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Because of intense transpiration and growth, the needs of plants for water can be immense. Yet water in the soil is most often heterogeneous if not scarce due to more and more frequent and intense drought episodes. The converse context, flooding, is often associated with marked oxygen deficiency and can also challenge the plant water status. Under our feet, roots achieve an incredible challenge to meet the water demand of the plant’s aerial parts under such dramatically different environmental conditions. For this, they continuously explore the soil, building a highly complex, branched architecture. On shorter time scales, roots keep adjusting their water transport capacity (their so-called hydraulics) locally or globally. While the mechanisms that directly underlie root growth and development as well as tissue hydraulics are being uncovered, the signalling mechanisms that govern their local and systemic adjustments as a function of water availability remain largely unknown. A comprehensive understanding of root architecture and hydraulics as a whole (in other terms, root hydraulic architecture) is needed to apprehend the strategies used by plants to optimize water uptake and possibly improve crops regarding this crucial trait. One consequence of climate change is an increased frequency of flood and drought episodes. This Perspective explores how water availability regulates root architecture and water transport capacity (hydraulics), from sensing mechanisms to novel responses.

Root water uptake is influenced by root system architecture, which is determined by root growth and branching and the hydraulics of root cells and tissues. The phytohormone abscisic acid (ABA) plays a major role in the adaptation of plants to water deficit (WD). Here we addressed at the whole-root level in Arabidopsis (Arabidopsis thaliana) the regulatory role of ABA in mechanisms that determine root hydraulic architecture. Root system architecture and root hydraulic conductivity (Lpr) were analyzed in hydroponically grown plants subjected to varying degrees of WD induced by various polyethylene glycol (PEG) concentrations. The majority of root traits investigated, including first- and second-order lateral root production and elongation and whole-root hydraulics, had a bell-shaped dependency on WD, displaying stimulation under mild WD conditions (25 g PEG L²¹) and repression under more severe conditions. These traits also showed a bell-shaped dependency on exogenous ABA, and their regulation by WD was attenuated in genotypes altered in ABA biosynthesis and response. Thus, we propose that ABA acts as a coordinator and an integrator of most root responses to mild and moderate WD, whereas responses to strong WD (150 g PEG L²¹) are largely ABA independent. We also found that roots exhibit different growth responses to both WD and ABA depending on their rank and age. Taken together, our results give further insights into the coordinated water acquisition strategies of roots deployed in relation to WD intensity.

Stomatal movements in response to environmental stimuli critically control the plant water status. Although these movements are governed by osmotically driven changes in guard cell volume, the role of membrane water channels (aquaporins) has remained hypothetical. Assays in epidermal peels showed that knockout Arabidopsis thaliana plants lacking the Plasma membrane Intrinsic Protein 2;1 (PIP2;1) aquaporin have a defect in stomatal closure, specifically in response to abscisic acid (ABA). ABA induced a 2-fold increase in osmotic water permeability (P f) of guard cell protoplasts and an accumulation of reactive oxygen species in guard cells, which were both abrogated in pip2;1 plants. Open stomata 1 (OST1)/Snf1-related protein kinase 2.6 (SnRK2.6), a protein kinase involved in guard cell ABA signaling, was able to phosphorylate a cytosolic PIP2;1 peptide at Ser-121. OST1 enhanced PIP2;1 water transport activity when coexpressed in Xenopus laevis oocytes. Upon expression in pip2;1 plants, a phosphomimetic form (Ser121Asp) but not a phosphodeficient form (Ser121Ala) of PIP2;1 constitutively enhanced the P f of guard cell protoplasts while suppressing its ABA-dependent activation and was able to restore ABA-dependent stomatal closure in pip2;1. This work supports a model whereby ABA-triggered stomatal closure requires an increase in guard cell permeability to water and possibly hydrogen peroxide, through OST1-dependent phosphorylation of PIP2;1 at Ser-121.

Soil water uptake by roots is a key component of plant performance and adaptation to adverse environments. Here, we use a genome-wide association analysis to identify the XYLEM NAC DOMAIN 1 (XND1) transcription factor as a negative regulator of Arabidopsis root hydraulic conductivity ( L p r ). The distinct functionalities of a series of natural XND1 variants and a single nucleotide polymorphism that determines XND1 translation efficiency demonstrate the significance of XND1 natural variation at species-wide level. Phenotyping of xnd1 mutants and natural XND1 variants show that XND1 modulates L p r through action on xylem formation and potential indirect effects on aquaporin function and that it diminishes drought stress tolerance. XND1 also mediates the inhibition of xylem formation by the bacterial elicitor flagellin and counteracts plant infection by the root pathogen Ralstonia solanacearum . Thus, genetic variation at XND1 , and xylem differentiation contribute to resolving the major trade-off between abiotic and biotic stress resistance in Arabidopsis . Soil water uptake is a major determinant of plant performance and stress tolerance. Here the authors show that, by affecting xylem formation in the root, natural variation at the Arabidopsis XND1 locus has contrasting effects on root hydraulics and drought tolerance versus pathogen resistance.

The endodermis is a key cell layer in plant roots that contributes to the controlled uptake of water and mineral nutrients into plants. In order to provide such functionality the endodermal cell wall has specific chemical modifications consisting of lignin bands (Casparian strips) that encircle each cell, and deposition of a waxy-like substance (suberin) between the wall and the plasma membrane. These two extracellular deposits provide control of diffusion enabling the endodermis to direct the movement of water and solutes into and out of the vascular system in roots. Loss of integrity of the Casparian strip-based apoplastic barrier is sensed by the leakage of a small peptide from the stele into the cortex. Here, we report that such sensing of barrier integrity leads to the rebalancing of water and mineral nutrient uptake, compensating for breakage of Casparian strips. This rebalancing involves both a reduction in root hydraulic conductivity driven by deactivation of aquaporins, and downstream limitation of ion leakage through deposition of suberin. These responses in the root are also coupled to a reduction in water demand in the shoot mediated by ABA-dependent stomatal closure.

A key impediment to studying water-related mechanisms in plants is the inability to non-invasively image water fluxes in cells at high temporal and spatial resolution. Here, we report that Raman microspectroscopy, complemented by hydrodynamic modelling, can achieve this goal - monitoring hydrodynamics within living root tissues at cell- and sub-second-scale resolutions. Raman imaging of water-transporting xylem vessels in Arabidopsis thaliana mutant roots reveals faster xylem water transport in endodermal diffusion barrier mutants. Furthermore, transverse line scans across the root suggest water transported via the root xylem does not re-enter outer root tissues nor the surrounding soil when en-route to shoot tissues if endodermal diffusion barriers are intact, thereby separating ‘two water worlds’. Existing methods for non-invasively monitoring water flow in plants have limited spatial/temporal resolution. Here, the authors report that Raman microspectroscopy, complemented by hydrodynamic modelling, can monitor hydrodynamics within living root tissues at cell- and sub-second-scale resolutions.

The endodermis represents the main barrier to extracellular diffusion in plant roots, and it is central to current models of plant nutrient uptake. Despite this, little is known about the genes setting up this endodermal barrier. In this study, we report the identification and characterization of a strong barrier mutant, schengen3 (sgn3). We observe a surprising ability of the mutant to maintain nutrient homeostasis, but demonstrate a major defect in maintaining sufficient levels of the macronutrient potassium. We show that SGN3/GASSHO1 is a receptor-like kinase that is necessary for localizing CASPARIAN STRIP DOMAIN PROTEINS (CASPs)—major players of endodermal differentiation—into an uninterrupted, ring-like domain. SGN3 appears to localize into a broader band, embedding growing CASP microdomains. The discovery of SGN3 strongly advances our ability to interrogate mechanisms of plant nutrient homeostasis and provides a novel actor for localized microdomain formation at the endodermal plasma membrane. Plant roots forage in the soil for minerals and water, but they must also provide a barrier that stops these nutrients leaking back out of the plant and stops microbes invading and causing disease. The endodermis—an inner layer of cells that surrounds the veins that run along the middle of a root—acts as such a barrier in young roots. Polymers that repel water are deposited between the cells in the roots of almost all vascular plants—which include ferns, conifers, and flowering plants—to form a band around the endodermis called the ‘Casparian strip’. This strip seals off the young roots and stops water moving through the gaps between plant cells, but still allows minerals, nutrients, and water to be transported through the root cells and into the plant. However, the importance of this structure has yet to be tested due to the lack of mutant plants without a Casparian strip. Pfister et al. now report that deleting the gene that encodes a protein called SCHENGEN3 in the model plant Arabidopsis thaliana causes the Casparian strip to be interrupted by irregularly sized holes. This protein is normally found at high levels in the root endodermis, where it is embedded into the cell membranes. Pfister et al. also showed that without the SCHENGEN3 protein, other proteins called CASPs—that normally mark out a stripe around the root cells where the Casparian strip will form—only accumulated in discontinuous patches. Further experiments revealed that deleting the gene for SCHENGEN3 does not cause general problems in delivering the CASP proteins to the cell membrane; instead, it specifically stops the CASP proteins from forming a single, uninterrupted stripe. Unexpectedly, disrupting the Casparian strip did not appear to hinder many of the functions of a root. The mutant plants could still take up water and nutrients, and the leaves of mutant plants had normal levels of many essential minerals—with the exception of potassium. The level of this mineral was much lower in mutant plants without the SCHENGEN3 protein. Pfister et al. suggest that in plants that lack an intact Casparian strip, potassium is continuously leaked from the root into the soil. These findings reveal that in Arabidopsis, at least, the Casparian strip might not be as important as once thought for helping the plant to take up and accumulate water and nutrients. Further work is now needed to uncover the as yet unknown backup systems that might be able to compensate for the loss of this structure.

PIP2;1 is an integral membrane protein that facilitates water transport across plasma membranes. To address the dynamics of Arabidopsis thaliana PIP2;1 at the single-molecule level as well as their role in PIP2;1 regulation, we tracked green fluorescent protein-PIP2;1 molecules by variable-angle evanescent wave microscopy and fluorescence correlation spectroscopy (FCS). Single-particle tracking analysis revealed that PIP2;1 presented four diffusion modes with large dispersion of diffusion coefficients, suggesting that partitioning and dynamics of PIP2;1 are heterogeneous and, more importantly, that PIP2;1 can move into or out of membrane microdomains. In response to salt stress, the diffusion coefficients and percentage of restricted diffusion increased, implying that PIP2;1 internalization was enhanced. This was further supported by the decrease in PIP2;1 density on plasma membranes by FCS. We additionally demonstrated that PIP2;1 internalization involves a combination of two pathways: a tyrphostin A23-sensitive clathrin-dependent pathway and a methyl-β-cyclodextrin-sensitive, membrane raft-associated pathway. The latter was efficiently stimulated under NaCI conditions. Taken together, our findings demonstrate that PIP2;1 molecules are heterogeneously distributed on the plasma membrane and that clathrin and membrane raft pathways cooperate to mediate the subcellular trafficking of PIP2;1, suggesting that the dynamic partitioning and recycling pathways might be involved in the multiple modes of regulating water permeability.

To gain insights into the natural variation of root hydraulics and its molecular components, genotypic differences related to root water transport and plasma membrane intrinsic protein (PIP) aquaporin expression were investigated in 13 natural accessions of Arabidopsis (Arabidopsis thaliana). The hydraulic conductivity of excised root systems (Lpr) showed a 2-fold variation among accessions. The contribution of aquaporins to water uptake was characterized using as inhibitors mercury, propionic acid, and azide. The aquaporin-dependent and -independent paths of water transport made variable contributions to the total hydraulic conductivity in the different accessions. The distinct suberization patterns observed among accessions were not correlated with their root hydraulic properties. Real-time reverse transcription-polymerase chain reaction revealed, by contrast, a positive overall correlation between Lpr and certain highly expressed PIP transcripts. Root hydraulic responses to salt stress were characterized in a subset of five accessions (Bulhary-1, Catania-1, Columbia-0, Dijon-M, and Monte-Tosso-0 [Mr-0]). Lpr was down-regulated in all accessions except Mr-0. In Mr-0 and Catania-1, cortical cell hydraulic conductivity was unresponsive to salt, whereas it was down-regulated in the three other accessions. By contrast, the five accessions showed qualitatively similar aquaporin transcriptional profiles in response to salt. The overall work provides clues on how hydraulic regulation allows plant adaptation to salt stress. It also shows that a wide range of root hydraulic profiles, as previously reported in various species, can be observed in a single model species. This work paves the way for a quantitative genetics analysis of root hydraulics.

The circadian clock regulates plant tissue hydraulics to synchronize water supply with environmental cycles and thereby optimize growth. The circadian fluctuations in aquaporin transcript abundance suggest that aquaporin water channels play a role in these processes. Here, we show that hydraulic conductivity (K ros) of Arabidopsis (Arabidopsis thaliana) rosettes displays a genuine circadian rhythmicity with a peak around midday. Combined immunological and proteomic approaches revealed that phosphorylation at two C-terminal sites (Ser280, Ser283) of PLASMA MEMBRANE INTRINSIC PROTEIN 2;1 (AtPIP2;1), a major plasma membrane aquaporin in rosettes, shows circadian oscillations and is correlated with K ros. Transgenic expression of phosphodeficient and phosphomimetic forms of this aquaporin indicated that AtPIP2;1 phosphorylation is necessary but not sufficient for K ros regulation. We investigated the supporting role of 14-3-3 proteins, which are known to interact with and regulate phosphorylated proteins. Individual knockout plants for five 14-3-3 protein isoforms expressed in rosettes lacked circadian activation of K ros. Two of these [GRF4 (14-3-3Phi); GRF10 (14-3-3Epsilon)] showed direct interactions with AtPIP2;1 in the plant and upon coexpression in Xenopus laevis oocytes and activated AtPIP2;1, preferentially when the latter was phosphorylated at its two C-terminal sites. We propose that this regulatory mechanism assists in the activation of phosphorylated AtPIP2;1 during circadian regulation of K ros.