1. Stomata and guard cell metabolism
Nurulhikma Md isa

2. Stomata and guard cell metabolism
Why plants do need stomata?
Stomata of a plant are an essential structure and are vital for photosynthesis
The word stoma means ‘mouth’ in Greek because it allows communication between the internal and external environments of the plant” 
Stomata consist of two specialized cells, also known as guard cells that surround a tiny pore called the stoma  

3. Stomata and guard cell metabolism
What is the main function of stomata?
The main function of the stomata is to allow gases- mainly carbon dioxide, water vapor and oxygen more rapidly in and out of the leaf of plants.
Typically, the plant epidermis is tightly sealed by wax-coated, interlocking epidermal pavement cells which protect the plant body from the dry atmosphere and UV-rays.
At the same time plants must be able to breathe, or exchange carbon dioxide and oxygen, for photosynthesis and respiration.
Stomata act as a gateway for efficient gas exchange and water movement from the roots through the vasculature to the atmosphere.

4. Stomata and guard cell metabolism
Stomata are found on all above ground parts of plants;
including the petals of flowers, petioles, stems and leaves. Stomata’s are formed during the initial stages of development of the plant;
therefore they reflect the environment and conditions in which they grow.
The stomata supplies things such as water and minerals to the entire plant system during transpiration.
However, there are times where some plants encounter conditions such as drought; at this time a plant hormone called abscisic acid alerts the stomata to shut tightly in order to prevent plants from dehydrating and wilting. The stomata are crucial for a plants survival.

5. Stomata and guard cell metabolism
There are multiple parts and functions of the stomata that work during the process of photosynthesis. The specialized cells in the stomata of plants are called guard cells; the most important part to the stomata.
Guard cells control opening and closing of the pores in the response of the environment.
They are undersurface of leaves for controlling gas exchange and water loss of the plant.
Guard cells are in pairs and shaped like a kidney bean so that stoma can exist between them. During warm weather, when a plant is likely to lose excessive water the guard cells close eliminating as much water evaporation from the interior of the leaf.

6. Stomata and guard cell metabolism
Stomatal openings are modulated by what is known as a “potassium pump
Potassium ions (K+) contained in the guard cells influence their osmotic properties.
As the K+ concentration increases, the cell osmotic potential drops. This pulls the water into the guard cells, opening the stomata.”

7. Stomata and guard cell metabolism
In figure A the guard cells are turgid, or swollen, and the stomatal opening is large.
This turgidity is caused by the accumulation of K+ (potassium ions) in the guard cells.
As K+ levels increase in the guard cells, the water potential of the guard cells drops and water enters the guard cells.

8. Stomata and guard cell metabolism
In figure B, the guard cells have lost water, which causes the cells to become flaccid and the stomatal opening to close.
This may occur when the plant has lost an excessive amount of water.
In addition, it generally occurs daily as light levels drop and the use of CO2 in photosynthesis decreases.

9. Stomata and guard cell metabolism
Photosynthesis is essential to the plant;
stomata’s are critical to the process of photosynthesis.
Without the stomata gasses such as carbon dioxide and oxygen could not get into the plant to make the nutrients and food the plant needs to live or the energy for the plant to grow.
But without guard cells the stomata would not be able to open and close to bring in what the plant needs and keep water in.
The guard cells need to work for the stomata and the stomata needs to provide a way in and out for gasses for photosynthesis to happen

10. Stomata and guard cell metabolism
Plant Vacuoles and the regulation of stomatal opening

11. Plant Vacuoles and the regulation of stomatal opening
Figure: Vacuoles in plant cells
Vacuolar proteins are synthesized and processed in the endoplasmic reticulum (ER)
transferred to vacuoles through various routes, for example, directly, via Golgi apparatus (G), or via prevacuolar compartment (PVC).
© 2004 Nature Publishing Group Brandizzi, F. & Hawes, C. A long and winding road. EMBO reports 5, 245–249

12. Plant Vacuoles and the regulation of stomatal opening
Figure: Vacuoles in plant cells
Tiny provacuoles form from budding and fusion of vesicles originating from the trans Golgi network.
With progression of cell expansion, provacuoles gradually fuse with each other and form a prominently larger vacuole.
There are two types of vacuoles, protein storage vacuoles with neutral pH and the lytic vacuoles of acidic pH.
© 2004 Nature Publishing Group Brandizzi, F. & Hawes, C. A long and winding road. EMBO reports 5, 245–249

13. Plant Vacuoles and the regulation of stomatal opening
How Do Vacuoles Change During Stomatal Opening and Closing?
One way to track dynamic changes in guard cell vacuoles during stomatal movements is to use cell imaging techniques, such as confocal microscopy and TEM.
In 2005, Gao et al. did this when they studied leaf epidermis from the plant Vicia faba using microscopy coupled with fluorescent dyes.
First, they removed strips of epidermal cells from leaves, then they stained guard cells with various fluorescent dyes.
They used two dyes that specifically attach to vacuoles due to their acidic pH. These dyes cause the vacuoles to glow fluorescent green or red. With the use of these compartment-specific dyes, they were able to observe the size, shape, and number of vacuoles at various time points during stomatal movements.

14. Plant Vacuoles and the regulation of stomatal opening
How Do Vacuoles Change During Stomatal Opening and Closing?
In their experiment, Gao et al. asked, what happens to the vacuoles and the cytoplasm during stomatal opening and closing?
They controlled stomatal action experimentally with known agents.
They induced opening with halogen cold-light, and closing with chemical abscisic acid (ABA).
During these inductions, they observed that, in the closed state, guard cells contain many small vacuoles, but during stomatal opening, these small vacuoles readily fuse with each other, or with bigger vacuoles.

15. Plant Vacuoles and the regulation of stomatal opening
How Do Vacuoles Change During Stomatal Opening and Closing?
The result is very large vacuoles in guard cells surrounding an open stoma.
Conversely, in closing stomata, the large vacuoles once again split into smaller ones, and generate many complex membrane structures.
Though these scientists observed a visual coincidence of vacuole changes and stomatal movements, are these dynamic changes necessary for stomatal movements to occur?

16. Plant Vacuoles and the regulation of stomatal opening
Does Stomatal Opening Require Vacuolar Fusion?
To test whether vacuole dynamics are necessary, Gao et al. asked, what would happen to stomatal movements if they experimentally disrupt vacuolar fusion?
To investigate this problem, they again turned to their test system, leaf epidermal peels.
They treated these peels with a membrane-permeable compound known to inhibit the fusion of endosomes with vacuoles, called E-64d ((2s,3s)-trans-epoxy-succinyl-L-leucylamido-3-methylbutane ethyl ester), and found that the treated guard cells had a greater number of vacuoles than untreated control guard cells.

17. Plant Vacuoles and the regulation of stomatal opening
Does Stomatal Opening Require Vacuolar Fusion?
They also observed that stomatal opening was slower in treated guard cells compared to the untreated controls.
To explain this, they concluded that interrupted vacuolar fusion has an effect of slowing stomatal opening, and therefore vacuolar fusion must be necessary for stomatal opening to properly function.

18. Plant Vacuoles and the regulation of stomatal opening
To explore the genetic basis for vacuolar dynamics, Gao et al. followed up this initial conclusion in Vicia faba with additional experiments using mutant plants.
Genetic manipulation in the plant Arabidopsis can produce a mutant that is defective in producing a protein named SGR3.
Previous work by other scientists established that SGR3 impacts the transport of vesicles into vacuoles and vacuolar fusion.

19. Plant Vacuoles and the regulation of stomatal opening
When Gao et al. compared SGR3 mutants to normal (wild type) plants, they found slower stomatal opening in response to light induction in the mutant plants.
With their knowledge of SGR3 function, and these observations, they again concluded that impaired stomatal movement was a consequence of reduced vacuolar fusion in guard cells.
All together, their results show that fusion of vacuoles is necessary for normal, rapid stomatal movements.

20. QUESTION!?
1) Why the guard cells regulate the opening and closing of stomata?
2) Why do plants spend energy on opening and closing these stomata, when they could leave them constantly open, and let CO2 flow freely?
3) What is the special feature of guard cells that allow the stomata movement (stomata opening and closing)?
4) How do guard cells change their volume to control this opening and closing?

21. QUESTION!?
Why the guard cells regulate the opening and closing of stomata?
Guard cells regulate this opening and closing in response to a wide variety of environmental signals, such as day/night rhythms, CO2 availability, and temperature
Why do plants spend energy on opening and closing these stomata, when they could leave them constantly open, and let CO2 flow freely?
The reason is that stomata also regulate the passage of water molecules. If the stomata were constantly open, plants would lose too much water via evaporation from the leaf surface, a process called transpiration.
What is the special feature of guard cells that allow the stomata movement (stomata opening and closing)?
A special feature of guard cells is that they can increase or decrease their volume, thereby changing their shape. This is the basis for the opening and closing of a stoma, known as stomatal movement, which controls gas exchange necessary for photosynthesis and limits water loss.
How do guard cells change their volume to control this opening and closing?
They do so by changing the osmotic pressure of their vacuoles, which then either take up or lose water, and consequently enlarge or shrink. Such changes in vacuolar volume are quite rapid and dramatic. This can be problematic because, unlike a quickly expanding balloon, biological membranes are more limited in their elasticity and do not allow over-stretching.

22. Guard Cell Signal Transduction Network: Advances in Understanding Abscisic Acid, CO2, and Ca2+ Signaling during stomata movement

23. Guard Cell Signal Transduction
Plants need to assimilate (incorporate) CO2 for photosynthesis while simultaneously preventing excessive loss of water.
Because the plant cuticle is impermeable to both water and CO2, transpirational water loss and CO2 influx in plants are tightly regulated by the opening and closing of stomatal pores in aerial tissues.
The transport of ions and water through channel proteins across the plasma and vacuolar membranes changes turgor and guard cell volume, thereby regulating stomatal aperture.
Guard cells continuously sense information from the leaf environment, including abiotic and biotic stimuli, as well as long-distance signals from roots.
Guard cells integrate all of these signals and convert them into appropriate turgor pressure changes.

24. Guard Cell Signal Transduction
Several important environmental factors induce stomatal opening in C3 and C4 plants, including blue and red light.
Stomates also open in response to high humidity and low CO2 in order to maintain CO2 intake.
Stomatal closure, on the other hand, is promoted by darkness in C3 and C4 plants.
In order to preserve water, CAM-plants do not close their stomates in response to darkness.
Instead, they accumulate CO2 during the nighttime by converting it into organic molecules such as malate.
Elevated CO2 leads to stomatal closure because less opening is required for efficient CO2 influx.
Stomata are also closed in response to drought, as well as elevated ozone, thus protecting the inside of leaves from ozone-induced oxidative damage to plants.

25. Guard Cell Signal Transduction
Drought causes production of the plant hormone:
abscisic acid (ABA),
which promotes stomatal closure and thereby reduces transpirational water loss.
Other plant hormones, including auxin, cytokinin, ethylene, brassinosteroids, jasmonates, and salicylic acid (in response to pathogenic bacteria), can have effects on stomatal function.

26. ION CHANNELS IN GUARD CELLS: An Overview of Guard Cell Ion Channels and Their Functions

27. ION CHANNELS IN GUARD CELLS: An Overview of Guard Cell Ion Channels and Their Functions
STOMATAL CLOSING
When guard cells perceive increased ABA levels,
their turgor and volume are REDUCED!!
BUT HOW!!??
by efflux of anions and potassium ions and by gluconeogenic conversion of malate into starch,
This causing stomatal closure.
ABA triggers cytosolic [Ca2+]cyt increases and enhances [Ca2+]cyt sensitivity, which activates two different types of anion channels:

28. ION CHANNELS IN GUARD CELLS: An Overview of Guard Cell Ion Channels and Their Functions
STOMATAL CLOSING
1. Slow-activating sustained (S-type) and
2. rapid-transient (R-type) anion channels
S-type anion channels generate slow and sustained anion efflux,
R-type anion channels are activated transiently within 50 ms,
suggesting that two different types of anion channels provide unique mechanisms for anion effluxe.

29. ION CHANNELS IN GUARD CELLS: An Overview of Guard Cell Ion Channels and Their Functions
Activation of anion channels at the plasma membrane of guard cells has been regarded as a critical step in stomatal closure.
Anion efflux via anion channels causes:
- membrane depolarization, which subsequently drives K+ efflux from guard cells through outward K+ out channels.
Among the solutes released from guard cells, more than 90% originate from vacuoles. [Ca2+]cyt-activated vacuolar K+ (VK) channels function in vacuolar K+ release.

30. ION CHANNELS IN GUARD CELLS: An Overview of Guard Cell Ion Channels and Their Functions
STOMATAL OPENING
Stomatal opening requires the activation of H+-ATPases in the plasma membrane of guard cells.
Membrane hyperpolarization caused by H+- ATPases, induces K+ uptake through inward K+ in channels.
Influx of K+ and production of malate from osmotically inactive starch increases turgor and volume in the guard cell and induces stomatal opening.
In guard cells, K+ is accumulated in vacuoles by H+/K+ antiporter activities, and anions can be transported into vacuoles through both low-affinity anion channels and a H+/anion exchange mechanism.
ABA inhibits stomatal opening through downregulation of K+ in channels and H+-ATPases.

31. ION CHANNELS IN GUARD CELLS: An Overview of Guard Cell Ion Channels and Their Functions
Updates on Ion Channels and Regulation during Stomatal Closure
Early cell signaling, and genetic studies suggested that S-type anion channels play a key role in stimulus-induced stomatal closure.
A gene encoding the anion-conducting subunit of S-type anion channels has recently been identified.
SLAC1 (SLOW ANION CHANNELASSOCIATED 1) was genetically isolated from independent mutant screens for stomatal closure mutants.
The SLAC1/SLAH (SLAC1 HOMOLOGUE) gene family encodes proteins with 10 predicted transmembrane domains, with similarity to bacterial and fungal dicarboxylate/malate transporters

32. ION CHANNELS IN GUARD CELLS: An Overview of Guard Cell Ion Channels and Their Functions
slac1 mutants exhibit reduced stomatal closure responses to ABA
In addition, Ca2+-and ABA-activation of S-type anion channels are impaired in slac1 guard cells, providing genetic evidence that SLAC1 encodes a major anion-transporting component of S-type anion channels in guard cells.
Furthermore, retention of R-type anion channel activities in slac1 provides genetic support for the model that two types of anion channels are present in guard cells.
slac1 mutant reduced stomatal closure: means SLAC1 function is needed for stomatal closing in response to ABA

33. CO2 SIGNALING IN GUARD CELLS
Elevated [CO2] activates anion channels and K+ out efflux channels in Vicia faba guard cells
CO2 triggers chloride release from guard cells and depolarization in intact leaves.
Ca2+ is required for CO2-induced stomatal closure and high CO2
Recently, mutant screening and functional characterizations in Arabidopsis have led to identification of plant mutants and genes that mediate CO2 control of stomatal movements.
The ABA-insensitive mutant gca2 (growth controlled by abscisic acid 2) is strongly impaired in CO2-induced stomatal closure in response to elevated CO2 (800 ppm) both in leaf epidermis and in intact leaves of plants
gca2, ABA insensitive mutant, means do not have any response to ABA (endogenous/exogenous)

34. CO2 SIGNALING IN GUARD CELLS
Together with previous research, showing that gca2 mutant plants are impaired in ABA-induced stomatal closure,
GCA2 likely functions downstream of the convergence point of CO2 and ABA signaling transduction networks.
SLAC1 protein is a positive mediator of the CO2-induced stomatal closure signaling pathway.
The HT1 (HIGH LEAF TEMPERATURE 1) protein kinase is the first identified molecular component that functions as a major negative regulator in the high CO2-induced stomatal closure pathway
Gca2 mutant impaired in ABA induced stomatal closure, impaired in CO2 induced stomatal closure. Thus it is assumed to act downstream of ABA and CO2

35. CO2 SIGNALING IN GUARD CELLS
A plasma membrane ABC malate uptake transporter, AtABCB14 (ABC TRANSPORTER B FAMILY MEMBER 14), in guard cells was identified as another negative regulator of CO2- induced stomatal closure.
CO2-induced stomatal closure in detached leaves was slightly accelerated in atabcb14 mutants and decreased in AtABCB14 overexpressing plants,
suggesting that malate uptake into guard cells by AtABCB14 plays a role in the CO2- induced regulation of stomatal closure

36. GUARD CELL ABA SIGNAL TRANSDUCTION

37. ABA SIGNAL TRANSDUCTION

38. GUARD CELL ABA SIGNAL TRANSDUCTION
The ABA-activated protein kinase OST1 and the V. faba homolog, AAPK (abscisic acid activated protein kinase), function as positive regulators of ABA-induced stomatal closure.
Interestingly, ABI1 (PP2C) interacts with OST1 in vitro and negatively regulates ABA-activated OST1 kinase activity.
Recent research has shown that the ABI1 protein phosphatase co-immunoprecipitates with the SnRK2.2 and SnRK2.3 protein kinases in Arabidopsis and that the ABI1/ABI2/HAB1 PP2Cs interact with the OST1 and SnRK2.3 protein kinases, confirming in vivo interactions between ABI1 and SnRK2s.