KOKETSO J MOGWE

ID:201402636

DETERMINE THE EFFECT OF INCREASING POTASSIUM CONCENTRATION OF

THE EXTERNAL SOLUTION ON THE SIZE OF THE STOMATAL APERTURE

BIO 316- Practical Lab 3

DETERMINE THE EFFECT OF INCREASING POTASSIUM CONCENTRATION OF

THE EXTERNAL SOLUTION ON THE SIZE OF THE STOMATAL APERTURE
INTRODUCTION
Stomata are formed by pairs of specialized epidermal guard cells which are generally surrounded
by subsidiary cells. They serve as major gateways both for CO2 influx from the atmosphere and
transpiration water loss of plants. Due to the impermeable wax layer on the epidermal surface,
the stomatal complex plays a major role in controlling gas exchange between plant and the
surrounding atmosphere. The guard cells are produced in pairs with a gap between them that
forms a stomatal pore.
The stomatal pores are largest when water is freely available and the guard cells turgid, and
closed when water availability is critically low and the guard cells become flaccid.
Photosynthesis depends on the diffusion of Carbon dioxide (CO2) from the air through the
stomata into the mesophyll tissues. Oxygen (O2), produced as a byproduct of photosynthesis,
exits the plant via the stomata. When the stomata are open, water is lost by evaporation and must
be replaced via the transpiration stream, with water taken up by the roots. Plants must balance
the amount of CO2 absorbed from the air with the water loss through the stomatal pores, and this
is achieved by both active and passive control of guard cell turgor and stomatal pore size
explains (Schroeder JI et. Al 2014).
Opening and closure of the stomatal pore is mediated by changes in the turgor pressure of the
two guard cells. According to (Zeiger, 2012) the turgor pressure of guard cells is controlled by
movements of large quantities of ions and sugars into and out of the guard cells. When guard
cells take up these solutes, the water potential () inside the cells increases (creating a
hypertonic solution), causing osmotic water flow into the guard cells. This leads to a turgor
pressure increase causing swelling of the guard cells and the stomatal pores open. The ions that
are taken up by guard cells are mainly potassium (K+) ions and chloride (Cl) ions. In addition
guard cells take up sugars that also contribute to opening of the stomatal pores.
Ion uptake into guard cells causes stomatal opening: The opening of gas exchange pores requires
the uptake of potassium ions into guard cells. Potassium channels and pumps have been
identified and shown to function in the uptake of ions and opening of stomatal apertures. Ion
release from guard cells causes stomatal pore closing: Other ion channels have been identified
that mediate release of ions from guard cells, which results in osmotic water efflux from guard
cells due to osmosis, shrinking of the guard cells, and closing of stomatal pores. Specialized
potassium efflux channels participate in mediating release of potassium from guard cells. Anion
channels were identified as important controllers of stomatal closing. Anion channels have
several major functions in controlling stomatal closing. They allow release of anions, such as
chloride and malate from guard cells, which is needed for stomatal closing.
Metabolism of guard cells continuously interacts with the environment, sensing biotic and
abiotic stimuli and signals coming from both the atmosphere and from roots and responding with
appropriate changes in pressure turgor. By this mechanism guard cells control stomata aperture
in accordance with changes in the plants environment, in order to preserve water and CO2

assimilation. Several environmental factors affect stomata opening, including hormone, light
quality and intensity, air humidity, atmospheric CO2 concentration, biotic and abiotic stresses.
The objective of the experiment was to determine the effect of increasing potassium
concentration of the external solution on the size of the stomatal aperture.

MATERIALS AND METHODS

A solution of KCL 25ml was prepared and used to incubate an epidermal peel from the leaf of
Rheo species provided in a petri dish. The peels were then illuminated for 10minutes using the
bench lamp, thereafter were they were mounted on a slide and covered. The prepared slide was
observed under a microscope. A calibrated eye piece was used to measure the maximum radius
pore and 5 replicates for each treatment were recorded in order to plot a graph of stomatal size
versus K+ ion concentration.

REFERENCES
1. Assmann SM, Simoncini L, & Schroeder JI (2014) Blue light activates electrogenic ion
pumping in guard cell protoplasts of Vicia faba. Nature 318:285-287.
2. Zeiger E (2012) Blue light-dependent proton extrusion by guard-cell protoplasts of Vicia
faba. Nature 319:324-326.
3. Neher E (2010) Voltage dependence of K+ channels in guard cell protoplasts. Proc. Natl.
Acad. Sci. USA 84:4108-4112.
4. Trentham DR (2009) Reversible inactivation of K+ channels of Vicia stomatal guard cells
following the photolysis of caged 1,4,5-trisphosphate. Nature 346:766-769.
5. Blatt MR (2008) Membrane transport in stomatal guard cells: The importance of voltage
control. J. Memb. Biol. 126:1-18.
6. Kwak JM, Murata Y, Baizabal-Aguirre VM, Merrill J, Wang M, Kemper A, Hawke SD,
Tallman G, & Schroeder JI (2013) Dominant negative guard cell K+ channel mutants
reduce inward-rectifying K+ currents and light-induced stomatal opening in Arabidopsis.
Plant Physiol. 127:473-485.

RESULTS

Fig. 1 Stomatal Size vs. Potassium Chloride Concentration (mM)

According to the figure above, it is observable that the stomatal size or diameter is directly
proportional to increase in potassium ion concentration but with a fluctuation at 800Mm.

DISCUSSION
According to the figure above, it is observable that the stomatal size is directly proportional to
increase in potassium ion concentration. The area of the cross section of the middle portion
of the guard cell may increase almost twofold during stomatal opening. The expansion of
the polar sections of the guard cells is made possible by a thickening and a fold in the
bottom cell wall, both of which expand when the stoma opens, (Neher, 2010). Optical
sectioning of intact stomata in the open and closed states and integration of the cross-sectional
areas made it possible to estimate the relationship between stomatal aperture and guard cell
volume. It seems this relationship is linear, and the volume of the lumina of guard cells
of leaf Rheo may almost double when a stoma opens from a closed state.
Guard cells vary considerably in size, even within one species. The walls of guard cells appear to
give when turgor increases, i.e. with increasing volume the increment in turgor needed to
produce a constant increment in volume decreases until; of course, stiffening occurs (Zeiger,
2012). This phenomenon may have two causes. One is that some of the volume changes are due
to shape changes. The other and perhaps more important cause is the composition of the
matrix of the guard cell walls.
According to (Blatt, 2008) the radial micellation of the guard cell walls appears early in the
ontogeny as a radial orientation of microtubules. (Blatt, 2008) also reports that the two guard
cells of a developing stomatal complex accumulate K+ and Cl- before they separate and form the
stomatal pore. Presumably the same mechanism which produces turgor in the functional stoma is
already used to exert the stress needed for splitting the cell walls between the two developing
guard cells.
Additionally the specific K+ effect in opening was observed in light but not in the dark, although
CO, free air was used in both cases. This work tends to support and enlarges upon the proposal
resulting from previous work that the basis for stomatal opening is the uptake of K+ by guard
cells, (Neher, 2010). Some of the present findings also indicate, though indirectly, that opening is
a result of ion uptake. The specific concentration dependent Light activated effect of K+ is
explained best by a highly selective active process, such as its uptake. Also the relation of
stomatal aperture to K+; concentration in light resembles a saturation curve of ion uptake versus
concentration.
K+ probably plays the same essential role in stomatal mechanisms in intact leaves as it does in
other plants. The proposed mechanism of stomatal opening would require that K+ be transported
in and out of guard cells for opening and closing. There is a need for a sizable reservoir in the
leaf to supply K+ to, and receive K+ from, the guard cells (Zeiger, 2012). It might be that
epidermal cells alone are not sufficient for this purpose and that the mesophyll also participates.
In plants K+ exists free in large quantities and is an activator for many enzymes. Its role as an

activator in most metabolic reactions can, however, is substituted with similar physiological ions.
Stomatal opening appears to be the first physiological process in higher plants in which a highly
specific requirement for K+ is demonstrated. Assuming that ion uptake is an integral part of the
opening process, the data would suggest that the guard cell has a light-activated ion uptake
mechanism of a degree of specificity previously unknown in higher plants.
K+ ion theory was proposed by Levitt in 1974. He states that, during light conditions starch is
converted to PEP ( phosphoenol pyruvic acid) which combines with CO to form oxaloacteic acid
(OAA) andconvert it to malic acid. This malic acid dissociates into malate anions and H ions in
guard cells. H ions are transported to subsidiary cells and in exchange of which K+ ion move
inside guard cells. This is called ion exchange. K+ ions are balanced by malate ions present in
guard cells and also by taking in some Cl- ions. This ion exchange occurs by the expenditure of
ATP energy. Increased concentration of K+ and malate ions in the guard cells vacuole will cause
sufficient osmotic pressure to absorb water from surrounding cells. This in turn will increase
turgor pressure of guard cells and lead to the opening of stomatal pores. During dark conditions
carbon-dioxide concentration increase in sub stomatal spaces because of initiation of respiration
which will prevent the H - K ion exchange. Due to this malate ions present in the vacuole of
guard cells combine with the H+ ions and form malic acid. Increase in the concentration of malic
acid will inhibit its synthesis. K+ ion move out of guard cells, osmotic pressure decrease. Water
will move out of guard cells into subsidiary cells, and cause the closure of stomatal pore.
Therefore it is not expected to find stomatal movements to be the same under light and dark
conditions.
Guard cells perceive and process environmental and endogenous stimuli such as light, humidity,
CO2 concentration, temperature, drought, and plant hormones to trigger cellular responses
resulting in stomatal opening or closure. These signal transduction pathways determine for
example how quickly a plant will lose water during a drought period. Guard cells have become a
model for single cell signaling (Neher, 2012).
Light is the most important environmental factor stimulating stomatal opening. The evidence that
light can induce stomatal opening in carbon dioxide concentration comes mainly from studies of
the extent of opening in different wavelength of light. However, a key achievement on the
understanding of stomatal mechanism was the light signal transduction pathway of guard cells.
The direct response of stomata to light was initially demonstrated. Stomatal response to white
light is the combined expression of two distinct photoreceptor systems: guard cell chloroplasts
and a blue light-dependent photosystem. A blue light photoreceptor for the signal transduction
chain in guard cells has been suggested to be the carotenoid pigment zeaxanthin. Phototropin has
also been postulated as a blue light photoreceptor ( Tienthen, 2009).
Blue light activates the plasma membrane H+-ATPase via phototropin and creates an
intracellular negative electrical potential across the membrane in guard cells. A number of
physiological data have demonstrated that proton efflux originating in guard cells precedes
stomatal opening, suggesting that proton efflux is a necessary precursor of stomatal opening
Therefore, when stomata open, protons are first pumped out of the guard cell, resulting in a
potential gradient across the plasmalemma. This gradient stimulates opening of inward K+

channels which may allow K+ influx to guard cells resulting in an increase of water potential
(Zeiger, 2010). Vacuoles are large intracellular storage organelles in plants cells. In addition to
the ion channels in the plasma membrane, vacuolar ion channels have important functions in
regulation of stomatal opening and closure because vacuoles can occupy up to 90% of guard
cells volume. Therefore, a majority of ions are released from vacuoles when stomata are closed
(Neher, 2012).

APPENDIX
Table 1: concentration of KCL corresponding diameter of stomata opening
0mM
rep 1
rep2
rep3
rep 4
rep 5
MEAN

1
0
1
1
1
0.8

200m
M
2
1
2
2
1
1.6

400m
M
2
2
2
2.2
2.1
2.06

600m
M
2
3
3
3
2.6

800m
M
3
2
3
2
2
2.4

1000m
M
2
3
3
2
2
2.4