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Glutathione is not involved in light-, Dark-, Ca- and H2O2- induced stomatal movement in Arabidopsis  [PDF]
Md Sarwar Jahan,Mohd Khairi Bin Che Lah,Mohd Nozulaidi Bin Nordin,Syed S.B. Syed Kamarulzaman
Journal of Stress Physiology & Biochemistry , 2012,
Abstract: Glutathione (GSH), is a thiol-containing tripeptide, maintains redox homeostasis in plants under normal and stressful conditions. In this study, we investigated whether GSH involved in light-, dark-, Ca- and H2O2-induced stomatal movement in Arabidopsis. Application of GSH and a GSH decreasing chemical (CDNB; 1-chloro-2,4-dinitrobenzene) did not affect stomatal aperture in guard cells of Arabidopsis. Dark induced stomatal closure and light induced stomatal opening but pre-treatment of GSH and CDNB did not alter dark- and light-induced stomatal aperture. Treatment of guard cells with Ca and H2O2 did not affect GSH contents in guard cells but induced stomatal closure in both wild type and chorinal-1 (ch1-1) mutant plants. In addition, pre-treatment of GSH and CDNB did not affect Ca and H2O2-induced stomatal closure in both plants. Taken together these results suggest that GSH might not directly affect light-, dark-, Ca- and H2O2-induced stomatal movement in guard cells of Arabidopsis.
Effects of the disaggregation of high-polymerized particles in guard cell vacuoles on osmoregulation of stomatal aperture (stomata opening)
Effects of the disaggregation of high—polymerized particles in guard cell vacuoles on osmoregulation of stomatal aperture(stomata opening)

YE Qing,ZHU Guoli,LOU Chenghou,
YEQing
,ZHUGuoli,LOUChenghou

科学通报(英文版) , 2003,
Abstract: Observation under an electron microscope reveals that in closed and open stomata of V. faba, the aver-age volume of particles in guard cell vacuoles (GCV) reduces about 3 orders in magnitude, while the distribution density of the particles increases about 2 orders of magnitude. By using the method of the ratio of fluorescent emissions with laser scanning confocal microscopy, the monitoring to sto-mata opening shows that during 10 to 30 s before the first distinguishable aperture of stomata, there is a change of pH in GCV about -0.5 units. A quick stomatal opening immedi-ately follows the changes of pH in GCV to reach a steady aperture about 12 mm in 100200 s. This work proposes a model for the osmoregulation in GCV for stomatal opening. The proposed osmoregulation is related to the disaggregation of some polymerized particles inside GCV, which is probably induced by a -DpH in the vacuole. This model describes a process of osmoregulation that avoids the massive energy consuming transportation across cell membranes, which is a foundation of the current chemiosmotic hypothesis. This model is a supplement to the multiple controlling hypothesis for the stomatal movement, which widens research principle ideas for other quick movements in plants.
Light-Induced Stomatal Opening Is Affected by the Guard Cell Protein Kinase APK1b  [PDF]
Nagat S. Elhaddad, Lee Hunt, Jennifer Sloan, Julie E. Gray
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0097161
Abstract: Guard cells allow land plants to survive under restricted or fluctuating water availability. They control the exchange of gases between the external environment and the interior of the plant by regulating the aperture of stomatal pores in response to environmental stimuli such as light intensity, and are important regulators of plant productivity. Their turgor driven movements are under the control of a signalling network that is not yet fully characterised. A reporter gene fusion confirmed that the Arabidopsis APK1b protein kinase gene is predominantly expressed in guard cells. Infrared gas analysis and stomatal aperture measurements indicated that plants lacking APK1b are impaired in their ability to open their stomata on exposure to light, but retain the ability to adjust their stomatal apertures in response to darkness, abscisic acid or lack of carbon dioxide. Stomatal opening was not specifically impaired in response to either red or blue light as both of these stimuli caused some increase in stomatal conductance. Consistent with the reduction in maximum stomatal conductance, the relative water content of plants lacking APK1b was significantly increased under both well-watered and drought conditions. We conclude that APK1b is required for full stomatal opening in the light but is not required for stomatal closure.
Effects of the disaggregation of high-polymerized particles in guard cell vacuoles on osmoregulation of stomatal aperture (stomata opening)
Qing Ye,Guoli Zhu,Chenghou Lou
Chinese Science Bulletin , 2003, DOI: 10.1007/BF03183249
Abstract: Observation under an electron microscope reveals that in closed and open stomata ofV. faba, the average volume of particles in guard cell vacuoles (GCV) reduces about 3 orders in magnitude, while the distribution density of the particles increases about 2 orders of magnitude. By using the method of the ratio of fluorescent emissions with laser scanning confocal microscopy, the monitoring to stomata opening shows that during 10 to 30 s before the first distinguishable aperture of stomata, there is a change of pH in GCV about -0.5 units. A quick stomatal opening immediately follows the changes of pH in GCV to reach a steady aperture about 12 μm in 100–200 s. This work proposes a model for the osmoregulation in GCV for stomatal opening. The proposed osmoregulation is related to the disaggregation of some polymerized particles inside GCV, which is probably induced by a-ΔpH in the vacuole. This model describes a process of osmoregulation that avoids the massive energy consuming transportation across cell membranes, which is a foundation of the current chemiosmotic hypothesis. This model is a supplement to the multiple controlling hypothesis for the stomatal movement, which widens research principle ideas for other quick movements in plants.
Coronatine inhibits stomatal closure and delays hypersensitive response cell death induced by nonhost bacterial pathogens  [PDF]
Seonghee Lee,Yasuhiro Ishiga,Kristen Clermont,Kirankumar?S. Mysore
PeerJ , 2013, DOI: 10.7717/peerj.34
Abstract: Pseudomonas syringae is the most widespread bacterial pathogen in plants. Several strains of P. syringae produce a phytotoxin, coronatine (COR), which acts as a jasmonic acid mimic and inhibits plant defense responses and contributes to disease symptom development. In this study, we found that COR inhibits early defense responses during nonhost disease resistance. Stomatal closure induced by a nonhost pathogen, P. syringae pv. tabaci, was disrupted by COR in tomato epidermal peels. In addition, nonhost HR cell death triggered by P. syringae pv. tabaci on tomato was remarkably delayed when COR was supplemented along with P. syringae pv. tabaci inoculation. Using isochorismate synthase (ICS)-silenced tomato plants and transcript profiles of genes in SA- and JA-related defense pathways, we show that COR suppresses SA-mediated defense during nonhost resistance.
RNAi-Directed Downregulation of Vacuolar H+-ATPase Subunit A Results in Enhanced Stomatal Aperture and Density in Rice  [PDF]
Huiying Zhang, Xiangli Niu, Jia Liu, Fangming Xiao, Shuqing Cao, Yongsheng Liu
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0069046
Abstract: Stomatal movement plays a key role in plant development and response to drought and salt stress by regulating gas exchange and water loss. A number of genes have been demonstrated to be involved in the regulation of this process. Using inverse genetics approach, we characterized the function of a rice (Oryza sativa L.) vacuolar H+-ATPase subunit A (OsVHA-A) gene in stomatal conductance regulation and physiological response to salt and osmotic stress. OsVHA-A was constitutively expressed in different rice tissues, and the fusion protein of GFP-OsVHA-A was exclusively targeted to tonoplast when transiently expressed in the onion epidermal cells. Heterologous expression of OsVHA-A was able to rescue the yeast mutant vma1Δ (lacking subunit A activity) phenotype, suggesting that it partially restores the activity of V-ATPase. Meanwhile, RNAi-directed knockdown of OsVHA-A led to a reduction of vacuolar H+-ATPase activity and an enhancement of plasma membrane H+-ATPase activity, thereby increasing the concentrations of extracellular H+ and intracellular K+ and Na+ under stress conditions. Knockdown of OsVHA-A also resulted in the upregulation of PAM3 (plasma membrane H+-ATPase 3) and downregulation of CAM1 (calmodulin 1), CAM3 (calmodulin 3) and YDA1 (YODA, a MAPKK gene). Altered level of the ion concentration and the gene expression by knockdown of OsVHA-A probably resulted in expanded aperture of stomatal pores and increased stomatal density. In addition, OsVHA-A RNAi plants displayed significant growth inhibition under salt and osmotic stress conditions. Taken together, our results suggest that OsVHA-A takes part in regulating stomatal density and opening via interfering with pH value and ionic equilibrium in guard cells and thereby affects the growth of rice plants.
Rearrangements of microtubule cytoskeleton in stomatal closure of Arabidopsis induced by nitric oxide
YongMei Zhang,ZhongYi Wu,XueChen Wang,Rong Yu
Chinese Science Bulletin , 2008, DOI: 10.1007/s11434-008-0142-7
Abstract: NO (nitric oxide), known as a key signal molecule in plant, plays important roles in regulation of stomatal movement. In this study, microtubule dynamics and its possible mechanism in the NO signal pathway were investigated. The results were as follows: (i) In vivo stomatal aperture assays revealed that both vinblastine (microtubule-disrupting drug) and SNP (exogenous NO donor) prevented stomatal opening in the light, and vinblastine even could enhance the inhibitory effect of SNP, whereas taxol (a microtubule-stabilizing agent) was able to reduce this effect; (ii) microtubules in the opening Arabidopsis guard cells expressing GFP:α-tubulin-6 (AtGFP:α-tubulin-6) were organized in parallel, straight and dense bundles, radiating from the ventral side to the dorsal side, and most of them were localized perpendicularly to the ventral wall; (iii) under the same environmental conditions, treated with SNP for 30 min, the radial arrays of microtubules in guard cells began to break down, twisted partially and became oblique or exhibited a random pattern; (iv) furthermore, the involvement of cytosolic Ca2+ in this event was tested. Stomatal aperture assays revealed that BAPTA-AM (a chelator of Ca2+) greatly suppressed the effect of NO on stomatal closure; however, it did not show the same function on stomatal closure induced by vinblastine. When BAPTA-AM was added to the SNP-pretreated solution, the SNP-induced disordered microtubulue cytoskeleton in guard cells underwent rearrangement in a time-dependent manner. After 30 min of treatment with BAPTA-AM, the cortical microtubules resumed the original radial distribution, almost the same as the control. All this indicates that NO may promote rearrangement of microtubule cytoskeleton via elevation of [Ca2+]cyt (free Ca2+ concentration in the cytoplasm), finally leading to stomatal closure.
Rearrangements of microtubule cytoskeleton in stomatal closure of Arabidopsis induced by nitric oxide

ZHANG YongMei,WU ZhongYi,WANG XueChen,YU Rong,

科学通报(英文版) , 2008,
Abstract: NO (nitric oxide), known as a key signal molecule in plant, plays important roles in regulation of stomatal movement. In this study, microtubule dynamics and its possible mechanism in the NO signal pathway were investigated. The results were as follows: (i) In vivo stomatal aperture assays revealed that both vinblastine (microtubule-disrupting drug) and SNP (exogenous NO donor) prevented stomatal opening in the light, and vinblastine even could enhance the inhibitory effect of SNP, whereas taxol (a microtubule-stabilizing agent) was able to reduce this effect; (ii) microtubules in the opening Arabidopsis guard cells expressing GFP:α-tubulin-6 (AtGFP:α-tubulin-6) were organized in parallel, straight and dense bundles, radiating from the ventral side to the dorsal side, and most of them were localized perpendicularly to the ventral wall; (iii) under the same environmental conditions, treated with SNP for 30 min, the radial arrays of microtubules in guard cells began to break down, twisted partially and became oblique or exhibited a random pattern; (iv) furthermore, the involvement of cytosolic Ca2+ in this event was tested. Stomatal aperture assays revealed that BAPTA-AM (a chelator of Ca2+) greatly suppressed the effect of NO on stomatal closure; however, it did not show the same function on stomatal closure induced by vinblastine. When BAPTA-AM was added to the SNP-pretreated solution, the SNP-induced disordered microtubulue cytoskeleton in guard cells underwent rearrangement in a time-dependent manner. After 30 min of treatment with BAPTA-AM, the cortical microtubules resumed the original radial distribution, almost the same as the control. All this indicates that NO may promote rearrangement of microtubule cytoskeleton via elevation of Ca2+]cyt (free Ca2+ concentration in the cytoplasm), finally leading to stomatal closure. These authors contributed equally to this work. Supported by the New Star Plan of Science & Technology of Beijin, China (Grant No. 2003B34) and National Natural Science Foundation of China (Grant Nos. 30600318 and 30400228)
Overexpression of the Mg-chelatase H subunit in guard cells confers drought tolerance via promotion of stomatal closure in Arabidopsis thaliana  [PDF]
Koji Takahashi,Toshinori Kinoshita
Frontiers in Plant Science , 2013, DOI: 10.3389/fpls.2013.00440
Abstract: The Mg-chelatase H subunit (CHLH) has been shown to mediate chlorophyll biosynthesis, as well as plastid-to-nucleus and abscisic acid (ABA)-mediated signaling. A recent study using a novel CHLH mutant, rtl1, indicated that CHLH specifically affects ABA-induced stomatal closure, but also that CHLH did not serve as an ABA receptor in Arabidopsis thaliana. However, the molecular mechanism by which CHLH engages in ABA-mediated signaling in guard cells remains largely unknown. In the present study, we examined CHLH function in guard cells and explored whether CHLH expression might influence stomatal aperture. Incubation of rtl1 guard cell protoplasts with ABA induced expression of the ABA-responsive genes RAB18 and RD29B, as also observed in wild-type (WT) cells, indicating that CHLH did not affect the expression of ABA-responsive genes. Earlier, ABA was reported to inhibit blue light (BL)-mediated stomatal opening, at least in part through dephosphorylating/inhibiting guard cell H+-ATPase (which drives opening). Therefore, we immunohistochemically examined the phosphorylation status of guard cell H+-ATPase. Notably, ABA inhibition of BL-induced phosphorylation of H+-ATPase was impaired in rtl1 cells, suggesting that CHLH influences not only ABA-induced stomatal closure but also inhibition of BL-mediated stomatal opening by ABA. Next, we generated CHLH-GFP-overexpressing plants using CER6 promoter, which induces gene expression in the epidermis including guard cells. CHLH-transgenic plants exhibited a closed stomata phenotype even when brightly illuminated. Moreover, plant growth experiments conducted under water-deficient conditions showed that CHLH transgenic plants were more tolerant of drought than WT plants. In summary, we show that CHLH is involved in the regulation of stomatal aperture in response to ABA, but not in ABA-induced gene expression, and that manipulation of stomatal aperture via overexpression of CHLH in guard cells improves plant drought tolerance.
Compound stress response in stomatal closure: a mathematical model of ABA and ethylene interaction in guard cells
Mariano Beguerisse-D?az, Mercedes C Hernández-Gómez, Alessandro M Lizzul, Mauricio Barahona, Radhika Desikan
BMC Systems Biology , 2012, DOI: 10.1186/1752-0509-6-146
Abstract: Toshed light on this unexplained behaviour, we have collected time course measurements of stomatal aperture and hydrogen peroxide production in Arabidopsis thaliana guard cells treated with abscisic acid, ethylene, and a combination of both. Our experiments show that stomatal closure is linked to sustained high levels of hydrogen peroxide in guard cells. When treated with a combined dose of abscisic acid and ethylene, guard cells exhibit increased antioxidant activity that reduces hydrogen peroxide levels and precludes closure. We construct a simplified model of stomatal closure derived from known biochemical pathways that captures the experimentally observed behaviour.Our experiments and modelling results suggest a distinct role for two antioxidant mechanisms during stomatal closure: a slower, delayed response activated by a single stimulus (abscisic acid ‘or’ ethylene) and another more rapid ‘and’ mechanism that is only activated when both stimuli are present. Our model indicates that the presence of this rapid ‘and’ mechanism in the antioxidant response is key to explain the lack of closure under a combined stimulus.Stomata are tiny pores located mainly in the lower epidermis of plant leaves. Each stoma is formed by two guard cells attached to each other by their extremes. When the guard cells are turgid, due to their vacuoles being full of water, the pore opens (Figure 1A). When the vacuoles are emptied and water exits the cells, the guard cells become flaccid and the pore closes (Figure 1B) [1]. Loss of turgor pressure (and the resulting closure of the stomatal pore) is a consequence of the efflux of ions out of the cell. Ion efflux may be caused by a variety of stimuli including different light conditions and atmospheric carbon dioxide (CO2) levels, or signalling hormones such as abscisic acid (ABA) and ethylene [2,3]. Open pores allow the plant to absorb CO2 from the air to perform photosynthesis and to release oxygen and water into the atmosphere. If the por
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