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The plant endosomal trafficking pathway controls the abundance of membrane-associated soluble proteins, as shown for abscisic acid (ABA) receptors of the PYRABACTIN RESISTANCE1/PYR1-LIKE/REGULATORY COMPONENTS OF ABA RECEPTORS (PYR/PYL/RCAR) family. ABA receptor targeting for vacuolar degradation occurs through the late endosome route and depends on FYVE DOMAIN PROTEIN REQUIRED FOR ENDOSOMAL SORTING1 (FYVE1) and VACUOLAR PROTEIN SORTING23A (VPS23A), components of the ENDOSOMAL SORTING COMPLEX REQUIRED FOR TRANSPORT-I (ESCRT-I) complexes. FYVE1 and VPS23A interact with ALG-2 INTERACTING PROTEIN-X (ALIX), an ESCRT-III-associated protein, although the functional relevance of such interactions and their consequences in cargo sorting are unknown. In this study we show that Arabidopsis ( Arabidopsis thaliana ) ALIX directly binds to ABA receptors in late endosomes, promoting their degradation. Impaired ALIX function leads to altered endosomal localization and increased accumulation of ABA receptors. In line with this activity, partial loss-of-function alix-1 mutants display ABA hypersensitivity during growth and stomatal closure, unveiling a role for the ESCRT machinery in the control of water loss through stomata. ABA-hypersensitive responses are suppressed in alix-1 plants impaired in PYR/PYL/RCAR activity, in accordance with ALIX affecting ABA responses primarily by controlling ABA receptor stability. ALIX-1 mutant protein displays reduced interaction with VPS23A and ABA receptors, providing a molecular basis for ABA hypersensitivity in alix-1 mutants. Our findings unveil a negative feedback mechanism triggered by ABA that acts via ALIX to control the accumulation of specific PYR/PYL/RCAR receptors., (© 2019 American Society of Plant Biologists. All rights reserved.)

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 (Pf) 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 Pf 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., (© 2015 American Society of Plant Biologists. All rights reserved.)

Zn is an essential microelement for all living cells and Zn deficiency is widespread in world's population. At the same time, high Zn concentration and low Cd concentration are toxic to the environment. Both Zn and Cd are transported in planta via Zn/Cd HMA transporters. Engineering of HMAs expression in plants may provide a way for Zn biofortification of food as well as phytoremediation of polluted soils. In the present study we have assessed the impact of Zn/Cd HMAs invalidation/overexpression in Arabidopsis thaliana on Zn and Cd translocation from the roots to the shoots and in Zn grain filling. Overexpression of AtHMA4 had a large impact on Zn and Cd translocation and resulted in a 3-fold higher potential of Cd and Zn extraction from an industrial soil highly contaminated by Zn, Pb and Cd. Despite AtHMA4 overexpressing lines presenting a higher Zn concentration in the shoot, the Zn content in the seeds was found to be lower than in wild type plants. Our results indicate that AtHMA4 overexpression is an efficient tool to increase the root to shoot translocation of Zn and Cd in plants. Concerning biofortification of seeds, this study underlines the need for specific promoters to drive an expression pattern of the transporters in favour of Zn grain filling.

Uranium is a natural element which is mainly redistributed in the environment due to human activity, including accidents and spillages. Plants may be useful in cleaning up after incidents, although little is yet known about the relationship between metal speciation and plant response. Here, J-Chess modeling was used to predict U speciation and exposure conditions affecting U bioavailability for plants. The model was confirmed by exposing Arabidopsis thaliana plants to U under hydroponic conditions. The early root response was characterized using complete Arabidopsis transcriptome microarrays (CATMA). Expression of 111 genes was modified at the three timepoints studied. The associated biological processes were further examined by real-time quantitative RT-PCR. Annotation revealed that oxidative stress, cell wall and hormone biosynthesis, and signaling pathways (including phosphate signaling) were affected by U exposure. The main actors in iron uptake and signaling (IRT1, FRO2, AHA2, AHA7 and FIT1) were strongly down-regulated upon exposure to uranyl. A network calculated using IRT1, FRO2 and FIT1 as bait revealed a set of genes whose expression levels change under U stress. Hypotheses are presented to explain how U perturbs the iron uptake and signaling response. These results give preliminary insights into the pathways affected by uranyl uptake, which will be of interest for engineering plants to help clean areas contaminated with U.

Plant mitogen-activated protein kinases (MAPKs) are involved in important processes, including stress signaling and development. In a functional yeast screen, we identified mutations that render Arabidopsis thaliana MAPKs constitutively active (CA). Importantly, CA-MAPKs maintain their specificity toward known activators and substrates. As a proof-of-concept, Arabidopsis MAPK4 (MPK4) function in plant immunity was investigated. In agreement with the phenotype of mpk4 mutants, CA-MPK4 plants were compromised in pathogen-induced salicylic acid accumulation and disease resistance. MPK4 activity was found to negatively regulate pathogen-associated molecular pattern-induced reactive oxygen species production but had no impact on callose deposition, indicating that CA-MPK4 allows discriminating between processes regulated by MPK4 activity from processes indirectly affected by mpk4 mutation. Finally, MPK4 activity was also found to compromise effector-triggered immunity conditioned by the Toll Interleukin-1 Receptor-nucleotide binding (NB)-Leu-rich repeat (LRR) receptors RPS4 and RPP4 but not by the coiled coil-NB-LRR receptors RPM1 and RPS2. Overall, these data reveal important insights on how MPK4 regulates plant defenses and establishes that CA-MAPKs offer a powerful tool to analyze the function of plant MAPK pathways.

Plant hormones, in addition to regulating growth and development, are involved in biotic and abiotic stress responses. To investigate whether a hormone signalling pathway plays a role in the plant response to the heavy metal cadmium (Cd), gene expression data in response to eight hormone treatments were retrieved from the Genevestigator Arabidopsis thaliana database and compared with published microarray analysis performed on plants challenged with Cd. Across more than 3000 Cd-regulated genes, statistical approaches and cluster analyses highlighted that gene expression in response to Cd and brassinosteroids (BR) showed a significant similarity. Of note, over 75% of the genes showing consistent (e.g. opposite) regulation upon BR and Brz (BR biosynthesis inhibitor) exposure exhibited a BR-like response upon Cd exposure. This phenomenon was confirmed by qPCR analysis of the expression level of 10 BR-regulated genes in roots of Cd-treated wild-type (WT) plants. Although no change in BR content was observed in response to Cd in our experimental conditions, adding epibrassinolide (eBL, a synthetic brassinosteroid) to WT plants significantly enhanced Cd-induced root growth inhibition, highlighting a synergistic response between eBL and the metal. This effect was specific to this hormone treatment. On the other hand, dwarf1 seedlings, showing a reduced BR level, exhibited decreased root growth inhibition in response to Cd compared with WT, reversed by the addition of eBL. Similar results were obtained on Brz-treated WT plants. These results argue in favour of an interaction between Cd and BR signalling that modulates plant sensitivity, and opens new perspectives to understand the plant response to Cd.

Molecular chaperones of the heat shock cognate 70 kDa (HSC70) family are highly conserved in all living organisms and assist nascent protein folding in normal physiological conditions as well as in biotic and abiotic stress conditions. In the absence of specific inhibitors or viable knockout mutants, cytosolic/nuclear HSC70-1 overexpression (OE) and mutants in the HSC70 co-chaperone SGT1 (suppressor of G(2)/M allele of skp1) were used as genetic tools to identify HSC70/SGT1 functions in Arabidopsis development and abiotic stress responses. HSC70-1 OE caused a reduction in root and shoot meristem activities, thus explaining the dwarfism of those plants. In addition, HSC70-1 OE did not impair auxin-dependent phenotypes, suggesting that SGT1 functions previously identified in auxin signalling are HSC70 independent. While responses to abiotic stimuli such as UV-C exposure, phosphate starvation, or seedling de-etiolation were not perturbed by HSC70-1 OE, it specifically conferred gamma-ray hypersensitivity and tolerance to salt, cadmium (Cd), and arsenic (As). Cd and As perception was not perturbed, but plants overexpressing HSC70-1 accumulated less Cd, thus providing a possible molecular explanation for their tolerance phenotype. In summary, genetic evidence is provided for HSC70-1 involvement in a limited set of physiological processes, illustrating the essential and yet specific functions of this chaperone in development and abiotic stress responses in Arabidopsis.

In the wild tomato Solanum habrochaites, the Sst2 locus on chromosome 8 is responsible for the biosynthesis of several class II sesquiterpene olefins by glandular trichomes. Analysis of a trichome-specific EST collection from S. habrochaites revealed two candidate genes for the synthesis of Sst2-associated sesquiterpenes. zFPS encodes a protein with homology to Z-isoprenyl pyrophosphate synthases and SBS (for Santalene and Bergamotene Synthase) encodes a terpene synthase with homology to kaurene synthases. Both genes were found to cosegregate with the Sst2 locus. Recombinant zFPS protein catalyzed the synthesis of Z,Z-FPP from isopentenylpyrophosphate (IPP) and dimethylallylpyrophosphate (DMAPP), while coincubation of zFPS and SBS with the same substrates yielded a mixture of olefins identical to the Sst2-associated sesquiterpenes, including (+)-alpha-santalene, (+)-endo-beta-bergamotene, and (-)-endo-alpha-bergamotene. In addition, headspace analysis of tobacco (Nicotiana sylvestris) plants expressing zFPS and SBS in glandular trichomes afforded the same mix of sesquiterpenes. Each of these proteins contains a putative plastid targeting sequence that mediates transport of a fused green fluorescent protein to the chloroplasts, suggesting that the biosynthesis of these sesquiterpenes uses IPP and DMAPP from the plastidic DXP pathway. These results provide novel insights into sesquiterpene biosynthesis and have general implications concerning sesquiterpene engineering in plants.

Oligomer-based DNA Affymetrix GeneChips representing about one-third of Arabidopsis (Arabidopsis thaliana) genes were used to profile global gene expression in a single cell type, guard cells, identifying 1309 guard cell-expressed genes. Highly pure preparations of guard cells and mesophyll cells were isolated in the presence of transcription inhibitors that prevented induction of stress-inducible genes during cell isolation procedures. Guard cell expression profiles were compared with those of mesophyll cells, resulting in identification of 64 transcripts expressed preferentially in guard cells. Many large gene families and gene duplications are known to exist in the Arabidopsis genome, giving rise to redundancies that greatly hamper conventional genetic and functional genomic analyses. The presented genomic scale analysis identifies redundant expression of specific isoforms belonging to large gene families at the single cell level, which provides a powerful tool for functional genomic characterization of the many signaling pathways that function in guard cells. Reverse transcription-PCR of 29 genes confirmed the reliability of GeneChip results. Statistical analyses of promoter regions of abscisic acid (ABA)-regulated genes reveal an overrepresented ABA responsive motif, which is the known ABA response element. Interestingly, expression profiling reveals ABA modulation of many known guard cell ABA signaling components at the transcript level. We further identified a highly ABA-induced protein phosphatase 2C transcript, AtP2C-HA, in guard cells. A T-DNA disruption mutation in AtP2C-HA confers ABA-hypersensitive regulation of stomatal closing and seed germination. The presented data provide a basis for cell type-specific genomic scale analyses of gene function.

Pharmacological studies have led to a model in which the phytohormone abscisic acid (ABA) may be positively transduced via protein phosphatases of the type 1 (PP1) or type 2A (PP2A) families. However, pharmacological evidence also exists that PP1s or PP2As may function as negative regulators of ABA signaling. Furthermore, recessive disruption mutants in protein phosphatases that function in ABA signal transduction have not yet been identified. A guard cell-expressed PP2A gene, RCN1, which had been characterized previously as a molecular component affecting auxin transport and gravity response, was isolated. A T-DNA disruption mutation in RCN1 confers recessive ABA insensitivity to Arabidopsis. The rcn1 mutation impairs ABA-induced stomatal closing and ABA activation of slow anion channels. Calcium imaging analyses show a reduced sensitivity of ABA-induced cytosolic calcium increases in rcn1, whereas mechanisms downstream of cytosolic calcium increases show wild-type responses, suggesting that RCN1 functions in ABA signal transduction upstream of cytosolic Ca(2+) increases. Furthermore, rcn1 shows ABA insensitivity in ABA inhibition of seed germination and ABA-induced gene expression. The PP1 and PP2A inhibitor okadaic acid phenocopies the rcn1 phenotype in wild-type plants both in ABA-induced cytosolic calcium increases and in seed germination, and the wild-type RCN1 genomic DNA complements rcn1 phenotypes. These data show that RCN1 functions as a general positive transducer of early ABA signaling.

In animal cells, ATP binding cassette (ABC) proteins are a large family of transporters that includes the sulfonylurea receptor and the cystic fibrosis transmembrane conductance regulator (CFTR). These two ABC proteins possess an ion channel activity and bind specific sulfonylureas, such as glibenclamide, but homologs have not been identified in plant cells. We recently have shown that there is an ABC protein in guard cells that is involved in the control of stomatal movements and guard cell outward K+ current. Because the CFTR, a chloride channel, is sensitive to glibenclamide and able to interact with K+ channels, we investigated its presence in guard cells. Potent CFTR inhibitors, such as glibenclamide and diphenylamine-2-carboxylic acid, triggered stomatal opening in darkness. The guard cell protoplast slow anion current that was recorded using the whole-cell patch-clamp technique was inhibited rapidly by glibenclamide in a dose-dependent manner; the concentration producing half-maximum inhibition was at 3 &mgr;M. Potassium channel openers, which bind to and act through the sulfonylurea receptor in animal cells, completely suppressed the stomatal opening induced by glibenclamide and recovered the glibenclamide-inhibited slow anion current. Abscisic acid is known to regulate slow anion channels and in our study was able to relieve glibenclamide inhibition of slow anion current. Moreover, in epidermal strip bioassays, the stomatal closure triggered by Ca2+ or abscisic acid was reversed by glibenclamide. These results suggest that the slow anion channel is an ABC protein or is tightly controlled by such a protein that interacts with the abscisic acid signal transduction pathway in guard cells.