Regulation Of Stomatal Opening Research Articles

MPK12 in stomatal CO2 signaling: function beyond its kinase activity.

Protein phosphorylation is a major molecular switch involved in the regulation of stomatal opening and closure. Previous research defined interaction between MAP kinase 12 and Raf-like kinase HT1 as a required step for stomatal movements caused by changes in CO2 concentration. However, whether MPK12 kinase activity is required for regulation of CO2 -induced stomatal responses warrants in-depth investigation. We apply genetic, biochemical, and structural modeling approaches to examining the noncatalytic role of MPK12 in guard cell CO2 signaling that relies on allosteric inhibition of HT1. We show that CO2 /HCO3 - -enhanced MPK12 interaction with HT1 is independent of its kinase activity. By analyzing gas exchange of plant lines expressing various kinase-dead and constitutively active versions of MPK12 in a plant line where MPK12 is deleted, we confirmed that CO2 -dependent stomatal responses rely on MPK12's ability to bind to HT1, but not its kinase activity. We also demonstrate that purified MPK12 and HT1 proteins form a heterodimer in the presence of CO2 /HCO3 - and present structural modeling that explains the MPK12:HT1 interaction interface. These data add to the model that MPK12 kinase-activity-independent interaction with HT1 functions as a molecular switch by which guard cells sense changes in atmospheric CO2 concentration.

PtrVCS2 Regulates Drought Resistance by Changing Vessel Morphology and Stomatal Closure in Populus trichocarpa.

Drought has severe effects on plant growth, forest productivity, and survival throughout the world. Understanding the molecular regulation of drought resistance in forest trees can enable effective strategic engineering of novel drought-resistant genotypes of tree species. In this study, we identified a gene, PtrVCS2, encoding a zinc finger (ZF) protein of the ZF-homeodomain transcription factor in Populus trichocarpa (Black Cottonwood) Torr. & A. Gray. ex Hook. Overexpression of PtrVCS2 (OE-PtrVCS2) in P. trichocarpa resulted in reduced growth, a higher proportion of smaller stem vessels, and strong drought-resistance phenotypes. Stomatal movement experiments revealed that the OE-PtrVCS2 transgenics showed lower stomata apertures than wild-type plants under drought conditions. RNA-seq analysis of the OE-PtrVCS2 transgenics showed that PtrVCS2 regulates the expression of multiple genes involved in regulation of stomatal opening and closing, particularly the PtrSULTR3;1-1 gene, and several genes related to cell wall biosynthesis, such as PtrFLA11-12 and PtrPR3-3. Moreover, we found that the water use efficiency of the OE-PtrVCS2 transgenic plants was consistently higher than that of wild type plants when subjected to chronic drought stress. Taken together, our results suggest that PtrVCS2 plays a positive role in improving drought adaptability and resistance in P. trichocarpa.

Functional diversity of RING E3 ligases of major cereal crops in response to abiotic stresses

Abiotic stresses significantly reduce the grain yield and productivity of cereal crops, especially rice, and this may affect food security in the future. Different abiotic stress adaptation pathways have been investigated and depicted in plants. Among these, the ubiquitin-proteasome system (UPS) has been studied as a key mechanism to understand the protein regulation pathways that enhance the adaptation and survival of plants under various environmental stresses such as drought, salinity, cold, and toxic metalloid exposure. RING E3 ligases constitute a highly diverse and important enzyme group that acts within the 26S UPS, and it also plays a crucial role as a central regulator of plant physiological and cellular processes. This review aimed to highlight recent findings and discoveries regarding the different stress-induced RING E3 ligase genes of major cereal crops and their functions via ubiquitination pathways under different environmental stress conditions. Such genes regulate different physiological responses including protein stabilization, cell membrane integrity, regulation of stomatal opening, and the maintenance of meristematic cells, and they also regulate reactive oxygen species and heavy metal levels via ubiquitination in plants. Hence, the ubiquitination process is considered a potential target for the development of abiotic stress-tolerant crops, and it might be used as an excellent mechanism for stress-tolerant crop improvement programs.

Guard cell-specific down-regulation of the sucrose transporter SUT1 leads to improved water use efficiency and reveals the interplay between carbohydrate metabolism and K+ accumulation in the regulation of stomatal opening

Sucrose is a key metabolite in guard cell (GC) regulation and has been proposed to form a key metabolic connection between mesophyll cells and GCs. However, the importance of sucrose transport across the GC plasma membrane in stomatal movements remains to be determined. Here, we characterized transgenic tobacco plants expressing an antisense gene construct for the SUCROSE TRANSPORTER 1 (SUT1) driven by the GC specific promoter KST1. GCs of transgenic plants contained less sucrose, more starch and had increases in K+ content. Lower GC sucrose content was associated with lower stomatal conductance that, in turn, led to a lower transpiration rate and higher carbon isotope composition ratio compared to wild type plants. Although the transgenic plants showed reduced growth under optimal water supply condition, they showed improved growth compared to wild type plants under osmotic stress and demonstrated lower water consumption that was correlated with higher relative water content under water-deficit conditions. This demonstrates the desiccation postponement phenotype of these plants together with an improved tolerance to water deficit. Taken together, our results indicate that SUT1 has an important role in stomatal movements and suggest that genetic manipulation of GC sucrose transport could be used to obtain plants with greater water use efficiency.

Flavonols Induced by 5-Aminolevulinic Acid Are Involved in Regulation of Stomatal Opening in Apple Leaves

Flavonols, the main flavonoids in plant leaves, have newly been proposed as a H2O2 scavenger in guard cells. However, whether 5-aminolevulinic acid (5-ALA) is involved in regulating stomatal movement of apple (Malus × domestica Borkh. ‘Fuji’) leaves through flavonol accumulation remains unclear. In this study, using diphenylboric acid 2-amino ethyl ester (DPBA, a flavonol fluorescent dye) and a laser scanning confocal microscope, we observed that 5-ALA pretreatment significantly improved flavonol accumulation in guard cells of apple leaves, especially around the nucleus. Then we demonstrated that 5-ALA pretreatment inhibited ABA-induced stomatal closure via decreasing reaction oxygen species (ROS) accumulation in guard cells. Pretreatment with quercetin or kaempferol, two main kinds of flavonols in plants, also inhibited ABA-induced stomatal closure by decreasing ROS content in the guard cells. Furthermore, exogenous flavonols could suppress H2O2-induced stomatal closure in apple leaves. Taken together, we conclude that ALA-induced flavonol accumulation in guard cells is involved in the inhibitory effect of ALA on ABA-induced ROS accumulation and stomatal closure in apple leaves.

Evolutionary potential and adaptation of Banksia attenuata (Proteaceae) to climate and fire regime in southwestern Australia, a global biodiversity hotspot.

Substantial climate changes are evident across Australia, with declining rainfall and rising temperature in conjunction with frequent fires. Considerable species loss and range contractions have been predicted; however, our understanding of how genetic variation may promote adaptation in response to climate change remains uncertain. Here we characterized candidate genes associated with rainfall gradients, temperatures, and fire intervals through environmental association analysis. We found that overall population adaptive genetic variation was significantly affected by shortened fire intervals, whereas declining rainfall and rising temperature did not have a detectable influence. Candidate SNPs associated with rainfall and high temperature were diverse, whereas SNPs associated with specific fire intervals were mainly fixed in one allele. Gene annotation further revealed four genes with functions in stress tolerance, the regulation of stomatal opening and closure, energy use, and morphogenesis with adaptation to climate and fire intervals. B. attenuata may tolerate further changes in rainfall and temperature through evolutionary adaptations based on their adaptive genetic variation. However, the capacity to survive future climate change may be compromised by changes in the fire regime.

Function of ABA in Stomatal Defense against Biotic and Drought Stresses.

The plant hormone abscisic acid (ABA) regulates many key processes involved in plant development and adaptation to biotic and abiotic stresses. Under stress conditions, plants synthesize ABA in various organs and initiate defense mechanisms, such as the regulation of stomatal aperture and expression of defense-related genes conferring resistance to environmental stresses. The regulation of stomatal opening and closure is important to pathogen defense and control of transpirational water loss. Recent studies using a combination of approaches, including genetics, physiology, and molecular biology, have contributed considerably to our understanding of ABA signal transduction. A number of proteins associated with ABA signaling and responses—especially ABA receptors—have been identified. ABA signal transduction initiates signal perception by ABA receptors and transfer via downstream proteins, including protein kinases and phosphatases. In the present review, we focus on the function of ABA in stomatal defense against biotic and abiotic stresses, through analysis of each ABA signal component and the relationships of these components in the complex network of interactions. In particular, two ABA signal pathway models in response to biotic and abiotic stress were proposed, from stress signaling to stomatal closure, involving the pyrabactin resistance (PYR)/PYR-like (PYL) or regulatory component of ABA receptor (RCAR) family proteins, 2C-type protein phosphatases, and SnRK2-type protein kinases.

A flowering integrator, SOC1, affects stomatal opening in Arabidopsis thaliana.

Stomatal movements are regulated by multiple environmental signals. Recent investigations indicate that photoperiodic flowering components, such as CRY, GI, CO, FT and TSF, are expressed in guard cells and positively affect stomatal opening in Arabidopsis thaliana. Here we show that SOC1, which encodes a MADS box transcription factor and integrates multiple flowering signals, also exerts a positive effect on stomatal opening. FLC encodes a potent repressor of FT and SOC1, and FRI acts as an activator of FLC. Thus, we examined stomatal phenotypes in FRI-Col, which contains an active FRI allele of accession Sf-2 by introgression. We found higher expression of FLC and lower expression of FT, SOC1 and TSF in guard cells from FRI-Col than in those from Col. Light-induced stomatal opening was significantly suppressed in FRI-Col. Interestingly, vernalization of FRI-Col partially restored light-induced stomatal opening, concomitant with a decrease of FLC and increase of FT, SOC1 and TSF. Furthermore, we observed the constitutive open-stomata phenotype in transgenic plants overexpressing SOC1-GFP (green fluorescent protein) in guard cells (SOC1-GFP overexpressor), and found that light-induced stomatal opening was significantly suppressed in a soc1 knockout mutant. RNA sequencing using epidermis from the SOC1-GFP overexpressor revealed that the expression levels of several genes involved in stomatal opening, such as BLUS1 and the plasma membrane H(+)-ATPases, were higher than those in background plants. From these results, we conclude that SOC1 is involved in the regulation of stomatal opening via transcriptional regulation in guard cells.

Role of H2O2 dynamics in brassinosteroid‐induced stomatal closure and opening in Solanum lycopersicum

Brassinosteroids (BRs) are essential for plant growth and development; however, their roles in the regulation of stomatal opening or closure remain obscure. Here, the mechanism underlying BR-induced stomatal movements is studied. The effects of 24-epibrassinolide (EBR) on the stomatal apertures of tomato (Solanum lycopersicum) were measured by light microscopy using epidermal strips of wild type (WT), the abscisic acid (ABA)-deficient notabilis (not) mutant, and plants silenced for SlBRI1, SlRBOH1 and SlGSH1. EBR induced stomatal opening within an appropriate range of concentrations, whereas high concentrations of EBR induced stomatal closure. EBR-induced stomatal movements were closely related to dynamic changes in H(2)O(2) and redox status in guard cells. The stomata of SlRBOH1-silenced plants showed a significant loss of sensitivity to EBR. However, ABA deficiency abolished EBR-induced stomatal closure but did not affect EBR-induced stomatal opening. Silencing of SlGSH1, the critical gene involved in glutathione biosynthesis, disrupted glutathione redox homeostasis and abolished EBR-induced stomatal opening. The results suggest that transient H(2)O(2) production is essential for poising the cellular redox status of glutathione, which plays an important role in BR-induced stomatal opening. However, a prolonged increase in H(2)O(2) facilitated ABA signalling and stomatal closure.

The Effect of Plant Growth Regulator for Phytoremediation

Phytoremediation is the use of vegetation for in situ treatment of contaminated soils, sediments, and water. It is best applied at sites with shallow contamination of metal pollutants. The term “assisted phytoextraction” usually refers to the process of applying a chemical additive to contaminated soil in order to increase the metal uptake by crop plants. In the process of phytoremediation, applying plant growth regulator can promote phytoextraction effects. This paper reviewed the effect of the plant growth regulator for the biological growth, plant transpiration and heavy metal stress. the increase in metal accumulation in upper parts of plant could be related to both the role of PGRs in the enhancement of plant resistance to stress (as toxic metals) and the increase in transpiration rate, i.e. flux of water-soluble soil components and contaminants by the regulation of stomatal opening.

An α-expansin, VfEXPA1, is involved in regulation of stomatal movement in Vicia faba L.

The wall loosening of guard cells differs from other types of plant cells. However, the regulation of wall loosening during stomatal movement is poorly understood. VfEXPA1 is an α-expansin gene cloned from Vicia faba epidermal strips. Expression of VfEXPA1 is regulated by darkness and submergence, and is not affected by light and abscisic acid (ABA). In situ hybridization showed that VfEXPA1 is expressed primarily in the guard cells. Overexpression of VfEXPA1 in transgenic tobacco accelerated light-induced stomatal opening, and increased both transpiration and photosynthetic rates under favorable growth conditions. Our results indicate the guard cell-expressed expansin VfEXPA1 plays an important role in regulation of stomatal opening.

Regulation of stomatal opening by the guard cell expansin AtEXPA1

Stomatal movement is strictly regulated by various intracellular and extracellular factors in response environmental signals. In our recent study, we found that an Arabidopsis guard cell expressed expansin, AtEXPA1, regulates stomatal opening by altering the structure of the guard cell wall. This addendum proposes a mechanism by which guard cell expansins regulate stomatal movement.

A viviparous mutant of maize exhibiting permanent water stress symptoms

In maize vivipary, the precocious germination of the seed while it is still attached to the ear is a reliable phenotype for the identification of mutants impaired in the biosynthesis or response to abscisic acid (ABA). Here we present the characterization of a new allele of vp10, a gene encoding for a cofactor (MoCo) required for the last step of ABA biosynthesis. The lesion in this gene leads to a reduction in the endogenous ABA level. Embryonic messenger RNAs of the ABA inducible genes glb1, lea3, and rab17 are barely detectable, although their level increases when stimulated by exogenous ABA administration. These findings confirm that the mutant can be ascribed to a defect in ABA biosynthesis. In the absence of water stress, mutant plants grow like wild-type siblings; however when mutant tissues are exposed to air they differ from non-mutant ones by showing a higher rate of water loss, of transpiration and of stomatal conductance. These events are restored to almost normal values by adding exogenous ABA. All these defects are ascribable to an impairment in the regulation of stomatal opening since, in contrast to wild-type, some of the mutant stomata exhibit partially or totally open rims. The defect in ABA biosynthesis is also associated with loss of regulation of the expression of rab17 and rab28, two genes expressed in vegetative tissues under abiotic stress. These genes are constitutively expressed in the mutant plant tissues independently of the water regime applied. Thus this mutant may provide a tool for the study of molecular mechanisms underlying drought-stress responses in crop plants.

Glycine‐rich RNA‐binding protein7 affects abiotic stress responses by regulating stomata opening and closing inArabidopsis thaliana

Despite the fact that glycine-rich RNA-binding proteins (GRPs) have been implicated in the responses of plants to environmental stresses, their physiological functions and mechanisms of action in stress responses remain largely unknown. Here, we assessed the functional roles of GRP7, one of the eight GRP family members in Arabidopsis thaliana, on seed germination, seedling growth, and stress tolerance under high salinity, drought, or cold stress conditions. The transgenic Arabidopsis plants overexpressing GRP7 under the control of the cauliflower mosaic virus 35S promoter displayed retarded germination and poorer seedling growth compared with the wild-type plants and T-DNA insertional mutant lines under high salinity or dehydration stress conditions. By contrast, GRP7 overexpression conferred freezing tolerance in Arabidopsis plants. GRP7 is expressed abundantly in the guard cells, and has been shown to influence the opening and closing of the stomata, in accordance with the prevailing stress conditions. GRP7 is localized to both the nucleus and the cytoplasm, and is involved in the export of mRNAs from the nucleus to the cytoplasm under cold stress conditions. Collectively, these results provide compelling evidence that GRP7 affects the growth and stress tolerance of Arabidopsis plants under high salt and dehydration stress conditions, and also confers freezing tolerance, particularly via the regulation of stomatal opening and closing in the guard cells.

Expression analysis of Arabidopsis vacuolar sorting receptor 3 reveals a putative function in guard cells

Vacuolar sorting receptors (VSRs) are responsible for the proper targeting of soluble cargo proteins to their destination compartments. The Arabidopsis genome encodes seven VSRs. In this work, the spatio-temporal expression of one of the members of this gene family, AtVSR3, was determined by RT-PCR and promoter::reporter gene fusions. AtVSR3 was expressed specifically in guard cells. Consequently, a reverse genetics approach was taken to determine the function of AtVSR3 by using RNA interference (RNAi) technology. Plants expressing little or no AtVSR3 transcript had a compressed life cycle, bolting approximately 1 week earlier and senescing up to 2 weeks earlier than the wild-type parent line. While the development and distribution of stomata in AtVSR3 RNAi plants appeared normal, stomatal function was altered. The guard cells of mutant plants did not close in response to abscisic acid treatment, and the mean leaf temperatures of the RNAi plants were on average 0.8 degrees C lower than both wild type and another vacuolar sorting receptor mutant, atvsr1-1. Furthermore, the loss of AtVSR3 protein caused the accumulation of nitric oxide and hydrogen peroxide, signalling molecules implicated in the regulation of stomatal opening and closing. Finally, proteomics and western blot analyses of cellular proteins isolated from wild-type and AtVSR3 RNAi leaves showed that phospholipase Dgamma, which may play a role in abscisic acid signalling, accumulated to higher levels in AtVSR3 RNAi guard cells. Thus, AtVSR3 may play an important role in responses to plant stress.

The effects of exogenous plant growth regulators in the phytoextraction of heavy metals

The term “assisted phytoextraction” usually refers to the process of applying a chemical additive to contaminated soil in order to increase the metal uptake by crop plants. In this study three commercially available plant growth regulators (PGRs) based on cytokinins (CKs) were used to boost the assisted phytoextraction of Pb and Zn in contaminated soil collected from a former manufactured gas-plant site. The effects of EDTA treatment in soil and PGR treatment in leaves of Helianthus annuus were investigated in terms of dry weight biomass, Pb and Zn accumulation in the upper parts of the plants, Pb and Zn phytoextraction efficiency and transpiration rate. Metal solubility in soil and its subsequent accumulation in shoots were markedly enhanced by EDTA. The combined effects of EDTA and cytokine resulted in an increase in the Pb and Zn phytoextraction efficiency (up to 890% and 330%, respectively, compared to untreated plants) and up to a 50% increase in foliar transpiration. Our results indicate that exogenous PGRs based on CKs can positively assist the phytoextraction increasing the biomass production, the metal accumulation in shoots and the plant transpiration. The observed increase in biomass could be related to its action in stimulation of cell division and shoot initiation. On the other hand, the increase in metal accumulation in upper parts of plant could be related to both the role of PGRs in the enhancement of plant resistance to stress (as toxic metals) and the increase in transpiration rate, i.e. flux of water-soluble soil components and contaminants by the regulation of stomatal opening.

A role for Arabidopsis cryptochromes and COP1 in the regulation of stomatal opening

Cryptochromes (CRY) are blue light photoreceptors that mediate various light-induced responses in plants and animals. Arabidopsis CRY (CRY1 and CRY2) functions through negatively regulating constitutive photomorphogenic (COP) 1, a repressor of photomorphogenesis. Water evaporation and photosynthesis are regulated by the stomatal pores in plants, which are closed in darkness but open in response to blue light. There is evidence only for the phototropin blue light receptors (PHOT1 and PHOT2) in mediating blue light regulation of stomatal opening. Here, we report a previously uncharacterized role for Arabidopsis CRY and COP1 in the regulation of stomatal opening. Stomata of the cry1 cry2 double mutant showed reduced blue light response, whereas those of the CRY1-overexpressing plants showed hypersensitive response to blue light. In addition, stomata of the phot1 phot2 double mutant responded to blue light, but those of the cry1 cry2 phot1 phot2 quadruple mutant hardly responded. Strikingly, stomata of the cop1 mutant were constitutively open in darkness and stomata of the cry1 cry2 cop1 and phot1 phot2 cop1 triple mutants were open as wide as those of the cop1 single mutant under blue light. These results indicate that CRY functions additively with PHOT in mediating blue light-induced stomatal opening and that COP1 is a repressor of stomatal opening and likely acts downstream of CRY and PHOT signaling pathways.

Participation of Phytohormones in the Stomatal Regulation of Gas Exchange During Water Stress

Almost all processes in the life of a plant are directly or indirectly affected by both stresses and phytohormones. Nevertheless, apart from abscisic acid, the role of phytohormones in plant response to water stress is far from being fully elucidated. This review tries to answer the question whether interactions between abscisic acid and some other phytohormones might be important in the regulation of stomatal opening during water stress and subsequent rehydration. Firstly, it describes the changes in the contents of individual endogenous phytohormones during water stress. Then, it deals with the effects of applied phytohormones on stomatal opening, and on transpiration and photosynthetic rates in different plants species. Finally, it focuses on the alleviation or stimulation of absicic acid-induced stomatal closure by application of other phytohormones.

Interaction of Cytokinins and Abscisic Acid During Regulation of Stomatal Opening in Bean Leaves

Effects of benzyladenine (BA) and abscisic acid (ABA) applied separately or simultaneously on parameters of gas exchange of Phaseolus vulgaris L. leaves were studied. In the first two experimental sets) 100 μM ABA and 10 μM BA were applied to plants sufficiently supplied with water. Spraying of leaves with ABA decreased stomatal conductance (gs) and in consequence transpiration rate (E) and net photosynthetic rate (PN) already 1 h after application, but 24 h after application the effect almost disappeared. 10 μM BA slightly decreased gas exchange parameters, but in simultaneous application with ABA reversed the effect of ABA. Immersion of roots into the same solutions markedly decreased gas exchange parameters and 24 h after ABA application the stomata were completely closed. The effect of ABA was ameliorated by simultaneous BA application, particularly after 1-h treatment. In the third experimental set, plants were pre-treated by immersing roots into water, 1 μM BA, or 100 μM ABA for 24 h and then the halves of split root system were dipped into different combinations of 1 μM BA, 100 μM ABA, and water. In plants pre-treated with ABA all gas exchange parameters were small and they did not differ in plants treated with H2O+H2O, H2O+BA, or BA+BA. In plants pre-treated with BA or H2O, markedly lower values of PN were found when both halves of roots were immersed in ABA. Further, the effects of pre-treatment of plants with water, 1 μM BA, 100 μM ABA, or ABA+BA on the development of water stress induced by cessation of watering and on the recovery after rehydration were followed. ABA markedly decreased gas exchange parameters at the beginning of the experiment, but in its later phase the effect was compensated by delay in development of water stress. BA also delayed development of water stress and increased PN in water-stressed leaves. BA reversed the effect of ABA at mild water stress. Positive effects of BA and ABA pre-treatments were observed also after rehydration.

Phot Receptors Open the Stomata

Phot1 and phot2 are sensors for the blue light-stimulated responses of phototropism and chloroplast relocation, respectively. Kinoshita et al. provide genetic evidence that phot1 and phot2 function redundantly in sensing blue light for the purpose of increasing stomatal aperture to increase transpiration in response to blue light. Intact Arabidopsis double-mutant plants, but not single-mutant plants, did not increase stomatal aperture in epidermal strips, nor increase transpiration in response to blue light. Furthermore, the defect was in light sensation, based on experiments showing that the downstream H + ATPase was present and could be activated by the fungal toxin fusicoccin and resulted in stomatal opening and H + extrusion in wild-type and mutant plants. Thus, phot1 and phot2 now have additional roles in blue light signal transduction as the sensor for stimulating stomatal opening. T. Kinoshita, M. Doi, N. Suetsugu, T. Kagawa, M. Wada, K.-i. Shimazaki, Phot1 and phot 2 mediate blue light regulation of stomatal opening. Nature 414 , 656-660 (2001). [Online Journal]