Guard cell

Guard cells are specialized plant cells in

the epidermis of leaves, stems and other
organs that are used to control gas
exchange. They 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
pressure and stomatal pore size.[1][2][3][4]
Opening and Closing of Stoma.

Guard cell function

Guard cells are cells surrounding each
stoma. They help to regulate the rate of
transpiration by opening and closing the
stomata. Light is the main trigger for the
opening or closing. Each guard cell has a
relatively thick cuticle on the pore-side
and a thin one opposite it. As water
enters the cell, the thin side bulges
outward like a balloon and draws the
thick side along with it, forming a
crescent; the combined crescents form
the opening of the pore.

Guard cells contain phototropin proteins

which are serine and threonine kinases
with blue-light photoreceptor activity.
Phototrophins contain two light, oxygen,
and voltage sensor (LOV) domains, and
are part of the PAS domain
superfamily.[5] The phototropins trigger
many responses such as phototropism,
chloroplast movement and leaf
expansion as well as stomatal opening.[5]
Not much was known about how these
photoreceptors worked prior to around
1998. The mechanism by which
phototropins work was elucidated
through experiments with broad bean
(Vicia faba). Immunodetection and far-
western blotting showed blue light
excites phototropin 1 and phototropin 2,
causing protein phosphatase 1 to begin a
phosphorylation cascade, which
activates H+-ATPase, a pump responsible
for pumping H+ ions out of the cell.[6] The
phosphorylated H+-ATPase allows the
binding of a 14-3-3 protein to an
autoinhibitory domain of the H+-ATPase
at the C terminus.[7] Serine and threonine
are then phosphorylated within the
protein, which induces H+-ATPase
activity.[5] The same experiment also
found that upon phosphorylation, a 14-3-
3 protein was bound to the phototropins
before the H+-ATPase had been
phosphorylated.[5] In a similar experiment
they concluded that the binding of 14-3-3
protein to the phosphorylation site is
essential for the activation of plasma
membrane H+-ATPase activity.[7] This
was done by adding phosphopeptides
such as P-950, which inhibits the binding
of 14-3-3 protein, to phosphorylated H+-
ATPase and observing the amino acid
sequence. As protons are being pumped
out, a negative electrical potential was
formed across the plasma membrane.
This hyperpolarization of the membrane
allowed the accumulation of charged
potassium (K+) ions and chloride (Cl−)
ions, which in turn, increases the solute
concentration causing the water
potential to decrease. The negative water
potential allows for osmosis to occur in
the guard cell, so that water entered,
allowing the cell to become turgid.

Opening and closure of the stomatal pore

is mediated by changes in the turgor
pressure of the two guard cells. The
turgor pressure of guard cells is
controlled by movements of large
quantities of ions and sugars into and
out of the guard cells. Guard cells have
cell walls of varying thickness and
differently oriented cellulose microfibers,
causing them to bend outward when they
are turgid, which in turn, causes stomata
to open. Stomata close when there is an
osmotic loss of water, occurring from the
loss of K+ to neighboring cells, mainly
potassium (K+) ions[8][9][10]

Water loss and water use

efficiency
Water stress (drought and salt stress) is
one of the major environmental problems
causing severe losses in agriculture and
in nature. Drought tolerance of plants is
mediated by several mechanisms that
work together, including stabilizing and
protecting the plant from damage
caused by desiccation and also
controlling how much water plants lose
through the stomatal pores during
drought. A plant hormone, abscisic acid
(ABA), is produced in response to
drought. A major type of ABA receptor
has been identified.[11][12] The plant
hormone ABA causes the stomatal pores
to close in response to drought, which
reduces plant water loss via transpiration
to the atmosphere and allows plants to
avoid or slow down water loss during
droughts. The use of drought-tolerant
crop plants would lead to a reduction in
crop losses during droughts. Since guard
cells control water loss of plants, the
investigation on how stomatal opening
and closure is regulated could lead to the
development of plants with improved
avoidance or slowing of desiccation and
better water use efficiency.[1] Research
done Jean-Pierre Rona shows that ABA
is the trigger for the closure of the
stomatal opening. To trigger this it
activates the release of anions and
potassium ions. This influx in anions
causes a depolarization of the plasma
membrane. This depolarization triggers
potassium plus ions in the cell to leave
the cell due to the unbalance in the
membrane potential. This sudden
change in ion concentrations causes the
guard cell to shrink which causes the
stomata to close which in turn decreases
the amount of water lost. All this is a
chain reaction according to his research.
The increase in ABA causes there to be
an increase in calcium ion concentration.
Although at first, they thought it was a
coincidence they later discovered that
this calcium increase is important. They
found Ca2+ ions are involved in anion
channel activation, which allows for
anions to flow into the guard cell. They
also are involved in prohibiting proton
ATPase from correcting and stopping the
membrane from being depolarized. To
support their hypothesis that calcium
was responsible for all these changes in
the cell they did an experiment where
they used proteins that inhibited the
calcium ions for being produced. If their
assumption that calcium is important in
these processes they'd see that with the
inhibitors they'd see less of the following
things. Their assumption was correct
and when the inhibitors were used they
saw that the proton ATPase worked
better to balance the depolarization.
They also found that the flow of anions
into the guard cells were not as strong.
This is important for getting ions to flow
into the guard cell. These two things are
crucial in causing the stomatal opening
to close preventing water loss for the
plant.[13]

Ion uptake and release

Ion channels and pumps regulating stomatal
opening and closure.

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.[10][14][15][16][17][18][19][20] 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
(Figures 1 and 2). Specialized potassium
efflux channels participate in mediating
release of potassium from guard
cells.[16][21][22][23][24] Anion channels were
identified as important controllers of
stomatal closing.[25][26][27][28][29][30][31]
Anion channels have several major
functions in controlling stomatal
closing:[26] (a) They allow release of
anions, such as chloride and malate from
guard cells, which is needed for stomatal
closing. (b) Anion channels are activated
by signals that cause stomatal closing,
for example by intracellular calcium and
ABA.[26][29][32] 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.[25][26][28][33]
Vacuolar ion transport
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
cell’s volume. Therefore, a majority of
ions are released from vacuoles when
stomata are closed.[34] Vascuolar K+ (VK)
channels and fast vacuolar channels can
mediate K+ release from
vacuoles.[35][36][37] Vacuolar K+ (VK)
channels are activated by elevation in the
intracellular calcium concentration.[35]
Another type of calcium-activated
channel, is the slow vacuolar (SV)
channel.[38] SV channels have been
shown to function as cation channels
that are permeable to Ca2+ ions,[35] but
their exact functions are not yet known in
plants.[39]

Guard cells control gas exchange and ion

exchange through opening and closing.
K+ is one ion that flows both into and out
of the cell, causing a positive charge to
develop. Malate is one of the main
anions used to counteract this positive
charge, and it is moved through the
AtALMT6 ion channel.[40] AtALMT6 is an
aluminum-activated malate transporter
that is found in guard cells, specifically in
the vacuoles. This transport channel was
found to cause either an influx or efflux
of malate depending on the
concentrations of calcium.[40] In a study
by Meyer et al, patch-clamp experiments
were conducted on mesophyll vacuoles
from arabidopsis rdr6-11 (WT) and
arabidopsis that were overexpressing
AtALMT6-GFP.[40] It was found from
these experiments that in the WT there
were only small currents when calcium
ions were introduced, while in the
AtALMT6-GFP mutant a huge inward
rectifying current was observed.[40] When
the transporter is knocked out from
guard cell vacuoles there is a significant
reduction in malate flow current. The
current goes from a huge inward current
to not much different than the WT, and
Meyer et al hypothesized that this is due
to residual malate concentrations in the
vacuole.[40] There is also a similar
response in the knockout mutants to
drought as in the WT. There was no
phenotypic difference observed between
the knockout mutants, the wild type, or
the AtALMT6-GFP mutants, and the exact
cause for this is not fully known. [40]

Signal transduction
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. Using
Arabidopsis thaliana, the investigation of
signal processing in single guard cells
has become open to the power of
genetics.[29] Cytosolic and nuclear
proteins and chemical messengers that
function in stomatal movements have
been identified that mediate the
transduction of environmental signals
thus controlling CO2 intake into plants
and plant water loss.[1][2][3][4] Research on
guard cell signal transduction
mechanisms is producing an
understanding of how plants can improve
their response to drought stress by
reducing plant water loss.[1][41][42] Guard
cells also provide an excellent model for
basic studies on how a cell integrates
numerous kinds of input signals to
produce a response (stomatal opening or
closing). These responses require
coordination of numerous cell biological
processes in guard cells, including signal
reception, ion channel and pump
regulation, membrane trafficking,
transcription, cytoskeletal
rearrangements and more. A challenge
for future research is to assign the
functions of some of the identified
proteins to these diverse cell biological
processes.

Development
During the development of plant leaves,
the specialized guard cells differentiate
from “guard mother cells”.[43][44] The
density of the stomatal pores in leaves is
regulated by environmental signals,
including increasing atmospheric CO2
concentration, which reduces the density
of stomatal pores in the surface of
leaves in many plant species by presently
unknown mechanisms. The genetics of
stomatal development can be directly
studied by imaging of the leaf epidermis
using a microscope. Several major
control proteins that function in a
pathway mediating the development of
guard cells and the stomatal pores have
been identified.[43][44]

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