GOGONTLE DINTWE

201403477
BIO 316
LAB PRACTICAL 3: STOMATAL OPENING
21/09/16

TITLE: INVESTIGATING STOMATAL MOVEMENTS IN THE PRESENCE OF

EXTERNAL ENVIRONMENT CONDITIONS
INTRODUCTION
Stomata are small adjustable pores in the leave surface that enable gas exchange. These pores
open to facilitate uptake of carbon dioxide for photosynthesis, while closure prevents excessive
loss of water during drought. During evolution, stomata enabled plants to survive in dry
environments. Celltocell signalling controls the development of stomata in the leaf surface.
Stomatal movements are forced by changes in the volume of two guard cells that are positioned
on either side of the pore. During stomatal opening, guard cells accumulate potassium salts,
causing an osmotically driven uptake of water (Zeiger, 2012)
Owing to the influx of water the guard cells swell and bend, thereby pushing each other apart
and creating an open pore in the middle. Various environmental signals, such as light, carbon
dioxide and humidity, influence the iontransport machinery within the guard cell and thus alter
stomatal movement. Because of their role in regulating carbon dioxide uptake and transpiration
of water, stomata are likely to play a key role in the adaption of plants to the anticipated global
warming (Neher, 2010).
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.
Photophosphorylation may provide ATP which is required to operate H+/K+ ions exchange
pumps in which H+ ions are pumped out and K+ ions, CI ions and organic anions are pumped
into the guard cells. These ions decrease water potential of guard cells. 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 (Trentham, 2009).
Specialized potassium efflux channels participate in mediating release of potassium from guard
cells. Anion channels are 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, the anion channels
are activated by signals that cause stomatal closing, for example by intracellular calcium (Blatt,
2008).
The resulting release of negatively charged anions from guard cells results in an electrical shift of
the membrane to more positive voltages (depolarization) at the intracellular surface of the guard
cell plasma membrane. This electrical depolarization of guard cells leads to activation of the
outward potassium channels and the release of potassium through these channels At least two
major types of anion channels have been characterized in the plasma membrane; S-type anion
channels and R-type anion channels (Zeiger, 2007).
The aim of the experiment is to find out the effect of potassium concentration of the external
solution of the size of the stomatal aperture

MATERIALS AND METHODS

25ml of KCL solutions were prepared with different concentrations. An epidermal peel from the
leaf of Rheo sp provided (the peel was removed from the side where stomata are more
numerous). Peels in different KCL solutions in petri dishes were incubated and light illuminated
for ten minutes using the bench lamp. The peel was then mounted on a slide and covered with a
cover slip. Observations were made under a microscope and a calibrated eye-piece was used to
measure radius of the pore. 5 replicates of each treatment were taken.

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
The figure above, shows that the stomatal size is directly proportional to increase in potassium
ion concentration. 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. The area of the cross section of the middle portion of the guard cell
may upsurge almost twofold during stomatal opening. The enlargement 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).
Guard cells differ considerably in size, i.e. with increasing volume the increment in turgor
needed to produce a constant increment in volume diminutions until stiffening occurs (Zeiger,
2012). This portent may have two reasons. 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. Furthermore 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 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 discoveries 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.
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 adequate enough 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 dark conditions carbondioxide 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. Whilst during
light conditions starch is converted to PEP ( phosphoenol pyruvic acid) which combines with CO
to form oxaloacteic acid (OAA) and convert 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. Thus it is not expected to find stomatal movements to be the same under light
and dark conditions.
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 (Neher, 2012).
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).
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 (Blatt, 2008).

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.

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