The science of the stomata of plants: a continuously growing list of references, abstracts and illustrations, helping researchers to data on publications.
Plants have developed strategies to circumvent limitations in water supply through the adjustment of stomatal aperture in relation to the photosynthetic capacity (water-use efficiency). The CO2 sensor of guard cells, reporting on the metabolic status of the photosynthetic tissue, is, however, as yet unknown.
We elucidated whether extracellular malate has the capability to serve as a signal metabolite in regulating the membrane properties of guard cells. Patch-clamp studies showed that slight variations in the external malate concentration induced major alterations in the voltage-dependent activity of the guard cell anion channel (GCAC1).
Superfusion of guard cell protoplasts with malate solutions in the physiological range caused the voltage-gate to shift towards hyperpolarized potentials (Km(mal) = 0.4 mM elicits a 38 mV shift). The selectivity sequence of the anion channel NO3- (4.2) > or = I- (3.9) > Br- (1.9) > Cl- (1) >> mal (0.1) indicates that malate is able to permeate GCAC1.
The binding site for shifting the gate is, however, located on the extracellular face of the channel since cytoplasmic malate proved ineffective.
Single-channel analysis indicates that extracellular malate affects the voltage-dependent mean open time rather than the unitary conductance of GCAC1.
In contrast to malate the rise in the extracellular Cl- concentration increases the unit conductance of the anion efflux channel.
We suggest that stomata sense changes in the intercellular CO2 concentration and thus the photosynthetic activity of the mesophyll via feedback regulation of anion efflux from guard cells through malate-sensitive GCAC1.
Stomatal regulation of plant carbon uptake and water loss under changing environmental conditions was a crucial evolutionary step in the colonization of land by plants. There are currently two conflicting models describing the nature of stomatal regulation across terrestrial vascular plants: the first is characterized by a fundamental mechanistic similarity across all lineages, and the second is characterized by the evolution of major differences in angiosperms compared with more ancient lineages. Specifically, the second model posits that stomata of ferns lack a response to elevated atmospheric CO2 concentration (ca) and therefore cannot regulate leaf intercellular CO2 concentration (ci).
We compared stomatal sensitivity to changes in ca in three distantly related fern species and a representative angiosperm species.
Fern and angiosperm stomata responded strongly and similarly to changes in ca. As a result, ci/ca was maintained within narrow limits during ca changes.
Our results challenge the model in which stomata of ferns generally lack a response to elevated ca and that angiosperms evolved new dynamic mechanisms for regulating leaf gas exchange that differ fundamentally from ferns. Instead, the results are consistent with a universal stomatal control mechanism that is fundamentally conserved across ferns and angiosperms, and therefore likely all vascular plant divisions.
As one of the first land plant groups to diversify, mosses are central in understanding the origin, diversification, and early function of stomata. Unlike tracheophytes that have stomata on anatomically complex leaves and stems, mosses bear stomata exclusively on spore-bearing organs (capsules). However, stomata do not occur in all mosses and, indeed, are absence in the earliest-divergent mosses (Takakia, Andreaea, Andreaeobryum and Sphagnum), suggesting that stomata originated in mosses independently of other plants. The occurrence of structurally unique pseudostomata in Sphagnum further confounds the resolution of homology of moss stomata with those of other plants. The five studies included in this dissertation are aimed at clarifying the structure, development and evolution of moss stomata. The first study focuses on the sporophyte anatomy and stomatal ultrastructure in two structurally and phylogenetically divergent mosses, Oedipodium and Ephemerum. Oedipodium is the sister to peristomate mosses and the first extant moss with true stomata. This monospecific genus has an elaborated capsule with an extended apophysis bearing numerous long-pored stomata. In contrast, Ephemerum nests within the peristomate mosses and has a reduced capsule that lacks an apophysis and has a few round-pored stomata. Ultrastructure of stomata is similar in these two mosses and comparable to that of tracheophytes, except that the stomata of mosses are not as structurally distinct from epidermal cells as are tracheophyte stomata. Anatomical features such as the presence of a cuticle, water-conducting cells, and spongy tissues with large areas for gas exchange are more pronounced in Oedipodium sporophytes and support the role of stomata in gas exchange and water transport during development and maturation. The second study examines changes in pectin composition during development in the model moss Funaria. Stomatal movement in tracheophytes requires guard cell walls to be strong, yet flexible, because they have to undergo reversible deformation to open and close the pore. Pectins are necessary for wall flexibility and proper stomatal functioning in seed plants. In this study of Funaria, immunogold-labeling using five antibodies to pectin epitopes was conducted on guard cell walls during development to relate these features to the limited movement of stomata in moss. Movement of Funaria stomata coincides with capsule expansion when guard cell walls are thin and pectinaceous. Walls dramatically increase in thickness after pore formation and the pectin content significantly decreases in mature guard cell walls, suggesting that a decrease in flexibility is responsible for the inability to open a close previously reported in older moss guard cells. Because this was the first study to demonstrate changes in pectin composition during stomatal development in any plant, a similar study was done on Arabidopsis to identify the main types of pectins in guard cell walls. Localization of pectins in guard cell walls of Arabidopsis is similar to mosses in the stage they can move, with homogeneous walls rich in arabinan pectins that are required for wall flexibility. This study extends knowledge of pectin composition from stomata of the moss Funaria with limited stomatal movement to an angiosperm in which stomatal activity is crucial to the physiological health of the plant. The fourth study describes stomata development and internal changes in sporophyte anatomy that lead to formation of air spaces in the moss Funaria. Developing sporophytes at different stages were examined using light, fluorescence and electron microscopy; immunogold-labeling was used to investigate the presence of pectin in the newly formed cavities. Stomata in mosses do not develop from a self-generating meristemoid like in Arabidopsis, but instead they originate from a protodermal cell that differentiates directly into a guard mother cell. Epidermal cells develop from protodermal or other epidermal cells, i.e., there are no stomatal lineage ground cells. This developmental pattern is congruent with the presence of a gene ortholog of FAMA, but not SPCH and MUTE, in Physcomitrella. The final study in this dissertation focuses on the enigmatic Sphagnum. Although true stomata are absent in early-divergent mosses, Sphagnum has specialized epidermal cells, pseudostomata, that partially separate but do not open to the inside. To further understand the structure, function and evolution of pseudostomata, capsule anatomy and ultrastructure of pseudostomata were detailed. As in moss stomata, pseudostomata wall architecture and behavior facilitate capsule dehydration, shape change, and dehiscence, supporting this common function. Unlike other moss stomata, pseudostomata collapse along their ventral walls and they lack a substomatal cavity. Similarities to true stomata include two modified epidermal cells with specialized cell walls that separate by cuticle deposition and respond to drying. Pseudostomata may be interpreted as modified stomata that suppressed substomatal cavity formation, which in turn eliminated pore development. However, clarification of the homology of pseudostomata and moss stomata will require genomic studies integrated with physiological and structural data. The studies described in this dissertation significantly advance our understanding of moss stomatal development and structure, and provide a comparison point to better evaluate the evolution of stomata. Moss capsule anatomy coupled with the exclusive existence of stomata on capsules supports the concept that stomata in moss are involve in gas exchange but also facilitate drying and dispersal of spores. Changes in wall architecture coupled with a decrease in total pectin explain the inability of mature stomata to move. Development and distribution of stomata in Funaria provides evidence of a direct and less elaborated mechanism for stomatal development than described in Arabidopsis. Resolving relationships among early land plants, especially hornworts and mosses, the only bryophyte groups with stomata, is critical to understanding stomata evolution. Evaluated together, the results of this dissertation are consistent with a single origin of stomata in land plants.
Using the patch-clamp technique we discovered that the voltage dependent anion channels in the plasma membrane of guard cells are activated by a rise in cytoplasmic Ca2+ in the presence of nucleotides.
Upon activation, these anion channels catalyse anion currents 10-20 times higher than in the inactivated state, thus shifting the plasma membrane from a K+ conducting state to an anion conducting state.
Prolonged stimulation by depolarizing voltages results in the inactivation of the anion current (t1/2 = 10-12 s).
We suggest that activation of the anion channel by Ca2+ and nucleotides is a key event in the regulation of salt efflux from guard cells during stomatal closure.
Ultraviolet B (UV-B) radiation is an important environmental signal for plant growth and development, but its signal transduction mechanism is unclear. UV-B is known to induce stomatal closure via hydrogen peroxide (H2O2), and to affect ethylene biosynthesis.
As ethylene is also known to induce stomatal closure via H2O2 generation, the possibility of UV-B-induced stomatal closure via ethylene-mediated H2O2 generation was investigated in Vicia faba by epidermal strip bioassay, laser-scanning confocal microscopy, and assays of ethylene production.
It was found that H2O2 production in guard cells and subsequent stomatal closure induced by UV-B radiation were inhibited by interfering with ethylene biosynthesis as well as ethylene signalling, suggesting that ethylene is epistatic to UV-B radiation in stomatal movement.
Ethylene production preceded H2O2production upon UV-B radiation, while exogenous ethylene induced H2O2production in guard cells and subsequent stomatal closure, further supporting the conclusion. Inhibitors for peroxidase but not for NADPH oxidase abolished H2O2 production upon UV-B radiation in guard cells, suggesting that peroxidase is the source of UV-B-induced H2O2production.
Taken together, our results strongly support the idea that ethylene mediates UV-B-induced stomatal closure via peroxidase-dependent H2O2 generation.
Previous studies have showed that UV-B can stimulate closure as well as opening of stomata. However, the mechanism of this complex effect of UV-B is not clear.
The purpose of this paper is to investigate the role and the interrelationship of H2O2 and NO in UV-B-induced stomatal closure in broad bean (Vicia fabaL.).
By epidermal strip bioassay and laser-scanning confocal microscopy, we observed that UV-B-induced stomatal closure could be largely prevented not only by NO scavenger c-PTIO or NO synthase (NOS) inhibitor l-NAME, but also by ascorbic acid (ASC, an important reducing substrate for H2O2 removal) or catalase (CAT, the H2O2 scavenger), and that UV-B-induced NO and H2O2 production in guard cells preceded UV-B-induced stomatal closure.
These results indicate that UV-B radiation induces stomatal closure by promoting NO and H2O2 production. In addition, c-PTIO, l-NAME, ASC and CAT treatments could effectively inhibit not only UV-B-induced NO production, but also UV-B-induced H2O2 production. Exogenous H2O2-induced NO production and stomatal closure were partly abolished by c-PTIO and l-NAME.
Similarly, exogenous NO donor sodium nitroprusside-induced H2O2 production and stomatal closure were also partly reversed by ASC and CAT.
These results show a causal and interdependent relationship between NO and H2O2 during UV-B-regulated stomatal movement. Furthermore, the l-NAME data also indicate that the NO in guard cells of Vicia faba is probably produced by a NOS-like enzyme.
Abscisic acid (ABA) is taken up by guard cells of isolated epidermata of Valerianella locustaonly at low external pH values. At pH 8.0, when nearly all ABA molecules are present as the union of ABA (ABA−), no uptake can be observed.
ABA-dependent movement of stomata was tested at external pH values between 5.0 and 8.0. Independent of the external pH, ABA induced stomatal closure at all tested ABA concentrations.
It is concluded that ABA need not be taken up into the cytosol of the guard cells in order to induce stomatal closure.
The primary site of ABA action at the guard cell plasmalemma must be located either at the outer surface of the plasmalemma or at least be easily accessible from outside. ABA− is as effective as undissociated ABA (ABAH).
We recently established an immunohistochemical method for the detection of blue light (BL)-induced and phototropin-mediated phosphorylation of plasma-membrane H+-ATPase in stomatal guard cells of Arabidopsis thaliana.
This technique makes it possible to detect the phosphorylation/activation status of guard-cell H+-ATPase in the epidermis of a single rosette leaf, without the need to prepare guard-cell protoplasts (GCPs) from a large number of plants. Moreover, it can detect guard-cell responses under more natural and stress-free conditions compared to using GCPs.
Taking advantage of these properties, we examined the effect of abscisic acid (ABA) on BL-induced phosphorylation of guard-cell H+-ATPase by using ABA-insensitive mutants. This revealed inhibition of BL-induced phosphorylation of guard-cell H+-ATPase via the early ABA-signaling components PYR/PYL/RCAR-PP2Cs-SnRK2s, which are known to be early ABA-signaling components for a wide range of ABA responses in plants.
Blue light (BL) receptor phototropins activate the plasma membrane H+-ATPase in guard cells through phosphorylation of a penultimate threonine and subsequent binding of the 14-3-3 protein to the phosphorylated C-terminus of H+-ATPase, mediating stomatal opening.
To date, detection of the phosphorylation level of the guard cell H+-ATPase has been performed biochemically using guard cell protoplasts (GCPs). However, preparation of GCPs from Arabidopsis for this purpose requires >5,000 rosette leaves and takes >8 h.
Here, we show that BL-induced phosphorylation of guard cell H+-ATPase is detected in the epidermis from a single Arabidopsis rosette leaf via an immunohistochemical method using a specific antibody against the phosphorylated penultimate threonine of H+-ATPase.
BL-induced phosphorylation of the H+-ATPase was detected immunohistochemically in the wild type, but not in a phot1-5 phot2-1 double mutant.
Moreover, we found that physiological concentrations of the phytohormone ABA completely inhibited BL-induced phosphorylation of guard cell H+-ATPase in the epidermis, and that inhibition by ABA in the epidermis is more sensitive than in GCPs.
These results indicate that this immunohistochemical method is very useful for detecting the phosphorylation status of guard cell H+-ATPase. Thus, we applied this technique to ABA-insensitive mutants (abi1-1, abi2-1 andost1-2) and found that ABA had no effect on BL-induced phosphorylation in these mutants.
These results indicate that inhibition of BL-induced phosphorylation of guard cell H+-ATPase by ABA is regulated by ABI1, ABI2 and OST1, which are known to be early ABA signaling components for a wide range of ABA responses in plants.
PLANT STOMATA ENCYCLOPEDIA 
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