monophosphate)V
÷V side chains of the cytokinins are made by the terpene pathway (IPP ² isoprene) and
added to the AMP by cytokinin synthaseV
÷V there is some speculation that surface and endophytic bacteria (?
, 


?)
may be the actual source of plant cytokinins. Rationale: (1) haven't isolated some of
the putative genes for cytokinin synthesis; (2) remove bacteria show impaired cytokinin
production.V
÷V tRNA nucleotides are modified to cytokinins after the tRNA is transcribed (post-
transcriptional processing)V

c 
V

÷V via xylem (transpiration stream)V

÷V in peas, a signal from the leaves may signal/regulate transport of cytokinins from the
rootsV
÷V zeatin ribosides are the main transport form; converted to the free base or glucosides in
the leavesV
÷V some cytokinin also moves in the phloem.V

$ 0% 
V

A. BioassayV

V Callus culture cell proliferation - not used too much because it takes too longV
è
V Expansion of radish or cocklebur cotyledonsV

V Inhibition of chlorophyll loss by detached oat leaves during senescenceV

B. Methods of Analysis ² liquid chromatography, mass spectroscopy; radioimmunoassaysV

c8
V

÷V forms conjugates with glucosides. major storage form of cytokinin, inactiveV

÷V cytokinin oxidase may be an important route of disposal, too.V
cc%
 V

A. Control morphogenesisV

÷V in plant tissue cultures, cytokinin is required for the growth of a callus (an undifferentiated,
tumor-like mass of cells):V

callus + auxin + no cytokininVńV little growth of callusV

callus + auxin + cytokinin VńV callus grows well, undifferentiatedV

÷V ratio of cytokinin and auxin are important in determining the fate of the callus:V

callus + low [cytokinin/auxin]VńV callus grows well, forms rootsV

callus + high [cytokinin/auxin] VńV callus grows well, forms meristem & shootsV

÷V some tissues become habituated during repeated cell culture ² loose the requirement
for cytokinin in the growth mediumV

B. Crown GallV

÷V tumor-like mass of undifferentiated cells that typically occurs near the crown (junction of
root and stem) of the plantV
÷V caused by the bacterium 
?

?
V
÷V carries a plasmid (Ti plasmid; a plasmid is a small loop of non-chromosomal DNA) with
loci/genes for auxin production (tms), zeatin production (tmr) and opines (are nitrogen-
containing molecules that provide food for the bacteria).V
÷V upon infection, the plasmid is incorporated into the plant cell genome which begins to
overproduce auxin and cytokininV
÷V stem forms an undifferentiated tumor (crown gall)V
÷V as predicted, if the tms genes (auxin production) are deleted from the plasmid, which
would increase the cytokinin/auxin ratio, the resultant crown gall is "shooty". If the tmr
genes are deleted the gall is "rooty".V

C. Regulates the cell cycle/cell division (hence, the name "cytokinins) ² especially by controlling
the transition from G2 à mitosis. This effect is moderated by cyclin-dependent protein kinases
(CDK's) and their subunits, cyclins.V

D. Delay senescenceV

÷V senescence is the programmed aging process that occurs in plants (and other organisms
for that matter).V
÷V loss of chlorophyll, RNA, protein and lipids.V
÷V cytokinin application to an intact leaf markedly reduces the extent and rate of
chlorophyll and protein degradation and leaf dropV
÷V correlation between cytokinin levels and senescence. For example, as detached leaves
senesce the cytokinin levels drop. And, when these leaves are treated with auxin to
stimulate rooting, when roots form the senescence process stops and cytokinin levels rise.V
 ÷V Tobacco plants + senescence promoter sequence + ipt gene (cytokinin gene) à causes
gene to become active on senescence à no senescenceV

The exact mechanism by which this occurs is unclear but likely involves the ability of cytokinins to
mobilize nutrients. Application of cytokinin to a leaf will cause it to act as a sink and nutrients will
be directed towards it.V

E. Greening
Promotes the light-induced formation of chlorophyll and conversion of etioplasts to
chloroplasts (greening process).V

F. Promote lateral bud development

Cytokinin application to dormant buds will cause them to develop. A witches· broom is
caused by a pathogen such as the bacterium 


?
?  (or 

?
)
that produces cytokinin which, in turn, causes stimulates lateral bud development (branching).
These results suggest that apical dominance may be related to cytokinin, too.V

For example, when tobacco cells are infected with the Ti-plasmid that has been modified to
possess the heat shock promoter, a heat treatment stimulates the cells to produce increased
amounts of cytokinin. These plants exhibit less apical dominance and remain green longer than
non-heat treated controls. Thus, these results support the conclusion that senescence and apical
dominance are related to cytokinin levels.V

G. Promote cell expansion

Cytokinins stimulate the expansion of cotyledons. This is the basis for the classical bioassay.
The mechanism is associated with increased plasticity of the cell wall, not associated with
acidification.V

cccc( 
 - Specific binding sites (receptor) for cytokinin are known. These may
be ribosomal proteins. Thus, it is not too surprising that cytokinins have been shown to regulate
protein synthesis. V

%%G%$%:V

c3 
V

÷V Bennet-Clark and Kefford (1953) described the presence of an inhibitor of coleoptile

elongation in oatsV
÷V about 10 years later, Addicott

 found a substance that stimulated abscission of fruits
in cotton and they named it abscisin IIV
÷V about the same time, Wareing found a substance in sycamore leaves that promoted
dormancy in buds and called it dorminV
÷V it was soon clear that these were the same substanceV
÷V a conference in 1967 straightened out the name and it was decided to call the hormone
abscisic acid (ABA).V

cc 
 V

÷V a single structure, not a family of related structures like the gibberellinsV

÷V sesquiterpene (?
., terpenoid) - C15 - made from 3 isoprene unitsV
÷V occurs as ? form; S-enantiomer is natural and active formV
 ÷V found in all green plants, also in some mosses, algae, and fungiV
÷V related to lunularic acid which is found in liverworts.V

ccc$
V

÷V plastidsV
÷V most tissues, especially leaves and seedsV
÷V terpene pathwayV
÷V IPP is the first intermediate; not derived from mevalonate (mevalonate-independent);
comes from intermediates of glycolysisV
÷V ABA derived from the breakdown of carotenoids. Evidence: maize mutants blocked in
carotene synthesis ń low levels of ABA ń vivipary (see below)V
÷V Pathway: IPP ń farnesyl pyrophosphate ń carotenoids (C40;
violaxanthin) ń xanthoxin ń ABAV


c$ 0% 
V

A. Bioassays ² there are several including:V

÷V inhibition of seed germinationV

÷V inhibition of GA induced alpha-amylase productionV

B. Analysis ² Gas chromatography, HPLC, and immunoassayV

8
0Œ

 V
÷V rapid changes in endogenous levels (up to 100x within a few days)V
÷V ABA levels can be regulated by: (a) degradation; (b) compartmentalization; (c)
transport; (d) conjugation to a sugar or other molecule; and (e) conversion (oxidation)
into phaseic acid and dihydrophaseic acid.V

c 
- xylem and phloem (greater amounts)V

cc%
 V

A. Growth Inhibitor
Widespread growth inhibitor; often antagonistic of GA actionsV

B. Maintains or "seals in" bud and seed dormancy (i.e., prevents germination)
In fact, ABA is made during the terminal stages of embryo development. Among it's roles in
seed dormancy is to: (1) provide desiccation tolerance of the embryo by promoting synthesis of
proteins involved in the process; and (b) promote accumulation of seed storage proteins.V

C. Prevents vivipary
Development of the embryo without a dormant period. Some evidence: viviparous mutants
have reduced [ABA]; and fluridone stimulates treatment stimulates vivipary (fluridone is an
inhibitor of carotenoid biosynthesis that blocks ABA production)V
D. Inhibits auxin induced growth (seems to block the H+ pump)V

E. Stomatal closure under water stress (remember our unit on gas exchange?)V

F. Abscission & senescence

Involved, though perhaps only a minor role


ccc( 
 V

A. Effects on plasma membraneV

B. Inhibits protein synthesisV

C. Regulation of genes (transcription)V

5

 V

c3 
V

÷V the Chinese may have been the first to observe the effects of ethylene when they noted
that burning incense increased fruit ripeningV
÷V in 1864 leaks in gas lights in street lamps were reported to stunt plant growth and
defoliate treesV
÷V in 1901, D. Neljubow realized that his dark-grown pea seedlings were short, fat and
negatively gravitropic (the triple response) because of a component in "laboratory air"
which he subsequently identified as ethyleneV
÷V Cousins (1910) first reported that ethylene occurred in plants.V

cc 
 V

÷V single compound (like ABA) and is not a family of related ones (?
., gibberellins)V
÷V CH2=CH2 V
÷V ethylene (MW 28) is similar in size/shape as waterV
÷V a gaseous plant hormoneV

ccc$
V

A. General:V

÷V made by most plants including angiosperms, gymnosperms, ferns, mosses, liverwortsV

÷V also synthesized by fungi and bacteriaV
÷V made by all parts of the plantV
÷V meristematic regions (shoot apex) and senescing tissues are rich sourcesV
÷V nodes make more ethylene than internodesV
÷V ethylene production is stimulated by physiological stresses including wounding,
anaerobic conditions, flooding, chilling, disease and drought.V
÷V during the climacteric ² which is the sudden surge of respiratory activity that occurs at
the peak of ripening in many fruits - lots of ethylene is madeV
B. Pathway of synthesisV

÷V the first precursor is methionine (one of the protein amino acids)V

÷V methionine + ATP à S-adenosyl-methionine (SAM) à ACC synthase à amino
cyclopropane carboxylic acid (ACC) à ACC oxidase à CH2=CH2 + HCN (hydrogen
cyanide) + CO2. To summarize the highlights:V

V ATP reacts with methionine to form SAMV

è
V SAM is essentially a carrier form of methionine (it is involved in other reactions in
the cell)V

V ACC synthase is the most crucial enzyme in the pathway. It is the rate limiting step
and induced by: (a) fruit ripening; (b) flower senescence; (c) in response to IAA;
(d) under wounding; (e) chilling injury; (f) drought; (g) flooding; (h) in response to
ethylene ("one bad apple spoils the whole bunch"). ACC synthase is cytosolic
and coded by a multi-gene family.V

V methionine is re-claimed via the Yang cycle.V

V ACC oxidase was formerly called "ethylene forming enzyme, abbreviated EFE." It
requires Fe2+ and ascorbate for activity ² which explains why it took awhile to
characterize this enzyme. It is also the product of a multigene family.V

V One of the products of ACC oxidase activity is HCN ² which explains why most
plants have enzyme systems for detoxifying/metabolizing cyanide. ACC oxidase
is inhibited by anaerobic conditions and cobalt ions but stimulated by ripening.V

cc 
V

÷V silver ions (Ag+), CO2 and KMnO4 inhibit ethylene actions. These bind to ethylene
receptors or otherwise interfere with the mechanism of ethylene action.V
÷V Aminovinylglycine (AVG) and aminooxyacetic acid (AOA) block the action of ACC
synthase. Since these compounds are knows to block pyridoxal enzymes, it suggested
that they participate in the process.V

8
V

÷V probably not a concern since it is a gasV

÷V in addition, malonate will bind to ACC forming N-malonyl ACC. Ënlike most conjugates
that can be hydrolyzed to produce the original compound, this one is NOT converted
back to ethylene.V
÷V a conjugate with glutamic acid (GACC) may be an important regulator of ethylene
levelsV

c%
 V

A. Fruit ripening
Ethylene triggers fruit ripening (?
., climacteric) - Flavr-Savr tomato.V

B. Abscission
This is the shedding of plant parts. Occurs at a specialized layer of cells ² the abscission layers.
Auxin apparently prevents leaf abscission by maintaining cells in the abscission zone insensitive
to ethylene. When auxin levels in the leaf decline, the tissues become sensitive to ethylene that
promotes abscission by producing and secreting cellulases, etc.V
C. Epinasty
Downward bending of leaves - common response to flooding or waterlogged soils.V

D. Triple Response
Pea seedlings treated with ethylene are short (inhibits internode elongation), fat (increase
stem thickness) and "stupid" (horizontal growth, no positive gravitropism). Further, they show little
leaf expansion and possess an apical hook.V

E. Thigmomorphogenesis
The change in growth form in response to a mechanical stimulation such as touch.V

F. Stimulates germination in cereals, peanuts; promotes sprouting in potato tubers and other
bulbs.V

G. Flower senescence
Stimulated by ethylene. Adding AVG to carnation can keep them fresh for weeks.V

cc 


 
Ethrel (Ethephon) liquid sprayed onto plants. It contains a dilute solution of 2-
chloroethylphosphonic acid that breaks down to give off ethylene. Among others things, it is
used to synchronize flowering and fruit set in pineapples. Commercial fruits are usually stored in
low O2 to inhibit ethylene biosynthesis or under high CO2 to prevent ethylene action as a
ripening promoter.V

( 


 0  V

c5


 
 

V

A. Table 6-1 from Salisbury and Ross.V

B. Conclusions: V

V 90+% of plant C, H, O, N;V

è
V over 60 different elements have been found in plants including gold, arsenic, mercury,
lead and uranium;V

V certain tissues concentrate elements (i.e., young tissues have higher levels of N, P and K);V

V similar elemental composition in different species.V

cc5

5

V

A. Definition - An element is considered essential if:V

V required for growth and development;V

è
V directly involved in plant metabolism;V

V an integral constituent of important constituents in the plants;V

V required for life cycle completion; and V

V no other element can substitute for it.V
B. SpecificsV

÷V There are species specific differencesV

÷V Not all of the elements that occur in a plant are essential (?
, some may be non-
selectively absorbed). V
÷V There are 17 essential elements (see table 5.2 from Taiz and Zeiger)V
÷V Mnemonic to remember: "C. Hopkins Cafe closing; mob coming with machine guns" or
in symbolic form - C HOPKNS CaFe ClZn; MoB CuMn Mg.V

C. Conclusions: V

V some elements are required in high concentration (macronutrients), others low

(micronutrients);V
è
V some supplied from the air (i.e., C and O via CO2), most absorbed by the roots from the
soil; V

V lot of O and H - shows the 
 
.V

D. Functions.
Check the text (Table 5.3 in Taiz and Zeiger) for a nice summary. Roughly speaking:V

V C, H, O, N, and S are required because they are the building blocks of the molecules of
life; V
è
V P, B (Si) involved in energy transfer reactions; V

V K, Na, Mg, Ca, Mn, Cl have various functions including maintaining
osmotic concentrations and structures of enzymes;V

V Fe Cu Zn Mo prosthetic groups, electron transfer reactions.V


ccc8
   




Ëse soil-less techniques. ‰ulius Sachs first showed could grow plants in solution culture in
1860. Term hydroponics coined by W.F.Gericke in 1930's (from Greek, hydro = water; ponos =
labor). Many variants of the technique so long as: (1) support plant; (2) supply proper elements;
(3) roots receive adequate aeration and moisture.

The techniques include: (1) solution culture (+/- aeration); (2) nutrient film techniques; (3)
aeration systems.

c =
Nitrogen, phosphorus and potassium are usually the most limiting elements because they are
required in the highest concentration and are least likely to be supplied in a soil/growth medium
in adequate amounts. Hence the need for fertilizers. Labels include the form and percent of
each; i.e, 14 - 14 -14 means that a fertilizer has 14% nitrogen, 14% phosphorus and 14%
potassium. Fertilizer labels also include micronutrients.V

In class we will see some slides of "The Land" exhibit from Epcot.V

 


 



;  
 

V

÷V use N because it is the most important element in houseplant fertilizersV

 ÷V Equation: $/kg N = Cost of fertilizer ($) x 1/%N x 1/pkg mass in kgV
÷V Example using RaPid Gro: $/kg = $1.99 x (1/0.23) x 1/0.227 = $38.10V
÷V Table 1 lists some common fertilizers, type, cost. Calculate the Nitrogen cost for the other
fertilizers. V

Comparing Costs of FertilizersV

BrandV TypeV N-P-KV Pkg Price ($)V Pkg Wt (kg)V Nitrogen
Cost ($/kg)V

RaPid GroV SV 23-19-17V 1.99V 0.227V 38.10V

Peter'sV SV 15-13-7V 1.19V 0.071V V

‰obe's spikesV CV 10-10-4V 1.49V 0.015V V

Knox GelatinV OV 15-0-0V 0.59V 0.028V V

Miracle-GroV SV 15-30-15V 2.19V 0.227V V

OsmocoteV CV 14-14-14V 4.95V 0.553V V

(key: S=soluble; C=controlled release; O=organic). V

Π:V

÷V This table is adapted from D. Hershey (1990) Science Activities 27:17-20.V

V


 
  ΠG3  
:V

V
c 
  
Recall our "plant way of life" discussions. We concluded that non-motile organisms, such as
plants, have a limited ability to position themselves in their environment since they cannot move
to a more favorable environment. The major positioning responses used by a plant, in
approximate order of need, are: (a) environmental positioning - mostly by chance/luck (i.e.,
seed germination mechanisms); (b) axis orientation - gravitropism; and (c) fine tuning
mechanisms- assorted responses to maximize environmental resource acquisition and utilization
(?
., phototropism). Here we focus on axis orientation and fine-tuning responses, which require
the plant to "move," at least to a small degree.V

V
cc(*
. Plants exhibit three major types of movements:V

V Hydration movements
These result from the uptake of water by non-living cells/tissues and is responsible for
actions such as the opening of a fern sorus, the opening of some types of fruits G?
 witch
hazel, wild cucumber), and the twisting of awns in porcupine grass (?) and other
grasses;
V
 è
V Turgor movements
Reversible; responsible for phenomena such as sleep movements (nyctinasty) and
movement of leaflets of the sensitive plant; and
V

V Growth movements
Irreversible; result from differential growth (elongation). There are several kinds of
growth movements including: (1) tropisms - unidirectional responses that are related to
the direction of stimulus (positive - toward stimulus; negative away). Examples include
phototropism and gravitropism; (2) Nastic responses - in this case the response is not
related to the direction of the stimulus (?
., thigmonasty, epinasty, seismonasty); and (3)
Nutations - rotary type movements of plant structures, particularly the shoot tip
(?

circumnutation).V

cccΠ 

 * 

Plants respond to signals from their environment. These signals are received by the plant and
then converted into a physiological response. This sequence of action can be diagrammed as
follows: environmental signal ń receptor ń transducing mechanism (includes amplifying the
signal) ń physiological response.V


V Signals
Plants respond to many stimuli or signals in their environment. These include light, wind,
and gravity. Overall, light is one of the most important environmental signals and it is
involved in many different responses. Photomorphogenesis is the fancy term for a plant
growth response to light.V

V Receptor
An environmental signal must be intercepted or received by the plant. The nature of
the receptor depends upon the stimulus. The receptor for any light-induced stimulus is a
pigment, which by definition is a molecule that absorbs light.V

V Transducing mechanism or chain
This refers to the sequence of events by which the activated receptor converts the
signal to a physiological response. The mechanism generally requires: (1) an
amplification of the signal; and (2) the plant must be in a physiological state to be able
to respond to the stimulus.V


c3*
V

A. Stimulus - gravity, hence the name gravitropism. It used to be called geotropism, but
gravitropism is more accurate since the response isn·t toward the earth ("geo")V

V The response varies - thus, the gravitropic response may be parallel to the direction of the
stimulus (?
 roots and shoots, called orthogravitropic), perpendicular to the stimulus
(diagravitropic, as in rhizomes and stolons) or at an angle other than perpendicular or
parallel (called plagiogravitropic; ?
 lateral roots)V
è
V How to study gravitropism on earth, especially considering there can·t be a "no gravity"
control? One answer - a clinostat. This device is essentially a phonograph turntable to
which a plant is attached. The entire unit is held horizontally and the plant rotated to
counteract the effect of gravity.V

V The threshold intensity - the minimum amount of stimulus required to induce the response.
Roots are more sensitive than shoots. Oat coleoptiles in clinostat experiments respond to
 0.0014 g, whereas roots require just 0.00014 g.
V

V The threshold dose - represents the minimum dose of stimulus required to induce a
response - it equals the product of stimulus intensity times the duration of the stimulus. The
threshold dosage for oat coleoptiles varies from 30-240 g s.
V

V Presentation (or reaction) time - minimum time necessary to induce a response which
can be as short as 12 seconds for cress roots. Ësually there is a lag of about 10 minutes
between the time of the dose and the response.V

B. ReceptorV

V Does one exist?

You bet. Plant on side ń place upright ń bends. One conclusion: the sites of stimulus
and perception are separated.V
è
V Location in root?
Root cap. Decapitated roots don·t respond to gravity. Interestingly, gravity seems to
be necessary for the regeneration of the root cap in §


 - when decapped in
microgravity (on the Space Shuttle) maize roots don·t regenerate a cap. This observation
suggests that a part of the root other than the cap also perceives gravity.V

V Location in shoot?
Not known precisely, though it seems to be associated with a starch sheath in stems,
such as sunflower hypocotyls and the dandelion scape (flowering stalk). Peeled
hypocotyls don·t respond to gravity suggesting the epidermal layers are important in
sunflower. The coleoptile tip is most responsive - but, even if you remove the tip,
coleoptiles still show some sensitivity to gravity.V

V Nature of the receptor - statoliths
These are small bodies that have a high specific gravity and presumably settle to the
bottom of cells (statocytes) to "tell" the plant which way is down. Statoliths are probably
amyloplasts (or starch grains). The evidence for this conclusion is: (a) Presentation time -
there is a correlation between the rate of amyloplast sedimentation and presentation
time; and (b) GA or kinetin treatment at elevated temperature (35 C), which causes
starch to disappear in the roots of cress seedlings, abolishes the gravitational response.
Recovery of gravitropism is correlated with the reappearance of amyloplasts.V

Specifically, which cells in the root cap are the statocytes? The root cap has three
layers of cells: (a) calyptrogen (which are meristematic producing new root cap cells; (b)
columella cells (named for their shape; filled with amyloplasts); and (c) peripheral cells
(secrete mucilage). Thus, the columella cells are the likely statoliths.V


V Nature of the receptor - Protoplast buoyancy? Stretch Receptors?

One major problem with the statolith hypothesis is that starchless mutants
of ?? are still gravitropic. This inconsistency (and others) have led to the
suggestion (see the series of three articles by Staves,

 in the 
?
, 



, volume 84, pp. 1516 and 1533, 1997) that buoyancy of the protoplast is the
means by which plants sense gravity. Recall our Chardokov experiment in which dye
drop either sank, floated or hovered, depending upon the density of the medium. Thus, if
the protoplast is suspended in a less density medium, like air, it will sink and press on the
bottom of the cell. Similarly, in a more dense medium, it will float and press on the top of
the cell. Ësing the green algae , Staves and colleagues observed that incubating
 the cells in media of different density affected the gravitropic response as predicted by
the buoyancy model. They further hypothesized that when the protoplast contacts the
wall it stimulates proteins (called integrins) which lead to a transduction response.V

C. Physiological response
Proton efflux on upper side of a gravistimulated root (and lower side of the shoot) causes the
upper root cells to be more plastic than the lower ones. Thus, these elongate more and shoots
bend up and roots bend down. This is rather similar to the way a vehicle with tracks, like a tank,
turns - one tread remains stationary while the other continues along (Mulkey

 - data from
acid efflux experiments with maize roots).V

D. Some observationsV

V The response is fast - the lag or presentation time is 7-60 min. Amyloplasts fall in
about 12 sec;
V
è
V A signal is transmitted from the site of perception (root cap) to the zone of
elongation where bending occurs;
V

V A growth inhibitor in the root or growth promoter in the shoot may be involved
(see data);
V

V Cholodny/Went hypothesis - suggested that auxin accumulates on the lower side
of gravistimulated tissues. The increased auxin content of the stem stimulates
elongation of cells on the lower side but inhibits roots;
V

V Placing a root on its side results in change in electrical potentials at the tip (see
data);
V

V There is a calcium gradient in gravistimulated roots and shoots - (a) in shoots there
is more calcium on the upper side, but more on the lower in roots; (b) in roots,
calcium moves through the mucilage. If you remove the mucilage by washing,
no response (see data). Further, aquatic plants have non-responsive roots; (c) the
asymmetrical distribution of calcium can cause bending (see data); (d) EDTA
binds (removes) calcium - bending away from EDTA application; and (e) IAA
moves toward calcium (Ca2+ )
V

V Roots show electrotropism - curve toward the anode (A‰B 77:446, 1990).
V

V Auxin is required for graviresponsiveness
V

V IAA is laterally transported downward in some tissues like maize coleoptiles there
are conflicting reports about the presence of an IAA gradient across shoots and
roots.
V

VSAËRS - small auxin up-regulated RNA's; within 20 minutes these accumulate on
the lower side of a horizontal stemV

D. The transducing mechanism

Plant on side ń amyloplast (or protoplast) settles ń associates with ER/microfilaments/proteins
or wall ń may stimulate calcium secretion and calmodulin ń stimulates electrical gradient ń
redistribution of Ca2+ secreted by peripheral cells ń calcium moves in mucilage toward tip ń
auxin follows (calcium acts as a sink) or calcium sensitizes tissue to the presence of auxin ń
stimulates proton pump ń increases cell wall plasticity ń unidirectional growth.V


 


Growth response to unidirectional light or a gradient (more on one side than the other) of
light. Some of the first studies by Darwin (Power of Movement in Plants, 1881). Plant response to
light varies (shoots - positively phototropic; roots - non-phototropic; leaves - plagiophototropic).
Some change during development (i.e., juvenile ivy - negatively phototropic, adult - positively
phototropic;
?
? - stems negatively phototropic after fertilization to "bury" the
pods).V

A. Stimulus - lightV

V Which kind?
The action spectrum suggests blue light (two peaks near 475 and 450 nm, and one in
ËV-A at 370) ² a "three finger response."V
è
V Dose response
Which is more important: (a) fluence rate (Ǎmol m-2 s-1); (b) duration (time) of the
exposure (sec); or (c) the total exposure (also called fluence, Ǎmol m-2 ) which equals the
exposure multiplied by the duration?V

For example, let·s assume that a 10 sec treatment with 1.4 Ǎmol m-2 s-1 blue light yields a
curvature of 15 degrees. Thus, the total exposure is 14 Ǎmol m-2 (= 10 s x 1.4 Ǎmol m-2 s-1). If the
plant is responding to the total exposure, then as long as we provide a total exposure of 14 Ǎmol
m-2, we should be able to vary the fluence rate and/or duration to yield the same results. Thus, a
100 sec treatment with 0.14 Ǎmol m-2s-1 light should yield the same response.V

Another way of looking at this: Fluence rate and time are reciprocals (Law of Reciprocity) of
one another if the system is proportional to total exposure. Thus, if reciprocity holds: (a)
increasing the duration of the exposure and decreasing the fluence; OR (b) decreasing the
duration and increasing the fluence - should both yield the same response.V

Does it? Yes, but....only at low total exposures. If phototropic curvature vs. log fluence (Ǎmol
m-2) is plotted the response is complex. Essentially there are two (or more) peaks. The first peak is
termed the first positive curvature and it can be followed by a second peak (second positive
curvature) and so on. Reciprocity only holds true for the first positive curvature (at low fluence).V

So, what does all this mean? These data suggest that light does two things: (a) it acts as a
trigger; and (b) decreases subsequent sensitivityV

B. ReceptorV

V Location
Shoot tip, young leaves. Evidence - remove the tip of coleoptiles and no response (see
data).V
è
V Nature of the receptor
Light responses are perceived by pigments. The action spectrum suggests a yellow
 (red) pigment is involved (see data). To determine which pigment plot absorption
spectra of potential candidates to find match with action spectrum. Two candidates:
riboflavin and/or carotene.V

Riboflavin is the probable receptor because: (a) carotene doesn·t absorb much below 400,
but riboflavin does; (b) carotene-less mutants still phototropic; (c) plants treated with fluridone
and norflurazon, which inhibit carotene synthesis, are still phototropic; (d) flavin inhibitors, like
potassium iodide or phenylacetic acid, reduce phototropic sensitivity. But, the absorption
spectrum of riboflavin is not a perfect match - a combination of the two seems even better.V

The actual pigment involved is called phototropin. There are two phototropins (1 & 2). The
gene is called phot (it used to be called npt = non-photoresponsive hypocotyls). Phototropin is
a flavoprotein. There is a gradient from tip to base, there the tip is more sensitive than the base.V

Phototropin protein capable of autophosphorylating protein kinases (add phosphates to

serine/threonine kinase). The N-terminal binds flavin, C-terminal kinase.V

C. Physiological response
Sunflower seedlings mounted in agar with dye show proton efflux from cells on the shaded
side of the stem.V

D. ObservationsV

V Cholodny-Went hypothesis - light causes a lateral redistribution of auxin to the shaded

side and elongate more than on the lighted side (see data). Evidence: (a) agar block
experiments (see data); (b) radioisotope redistribution experiments in maize (see data);
(c) elongation should be slower lighted side, faster on shaded because of auxin
redistribution - it is based on experiments in which small glass beads were glued onto
coleoptiles and photographed (see data).
V
è
V Blaauw Inhibitor hypothesis - an inhibitor is synthesized on the lighted side of the stem.
Evidence: (a) experiments in which sunflower cotyledons are shaded; (b) a xanthoxin
gradient is observed in photostimulated radish and sunflower (see data). But there are
some contradictory data.V

E. Transducing Mechanism
Blue light stimulates redistribution of auxin across stem to shaded side resulting in increased
proton efflux and hence elongation.


cc 
Plant growth response to a mechanical stimulation such as rubbing, wind, raindrops, etc. The
termed was first coined by M. ‰affee. Seismomorphogenesis is specifically the response to
shaking.V

Compared to unstimulated plants, mechanically-stimulated plants: (a) grow more slowly; (b)
increase more in diameter. In essence, they are shorter and fatter. This response makes "sense" to
minimize the risk of breaking which is especially true for plants in the mountains. As an example,
compare plants grown in indoors (houseplants, greenhouse) with those grown outdoors.V
 This phenomenon is due to ethylene (the triple response) for the following reasons: (a)
ethylene concentrations increase in response to mechanical stimulation; and (b) ethylene
treatment mimics these effects, i.e., inhibits shoot elongation and induce stem swelling.V

mRNA synthesis is stimulated shortly after mechanical stimulation. Four or five genes are
activated, one of which is the gene for calmodulin.V

ccc 
- sensitive plant (?
?)

Leaves fold upon touch, or being burned or otherwise stimulated. This response is very rapid,
the plant responds within a second or two. In addition, the stimulus can be transmitted to other
parts of the plant. The transmission occurs by:V

V Electrical potential
The membrane is depolarized and generates an action potential which is similar to
that in an animal nerve cell, only slower. It travels from cell to cell at the rate of about 2
cm/s. It may travel via the phloem.V
è
V Chemical factor
It is clear that one is released to move from one leaflet to another. The substance must
pass through vascular system. Ricca·s Factor - substance isolated that induced response
in other leaves. Turgorins, gallic acid derivatives, are hormones that may give rise to
action potentials - like the animal neurotransmitter acetylcholine.V

The action potential/chemical factor would stimulate rapid unloading of potassium into the
apoplast and the leaves fold.V

c, 
 
'

*

Many plants, especially legumes with compound leaves, fold up their leaves at night. This may
serve to: (a) prevent moonlight from inducing phytochrome responses; or (b) conserve
water/heat.V

A. Stimulus - blue lightV

B. Receptor.
Is not known, but may be the blue-light absorbing pigment (cryptochrome) such as in
phototropism and/or phytochrome.V

C. Transducing mechanism
Leaf movements arise when cells (called motor cells) in the pulvinus, which is a swollen area at
the base of the leaf and each leaflet, undergo turgor changes. Swelling of motor cells on one
side of the pulvinus (extensor cells) and shrinking on the other (flexor) causes the leaves to open
"up" (perpendicular to incoming light). When the leaves "go to sleep" or close "down", parallel to
the stem, the reverse process occurs.V

The turgor changes are caused by the influx/efflux of potassium ions through membrane
channels, in a manner reminiscent of guard cell opening/closing. Chloride ions also enter the
pulvinus cells.V

An ATP-requiring proton pump is apparently involved. The pH of the apoplast decreases

during potassium uptake. This perhaps establishes an electrical gradient which drives the uptake
of potassium.V
 
  
 V

c

 
*

Plants, like all organisms, must be able to monitor and respond to environmental conditions.
For example, a dark-grown seedling "knows" that it has a limited time to get to light until it runs
out of energy. In response, an "etiolated" plant exhibits a variety of features in response to
darkness including expanded internodes for rapid growth, an apical hook (in eudicots),
unexpanded leaves, no chlorophyll. A brief exposure to light causes internode elongation to
slow, the hook to uncurl, leaves to expand and chlorophyll synthesis to begin. Thus, light has an
obvious impact on the form of the plant (  

?).V

V
cc 
-+'


In many photomorphogenetic responses, red and far-red light are important environmental
signals. For example, from studies of light sensitive lettuce seed germination (see Table 1),
Borthwick and Hendricks concluded that: (1) germination is dependent upon which wavelength
of light is received last; and that (2) this response is the product of a photo-reversible pigment.V

Table 1: Germination of Grand Rapids Lettuce

Seeds after brief exposures to red or far-red
lightV

TreatmentV Percent GerminationV

noneV 8.5V

redV 98V

red, far redV 54V

r, fr, rV 100V

r, fr, r, frV 43V

r, fr, r, fr, rV 99V

r, fr, r, fr, r, frV 54V

r, fr, r, fr, r, fr, rV 98V


ccc
- 

The best characterized, and most important receptor for light-induced growth responses is
phytochrome. The absorption spectrum of phytochrome closely matches the action spectrum
implicating it in these processes. V

? +?


V

c

V

A. Chemistry
Phytochrome is a pigment-protein complex (called a holoprotein = chromophore +
apoprotein)V

V Pigment (chromophore)V

÷V blue-greenV
÷V open chain tetrapyrolle; called phytochromobilin (overhead)V
÷V made in the plastidsV

è
V Protein (apoprotein)V

÷V glycoproteinV
÷V solubleV
÷V dimer (MW 240,000 D = 240 kD); each of the two peptides are identical with a
MW . 124,000 D and comprised of  1128 amino acidsV
÷V gene(s) have been cloned and the amino acid sequence is known; large
proportion of hydrophobic amino acids; suggests phytochrome is associated with
membranesV
÷V tetrapyrolle is covalently-bonded to the protein via a thioether linkage involving a
cysteineV
÷V one chromophore per dimerV
÷V holochrome apparently "self assembles" (autocatalytic) after the individual
components are synthesized. For example, the protein is made by ribosomes
associated with the ER and the tetrapyrolle is synthesized in the plastids, which is
not too surprising considering the similarity it has to the structure of chlorophyll.V
÷V coded by phy genesV


V Types of phytochrome - phytochrome in dark-grown seedlings is distinctly different than

that in light-grown plants. These two forms are:V

Type I - found in dark grown, etiolated seedlings; most studied (MW 124 kD; maximum absorption
- Pr 666 nm, Pfr 730 nm)V

Type II - found in light grown plants; (118 kD, Pr 654; Pfr 724)V


V ConclusionsV

÷V phytochrome in etiolated plants (type I) is slightly larger and absorbs light

maximally at a longer wavelength than phytochrome from light-grown plants
(type II).V
÷V differ mainly in apoproteinV
÷V at least five different proteins that can be identified immunologically; proteins are
about 50% similar to one another.V
÷V proteins are coded by 5 different genes called phy A - phy E. The phytochrome
they make is called PHY A etcV
÷V PHY A is found only in dark-grown seedlings (Type 1); the other four types occur in
both etiolated and green (light-grown) plants. Interestingly, PHY A is unstable. The
PHY A is probably important for detecting the presence of light while the others
monitor its quality.V
B. Photoreversibility
The unique feature of phytochrome is that it exhibits photoreversibility; it exists in two forms that
are interchangeable. Pr - red light absorbing form and Pfr - far red light absorbing form. When Pr
absorbs red light (. 660 nm) it is converted into Pfr. When Pfr absorbs far red light (. 730 nm)
it is converted into Pr. In short, phytochrome acts like a light switch. This can be depicted:
Pr ņ PfrV

The absorption spectrum for phytochrome will be provided. Note that there is some overlap in
the spectra and also note that there is some absorption of blue light.V

C. Chemical Changes during photoreversibility

The main difference between the two forms is a cis-trans isomerization that occurs between
one pair of tetrapyrolles. This change has the effect of extending or opening up the
chromophore. The protein also undergoes a conformation change. One piece of evidence
that supports this is that the protein is more readily digested in the Pfr form.V

D. Efficiency of photoconversion
Phytochrome acts like a weird light switch that only turns off/on a portion of the lights. In other
words, red light treatment of Pr results in about 85% Pfr + 15% Pr; far red light treatment of Pfr
results in 97% Pr + 3% Pfr. Thus, at photo-equilibrium not all the phytochrome is interconverted.
The reason for this is because the absorption spectra for the two pigments overlap and they are
essentially competing reactions.V

A measure of the efficiency conversion is the ratio of the Pfr to the total which is expressed as
follows: V

efficiency = Pfr/(Pr+Pfr = phytochrome total)V

In red light = 0.85; far red 720 nm = 0.03; this varies with the environment (see text and
overhead)V

E. Intermediates
There are a series of intermediates in the conversion from Pr to Pfr. The intermediates are
unstable but there is probably always a small pool of them available. There are apparently three
intermediate stages in conversion of Pr to Pfr; and 2 stages in conversion of Pfr to Pr. Thus we can
modify the original equation:V

Pr ń red light ń Pr* ń [1] ń [2] ń [3] ń Pfr ń far red ń Pfr* [1] ń [2] ń Pr
V
G

ü
ü




? ??

?





 



?


  V

Œ

  

*
V

A. Synthesis
Phytochrome makes up about 0.2% of the total protein in a dark grown plant. And, there is
about 50x more phytochrome in an etiolated plant than a green one. Pr is the form synthesized
by the plant; only form in the dark; light inhibits synthesis of Pr. Thus:V

phy gene ń mRNA ń Pr ņ PfrV

B. Destruction
Both are degraded ?
? . Pfr is more labile (unstable). Protease digestion is an important
route of hydrolysis; Pfr is more accessible to hydrolysis, perhaps because it gets tagged by
proteins like ubiquitin for disposal. Thus: V

phy gene ń mRNA ń Pr ņ Pfr ń destructionV


c 

*
The first hint of this came from lettuce seed germination experiments. Since the seeds only
germinated in the light and since Pr is only found in dark grown plants, Pfr must be the active
form. Thus:V

phy gene ń mRNA ń Pr ņ Pfr ń destruction or physiological actionV


cc
;
 V
÷V wide distribution (including angiosperms, gymnosperms, algae, bryophytes)V
÷V meristems and leavesV
÷V found throughout cell (in cytoplasm and associated with organelles)V
÷V Pr seems to be dispersed through cytosol and in nuclei and plastidsV
÷V Pfr seems to be sequestered in clumps - granules about 300 nm in size with no
membranesV


ccc%
  - there are many phytochrome-mediated responses (see listing). These can be
roughly grouped into three major categories:V

A. Induction-Reversion Responses or, Low Fluence (LF) Responses

These are the "classic" responses that are induced by red light and reversed by far red. These
responses are sensitive to 1 Ǎmol m-2 , are photoreversible, and saturate at 1000 Ǎmol m-2.
Examples include:V

V Light-sensitive seed germination

Note that many seeds require light for germination (like lettuce), whereas others are
inhibited by light (wild oats, m 
?, Royal Paulownia). Phytochrome presumably
stimulates GA synthesis/release which in turn, stimulates the mobilization of stored
reserves (recall the GA lecture). This provides energy for the germinating seed and also
decreases the solute potential for water uptake necessary for providing the force for the
radicle to push its way through the seed.V
è
V Reversal of etiolation
Recall that etiolated plants are those that have been grown in the dark and exhibit a
series of characteristics including elongated internodes, no chlorophyll, apical hook or
unopened coleoptile, unexpanded or coiled leaves. These are all adaptations to save
energy and get to the light asap. Light stimulates increased [cytokinin] which stimulates
cell division and greening; increases [GA] which presumably stimulates IAA oxidase
production to get rid of excess IAA that causes the long spindly growth. Also, IAA
stimulates ethylene synthesis which maintains the apical hook. Treating the apical hook
with red light + ethylene maintains the hook.V
 
V Change surface charge
In a classic experiment, Tanada (1968) observed that red light-treated barley root tips
adhered to the sides of a beaker (with a negatively charged surface) but released after
far red treatment. It was subsequently show that red light caused the surface to become
positively charged and far red light negatively charged. Red light causes a
depolarization of membranes and far-red reverses or even causes a slight
hyperpolarization.V

V Chloroplast rotation in 
? (Haupt)V

B. Very Low Fluence (VLF) Responses

These are extremely sensitive to low levels of light (0.1 nmol m-2. Less than 0.02% is Pfr). These
are not photo-reversible and saturate at 50 nmol m-2. Some examples includeV

V mesocotyl elongation in cereals; and V

è
V ?? seed germinationV

C. High fluence (HF) or High Irradiance Response (HIR)

Prolonged or continuous light (10 mmol m-2); fluence rate is important; and not fully
photoreversible. Apparently what is important is maintaining a low level of Pfr over time. Another
pigment is likely involved - a blue-ËVA receptor (cryptochrome?). Examples of phenomena
involved in this reaction include: V

÷V anthocyanin synthesis in seedlings and apple skinsV

÷V inhibition of hypocotyl elongation in mustard, lettuce and petuniaV
÷V plume hook opening in lettuceV
÷V cotyledon enlargement in lettuceV
÷V ethylene production in sorghumV

c( %
 V
A. Stimulates transcription
‰affee (1969) found RNA levels in pea increased after red light treatment. Rubisco is light-
stimulated, other genes inhibited. Phytochrome involved at the transcriptional level.V

The mechanism of action is not clear, but the Pfr may activate an inactive regulatory protein
that moves into the nucleus to bind to a site on the DNA near the genes of interest to promote
their transcription.V

B. Calmodulin
This calcium binding protein may be involved in phytochrome action. Pfr has been shown to
stimulate calcium uptake in 
?. Calcium complexes with calmodulin and can stimulate
enzymes.V

Predictions:V

V Calcium should increase after red light treatment - it does (

?)V
è
V Calcium ionophores like A23187 can substitute for red light - it doesV

V Valinomycin - a potassium ionophore has no effectV
 
V Calmodulin treatment should stimulate activity in absence of red lightV

V Calmodulin inhibitors should prevent the response - they doV


5
V

1. Examine the data provided in Table 1. Based on these data:V

÷V is germination a phytochrome response? (cite evidence to support your answer)V

÷V if the phenomenon is caused by phytochrome, is this a VLFR, LFR or HIR response?
Explain.V
÷V after a five minute exposure, in which of the following treatments would you expect the
lettuce seeds to germinate: white light? green light? blue light? far red? Explain.V


6-3 
 3 Œ




'

V

TreatmentV % GerminationV

noneV 8.5V

red lightV 98V

red followed by far-red light treatmentV 54V

r, fr, rV 100V

r, fr, r, frV 43V

r, fr, r, fr, rV 99V

r, fr, r, fr, r, frV 54V

r, fr, r, fr, r, fr, rV 98V

2. Examine the data in Table 2. V

÷V is this an example of a phytochrome effect? Explain.V
÷V explain the interrelation of kinetin and light.V


7-5
. 
  

 
 
. 
>?8
 #(



  
@6-@6?G6ABC:V

[Kinetin] LightV Increase in diameter (mm)V

(ǍM)V
 0V noneV 1.05V

0.5V noneV 2.48V

0V 5 min redV 2.58V

0V 5 min fr-redV 1.01V

0V 5 min red, then 5 min far-redV 1.17V

0.5V 5 min far redV 2.49V

3. Examine the data in Table 3. V

÷V describe the effect of red, far-red, and blue light treatments on anthocyanin synthesisV
÷V is this a phytochrome effect? Explain. V
÷V If this effect is mediated by phytochrome, is it an example of a VLFR, LFR or HIR
response? Explain.V


@-%
   
  
Gº

 
:
 

 .
 
.
 

 



  

 * 
8
. G6AAB:V

TreatmentV [Anthocyanin] (A510)V

27 hours redV 0.0V

27 hours far-redV 0.0V

3 hours blue + 24 hours darkV 0.19V

3 hours blue + 5 min R + 24 hours darkV 0.19V

3 hours blue + 5 min FR + 24 hours darkV 0.05V

3 hours blue + 5 min FR + 5 min R + 24 hours 0.19V

darkV

4. The data in Table 4 provide an indication of how hormones may mediate the phytochrome
response. Examine the data in the table and then answer the following questions:V

V What is the effect of GA on lettuce seed germination?V

è
V What is the effect of kinetin on lettuce seed germination?V

V What is the effect of ABA on lettuce seed germination?V

V Describe the interaction of GA, kinetin and ABA in lettuce seed germination.V

V Develop a model for light sensitive seed germination that includes phytochrome and
hormone.V
 
>-5
  


 
  
.V
% GerminationV
[GA] mMV Control (no
+ kinetinV + ABAV + ABA + kinetinV
addition)V
0V 10V 15V 0V 0V
0.05V 21V 27V 0V 17V
0.5V 66V 69V 0V 57V
5V 95V 97V 0V 73V
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Photosynthesis can be defined as the light-driven synthesis of carbohydrate. The equation for
this reaction, that you·ve seen many times is:V

CO2 + H2O + light + chloroplast ń (CH20)n + O2

From this simple equation we can make some elegant conclusions:

A. Photosynthesis is a redox reaction.

1.V Some definitions: (a) Reduction - gain of electrons; (b) Oxidation - loss of electrons; (c)
helpful mnemonics to remember: "oil rig" - oxidation is loss,reduction is gain, or "Leo says
grrrr" - loss equals oxidation, gain reduction; (d) Redox reaction - reaction in which one
component is oxidized and the other is reduced. Obviously, electrons must come from
somewhere and go somewhere.
è
V The reduction sequence of carbon: carbon dioxide (most oxidized form of carbon) ń
carboxyl (organic acid) ń carbonyl (aldehydes, ketones) ń hydroxyl (alcohols) ń
methyl ń methane (most reduced form of carbon). Note: each step requires the
addition (or removal) of two electrons and two protons for reduction (oxidation). Two
steps also require the addition/removal of water.V
 V How can you tell if a molecule has been oxidized or reduced? (1) look for a change in
valence (?
., Fe2+ ń Fe3+ represents an oxidation because an electron was lost,
increasing the total positive charge); (2) In many biological redox reactions, oxidation is
usually accompanied by a loss of protons (hydrogen ions) and reduction is
accompanied by a gain of protons; and (3) look for a decrease in the number of oxygen
atoms.V
4.V Biological redox reactions may require electron donors and/or acceptors. These include:
(1) NAD+; (2) NADP+; and (3) FAD; which are coenzymes (organic compounds, other than
the substrate, required by an enzyme for activity):

NAD(P) + (ox) + 2e- + 2H+ ń NAD(P)H (red) + H+

FAD(ox) + 2e- + 2H+ ń FADH2 (red)V

5.V Reducing Potential - potential for components to participate in a redox reaction; to

predict the direction and tendency of electrons to flow between two electron carriers.
The take-home-lessons are: (1) the more negative the reducing potential the better the
 electron donor; (2) the more positive the reducing potential the better the electron
acceptor; (3) spontaneous electron passage occurs from a carrier with more negative
reducing potential to one with a more positive reducing potential.

B. CO2 is reduced to a carbohydrate.

C. Water is oxidized (to oxygen).

D. Water supplies the electrons for the reduction; water is cleaved in the process yielding
oxygen as a byproduct.

E. Light provides the energy for the reduction.

F. Photosynthesis is an energy conversion process that ultimately converts light energy to

chemical energy (carbohydrate). In a broad sense, it is an example of the 1st Law of
Thermodynamics - energy cannot be created nor destroyed, but it can be changed from one
form to another.

G. BLACK BOX summary model for photosynthesis. Diagram in class that shows two boxes (light
dependent & light independent reactions). This model further shows that during the light-
dependent and light-independent reactions that there are three major types of energy
conversions during photosynthesis:

Conversion 1: Radiant energy (sunlight) ń electrical energy (passage of electrons via a series of
carrier). This reaction series is part of the light-dependent reactions (z-scheme, non-cyclic
electron flow)V

Conversion 2: Electrical energy ń "Labile" chemical energy (ATP, NADPH; unstable, not readily
stored). During this step, ATP and NADPH are produced as the end result of non-cyclic electron
flow.

Conversion 3: "Labile" chemical energy ń Stable chemical energy (carbohydrate). This last step
is the light-independent reactions or Calvin-Benson cycle. This process requires ATP and NADPH.

cc 


 - 
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?

A. Structure.
Remember the cell unit? To jog your memory, reread Chapter 1. Terms that you should know
are thylakoid (or lamellae), lumen (intermembrane space), envelope, double membrane,
stroma, granum, granal thylakoids (or lamellae), stromal thylakoids (lamellae), and starch grains.
Chloroplasts may contain fat globules (plastoglobuli). Stacked (or appressed) regions - portion of
granum in which thylakoids are adjacent to one another. Non-stacked (non-appressed) regions
- regions of the chloroplast where the thylakoids are not adjacent to another.

B. Ontogeny and phylogeny - recall the cell unit?

C. Chemistry - 
 ?

1.V DNA - circular loop; 120-160 kilobases that code for about 120 proteins;
2.V RNA;
 3.V ribosomes;
4.V proteins - some are coded by the nuclear genome, others by the chloroplastic
genome. For example, rubisco, an important enzyme, has 2 different subunits,
one from each source. The nuclear genes are essential for chloroplast function;
5.V pigments - make up about 7% of the chloroplast. These are molecules with a
color that absorb light. Two major groups of pigments in higher plants, chlorophylls
and carotenoids/xanthophylls. These occur in the thylakoids because they are
highly hydrophobic (fat soluble)

D. Pigments

1. Chlorophylls
These molecules look like a tennis racket. The head of the racket is a   ring system,
made of four  

 units linked together (

 

). It has a long hydrocarbon tail,

called 

(C-20), that is derived from the
  pathway (diterpene), built from
the  skeleton. (  is chelated in the ring. The tail is important for orienting the
molecule in the membrane. The interaction of the chlorophyll with the membrane is non-
covalent and is important because it ultimately determines the physical properties of the
chlorophyll.

÷V chlorophyll a - methyl group

÷V chlorophyll b - formyl group
÷V phaeophytin - chlorophyll without the magnesium
÷V chlorophyllide - chlorophyll without the tail

2. Carotene/xanthophylls
Both are terpenoid pigments,


  (C-40). Carotenes are hydrocarbons,
xanthophylls are oxygenated. These pigments are orange and yellow in color.

3. Chlorophyll biosynthesis - 

!
 

 


V ALA (Ʀ-aminolevulinic acid) is the first well-established precursorV

b.V ALA is derived from ǂ-ketoglutarate (or glutamate) (a Kreb's cycle
intermediate, from the mitochondrion)
c.V 2 ALA condense to form a unit of pyrolle
d.V 4 pyrolles condense to form porphyrin (tetrapyrolle)
e.V Magnesium is inserted


V A photoreduction step occurs (converts protochlorophyllide ń
chlorophyllide)V

V the tail is addedV
4. Light and the Greening Process
Recall that etiolated plants (grown in the dark) are yellowish but turn green rapidly when
placed in the light. Light is required, among reasons, to:

› V convert etioplasts ń chloroplasts;V

V photo-reduce protochlorophyllide to chlorophyllide; andV
 V activate enzymes for ALA synthesis.V
ccc  * 6- 
 


 

A. Nature of light
Light is part of the electromagnetic spectrum - radiation emitted by sun. Acts as discrete
particles (called photons) traveling as waves. Wavelength - distance between any two crests (or
troughs). Symbolized by lambda (nj); frequency - number of waves passing a point in one
second (ǖ). Frequency is inversely related to wavelength ǖ = c/nj where c = speed of light (3 x
1010 cm sec-1). The energy of a photon is a quantum. V

B. Which photons are important in photosynthesis?

Run an action spectrum (plot of a physiological process vs. wavelength).


  

    

Conclusion: radiations between 400-700 nm are photosynthetically active (termed PAR).

Specifically, red (600·s) and blue (400·s) light are important.

C. Photons must be absorbed to be used in a photochemical reaction.

In other words, only those molecules that absorb quanta participate in photosynthesis. So,
which molecules absorb the red and blue light? Run an absorption spectrum of potential
pigment candidates (plot of light absorption vs. wavelength) and compare it to the action
spectrum.



  

    


Chlorophyll a & b absorb light in the red and blue regions of the visible spectrum. Note that
the absorption spectra match the action spectrum of photosynthesis and hence, implicates
(though doesn·t prove) that they are involved in the process. (Subsequent work has shown the
chlorophylls to be the major photosynthetic pigments).

D. Quantity vs. Quality

1.V Light quality - refers to the wavelengths of light that are important.
Photosynthetically active radiations (PAR) range from 400 - 700 nm with peaks in
the red and blue.
2.V Light quantity - refers to the amount of light (PAR) received; units of mol m-2 s-1 ,
called the photon fluence rate; or units of energy, ‰ m-2 s-1.

E. What happens when chlorophyll absorbs light?

The chlorophyll molecule becomes excited (this takes only 10-15 sec = femptosec) and an
electron moves to an outer energy level. This is diagrammed:

CHL (ground state) ń CHL* (excited state)

Blue light excites an electron to a higher energy level than red light. Imagine the "bell ringer"
at a carnival. The electrons change spin at the first (S1) and second (S2) excited singlet states.
Electrons don·t stay excited long (10-9 sec), because they either:

1.V return to the ground state and release their absorbed energy as heat (


  );
 2.V return to ground state and release their extra energy as light (
  );
3.V transfer their energy to another molecule; kind of like hitting pool balls (
  


); or
4.V change spin and revert to a triplet state (same spin as ground state) and be used
in a photochemical reaction (  

).

F. Why excite electrons?

The ultimate purpose of exciting electrons from chlorophyll is to provide the energy needed to
transfer electrons from water to NADP+. Recall that spontaneous electron transfers proceed
from a carrier with a more negative redox potential to a more positive one. The redox potential
of water/oxygen = +0.82 eV while for NADP/H = -0.32 eV. Thus, photosynthetic electron flow is
not a spontaneous process and requires an energy.

G. How much energy is required to transfer electrons from water to NADP+?

First, let's calculate the actual redox difference (ƦEm) between water and NADPH:V

ƦEm = Em (acceptor) - Em (donor). Or, ƦEm = -0.320 - (0.820) = -1.14 = ca. -1.2 eV.V

The actual amount of energy involved is calculated from the equation:V

ƦG = -n F Em

where F = Faraday constant = 96,000 ‰/coulombs, and n = number of electrons involved in the
reaction (which equals one for each photon). Substituting in the equation:V

ƦG = - (1) x 96000 x (-1.14) = 109440 ‰ mol-1 (=109.4 k‰ mol-1 )

To summarize, approx. 110 k‰ mol-1 is required to reduce NADPH from water.

H. Do red and blue photons have enough energy?

Let's calculate the energy in red photons. Assume red photons have a wavelength of 660 nm
= 6.6x10-5 cm.

The energy of a photon is expressed by the following equation:

E = hǖ

where h = Planck·s constant which relates energy to frequency of oscillation and is 6.6255 x 10-
34 ‰ sec photon-1; and ǖ = pulses sec-1.

Since ǖ = c/nj (see A above), we can substitute back in original equation:

E = hc/njV

!





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?



? 

?


 
 



? 







 


? 

E = ((6.625x10-34 j sec photon-1)(3x1010 cm sec-1))/6.6x10-5 cm

= 3.01x10-19 j photon-1

multiply by Avogadro·s number

= 3.01x10-19 j photon-1 x 6.02 x 1023 photon mol-1

= 181,000 j mol-1

= 181 kj mol-1V

!





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"V

c 



:
There are four major complexes in the chloroplast. These are physically distinct from one
another and can be isolated from the chloroplast by electrophoresis and ultracentrifugation.V

A. Photosystem II (PSII) Complex

1.V large multi-subunit protein complex

2.V occurs in the stacked regions of the granal thylakoids
3.V integral proteins - coded by the chloroplast genome; including D1 (33 k) and D2
(31 KD)
4.V peripheral proteins - coded by nuclear genome; bind Ca2+ and Cl-
5.V P680 reaction center - a unique chlorophyll a, maximum red light absorption at
680nm; maybe two chlorophyll a molecules; this is  chlorophyll that "looses"
electrons
6.V manganese ions (Mn2+
)

7.V phaeophytin, plastoquinone

8.V LHCII - Light harvesting pigment complex associated with PSII. It is comprised of
(a) 250 chlorophyll a & b, in approximately equal amounts; (b) several
carotenoids; (c) proteins - each pigment is associated with protein (. 15
pigments/protein); the protein is coded by the nuclear genome

B. Cytochrome b/f Complex

1. occurs in stacked and non-stacked regions

2. cytochrome b (b-type cytochrome, not associated with protein)
3. cytochrome f (c-type cytochrome, associated with protein)
4. non heme iron-sulfur protein (Fe-SR)

C. Photosystem I (PSI) Complex

1.V occurs in non-stacked regions (stromal thylakoids)

 2.V about 11 polypeptides - including 1a & 1b that are coded by a single operon in
the chloroplast genome, bind p700
3.V 50-100 chl a
4.V electron carriers
5.V LHCI - contains about 100 chlorophylls; 4:1 ratio of chl a: chl b.; the protein is
encoded by nuclear genome
6.V P700 reaction center chlorophyll a
D. ATP synthase/Coupling Factor Complex

1. occurs in non-stacked regions

2. stalk - CFo (4 polypeptides)
3. head - CF1 (5 polypeptides)
4. nine polypeptides, some nuclear, some chloroplastic

D' (Or, the Light-Dependent Reactions; Or, Non-cyclic photophosphorylation).

A. Overview
During the light-dependent reactions of photosynthesis, electrons are transferred from water
to NADP+. This reaction is depicted as follows:

H2O ń NADP+V

As the electrons move from water to NADP+, they pass through three of the four complexes
described above - Photosystem II (PSII), a cytochrome b/f complex (cyt b/f), and Photosystem
I (PSI). After electrons are removed from water, they are sequentially shuttled from PSII to the
cyto b-f complex to PSI and then finally to NADP+. Thus:

H2O ń PSII ń Cytb/f ń PSI ń NADP+V

Since PSII, cyt b/f, and PSI are physically separated from one another, there must be a means
to transfer electrons between the complexes. A mobile form of plastoquinone (PQ) transfers
electrons from PSII to cyt b-f. A copper-containing protein, plastocyanin (PC), transfers electrons
from the cytochrome b-f complex to PSI. Thus, the reaction sequence is modified as follows:

H2O ń PSII ń PQ ń Cytb/f ń PC ń PSI ń NADP+V

The transfer of electrons from PSI to NADP+ is mediated by a soluble complex found in the
stroma, ferredoxin (Fd). Thus our revised equation:

H2O ń PSII ń PQ ń Cytb/f ń PC ń PSI ń Fd ń NADP+V

The transfer of electrons from water to PSII involves an "oxygen evolving complex" (OEC), part of
PSII, that is rich in chloride and manganese ions. Thus,

H2O ń OEC ń PSII ń PQ ń Cytb/f ń PC ń PSI ń Fd ń NADP+

B. Origin of the name
Derived from the zig-zag arrangement of components with regard to redox potential. But, why
don·t we call it the N-scheme?

C. Oxygen evolving complex

The energy of a single photon is not sufficient to split water. Experiments suggest that 4
photons are required to split two water molecules. Since only one electron can be excited at a
time (Einstein Law of Photochemical equivalents), this presents a minor problem.

The solution ² a water oxidizing "clock". Single electrons are transferred through a series of
intermediate stages sequentially increasing the electron deficit to a total of four. At this point the
original oxidation state is restored by extracting four electrons from water.

Diagram of the water-oxidizing clock - in class

Take-Home-Lessons:

1.V A series of five intermediate states, S0 - S4 are postulated;

2.V Initially the clock is in the So state, and may be associated with Mn II
3.V S1, which may be associated with Mn III, is the most stable form;
4.V S2 may be associated with Mn IV;
5.V S3 may be associated with a histidine (one of the amino acids in the D1
protein);
6.V The nature of S4 isn·t clear;
7.V Conversion from one state to the next requires one photon and results in
the loss of one electron to P680; and
8.V the loss of 4 total electrons generates a strong enough potential to split
water.

Evidence:

1.V after a dark equilibration period, oxygen is released after the third light
flash and then after every fourth flash;
2.V explains occurrence of Mn in photosystem II.
D. PQ Shuttle (Q cycle)

1.V PQH2 is reduced on the stromal side of the thylakoid in PSII

2.V PQH2 shuttles over to the lumen side of thylakoid and gets oxidized when it
transfers its electrons to the cyt b/f complex
3.V One electron is given to an Fe-S protein, which in turn, passes it to cyt f and then
to PC. The other electron is given to cyt b which then partially reduces another
PQ.
4.V The "leftover" protons are dumped into the lumen
5.V A second PQH2 from PSII shuttles to the cyto b/f complex and essentially repeats
step 3 - one electron is passed to Fe-S then to cyt f and to PC. The other electron
 from PQH2 is given to cyt b and then to PQ to fully reduce it to PQH2 which must
also grab two protons from the stroma.

Take-Home-Lessons: for every two electrons shuttled to PSI, four protons are moved across the
membrane! The stoichiometry requires more than 2 protons per electron pair to account for ATP
synthesis.

E. Herbicides and electron transport

1.V Ërea derivatives - DCMË (diuron) blocks electron flow at a point after Q. They
bind to the Qb binding site, preventing PQ from doing so. Interestingly, there are
resistant varieties, that have a single amino acid substitution in the D1 binding
protein.
2.V Viologen dyes - paraquat/diquat - accept electrons from the reducing side of PSI
- thus, they interrupt electron flow and also convert oxygen to superoxide - which
causes damage to membranes, etc.

c 





÷V Literal translation - "the light driven synthesis of ATP"

÷V Occurs by the same mechanism, Chemiosmotic hypothesis, that occurs during ATP
synthesis in the mitochondria

A. Non-cyclic
Electrons are passed in a single direction, one-way, no backtracking, from water to NADP+.

B. Cyclic
Electrons cycle through PSI. This occurs when carriers get backed up with electrons but still
want to get rid of electrons. This is a mechanism for generating additional ATP.

Features:

1.V asymmetric distribution of electron carriers in the thylakoids;

2.V some carriers require only electrons (i.e., cytochromes, chlorophyll), others both
(i.e., PQ);
3.V at the transitions, protons will be extracted or expelled;
4.V the transitions occur at the surfaces of the membrane;
5.V PQ is the major source of the proton gradient;
6.V reduction of PQ occurs at the stroma side of the thylakoid and removes 2 protons
from the stroma;
7.V PQ shuttles electrons to the cyt b/f complex on the lumen side of the thylakoid.
Only electrons are passed to the cyt b/f complex, the protons are expelled into
the lumen. Thus, PQ is oxidized on the lumen side of the membrane;
8.V the Q cycle shuttles additional protons across the membrane;
9.V the thylakoid is impermeable to protons;
10.V a pH gradient is generated across the membrane, the stoma pH is  8
while the lumen is  5;
 11.V the pH gradient provides the energy for the synthesis of ATP;
12.V ATP is synthesized by an ATPase associated with the CF complex; and
13.V protons escaping through the channel drives ATP synthesis something like
water turning a mill.

Evidence: lots!

1.V a pH gradient exists in the chloroplast. Discharging the gradient with buffers
prevents ATP synthesis;
2.V artificially creating a gradient allows for ATP synthesis;
3.V uncouplers like DNP "poke holes" in the thylakoid making it "leaky" and
discharging the gradient preventing ATP synthesis. Note that this doesn·t stop
electron flow - in fact, it usually increases the rate.

cc 
 


Emerson observed that the rate of photosynthesis was greater than the sum of the rates when
red light (660 nm) and far red light (710 nm) were given separately. This synergistic effect, called
the Emerson enhancement Effect, suggested two cooperating systems which has been the
conventional wisdom for a long time.

cccΠ:

÷V Govindjee and Krogmann, D (2004). Discoveries in oxygenic photosynthesis

(1727-2003): a perspective. Photosynthesis Research 80: 15 - 57.
÷V Orr, L & Govindjee. Photosynthesis and the web. (revised Oct 4, 2004)
÷V Arizona State Ëniversity Center for the Study of Early Events of Photosynthesis.V
V

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÷V also called "dark reactions" because the reactions don·t require light - however, note
that these reactions can (and normally do) occur in the light. In one sense they can be
considered "light-dependent" since they require the ATP and NADPH generated during
the Z scheme.
÷V called the Calvin cycle - after the fellow and his colleagues who worked out most of the
reactions. If you had done it, you too, would own a Nobel Prize.
÷V occurs in the stroma
÷V there are three major steps: fixation ń reduction ń rearrangement/recharging/releaseV

A. Carbon dioxide fixation

Carbon dioxide is fixed (trapped, bound) to form an organic compound (phosphoglyceric
acid, PGA)

÷V carbon dioxide condenses with RuBP (ribulose bisphosphate; C5) to form 2 molecules of
PGA (C3)
÷V first product of carbon fixation is PGA (Calvin·s experiments)
÷V catalyzed by the enzyme ribulose bisphosphate carboxylase (rubisco).
 ÷V rubisco is the most abundant protein on earth; it makes up 50% of leaf protein
÷V essentially, the carbon dioxide binds to the keto-carbon and then the resultant molecule
splits

B. Reduction
Step in which the temporary chemical (ATP) and reducing (NADPH) potentials that were
generated in the light-dependent reactions are used to reduce the PGA (an acid) to a carbonyl
(glyceraldehyde 3-phosphate; abbreviated G3P or GAP)

÷V PGA is reduced to G3P

÷V this is a two-step reaction sequence
÷V first, PGA is phosphorylated with ATP to 1,3-bisphosophoglycerate which is subsequently
reduced to G3P (note a phosphate is lost during this reaction). NADPH provides the
electrons for the reduction
÷V energy requirements - at this point in the cycle, for each carbon dioxide fixed, two ATP
and two NADPH are required (one for each of the two PGA·s)

C. Rearrangement/Recharging/Release
Complex series of reactions (rearrangment) that result in the net removal of a C3
carbohydrate from the cycle (release) and the production of the precursor to the starting
material (recharging):

÷V see overhead and diagram in text for details

÷V the cycle must turn 3 times for the production of one net triose
÷V the end product of the cycle is ribulose-5-P (RuP)
÷V ATP converts ribulose-5-P to RuBP
÷V ATP comes from the Z scheme

E. Summary
The fixation of 1 carbon dioxide requires: 3 ATP and 2 NADPH.

ccŒ

 
 
*  

We will not cover this in class except to say that regulation of the cycle is obviously important.
There are several regulatory controls:

1.V rubisco - light activated;

2.V allosteric regulation - rubisco has a binding site for CO2;
3.V rubisco activase - protein that "activates" rubisco; and
4.V Fd/thioredoxin - several enzymes require a reduction to become activated.
ccc @


Plants that exhibit the type of photosynthetic carbon reduction that we described above are
termed C3 plants. In other words, the first product of carbon dioxide fixation is a 3-carbon
compound (PGA). Thus, when radioactively labeled carbon dioxide is fed to a plant, the first
place that it shows up is PGA.
c 

 
Light stimulated production of carbon dioxide in the presence of oxygen

÷V not associated with mitochondrial respiration

÷V requires light
÷V not accompanied by ATP synthesis
÷V wastes energy (i.e., ATP, NADPH)

A. Observations on photorespiration

1.V Not all plants photorespire - see plot of carbon dioxide production (= respiration rate) vs.
time for tobacco and maize. Note the dark rates are the same, but the light rate is much
greater in tobacco.
2.V Plants that photorespire typically show light saturation - points to note: (a) plot of carbon
dioxide uptake (=Ps rate) vs. fluence for tobacco and maize in ambient oxygen (21%);
(b) light saturation point - point at which increasing fluence yields a constant amount of
photosynthesis; (c) light compensation point - fluence at which the amount of
photosynthesis just equals the amount of respiration; and (d) note that plants that
photorespire (like tobacco) have a higher light compensation point and light saturate.
3.V Plants that photorespire have a higher CO2 compensation point. In other words, it takes a
greater amount of carbon dioxide to break even.
4.V Oxygen inhibits photosynthesis in plants that photorespire (called the Warburg effect) -
plot carbon dioxide uptake vs. fluence for maize and tobacco at 1.5% oxygen
5.V Carbon dioxide is limiting to plants that photorespire - for evidence see: (a) plot of
carbon dioxide fixation vs. carbon dioxide concentration for maize and red clover
(photorespires); and (b) plot of carbon dioxide uptake vs. fluence for tobacco and
maize at ambient oxygen at varying carbon dioxide levels.

B. Making sense of the data

The data cited above suggest that carbon dioxide and oxygen have antagonistic (opposite)
actions in photosynthesis and act in a competitive manner.

C. The problem - rubisco

Ënlike most enzymes, rubisco is not substrate specific - it also has an oxygenase function. In
addition to its normal substrate (carbon dioxide) rubisco also binds oxygen to RuBP. Although
rubisco has a higher affinity for binding carbon dioxide (Km = 9 µM), if enough oxygen is present,
it acts as a competitive inhibitor (the Km for oxygen is 535 µM).

D. The reaction catalyzed by ribulose bisphosphate carboxylase/ oxygenase

When rubisco binds oxygen to RuBP, the RuBP is essentially split in half to a 3 carbon piece and
a 2 carbon fragment according to the following reaction:

RuBP + oxygen + rubisco ń PGA (C3)+ phosphoglycolate (C2)V

Compare this to the normal reaction:

RuBP + oxygen + rubisco ń 2 PGA (C3)V

Thus, rubisco has oxygenase activity as well as a carboxylase.

E. What determines which process will occur? Oxygenase activity occurs when:

1.V carbon dioxide levels are low - during periods of active photosynthesis; and
2.V oxygen levels are high - due to the activity of PSII; high light intensity.
The ratio of [carbon dioxide]/[oxygen] ultimately determines the product of the rubisco reaction.

if [carbon dioxide/oxygen] = high; then it favors normal Calvin cycle

if [carbon dioxide/oxygen] = low; then it favors oxygenase activity

 


 
 G #:""3



 

The purpose of this pathway is to metabolize and reclaim the carbon in phosphoglycolate

A. Overview of the major steps:

1.V The products of rubisco oxygenase activity are phosphoglycolate and PGA;
2.V PGA enters the Calvin cycle as normal;
3.V Phosphoglycolate is dephosphorylated to glycolate and is then shuttled out of
the chloroplast into the peroxisome;
4.V Recall that peroxisomes are single membrane-bound organelles that contain
catalase. They also have the marker enzyme glycolate oxidase;
5.V In the peroxisome, the glycolate is oxidized to glyoxylate by glycolate oxidase.
This is a redox reaction. Oxygen gets reduced to hydrogen peroxide;
6.V Catalase converts the potentially destructive hydrogen peroxide to oxygen and
water;
7.V Glyoxylate is converted to glycine (an amino acid) by a transamination reaction.
Glycine is transported out of the peroxisome into the mitochondrion. Two glycine
molecules condense to form serine releasing carbon dioxide. This process requires
NADH;
8.V Serine is further metabolized in the peroxisome to glycerate;
9.V Glycerate enters the chloroplast, is phosphorylated and enters the Calvin cycle;

B. The Highlights - The glycolate cycle:

÷V is oxidative;
÷V occurs in three organelles;
÷V reclaims some (75%), but not all, of the carbon from glycolate;
÷V carbon dioxide is released in the mitochondria and is hence the reason this is a
type of "respiration".

C. Why do plant photorespire?

From a Darwinian perspective, we·d expect that this process would have been selected
against. However, the fact that so many plants do it, suggests that it may have an
unappreciated function. Possibilities include: (a) salvage the carbon lost during rubisco
oxygenase action; (b) mechanism to help prevent destruction by excess light.

c > 

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Plants that avoid photorespiration have a unique modification of photosynthesis. They are
called C4 plants because the first product of carbon dioxide fixation is a 4-carbon compound,
not PGA as it is in C3 plants.

Examples: There are many plants that have this specialized modification. Found in many
different and unrelated groups of plants which indicates that it apparently evolved
independently several times. Even within a genus, some members can be C4 others C3.

C4 photosynthesis is common in grasses like maize, sorghum, crabgrass and members of the
Centrospermae (a closely related group of plants that includes Chenopodiaceae,
Amaranthaceae, Aizoaceae, Nyctaginaceae, Portulaceae, Zygophyllaceae). Not all grasses
are C4; for example, Kentucky blue grass (m

?; common lawn grass) is C3.

A. How do C4 plants avoid photorespiration?

The answer is simple - C4 plants separate the site of oxygen production (PSII) from rubisco
(Calvin cycle). But how? PSII and rubisco are placed in different:

1.V Cells. In typical C3 plants the chloroplasts are dispersed throughout the
mesophyll. Ësually there is a well-defined palisade and spongy layer. In contrast,
C4's have a more or less uniform mesophyll layer with a well-developed bundle
sheath around each vein. This is called Kranz anatomy, because the bundle
sheaths appears like a wreath surrounding the vein. In C4 plants, the Calvin cycle
activity occurs primarily in the bundle sheath cells, whereas PSII activity occurs in
the mesophyll cells.
2.V Chloroplasts - The chloroplasts of C4 are dimorphic. Bundle sheath cell (BSC)
chloroplasts are agranal. Recall that PSII occurs in the appressed regions of the
chloroplasts. Thus, agranal chloroplasts have little PSII activity; but, they do have
hi PSI activity. The mesophyll cell (MC) chloroplasts have typical granal stacking,
but low rubisco activity. Thus, most carbon fixation (carbohydrate production)
occurs in the bsc. Smart, eh?

B. Since C4 plants have separated the Calvin cycle PSII, there must be a mechanism to get
carbon dioxide into the BSC since:

1.V there is relatively slow diffusion to deep, interior regions of the leaf, especially
considering;
2.V the ambient level of carbon dioxide is low.
In order to solve this problem, plants required a mechanism to:

1.V fix carbon dioxide in regions of the leaf where it occurs in high concentration (i.e.,
MC). The enzyme that catalyzes this reaction is phosphoenolpyruvate
carboxylase (PEPcase). This enzyme binds carbon dioxide (actually bicarbonate)
to PEP to form oxaloacetate (reaction diagram). This reaction occurs in the
 cytoplasm. Note that OAA is a C4 compound. Hence these plants are called C4 -
because the first product of carbon fixation is a four carbon compound.
2.V transport the fixed carbon dioxide (which is in the form of a C4 compound like
malate or aspartate) from the MC to the BSC. OAA is converted to another C4
compound that, in turn, migrates to the BSC where it is decarboxylated and used
in the Calvin cycle. The "leftover" C3 shuttles back to the MC to pick up another
carbon dioxide and repeat the process.

C. General scheme - on overhead, covered in class

D. Details
Note that there are at least three different types of C4 plants. They differ in specific form in
which carbon dioxide is transported.

E. Advantages of C4 metabolism
Plants that exhibit this type of photosynthesis are characteristic of hot, tropical environments
that have a high light fluence. The advantage of C4 in these circumstances is that C4
metabolism:

1.V avoids the photorespiratory loss of carbon

2.V improves the water use efficiency of the plants
3.V results in higher rates of photosynthesis at high temperatures
4.V improves the efficiency of nitrogen utilization (because C3 require lots of rubisco)
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A. Origin of the name



refers to the Stonecrop family (Crassulaceae) and related succulents in which
this process is common. To date, plants in more than 18 different families including Cactaceae
(Cactus family) and Bromeliaceae (Pineapple family) have been shown to carry out CAM
metabolism. ? is derived from the observation that these plants accumulate large amounts
of organic acids in the dark.

Plants with CAM metabolism evolved in dry, hot, high light environments. This is largely a
mechanism to conserve water. Recall the photosynthesis-transpiration compromise (paradox)?
Plants in dry environments can·t afford to compromise - they loose too much water opening
their stomates during the day. CAM plants solved this problem by opening up the stomates at
night to obtain carbon dioxide. This strategy is just the reverse of "normal" plants. But, this presents
another problem - ATP & NAPDH, which are products of the light dependent reactions, are not
available when the carbon dioxide is fixed. The solution to this problem was to store the carbon
dioxide during the night until ATP and NADPH were available the following day. Thus, there is a
temporal separation of initial carbon fixation via PEPcase and the Calvin cycle (C4 plants have
a spatial separation).

B. PEPcase
This is the initial enzyme that fixes carbon dioxide. The product is ultimately malate which
accumulates in the vacuole during the night (hence the "acid" term).

C. Sequence of events.
Night ń stomates open ń nocturnal transpiration (lower than diurnal) and carbon fixation by
PEPcase ń OAA produced ń reduced with NADPH to malate ń shuttled into vacuole ń acid
content of vacuole increases ń starch depleted to provide PEP for
carboxylation ń day ń stomates close ńtranspiration decreased ń acid content
decreases ń malate decarboxylated to provide carbon dioxide for Calvin cycle ń starch
content increasesV

V
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V

FeatureV C3V C4V CAMV

Ësually no
no distinct bundle
Leaf anatomyV Kranz anatomyV palisade cells,
sheathV
large vacuolesV

Initial carboxylating enzymeV rubiscoV PEPcaseV PEPcaseV

Product of CO2 fixationV PGA (C3)V OAA (C4)V OAA (C4)V

ChloroplastsV one typeV dimorphicV one typeV

Theoretical energy requirements

1: 3: 2V 1: 5 : 2V 1: 6.5: 2V
(CO2: ATP: NADPH)V

Transpiration ratio (g H2O/g dry

450-950V 250-350V 18-125V
wt)V

Photosynthesis rate (mg CO2fixed

15 - 30V 40 - 80V (low)V
dm-2 h-1)V

chl a/b ratioV 2.8V 3.9V 2.5 - 3.0V

Requirement for sodium as a

NoV YesV NoV
micronutrient?V

Carbon dioxide compensation

50 - 150 (Hi)V 0-10 (low)V 0-5 (in dark)V
point (ppm)V

Light saturation
Response to lightV No light saturationV -V
easily achievedV

Photosynthesis inhibited by
YesV NoV YesV
oxygen?V

Only in bundle
Photorespiration detectable?V YesV Late afternoonV
sheathV
Temperature optimum for
15-25V 30-47V 35V
photosynthesisV

Low (26 ²
Dry matter production High (87 ² maize;
soybean; 30 ² low, variableV
(bushels/acre)V 50 ² sorghum)V
wheat)V