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Date palm Phoenix dactylifera is a desert crop well adapted to survive and produce fruits under extreme drought and heat. How are palms under such harsh environmental conditions able to limit transpirational water loss? Here, we analysed the cuticular waxes, stomata structure and function, and molecular biology of guard cells from P.dactylifera. To understand the stomatal response to the water stress phytohormone of the desert plant, we cloned the major elements necessary for guard cell fast abscisic acid (ABA) signalling and reconstituted this ABA signalosome in Xenopus oocytes. The PhoenixSLAC1-type anion channel is regulated by ABA kinase PdOST1. Energy-dispersive X-ray analysis (EDXA) demonstrated that date palm guard cells release chloride during stomatal closure. However, in Cl(-) medium, PdOST1 did not activate the desert plant anion channel PdSLAC1 perse. Only when nitrate was present at the extracellular face of the anion channel did the OST1-gated PdSLAC1 open, thus enabling chloride release. In the presence of nitrate, ABA enhanced and accelerated stomatal closure. Our findings indicate that, in date palm, the guard cell osmotic motor driving stomatal closure uses nitrate as the signal to open the major anion channel SLAC1. This initiates guard cell depolarization and the release of anions together with potassium.

The Venus flytrap Dionaea muscipula captures insects and consumes their flesh. Prey contacting touch-sensitive hairs trigger traveling electrical waves. These action potentials (APs) cause rapid closure of the trap and activate secretory functions of glands, which cover its inner surface. Such prey-induced haptoelectric stimulation activates the touch hormone jasmonate (JA) signaling pathway, which initiates secretion of an acidic hydrolase mixture to decompose the victim and acquire the animal nutrients. Although postulated since Darwin’s pioneering studies, these secretory events have not been recorded so far. Using advanced analytical and imaging techniques, such as vibrating ion-selective electrodes, carbon fiber amperometry, and magnetic resonance imaging, we monitored stimulus-coupled glandular secretion into the flytrap. Trigger-hair bending or direct application of JA caused a quantal release of oxidizable material from gland cells monitored as distinct amperometric spikes. Spikes reminiscent of exocytotic events in secretory animal cells progressively increased in frequency, reaching steady state 1 d after stimulation. Our data indicate that trigger-hair mechanical stimulation evokes APs. Gland cells translate APs into touch-inducible JA signaling that promotes the formation of secretory vesicles. Early vesicles loaded with H+ and Cl− fuse with the plasma membrane, hyperacidifying the "green stomach"-like digestive organ, whereas subsequent ones carry hydrolases and nutrient transporters, together with a glutathione redox moiety, which is likely to act as the major detected compound in amperometry. Hence, when glands perceive the haptoelectrical stimulation, secretory vesicles are tailored to be released in a sequence that optimizes digestion of the captured animal.

Stomata are microscopic pores found on the surfaces of leaves that act to control CO2 uptake and water loss. By integrating information derived from endogenous signals with cues from the surrounding environment, the guard cells, which surround the pore, 'set' the stomatal aperture to suit the prevailing conditions. Much research has concentrated on understanding the rapid intracellular changes that result in immediate changes to the stomatal aperture. In this study, we look instead at how stomata acclimate to longer timescale variations in their environment. We show that the closure-inducing signals abscisic acid (ABA), increased CO2, decreased relative air humidity and darkness each access a unique gene network made up of clusters (or modules) of common cellular processes. However, within these networks some gene clusters are shared amongst all four stimuli. All stimuli modulate the expression of members of the PYR/PYL/RCAR family of ABA receptors. However, they are modulated differentially in a stimulus-specific manner. Of the six members of the PYR/PYL/RCAR family expressed in guard cells, PYL2 is sufficient for guard cell ABA-induced responses, whereas in the responses to CO2, PYL4 and PYL5 are essential. Overall, our work shows the importance of ABA as a central regulator and integrator of long-term changes in stomatal behaviour, including sensitivity, elicited by external signals. Understanding this architecture may aid in breeding crops with improved water and nutrient efficiency. ispartof: NATURE PLANTS vol:5 issue:9 pages:1002-1011 ispartof: location:England status: published

Highlight bam1 mutant plants impaired in stomatal starch degradation showed an improved drought tolerance associated with a down-regulation of guard cell-specific gene expression involved in water uptake and cell expansion., In plants, drought stress is a major growth limiting factor causing cell water loss through open stomata. In this study, guard cell-specific transcripts from drought-stressed Arabidopsis plants were analysed and a down-regulation of β-amylase 1 (BAM1) was found. In previous studies, BAM1 was shown to be involved in stomatal starch degradation under ambient conditions. Impaired starch breakdown of bam1 mutant plants was accompanied by decreased stomatal opening. Here, it is shown that drought tolerance of bam1 mutant plants is improved as compared with wild-type controls. Microarray analysis of stomata-specific transcripts from bam1 mutant plants revealed a significant down-regulation of genes encoding aquaporins, auxin- and ethylene-responsive factors, and cell-wall modifying enzymes. This expression pattern suggests that reduced water uptake and limited cell wall extension are associated with the closed state of stomata of bam1 mutant plants. Together these data suggest that regulation of stomata-specific starch turnover is important for adapting stomata opening to environmental needs and its breeding manipulation may result in drought tolerant crop plants.

SummaryStomata are pores on the leaf surface, bounded by two guard cells, which control the uptake of CO2 for photosynthesis and the concomitant loss of water vapor. In 1898, Francis Darwin [1] showed that stomata close in response to reduced atmospheric relative humidity (rh); however, our understanding of the signaling pathway responsible for coupling changes in rh to alterations in stomatal aperture is fragmentary. The results presented here highlight the primacy of abscisic acid (ABA) in the stomatal response to drying air. We show that guard cells possess the entire ABA biosynthesis pathway and that it appears upregulated by positive feedback by ABA. When wild-type Arabidopsis and the ABA-deficient mutant aba3-1 were exposed to reductions in rh, the aba3-1 mutant wilted, whereas the wild-type did not. However, when aba3-1 plants, in which ABA synthesis had been specifically rescued in guard cells, were challenged with dry air, they did not wilt. These data indicate that guard cell-autonomous ABA synthesis is required for and is sufficient for stomatal closure in response to low rh. Guard cell-autonomous ABA synthesis allows the plant to tailor leaf gas exchange exquisitely to suit the prevailing environmental conditions.

Stomatal opening and closing are driven by ion fluxes that cause changes in guard cell turgor and volume. This process is, in turn, regulated by environmental and hormonal signals, including light and the phytohormone abscisic acid (ABA). Here, we present genetic evidence that expression of PHO1 in guard cells of Arabidopsis thaliana is required for full stomatal responses to ABA. PHO1 is involved in the export of phosphate into the root xylem vessels and, as a result, the pho1 mutant is characterized by low shoot phosphate levels. In leaves, PHO1 was found expressed in guard cells and up-regulated following treatment with ABA. The pho1 mutant was unaffected in production of reactive oxygen species following ABA treatment, and in stomatal movements in response to light cues, high extracellular calcium, auxin, and fusicoccin. However, stomatal movements in response to ABA treatment were severely impaired, both in terms of induction of closure and inhibition of opening. Micro-grafting a pho1 shoot scion onto wild-type rootstock resulted in plants with normal shoot growth and phosphate content, but failed to restore normal stomatal response to ABA treatment. PHO1 knockdown using RNA interference specifically in guard cells of wild-type plants caused a reduced stomatal response to ABA. In agreement, specific expression of PHO1 in guard cells of pho1 plants complemented the mutant guard cell phenotype and re-established ABA sensitivity, although full functional complementation was dependent on shoot phosphate sufficiency. Together, these data reveal an important role for phosphate and the action of PHO1 in the stomatal response to ABA.

International audience; Stomatal opening and closing are driven by ion fluxes that cause changes in guard cell turgor and volume. This process is, in turn, regulated by environmental and hormonal signals, including light and the phytohormone abscisic acid (ABA). Here, we present genetic evidence that expression of $PHO1$ in guard cells of Arabidopsis thaliana is required for full stomatal responses to ABA. $PHO1$ is involved in the export of phosphate into the root xylem vessels and, as a result, the $pho1$ mutant is characterized by low shoot phosphate levels. In leaves, $PHO1$ was found expressed in guard cells and up‐regulated following treatment with ABA. The $pho1$ mutant was unaffected in production of reactive oxygen species following ABA treatment, and in stomatal movements in response to light cues, high extracellular calcium, auxin, and fusicoccin. However, stomatal movements in response to ABA treatment were severely impaired, both in terms of induction of closure and inhibition of opening. Micro‐grafting a $pho1$ shoot scion onto wild‐type rootstock resulted in plants with normal shoot growth and phosphate content, but failed to restore normal stomatal response to ABA treatment. $PHO1$ knockdown using RNA interference specifically in guard cells of wild‐type plants caused a reduced stomatal response to ABA. In agreement, specific expression of $PHO1$ in guard cells of $pho1$ plants complemented the mutant guard cell phenotype and re‐established ABA sensitivity, although full functional complementation was dependent on shoot phosphate sufficiency. Together, these data reveal an important role for phosphate and the action of $PHO1$ in the stomatal response to ABA.

*† SUMMARY Uptake of CO2 by the leaf is associated with loss of water. Control of stomatal aperture by volume changes of guard cell pairs optimizes the efficiency of water use. Under water stress, the protein kinase OPEN STOMATA 1 (OST1) activates the guard-cell anion release channel SLOW ANION CHANNEL-ASSOCIATED 1 (SLAC1), and thereby triggers stomatal closure. Plants with mutated OST1 and SLAC1 are defective in guard-cell turgor regulation. To study the effect of stomatal movement on leaf turgor using intact leaves of Arabidopsis, we used a new pressure probe to monitor transpiration and turgor pressure simultaneously and non-invasively. This probe permits routine easy access to parameters related to water status and stomatal conductance under physiological conditions using the model plant Arabidopsis thaliana. Long-term leaf turgor pressure recordings over several weeks showed a drop in turgor during the day and recovery at night. Thus pressure changes directly correlated with the degree of plant transpiration. Leaf turgor of wild-type plants responded to CO2, light, humidity, ozone and abscisic acid (ABA) in a guard cell-specific manner. Pressure probe measurements of mutants lacking OST1 and SLAC1 function indicated impairment in stomatal responses to light and humidity. In contrast to wild-type plants, leaves from well-watered ost1 plants exposed to a dry atmosphere wilted after light-induced stomatal opening. Experiments with open stomata mutants indicated that the hydraulic conductance of leaf stomata is higher than that of the root–shoot continuum. Thus leaf turgor appears to rely to a large extent on the anion channel activity of autonomously regulated stomatal guard cells.

Mobilities of selected pesticides in plant cuticles have been measured using unilateral desorption from the outer surface (UDOS). The outer layer of the cuticle limits the rate of foliar uptake and solute mobilities in this layer have been determined. The primary parameter measured was the first-order rate constant of desorption. From these rate constants, permeances, half-times of penetration and diffusion coefficients could be calculated. Rate constants were lowest in bitter-orange (Citrus aurantium L.) leaf cuticles and this cuticle turned out to be very size-selective, as rate constants decreased by a factor of 264 when molar volumes increased by only a factor of about three. Rate constants measured using green-pepper (Capsicum annuum L. cv. Bell Boy) fruit cuticles were larger by one to two orders of magnitude and this cuticle was considerably less size-selective. A good correlation between rate constants and molar volumes was observed for cuticles from both plant species, which means that rate constants can be estimated from molar volumes of pesticides. Rate constants of desorption are mobility parameters which do not depend on lipophilicity of solutes. Thus, it is now possible to separate the contributions of mobilities and solubilities in cuticles to rates of foliar uptake of pesticides.

In response to drought stress the phytohormone ABA (abscisic acid) induces stomatal closure and, therein, activates guard cell anion channels in a calcium-dependent as well as-independent manner. Two key components of the ABA signaling pathway are the protein kinase OST1 (open stomata 1) and the protein phosphatase ABI1 (ABA insensitive 1). The recently identified guard cell anion channel SLAC1 appeared to be the key ion channel in this signaling pathway but remained electrically silent when expressed heterologously. Using split YFP assays, we identified OST1 as an interaction partner of SLAC1 and ABI1. Upon coexpression of SLAC1 with OST1 in Xenopus oocytes, SLAC1-related anion currents appeared similar to those observed in guard cells. Integration of ABI1 into the SLAC1/OST1 complex, however, prevented SLAC1 activation. Our studies demonstrate that SLAC1 represents the slow, deactivating, weak voltage-dependent anion channel of guard cells controlled by phosphorylation/dephosphorylation.

Solute mobilities of 28 compounds in isolated cuticular membranes (CM) from Capsicum annuum L. fruit, Citrus aurantium L. and Pyrus communis L. leaves were studied using unilateral desorption from the outer surface. First-order rate constants of desorption (k*), which are directly proportional to the diffusion coefficient in the waxy outer limiting skins of cuticles were measured. When log k* was plotted vs. molar volumes of test compounds linear graphs were obtained. The y-intercepts of these graphs (k*∘) represent the mobility of a hypothetical molecule having zero molar volume and the slopes of the graphs (β′) represent the size selectivity of the barrier and are related to the free volume available for diffusion. Thus, solute mobilities in cuticles are composed of two independent terms which are subtractive. If k*∘ and β′ are known, k* can be estimated for any solute from its molar volume (Vx) using the equation log k*=log k*∘ −β′Vx. These parameters were used to analyse the effects of plant species, extraction of cuticular waxes and molecular structure of solutes on solute mobilities in plant cuticles. For aliphatic solutes, k*∘ was a factor of 10 smaller than for cyclic compounds, while β′ was 0.011 and 0.012, respectively. The k*-values for CM of the three species were very similar, but β′ was higher for bitter-orange CM (0.012) than for those of pepper fruits and pear leaves (0.009). This has the consequence that differences in solute mobilities (k*∘) among cuticles from different plan species increase with increasing molar volumes of solutes. Our data and our analysis provide evidence that constituents of cuticular waxes are mobile, at least in the solid amorphous wax fraction, but mobility decreases rapidly with increasing molar volume. For instance, if β′ amounts to 0.01, mobilities of wax monomers decrease by a factor of 10 for every increase in molar volume of 100 cm3 · mol−1. Thus, hexadecanoic acid is quite mobile in the amorphous wax fraction of Citrus (k*=1.5×10−6·s−1), but for dotriacontane having twice the molar volume, k* was only 2.5×10−9·s−1, which is almost three orders of magnitude smaller. Wax esters have even higher molar volumes and their mobilities will be even smaller (about 4×10−12·s−1 for a C48-ester). Since low chain mobilities are a prerequisite for low mobilities and permeabilities, the selective advantage of high-molecular-weight wax monomers in plant cuticular waxes becomes obvious. Extracting cuticular waxes from pear leaf CM increased solute mobilities by a factor of 182, but it had no effect on size selectivity. We interpret this result as evidence to the effect that cuticular waxes reduce mobility by increasing tortuosity of the diffusion path, rather than by decreasing the mean free path of diffusional jumps and jump frequencies of diffusants.