A hydraulic signal in root-to-shoot signalling of water shortage

Photosynthesis and biomass production of plants are controlled by the water status of the soil. Upon soil drying, plants can reduce water consumption by minimizing transpiration through stomata, the closable pores of the leaf. The phytohormone abscisic acid (ABA) mediates stomatal closure, and is the assigned signal for communicating water deficit from the root to the shoot. However, our study does not support ABA as the proposed long-distance signal. The shoot response to limited soil water supply is not affected by the capacity to generate ABA in the root; however, the response does require ABA biosynthesis and signalling in the shoot. Soil water stress elicits a hydraulic response in the shoot, which precedes ABA signalling and stomatal closure. Attenuation of the hydraulic response in various plants prevented long-distance signalling of water stress, consistent with root-to-shoot communication by a hydraulic signal.     

Cell-specific mechanisms and systemic signalling as emerging themes in light acclimation of C3 plants

Chloroplasts perform essential signalling functions in light acclimation and various stress responses in plants. Research on chloroplast signalling has provided fundamental information concerning the diversity of cellular responses to changing environmental conditions. Evidence has also accumulated indicating that different cell types possess specialized roles in regulation of leaf development and stress acclimation when challenged by environmental cues. Leaf veins are flanked by a layer of elongated chloroplast-containing bundle sheath cells, which due to their central position hold the potential to control the flux of information inside the leaves. Indeed, a specific role for bundle sheath cells in plant acclimation to various light regimes is currently emerging. Moreover, perception of light stress initiates systemic signals that spread through the vasculature to confer stress resistance in non-exposed parts of the plant. Such long-distance signalling functions are related to unique characteristics of reactive oxygen species and their detoxification in bundle sheath cells. Novel techniques for analysis of distinct tissue types, together with Arabidopsis thaliana mutants with vasculature-specific phenotypes, have proven instrumental in dissection of structural hierarchy among regulatory processes in leaves. This review emphasizes the current knowledge concerning the role of vascular bundle sheath cells in light-dependent acclimation processes of C3 plants.     

Arabidopsis mutants of AtABCG22, an ABC transporter gene, increase water transpiration and drought susceptibility

In plants, water vapour is released into the atmosphere through stomata in a process called transpiration. Abscisic acid (ABA) is a key phytohormone that facilitates stomatal closure through its action on guard cells. Recently, ATP-binding cassette (ABC) transporter genes, AtABCG25 and AtABCG40, were shown to be involved in ABA transport and responses. However, the functions of many other AtABCG family genes are still unknown. Here, we identified another ABCG gene (AtABCG22) that is required for stomatal regulation in Arabidopsis. The atabcg22 mutant plants had lower leaf temperatures and increased water loss, implying elevated transpiration through an influence on stomatal regulation. We also found that atabcg22 plants were more suspectible to drought stress than wild-type plants. AtABCG22 was expressed in aerial organs, mainly guard cells, in which the gene expression pattern was consistent with the mutant phenotypes. Using double mutants, we investigated the genetic relationships between the mutations. The atabcg22 mutation further increased the water loss of srk2e/ost1 mutants, which were defective in ABA signalling in guard cells. Also, the atabcg22 mutation enhanced the phenotype of nced3 mutants, which were defective in ABA biosynthesis. Accordingly, the additive roles of AtABCG22 functions in ABA signalling and ABA biosynthesis are discussed.     

The plant multidrug resistance ABC transporter AtMRP5 is involved in guard cell hormonal signalling and water use

Carbon dioxide uptake and water release through stomata, controlling the opening and closure of stomatal pore size in the leaf surface, is critical for optimal plant performance. Stomatal movements are regulated by multiple signalling pathways involving guard cell ion channels. Using reverse genetics, we recently isolated a T-DNA insertion mutant for the Arabidopsis ABC-transporter AtMRP5 (mrp5-1). Guard cells from mrp5-1 mutant plants were found to be insensitive to the sulfonylurea compound glibenclamide, which in the wild type induces stomatal opening in the dark. Here, we report that the knockout in AtMRP5 affects several signalling pathways controlling stomatal movements. Stomatal apertures of mrp5-1 and wild-type Ws-2 were identical in the dark. In contrast, opening of stomata of mrp5-1 plants was reduced in the light. In the light, stomatal closure of mrp5-1 was insensitive to external calcium and abscisic acid, a phytohormone responsible for stomatal closure during drought stress. In contrast to Ws-2, the phytohormone auxin could not stimulate stomatal opening in the mutant in darkness. All stomatal phenotypes were complemented in transgenic mrp5-1 plants transformed with a cauliflower mosaic virus (CaMV) 35S-AtMRP5 construct. Both whole-plant and single-leaf gas exchange measurements demonstrated a reduced transpiration rate of mrp5-1 in the light. Excised leaves of mutant plants exhibited reduced water loss, and water uptake was strongly decreased at the whole-plant level. Finally, if plants were not watered, mrp5-1 plants survived much longer due to reduced water use. Analysis of CO2 uptake and transpiration showed that mrp5-1 plants have increased water use efficiency. Mutant plants overexpressing AtMRP5 under the control of the CaMV 35S promoter again exhibited wild-type characteristics. These results demonstrate that multidrug resistance-associated proteins (MRPs) are important components of guard cell functioning.     

Arabidopsis HT1 kinase controls stomatal movements in response to CO2

Guard cells, which form stomata in leaf epidermes, sense a multitude of environmental signals and integrate this information to regulate stomatal movements. Compared with the advanced understanding of light and water stress responses in guard cells, the molecular mechanisms that underlie stomatal CO(2) signalling have remained relatively obscure. With a high-throughput leaf thermal imaging CO(2) screen, we report the isolation of two allelic Arabidopsis mutants (high leaf temperature 1; ht1-1 and ht1-2) that are altered in their ability to control stomatal movements in response to CO(2). The strong allele, ht1-2, exhibits a markedly impaired CO(2) response but shows functional responses to blue light, fusicoccin and abscisic acid (ABA), indicating a role for HT1 in stomatal CO(2) signalling. HT1 encodes a protein kinase that is expressed mainly in guard cells. Phosphorylation assays demonstrate that the activity of the HT1 protein carrying the ht1-1 or ht1-2 mutation is greatly impaired or abolished, respectively. Furthermore, dominant-negative HT1(K113W) transgenic plants, which lack HT1 kinase activity, show a disrupted CO(2) response. These findings indicate that the HT1 kinase is important for regulation of stomatal movements and its function is more pronounced in response to CO(2) than it is to ABA or light.     

Stomatal movements and long-distance signaling in plants

As the nerve-mediated signaling in animals, long-distance signaling in plants is a prerequisite for plants to be able to perceive environmental stimuli and initiate adaptive responses. While intracellular signal transduction has been attracting considerable attentions, studies on long-distance signaling in plants has been relatively overlooked. Stomatal movements are well recognized as a model system for studies on cellular signal transduction. It has been demonstrated that the stomatal movements may be frequently tuned by long-distance signaling under various environmental stimuli. Stomatal movements can not only respond to persistent stress stimuli but also respond to shock stress stimuli. Stomatal responses to drought stress situations may be best characterized in terms of interwoven networks of chemical signaling pathways playing predominant roles in these adaptive processes. In cases of shock stress stimuli, stomatal movements can be more sensitively regulated through the long-distance signaling but with distinctive patterns not observed for drought or other persistent stresses. Here, the fundamental characteristics of stomatal movements and associated long-distance signaling are reviewed and the implications for plant responses to environmental stresses are discussed.Key words: stomatal movement, long-distance signaling, environmental stresses, abscisic aci, pH signaling, hydraulic signaling, cytokinins, acetylcholine, heat-shock, electric signal     

Stomatal development and patterning in Arabidopsis leaves

The functional unit for gas exchange between plants and the atmosphere is the stomatal complex, an epidermal structure composed of two guard cells, which delimit a stomatal pore, and their subsidiary cells. In the present work, we define the basic structural unit formed in Arabidopsis thaliana during leaf development, the anisocytic stomatal complex. We perform a cell lineage analysis by transposon excision founding that at least a small percentage of stomatal complexes are unequivocally non-clonal. We also describe the three-dimensional pattern of stomata in the Arabidopsis leaf. In the epidermal plane, subsidiary cells of most stomatal complexes contact the subsidiary cells of immediately adjacent complexes. This minimal distance between stomatal complexes allows each stoma to be circled by a full complement of subsidiary cells, with which guard cells can exchange water and ions in order to open or to close the pore. In the radial plane, stomata (and their precursors, the meristemoids) are located at the junctions of several mesophyll cells. This meristemoid patterning may be a consequence of signals that operate along the radial axis of the leaf, which establish meristemoid differentiation precisely at these places. Since stomatal development is basipetal, these radially propagated signals may be transmitted in the axial direction, thus guiding stomatal development through the basal end of the leaf.     

Abnormal Stomatal Behavior and Hormonal Imbalance in flacca, a Wilty Mutant of Tomato: I. Root Effect and Kinetin-like Activity

The wilty tomato mutant, flacca, and the normal variety, Rheinlands Ruhm, were compared for kinetin-like activity in ontogeny. The mutant wilts easily because its stomata resist closure. This stomatal resistance decreases with age. The occurrence of a root factor which induces stomatal opening was inferred from grafting experiments. It was hypothesized that the excessive stomatal openings in the mutant may result from excess of kinetin-like activity in the leaf of that plant. In addition, it was suggested that the closure of stomata in the aging mutant is due to a decrease of kinetin-like activity with age. Kinetin-like activity in the leaf was determined by incorporation of labeled leucine. The concentration of cytokinins in root exudate and leaf extract was determined by the soybean callus assay. Evidence was presented of higher kinetin-like activity in the leaves of the mutant and higher cytokinin concentration in its root exudate. Cytokinin concentration in the shoot was found to be only slightly higher in the mutant than in the normal plants. Kinetin-like activity in the leaf and cytokinin concentration of root exudate decreased with age in both mutant and normal plants. Kinetin-like activity in the leaves of mutant plants, which phenocopy the normal variety as a result of continuous application of abscisic acid, was lower than in control mutant plants. The significance of these findings per se and in connection with stomatal behavior is discussed.     

cGMP-dependent ABA-induced stomatal closure in the ABA-insensitive Arabidopsis mutant abi1-1

? The drought hormone abscisic acid (ABA) is widely known to produce reductions in stomatal aperture in guard cells. The second messenger cyclic guanosine 3', 5'-monophosphate (cGMP) is thought to form part of the signalling pathway by which ABA induces stomatal closure. ? We have examined the signalling events during cGMP-dependent ABA-induced stomatal closure in wild-type Arabidopsis plants and plants of the ABA-insensitive Arabidopsis mutant abi1-1. ? We show that cGMP acts downstream of hydrogen peroxide (H(2) O(2) ) and nitric oxide (NO) in the signalling pathway by which ABA induces stomatal closure. H(2) O(2) - and NO-induced increases in the cytosolic free calcium concentration ([Ca(2+) ](cyt) ) were cGMP-dependent, positioning cGMP upstream of [Ca(2+) ](cyt) , and involved the action of the type 2C protein phosphatase ABI1. Increases in cGMP were mediated through the stimulation of guanylyl cyclase by H(2) O(2) and NO. We identify nucleoside diphosphate kinase as a new cGMP target protein in Arabidopsis. ? This study positions cGMP downstream of ABA-induced changes in H(2) O(2) and NO, and upstream of increases in [Ca(2+) ](cyt) in the signalling pathway leading to stomatal closure.     

The Arabidopsis small G-protein AtRAN1 is a positive regulator in chitin-induced stomatal closure and disease resistance

Chitin, a fungal microbial-associated molecular pattern, triggers various defence responses in several plant systems. Although it induces stomatal closure, the molecular mechanisms of its interactions with guard cell signalling pathways are unclear. Based on screening of public microarray data obtained from the ATH1 Affymetrix and Arabidopsis eFP browser, we isolated a cDNA encoding a Ras-related nuclear protein 1 AtRAN1. AtRAN1 expression was enriched in guard cells in a manner consistent with involvement in the control of the stomatal movement. AtRAN1 mutation impaired chitin-induced stomatal closure and accumulation of reactive oxygen species and nitric oxide in guard cells. In addition, Atran1 mutant plants exhibited compromised chitin-enhanced plant resistance to both bacterial and fungal pathogens due to changes in defence-related genes. Furthermore, Atran1 mutant plants were hypersensitive to drought stress compared to Col-0 plants, and had lower levels of stress-responsive genes. These data demonstrate a previously uncharacterized signalling role for AtRAN1, mediating chitin-induced signalling.     

Phospholipase A2beta mediates light-induced stomatal opening in Arabidopsis

Phospholipase A(2) (PLA(2)) catalyses the hydrolysis of phospholipids into lysophospholipids and free fatty acids. Physiological studies have indicated that PLA(2) is involved in stomatal movement. However, genetic evidence of a role of PLA(2) in guard cell signalling has not yet been reported. To identify PLA(2) gene(s) that is (are) involved in light-induced stomatal opening, stomatal movement was examined in Arabidopsis thaliana plants in which the expression of PLA(2) isoforms was reduced or knocked-out. Light-induced stomatal opening in PLA(2)alpha knockout plants did not differ from wild-type plants. Plants in which PLA(2)beta was silenced by RNA interference exhibited delayed light-induced stomatal opening, and this phenotype was reversed by exogenous lysophospholipids, which are products of PLA(2). Stomatal opening in transgenic plants that over-expressed PLA(2)beta was faster than wild-type plants. The expression of PLA(2)beta was localized to the endoplasmic reticulum of guard cells, and increased in response to light in the mature leaf. Aristolochic acid, which inhibits light-induced stomatal opening, inhibited the activity of purified PLA(2)beta. Collectively, these results provide evidence that PLA(2)beta is involved in light-induced stomatal opening in Arabidopsis.     

Assessing Stomatal Response to Live Bacterial Cells using Whole Leaf Imaging

Stomata are natural openings in the plant epidermis responsible for gas exchange between plant interior and environment. They are formed by a pair of guard cells, which are able to close the stomatal pore in response to a number of external factors including light intensity, carbon dioxide concentration, and relative humidity (RH). The stomatal pore is also the main route for pathogen entry into leaves, a crucial step for disease development. Recent studies have unveiled that closure of the pore is effective in minimizing bacterial disease development in Arabidopsis plants; an integral part of plant innate immunity. Previously, we have used epidermal peels to assess stomatal response to live bacteria (Melotto et al. 2006); however maintaining favorable environmental conditions for both plant epidermal peels and bacterial cells has been challenging. Leaf epidermis can be kept alive and healthy with MES buffer (10 mM KCl, 25 mM MES-KOH, pH 6.15) for electrophysiological experiments of guard cells. However, this buffer is not appropriate for obtaining bacterial suspension. On the other hand, bacterial cells can be kept alive in water which is not proper to maintain epidermal peels for long period of times. When an epidermal peel floats on water, the cells in the peel that are exposed to air dry within 4 hours limiting the timing to conduct the experiment. An ideal method for assessing the effect of a particular stimulus on guard cells should present minimal interference to stomatal physiology and to the natural environment of the plant as much as possible. We, therefore, developed a new method to assess stomatal response to live bacteria in which leaf wounding and manipulation is greatly minimized aiming to provide an easily reproducible and reliable stomatal assay. The protocol is based on staining of intact leaf with propidium iodide (PI), incubation of staining leaf with bacterial suspension, and observation of leaves under laser scanning confocal microscope. Finally, this method allows for the observation of the same live leaf sample over extended periods of time using conditions that closely mimic the natural conditions under which plants are attacked by pathogens.     

ABA-based chemical signalling: the co-ordination of responses to stress in plants

There is now strong evidence that the plant hormone abscisic acid (ABA) plays an important role in the regulation of stomatal behaviour and gas exchange of droughted plants. This regulation involves both long-distance transport and modulation of ABA concentration at the guard cells, as well as differential responses of the guard cells to a given dose of the hormone. We will describe how a plant can use the ABA signalling mechanism and other chemical signals to adjust the amount of water that it loses through its stomata in response to changes in both the rhizospheric and the aerial environment. The following components of the signalling process can play an important part in regulation: (a) ABA sequestration in the root; (b) ABA synthesis versus catabolism in the root; (c) the efficiency of ABA transfer across the root and into the xylem; (d) the exchange of ABA between the xylem lumen and the xylem parenchyma in the shoot; (e) the amount of ABA in the leaf symplastic reservoir and the efficiency of ABA sequestration and release from this compartment as regulated by factors such as root and leaf-sourced changes in pH; (f) cleavage of ABA from ABA conjugates in the leaf apoplast; (g) transfer of ABA from the leaf into the phloem; (h) the sensitivity of the guard cells to the [ABA] that finally reaches them; and lastly (i) the possible interaction between nitrate stress and the ABA signal.     

Long-distance movement factor: a transport function of the potyvirus helper component proteinase.

Transport of viruses from cell to cell in plants typically involves one or more viral proteins that supply dedicated movement functions. Transport from leaf to leaf through phloem, or long-distance transport, is a poorly understood process with requirements differing from those of cell-to-cell movement. Through genetic analysis of tobacco etch virus (TEV; potyvirus group), a novel long-distance movement factor was identified that facilitates vascular-associated movement in tobacco. A mutation in the central region of the helper component proteinase (HC-Pro), a TEV-encoded protein with previously described activities in aphid-mediated transmission and polyprotein processing, inactivated long-distance movement. This mutant virus exhibited only minor defects in genome amplification and cell-to-cell movement functions. In situ histochemical analysis revealed that the mutant was capable of infecting mesophyll, bundle sheath, and phloem cells within inoculated leaves, suggesting that the long-distance movement block was associated with entry into or exit from sieve elements. The long-distance movement defect was specifically complemented by HC-Pro supplied in trans by a transgenic host. The data indicate that HC-Pro functions in one or more steps unique to long-distance transport.     

伤胁迫对蚕豆叶片中茉莉酸分布的影响

在植物应对伤害等环境刺激的反应中,已知茉莉酸(JA)作为一种重要的信号分子在植物体内长距离运输,但目前对JA的细胞和亚细胞定位知之甚少。本研究用免疫荧光显微镜技术和免疫胶体金电镜技术证明茉莉酸分布在蚕豆叶片叶肉细胞的叶绿体、表皮细胞的细胞壁、保卫细胞的细胞壁、细胞质、叶绿体和细胞核上。其中保卫细胞的叶绿体和细胞核是JA分布的主要场所。叶片的局部灼伤可提高JA在质外体和气孔保卫细胞中的水平。由此推测,伤胁迫下JA分配的改变可能与植物体防御反应密切相关,并参与了对气孔运动的调控。     

Drawing the future: Stomatal response to CO2 levels

Gas exchange between the plant and the atmosphere is regulated by controlling both the stomatal density and the aperture of the stomatal pore. Environmental factors such as light, the level of atmospheric CO₂ and hormones regulate stomatal development and/or function. Because atmospheric CO₂ levels have been rising since the Industrial Revolution, and it is predicted that they will continue doing so in the future, an understanding of the CO₂ signalling mechanisms in the stomatal responses will help to know how plants were in the past and will allow predicting how they will respond to climate change in the near future. This article covers the recent knowledge of the CO₂ signalling mechanisms that regulate both stomatal function and development.Key words: Arabidopsis, CO₂, development, epidermis, gas exchange, leaf, patterning, stoma     

Respiratory complex I deficiency induces drought tolerance by impacting leaf stomatal and hydraulic conductances

To investigate the role of plant mitochondria in drought tolerance, the response to water deprivation was compared between Nicotiana sylvestris wild type (WT) plants and the CMSII respiratory complex I mutant, which has low-efficient respiration and photosynthesis, high levels of amino acids and pyridine nucleotides, and increased antioxidant capacity. We show that the delayed decrease in relative water content after water withholding in CMSII, as compared to WT leaves, is due to a lower stomatal conductance. The stomatal index and the abscisic acid (ABA) content were unaffected in well-watered mutant leaves, but the ABA/stomatal conductance relation was altered during drought, indicating that specific factors interact with ABA signalling. Leaf hydraulic conductance was lower in mutant leaves when compared to WT leaves and the role of oxidative aquaporin gating in attaining a maximum stomatal conductance is discussed. In addition, differences in leaf metabolic status between the mutant and the WT might contribute to the low stomatal conductance, as reported for TCA cycle-deficient plants. After withholding watering, TCA cycle derived organic acids declined more in CMSII leaves than in the WT, and ATP content decreased only in the CMSII. Moreover, in contrast to the WT, total free amino acid levels declined whilst soluble protein content increased in CMSII leaves, suggesting an accelerated amino acid remobilisation. We propose that oxidative and metabolic disturbances resulting from remodelled respiration in the absence of Complex I activity could be involved in bringing about the lower stomatal and hydraulic conductances.     

盾叶秋海棠叶表皮气孔簇的发育及分布格局

气孔是植物控制气体交换和调节水分散失的门户。大部分高等植物气孔的分布格局是相邻气孔之间被一至多个表皮细胞所间隔。而在有限分布的几个科属的植物种中发现气孔成簇分布的现象 ,即由 2至多个紧密相邻的气孔器组成相对独立的单元 ,称为气孔簇 (stomatalcluster)。以中国原产的盾叶秋海棠 (BegoniapeltatifoliaLi)为研究对象 ,探讨了叶表皮气孔簇的发育机制及其分布格局。结果表明 :气孔发育初期 ,气孔拟分生组织的成簇 (相邻紧密 )排列可能是气孔簇形成的主要机制 ;气孔副卫细胞恢复分裂形成的卫星拟分生组织也对气孔簇的形成起一定的作用。把气孔簇和单个气孔视为一个气孔单元发现 ,盾叶秋海棠气孔单元密度 (单位面积中气孔单元数 )和气孔单元大小 (气孔单元所包含气孔数 )在叶片上呈有规律的分布 :前者由叶片中部向叶尖、叶缘逐圈增多 ,而后者逐圈减少。对这种分布格局的成因进行了讨论     

Signalling drought in guard cells

A number of environmental conditions including drought, low humidity, cold and salinity subject plants to osmotic stress. A rapid plant response to such stress conditions is stomatal closure to reduce water loss from plants. From an external stress signal to stomatal closure, many molecular components constitute a signal transduction network that couples the stimulus to the response. Numerous studies have been directed to resolving the framework and molecular details of stress signalling pathways in plants. In guard cells, studies focus on the regulation of ion channels by abscisic acid (ABA), a chemical messenger for osmotic stress. Calcium, protein kinases and phosphatases, and membrane trafficking components have been shown to play a role in ABA signalling process in guard cells. Studies also implicate ABA-independent regulation of ion channels by osmotic stress. In particular, a direct osmosensing pathway for ion channel regulation in guard cells has been identified. These pathways form a complex signalling web that monitors water status in the environment and initiates responses in stomatal movements.     

Natural Variation in Stomatal Responses to Environmental Changes among Arabidopsis thaliana Ecotypes

Stomata are small pores surrounded by guard cells that regulate gas exchange between plants and the atmosphere. Guard cells integrate multiple environmental signals and control the aperture width to ensure appropriate stomatal function for plant survival. Leaf temperature can be used as an indirect indicator of stomatal conductance to environmental signals. In this study, leaf thermal imaging of 374 Arabidopsis ecotypes was performed to assess their stomatal responses to changes in environmental CO2 concentrations. We identified three ecotypes, Köln (Kl-4), Gabelstein (Ga-0), and Chisdra (Chi-1), that have particularly low responsiveness to changes in CO2 concentrations. We next investigated stomatal responses to other environmental signals in these selected ecotypes, with Col-0 as the reference. The stomatal responses to light were also reduced in the three selected ecotypes when compared with Col-0. In contrast, their stomatal responses to changes in humidity were similar to those of Col-0. Of note, the responses to abscisic acid, a plant hormone involved in the adaptation of plants to reduced water availability, were not entirely consistent with the responses to humidity. This study demonstrates that the stomatal responses to CO2 and light share closely associated signaling mechanisms that are not generally correlated with humidity signaling pathways in these ecotypes. The results might reflect differences between ecotypes in intrinsic response mechanisms to environmental signals.