Thierry Simonneau

Functional analysis of AtHKT1 in Arabidopsis shows that Na+ recirculation by the phloem is crucial for salt tolerance

Two allelic recessive mutations of Arabidopsis, sas2‐1 and sas2‐2, were identified as inducing sodium overaccumulation in shoots. The sas2 locus was found (by positional cloning) to correspond to the AtHKT1 gene. Expression in Xenopus oocytes revealed that the sas2‐1 mutation did not affect the ionic selectivity of the transporter but strongly reduced the macro scopic (whole oocyte current) transport activity. In Arabidopsis, expression of AtHKT1 was shown to be restricted to the phloem tissues in all organs. The sas2‐1 mutation strongly decreased Na+ concentration in the phloem sap. It led to Na+ overaccumulation in every aerial organ (except the stem), but to Na+ underaccumulation in roots. The sas2 plants displayed increased sensitivity to NaCl, with reduced growth and even death under moderate salinity. The whole set of data indicates that AtHKT1 is involved in Na+ recirculation from shoots to roots, probably by mediating Na+ loading into the phloem sap in shoots and unloading in roots, this recirculation removing large amounts of Na+ from the shoot and playing a crucial role in plant tolerance to salt.

An overview of models of stomatal conductance at the leaf level

Stomata play a key role in plant adaptation to changing environmental conditions as they control both water losses and CO(2) uptake. Particularly, in the context of global change, simulations of the consequences of drought on crop plants are needed to design more efficient and water-saving cropping systems. However, most of the models of stomatal conductance (g(s)) developed at the leaf level link g(s) to environmental factors or net photosynthesis (A(net)), but do not include satisfactorily the effects of drought, impairing our capacity to simulate plant functioning in conditions of limited water supply. The objective of this review was to draw an up-to-date picture of the g(s) models, from the empirical to the process-based ones, along with their mechanistic or deterministic bases. It focuses on models capable to account for multiple environmental influences with emphasis on drought conditions. We examine how models that have been proposed for well-watered conditions can be combined with those specifically designed to deal with drought conditions. Ideas for future improvements of g(s) models are discussed: the issue of co-regulation of g(s) and A(net); the roles of CO(2), absissic acid and H(2)O(2); and finally, how to better address the new challenges arising from the issue of global change.

The Arabidopsis outward K+ channel GORK is involved in regulation of stomatal movements and plant transpiration

Microscopic pores present in the epidermis of plant aerial organs, called stomata, allow gas exchanges between the inner photosynthetic tissue and the atmosphere. Regulation of stomatal aperture, preventing excess transpirational vapor loss, relies on turgor changes of two highly differentiated epidermal cells surrounding the pore, the guard cells. Increased guard cell turgor due to increased solute accumulation results in stomatal opening, whereas decreased guard cell turgor due to decreased solute accumulation results in stomatal closing. Here we provide direct evidence, based on reverse genetics approaches, that the Arabidopsis GORK Shaker gene encodes the major voltage-gated outwardly rectifying K+ channel of the guard cell membrane. Expression of GORK dominant negative mutant polypeptides in transgenic Arabidopsis was found to strongly reduce outwardly rectifying K+ channel activity in the guard cell membrane, and disruption of the GORK gene (T-DNA insertion knockout mutant) fully suppressed this activity. Bioassays on epidermal peels revealed that disruption of GORK activity resulted in impaired stomatal closure in response to darkness or the stress hormone azobenzenearsonate. Transpiration measurements on excised rosettes and intact plants (grown in hydroponic conditions or submitted to water stress) revealed that absence of GORK activity resulted in increased water consumption. The whole set of data indicates that GORK is likely to play a crucial role in adaptation to drought in fluctuating environments.

Drought and Abscisic Acid Effects on Aquaporin Content Translate into Changes in Hydraulic Conductivity and Leaf Growth Rate: A Trans-Scale Approach

The effects of abscisic acid (ABA) on aquaporin content, root hydraulic conductivity (Lpr), whole plant hydraulic conductance, and leaf growth are controversial. We addressed these effects via a combination of experiments at different scales of plant organization and tested their consistency via a model. We analyzed under moderate water deficit a series of transformed maize (Zea mays) lines, one sense and three antisense, affected in NCED (for 9-cis-epoxycarotenoid dioxygenase) gene expression and that differed in the concentration of ABA in the xylem sap. In roots, the mRNA expression of most aquaporin PIP (for plasma membrane intrinsic protein) genes was increased in sense plants and decreased in antisense plants. The same pattern was observed for the protein contents of four PIPs. This resulted in more than 6-fold differences between lines in Lpr under both hydrostatic and osmotic gradients of water potential. This effect was probably due to differences in aquaporin activity, because it was nearly abolished by a hydrogen peroxide treatment, which blocks the water channel activity of aquaporins. The hydraulic conductance of intact whole plants was affected in the same way when measured either in steady-state conditions or via the rate of recovery of leaf water potential after rewatering. The recoveries of leaf water potential and elongation upon rehydration differed between lines and were accounted for by the experimentally measured Lpr in a model of water transfer. Overall, these results suggest that ABA has long-lasting effects on plant hydraulic properties via aquaporin activity, which contributes to the maintenance of a favorable plant water status.

The dual effect of abscisic acid on stomata

The classical view that the drought-related hormone ABA simply acts locally at the guard cell level to induce stomatal closure is questioned by differences between isolated epidermis and intact leaves in stomatal response to several stimuli. We tested the hypothesis that ABA mediates, in addition to a local effect, a remote effect in planta by changing hydraulic regulation in the leaf upstream of the stomata. By gravimetry, porometry to water vapour and argon, and psychrometry, we investigated the effect of exogenous ABA on transpiration, stomatal conductance and leaf hydraulic conductance of mutants described as ABA-insensitive at the guard cell level. We show that foliar transpiration of several ABA-insensitive mutants decreases in response to ABA. We demonstrate that ABA decreases stomatal conductance and down-regulates leaf hydraulic conductance in both the wildtype Col-0 and the ABA-insensitive mutant ost2-2. We propose that ABA promotes stomatal closure in a dual way via its already known biochemical effect on guard cells and a novel, indirect hydraulic effect through a decrease in water permeability within leaf vascular tissues. Variability in sensitivity of leaf hydraulic conductance to ABA among species could provide a physiological basis to the isohydric or anisohydric behaviour.

Control of leaf growth by abscisic acid: hydraulic or non‐hydraulic processes?

Abscisic acid (ABA) affects plant metabolism and water transfers via multiple mechanisms at cell, organ and whole plant levels. These mechanisms translate into contradictory effects on leaf growth, so the literature reports positive, null or negative effects of ABA on leaf growth upon water deficit. We review evidences based on genetic manipulations of ABA biosynthesis, feeding the plant with artificial ABA or partial root drying and provide elements to avoid confusions of effects. We propose that ABA has mainly three effects on growth. (i) Via its controlling effect on stomatal aperture and transpiration rate, an increased concentration of ABA tends to buffer the day-night alternations of leaf growth rate and the negative effect of evaporative demand. (ii) ABA tends to improve leaf growth via an increase in the conductance to water transfer in the plant as a result of increased tissue hydraulic conductivity. (iii) ABA has also a modest non-hydraulic effect which is negative in plants subjected to water deficit, either manipulated for ABA synthesis or fed with artificial ABA, but can be positive in well watered plants deficient of ABA. The overall effect of increasing ABA biosynthesis depends on the relative weight of each of these effects under different environmental scenarios.

Aquaporin-mediated reduction in maize root hydraulic conductivity impacts cell turgor and leaf elongation even without changing transpiration

Root hydraulic conductivity in plants (Lpr) exhibits large variations in response to abiotic stimuli. In this study, we investigated the impact of dynamic, aquaporin-mediated changes of Lpr on leaf growth, water potential, and water flux throughout the plant. For this, we manipulated Lpr by subjecting roots to four independent treatments, with aquaporin inhibitors applied either to transpiring maize (Zea mays) plants grown in hydroponics or to detopped root systems for estimation of Lpr. The treatments were acid load at pH 6.0 and 5.0 and hydrogen peroxide and anoxia applied for 1 to 2 h and subsequently reversed. First, we established that acid load affected cell hydraulic conductivity in maize root cortex. Lpr was reduced by all treatments by 31% to 63%, with half-times of about 15 min, and partly recovered when treatments were reversed. Cell turgor measured in the elongating zone of leaves decreased synchronously with Lpr, and leaf elongation rate closely followed these changes across all treatments in a dose-dependent manner. Leaf and xylem water potentials also followed changes in Lpr. Stomatal conductance and rates of transpiration and water uptake were not affected by Lpr reduction under low evaporative demand. Increased evaporative demand, when combined with acid load at pH 6.0, induced stomatal closure and amplified all other responses without altering their synchrony. Root pressurization reversed the impact of acid load or anoxia on leaf elongation rate and water potential, further indicating that changes in turgor mediated the response of leaf growth to reductions in Lpr.

Coming of leaf age: control of growth by hydraulics and metabolics during leaf ontogeny

Leaf growth is the central process facilitating energy capture and plant performance. This is also one of the most sensitive processes to a wide range of abiotic stresses. Because hydraulics and metabolics are two major determinants of expansive growth (volumetric increase) and structural growth (dry matter increase), we review the interaction nodes between water and carbon. We detail the crosstalks between water and carbon transports, including the dual role of stomata and aquaporins in regulating water and carbon fluxes, the coupling between phloem and xylem, the interactions between leaf water relations and photosynthetic capacity, the links between Lockharts hydromechanical model and carbon metabolism, and the central regulatory role of abscisic acid. Then, we argue that during leaf ontogeny, these interactions change dramatically because of uncoupled modifications between several anatomical and physiological features of the leaf. We conclude that the control of leaf growth switches from a metabolic to a hydromechanical limitation during the course of leaf ontogeny. Finally, we illustrate how taking leaf ontogeny into account provides insights into the mechanisms underlying leaf growth responses to abiotic stresses that affect water and carbon relations, such as elevated CO2, low light, high temperature and drought.

Control of leaf expansion: a developmental switch from metabolics to hydraulics

Leaf expansion is the central process by which plants colonize space, allowing energy capture and carbon acquisition. Water and carbon emerge as main limiting factors of leaf expansion, but the literature remains controversial about their respective contributions. Here, we tested the hypothesis that the importance of hydraulics and metabolics is organized according to both dark/light fluctuations and leaf ontogeny. For this purpose, we established the developmental pattern of individual leaf expansion during days and nights in the model plant Arabidopsis (Arabidopsis thaliana). Under control conditions, decreases in leaf expansion were observed at night immediately after emergence, when starch reserves were lowest. These nocturnal decreases were strongly exaggerated in a set of starch mutants, consistent with an early carbon limitation. However, low-light treatment of wild-type plants had no influence on these early decreases, implying that expansion can be uncoupled from changes in carbon availability. From 4 d after leaf emergence onward, decreases of leaf expansion were observed in the daytime. Using mutants impaired in stomatal control of transpiration as well as plants grown under soil water deficit or high air humidity, we gathered evidence that these diurnal decreases were the signature of a hydraulic limitation that gradually set up as the leaf developed. Changes in leaf turgor were consistent with this pattern. It is concluded that during the course of leaf ontogeny, the predominant control of leaf expansion switches from metabolics to hydraulics. We suggest that the leaf is better armed to buffer variations in the former than in the latter.

RD20, a stress-inducible caleosin, participates in stomatal control, transpiration and drought tolerance in Arabidopsis thaliana

Plants overcome water deficit conditions by combining molecular, biochemical and morphological changes. At the molecular level, many stress-responsive genes have been isolated, but knowledge of their physiological functions remains fragmentary. Here, we report data for RD20, a stress-inducible Arabidopsis gene that belongs to the caleosin family. As for other caleosins, we showed that RD20 localized to oil bodies. Although caleosins are thought to play a role in the degradation of lipids during seed germination, induction of RD20 by dehydration, salt stress and ABA suggests that RD20 might be involved in processes other than germination. Using plants carrying the promoter RD20::uidA construct, we show that RD20 is expressed in leaves, guard cells and flowers, but not in root or in mature seeds. Water deficit triggers a transient increase in RD20 expression in leaves that appeared predominantly dependent on ABA signaling. To assess the biological significance of these data, a functional analysis using rd20 knock-out and overexpressing complemented lines cultivated either in standard or in water deficit conditions was performed. The rd20 knock-out plants present a higher transpiration rate that correlates with enhanced stomatal opening and a reduced tolerance to drought as compared with the wild type. These results support a role for RD20 in drought tolerance through stomatal control under water deficit conditions.