Plant Physiology I

Vegetative development:
Trichome, root hair, and stomatal Development:
Epidermal cell fate: Hitherto we have discussed cellular differentiation in the context of the
maturation, from a ground meristem, of a contiguous array of cells comprising a particular tissue
type. Assemblages of epidermal cells provide an interesting contrast in that there are two types of
highly specialized cells present throughout the epidermis that develop in isolation, surrounded by
pavement cells. These are the guard cells (making up the stomata), and the trichomes/root hairs.
Two fundamental questions can be asked 1) how are the cells destined to differentiate into guard
cells or trichomes/root hairs selected? 2) How is the spatial patterning of stomata and
trichome/root hair placement in the pavement controlled?
Stomatal development:
Stomata are essential to the life of terrestrial plants. There is usually a stomata free zone
surrounding each stoma in normal plants. This intervention of subsidiary (when they exist) or
epidermal cells between stomata is thought to provide a pool of ions for the guard cells and
optimize the access of the underlying mesophyll cells to the external environment. Two key
features combine to influence stomatal patterning; internal anatomy (cell type, position of the
vasculature), and contact between stomata. Little is known respecting why stomata only rarely
develop over the vasculature. Even less is known about how certain underlying cell types seem to
inhibit stomata from developing over them. Hence, we will discuss the control of stomatal
patterning such that stoma rarely develop side-by-side.
Stomatal initials form by asymmetric cell division of precursor cells such that the smaller cell
becomes a stomatal precursor. The stomatal initial can produce both the guard cells and ordinary
epidermal cells. Considerable cell-to-cell communication takes place leading to the coordination
of the plane of cell division as well as the regulation of precursor cell activity. Additionally, the
timing of when a cell is competent to become a stomatal precursor and where such precursors
can initiate are highly controlled.
In both dicots and monocots, the cell divisions leading to the production of stomata are highly
asymmetric both in geometry and in the fate of the cells thus produced. In monocots, the first
asymmetric division results in the smaller, usually rectangular cell becoming a guard mother cell
which divides symmetrically producing two guard cells. In dicots, the smaller cell is usually
triangular and continues to divide after the surrounding cells have ceased to do so. Eventually, it
converts into a guard mother cell and divides symmetrically to produce two guard cells. Since
dicot stomatal initials continue to divide after the surrounding epidermal cells have ceased they
have been termed meristemoid cells to emphasize their ability to continue cell division. In dicots
there are two types, primary meristemoids and secondary (satellite) meristemoids, the latter
produced by an asymmetric division of a neighboring cell to the stomata. Rectangular initials
(a.k.a. short cells) in monocots comprise the three types of stomatal initial found in the
angiosperms. How stomatal pattern is initiated depends on the type of initial.
Primary meristemoid positioning appears to be random, with the exception that it obeys the rule
that two stomata should not be adjacent. Secondary meristemoids and stomatal initials in
monocots are placed in a highly regular fashion crucial to stomatal patterning.
The asymmetric division producing the stomatal initial in monocots occurs so that the initial is
placed distal from the base of the leaf. Upon the formation of the initial, asymmetric divisions
occur in cells adjacent to the new initial to form two subsidiary cells. Finally, the guard mother cell
divides symmetrically to form two guard cells surrounding a pore.

Primary meristemoids arise from protodermal cells. These cell undergo a series of asymmetric
divisions that produces a small, triangular initial situated approximately in the middle of the future
stomatal complex. The meristemoid next alters shape to an oval guard mother cell. Finally, as in
monocots, the mother cell divides symmetrically and produces two guard cells.
Satellite meristemoids arise once a primary meristemoid has completed development into a
stoma. One of the surrounding cells maintains meristemoid identity and undergoes asymmetric
division such that the guard mother cell is formed distal from the existing stoma. Upon several
asymmetric divisions, the meristemoid, now separated from the existing guard cells by epidermal
cells, also converts to an oval guard mother cell and divides symmetrically.
Timing of stomatal development:
In arabidopsis at least, stomata develop after trichomes have formed.
Cell-to-cell communication during stomatal development:
There is a wide array of opportunities for signals to be traversing between cells thus
coordinating stomatal development. Evidence for cell-to-cell communication include the inhibition
of stomata to form above veins in the leaf, the formation of satellite meristemoids situated away
from existing stomata, the arrested development of meristemoids located proximally to each other
or to stomata, the orientation of subsequent cell divisions to separate meristemoids when two do
happened to develop beside each other and, the initiation of subsidiary cell formation in
monocots.
A genetic analysis of stomatal patterning:
There are two mutants identified that violate the rule that no two stomates should be in
contact. However, the four lips (flp) and too many mouths (tmm) mutations transgress by two
different developmental mechanisms. Tmm mutants have far greater numbers of stoma in rosette
leaves, cotyledons, and abaxial sepal surface. Other tissues of tmm plants (inflorescence stem,
adaxial sepal epidermis, silique tips) entirely lack stomata whereas wild type plants possess
them. The flower pedicle shows an abnormal gradation of stomatal frequency from none proximal
to the stem to abnormally greater numbers near the flower base. Hence, TMM is thought to
control the entry of protodermal cells into the stomatal developmental process and appears to
have opposite effects on meristemoids depending on the tissue (compare rosette leaves with
sepal epidermis).
The tmm mutation appears to increase the formation of satellite meristemoids as well as to alter
the polarity of cell divisions such that meristemoids can now form next to guard cells.
The flp mutation can produce unpaired guard cells as well as result in stomatal
twinning. Unlike tmm mutants, flp mutants do not exhibit increased numbers of guard cells.
Analysis of stomatal development in flp mutants suggests that stomata develop normally until the
guard mother cell stage. At this stage the guard mother cell undergoes a symmetric division and
the resulting daughter cells divide symmetrically again to produce two, contiguous stomates.
Hence FLP may control guard mother cell identity, or regulate the number of symmetric divisions
a guard mother cell can undergo.
The R-558 mutation causes an increase in stomatal density throughout the plant and
stomatal clustering can also occur.
Trichome Development:
Trichomes are present on the aerial portions of almost every terrestrial plant. Although some
plants have but a single type of trichome, trichomes exist in several different forms, often on the
same plant. Tomato has six different types of trichome. Trichomes are thought to perform a

variety of functions, from plant defense (physical obstruction to marauding insects, to site of
synthesis and display of complex chemical inhibitors and repellents) to creation of a boundary
layer of moister air to reduce transpirational loss of water. The nucleus of the cell resides in the
base of the trichome and a thin strand of cytoplasm is continuous into the cell extension which is
highly vacuolate. The cell wall of the trichome is thick and covered with numerous papillae the
function of which is unknown. The most impressive of trichomes is that of the cotton ovule. The
single cells of the ovule are capable of supporting a rate of cell extension (trichome elongation) in
excess of 2 mm a day, leading to a final single cell length of 30-35 mm!!!
Trichomes mature basipetally. They expand laterally as the cell extension elongates so
that the girth as well as the length of a mature trichome is much greater than that of the
undifferentiated trichoblast. Additionally, a rosette of normal atrichblast epidermal cells form a
group of support cells at the base of the trichome stalk. There are 10 known mutations that affect
trichome development, six of which seem to affect only trichome occurrence and shape. The first
of these 6 is the GLABRA1 (GL1) gene (glabrous = "free from hair") which, when defective (gl1),
results in the loss of trichomes on most surfaces. This gene is thought to encode a DNA binding
protein of the Myb family. The second is the glabra3 (gl3) mutant that has several aborted
trichome-like cells present not being as severe as gl1. The DISTORTED1 and 2 genes (DIS1,
DIS2) when disfunctional (dis1, dis2) result in shorter than normal trichomes that are twisted,
branchless, and bulbous relative to wild type trichomes. Despite the similarity of the phenotype
mutations in these genes elicit, they are not allelic. The fifth gene is STALKLESS (STL).
Mutations in this gene result in trichomes that have very short stalks that support a normal
looking, branching trichome tip. The last of the six genes is UNDERDEVELOPED TRICHOME
(UDT). Mutations in this gene result in a lower number of branches per trichome.
The timing of trichome development varies among plants. In arabidopsis leaves the trichomes are
the first cells to terminally differentiate while on the ovules of cotton, the trichomes (which
ultimately become single-celled cotton fibers) do not commence elongation until after all other
epidermal cells have ceased to divide.
So, how is trichome spacing determined? There are two models. One suggests that precursor
cells produce inhibitory signals that prevent other, neighboring cells, from going down the same
developmental pathway. The second suggests that, once a precursor cell is induced to become a
trichome it undergoes several divisions that separate it from other trichomes by a group of cells
all derived from the same precursor daughter cell.
The second hypothesis has been refuted by cell lineage experiments using a transposon
interrupted -glucuronidase (GUS) gene. With the transposon in place the gene does not make a
functional enzyme and cells expressing the interrupted GUS gene stay clear in the presence of
substrate. In cells that have had a transposition event occur, the excision of the transposon leads
to the production of functional GUS from the now transposon-free gene and cells in which this
occurs can turn the substrate blue. The transposition event can be induced in some cells prior to
their mitoses in the expanding epidermis and thereafter, all daughter cells produced from the
affected cell will be able to generate a blue color from the substrate. It was determined that the
boundary of the cells developing from such transposition-positive cells passed through trichome
producing and non-producing cells randomly, debunking the second hypothesis.
Another way in which trichome spacing may be maintained is that the precursor cell, undergoes
asymetric division with the different sized cells resulting in different developmental fates. Although
this does occur in some plants (e.g. many monocots) it is not a universal method of determining
trichome spacing. Additionally, epidermal cells of arabidopsis can be artificially induced to form
trichomes without the requirement of undergoing cell division.
However, neither is the hypothesis of trichome cell signaling to surrounding cells to prevent them
becoming trichomes without pitfalls. Mutagenises of heterozygous glabra1 gl1 plants producing
normally spaced and sized trichomes, generated some plants on which there developed glabrous

sectors. This signifies that signal generated by normal heterozygous cells outside the sector
affected cannot generate a transported signal sufficient to overcome the affect of the gl1 mutation
(i.e. a proliferation of trichomes). These findings argue that the effect of the GL1 protein is cell
autonomous.
The timing of trichome development:
Fusing the nuclear targeted maize R gene that promotes trichome formation, to a steroid
receptor from mice permitted the inducible expression of the R gene in the trichome-less
transparent testa glabra (ttg) mutant. With no steroid application, the R gene remained
cytosolic and the mutant remained trichome-less. Upon application of the steriod, the R gene
product traversed the nucleous and the mutant reverted to a trichomed phenotype. When
application of steroid was delayed, the more distal, older, sections of expanding leaves failed to
revert, signifying that the trichome precursor cells are determined early in leaf expansion.
Recently, the pleiotropic ttg mutant locus was identified by positional cloning as encoding a WD40
repeat protein (Walker et al. The Plant Cell 11: 1337-1349).
Additionally, the reduced trichome number (rtn) gene may be responsible for determining the
duration a particular cell is competent to make trichomes. This gene has been shown to be an
allelic variant between Landsberg erecta and Columbia strains of arabidopsis. The Ler strain
typically has fewer trichomes than the Col strain and this was demonstrated to be due to a shorter
period of trichome initiation during the development of a Ler leaf. Hence, RTN may control the
expression of either TTG or GL1 or control the period during which epidermal cells are receptive
to TTG or GL1.
There is also some evidence that TTG and/or GL1 may be involved in determining the spacing or
trichomes. Weak mutant allels of both TTG and GL1 have been identified that do not completely
suppress trichome development. In these plants, trichomes appear in clusters. Additionally,
heterozygous TTG/ttg plants that ectopically express GL1 often develop numerous clusters of
trichomes. This suggests that a stoichiometric balance between TTG and GL1 must be
maintained to prevent trichome clustering. An additional gene, Tryptychon (TRY) may act
downstream of TTG and GL1. Mutations in this gene increases the numbers of trichomes that
occur in clusters.
The results presented above has led to the proposition of the following model to explain the
control over trichome initiation and spacing in arabidopsis:
A.
TRY
GL1/TTG

GL1/TTG
TRY

Competent cells: mutual inhibition

B.
TRY
GL1/TTG

GL1/TTG
TRY

All protodermal cells are assumed to have equal potential to

develop into trichomes. Both GL1 and TTG proteins associate to
form a heterodimeric transcription factor responsible for
stimulating entry into the trichome differentiation pathway. The
level of commitment of a cell to the trichome pathway is directly
proportional to the amounts of the heterodimer in that cell. They in
turn act upon TRY. TRY expression is inhibitory to neighboring
cells entering the trichome differentiation pathway and a state of
mutual inhibition among cells exists (Fig. A). Due to random
chance, some cells are able to escape from this lateral inhibition
and produce more of the heterodimer than their neighbors. This
serves to positively reinforce the trichome fate of the precursor cell
through a positive feedback loop for at least GL1 and possibly
TTG, while inhibiting neighboring cells from becoming trichomes
(Fig. B).

Precursor cell: lateral inhibition

Redrawn from Plant Cell 9: 1109-1120.

Root hair development:

Root hairs have been hypothesized to expand the surface area of a root in order to
enhance the water and nutrient uptake efficiency of the root. They are also the site of attachment
of soil mycorhiza. Root hair development parallels trichome development in many aspects. Like
trichomes root hairs arise from epidermal cells (trichoblasts) that are over anticlinal cell walls of
the underlying cortex.
Microscopically, trichoblasts can be discerned from atrichoblasts early in their
development, prior to the elongation of the root hair, by having simpler plastids, larger nuclei and
nucleoli, greater amounts of protein, and RNA. This gives the trichoblasts a more cytoplasmically
dense appearance under normal light microscopy indicative of a delayed maturation as the cell
prepares to put forth the cell extension, the root hair. The growth of the root hair has much in
common with the growth of a pollen tube in that it occurs through the phenomenon of tip growth
as distinguished from the diffuse growth of the vast majority of cells. In tip growth, the cell wall
components required for wall synthesis to permit continued elongation are deposited at the very
tip of the trichoblast by fusion of dictyosome vesicules with the plasmamembrane at the apex of
the trichoblast. The newly deposited wall material is intercalated into the existing wall and is
displaced towards the periphery of the cell and down along the sides of the cell extension as the
trichoblast increases in length. The wall components subsequently undergo modification such as
desterification of pectin as new waves of wall material deposition displace the former tip back
along the trichoblast.
Root hairs elongate at a defined period of root maturation. In arabidopsis, the so called
"root hair zone" of the elongating root is located about a millimeter behind the root tip. Cells in this
region first commence putting forth root hairs. Root hairs can eventually attain lengths of 1.5 mm
(small in relation to a cotton ovule trichome but still impressive for a single cell) elongating at a
peak rate of up to 100 m an hour!