S-acylated and nucleus-localized SALT OVERLY SENSITIVE3/CALCINEURIN B-LIKE4 stabilizes GIGANTEA to regulate Arabidopsis flowering time under salt stress.

The precise timing of flowering in adverse environments is critical for plants to secure reproductive success. We report a mechanism in Arabidopsis (Arabidopsis thaliana) controlling the time of flowering by which the S-acylation-dependent nuclear import of the protein SALT OVERLY SENSITIVE3/CALCINEURIN B-LIKE4 (SOS3/CBL4), a Ca2+-signaling intermediary in the plant response to salinity, results in the selective stabilization of the flowering time regulator GIGANTEA inside the nucleus under salt stress, while degradation of GIGANTEA in the cytosol releases the protein kinase SOS2 to achieve salt tolerance. S-acylation of SOS3 was critical for its nuclear localization and the promotion of flowering, but partly dispensable for salt tolerance. SOS3 interacted with the photoperiodic flowering components GIGANTEA and FLAVIN-BINDING, KELCH REPEAT, F-BOX1 and participated in the transcriptional complex that regulates CONSTANS to sustain the transcription of CO and FLOWERING LOCUS T under salinity. Thus, the SOS3 protein acts as a Ca2+- and S-acylation-dependent versatile regulator that fine-tunes flowering time in a saline environment through the shared spatial separation and selective stabilization of GIGANTEA, thereby connecting two signaling networks to co-regulate the stress response and the time of flowering.

Non-CG DNA methylation-deficiency mutations enhance mutagenesis rates during salt adaptation in cultured Arabidopsis cells.

Much has been learned about how plants acclimate to stressful environments, but the molecular basis of stress adaptation and the potential involvement of epigenetic regulation remain poorly understood. Here, we examined if salt stress induces mutagenesis in suspension cultured plant cells and if DNA methylation affects the mutagenesis using whole genome resequencing analysis. We generated suspension cell cultures from two Arabidopsis DNA methylation-deficient mutants and wild-type plants, and subjected the cultured cells to stepwise increases in salt stress intensity over 40 culture cycles. We show that ddc (drm1 drm2 cmt3) mutant cells can adapt to grow in 175 mM NaCl-containing growth medium and exhibit higher adaptability compared to wild type Col-0 and nrpe1 cells, which can adapt to grow in only 125 mM NaCl-containing growth medium. Salt treated nrpe1 and ddc cells but not wild type cells accumulate more mutations compared with their respective untreated cells. There is no enrichment of stress responsive genes in the list of mutated genes in salt treated cells compared to the list of mutated genes in untreated cells. Our results suggest that DNA methylation prevents the induction of mutagenesis by salt stress in plant cells during stress adaptation.

BONZAI Proteins Control Global Osmotic Stress Responses in Plants.

Hyperosmotic stress caused by drought and salinity is a significant environmental threat that limits plant growth and agricultural productivity. Osmotic stress induces diverse responses in plants including Ca2+ signaling, accumulation of the stress hormone abscisic acid (ABA), reprogramming of gene expression, and altering of growth. Despite intensive investigation, no global regulators of all of these responses have been identified. Here, we show that the Ca2+-responsive phospholipid-binding BONZAI (BON) proteins are critical for all of these osmotic stress responses. A Ca2+-imaging-based forward genetic screen identified a loss-of-function bon1 mutant with a reduced cytosolic Ca2+ signal in response to hyperosmotic stress. The loss-of-function mutants of the BON1 gene family, bon1bon2bon3, are impaired in the induction of gene expression and ABA accumulation in response to osmotic stress. In addition, the bon mutants are hypersensitive to osmotic stress in growth inhibition. BON genes have been shown to negatively regulate plant immune responses mediated by intracellular immune receptor NLR genes including SNC1. We found that the defects of the bon mutants in osmotic stress responses were suppressed by mutations in the NLR gene SNC1 or the immunity regulator PAD4. Our findings indicate that NLR signaling represses osmotic stress responses and that BON proteins suppress NLR signaling to enable global osmotic stress responses in plants.

Abscisic acid dynamics, signaling, and functions in plants.

Abscisic acid (ABA) is an important phytohormone regulating plant growth, development, and stress responses. It has an essential role in multiple physiological processes of plants, such as stomatal closure, cuticular wax accumulation, leaf senescence, bud dormancy, seed germination, osmotic regulation, and growth inhibition among many others. Abscisic acid controls downstream responses to abiotic and biotic environmental changes through both transcriptional and posttranscriptional mechanisms. During the past 20 years, ABA biosynthesis and many of its signaling pathways have been well characterized. Here we review the dynamics of ABA metabolic pools and signaling that affects many of its physiological functions.

AtPR5K2, a PR5-Like Receptor Kinase, Modulates Plant Responses to Drought Stress by Phosphorylating Protein Phosphatase 2Cs.

Cell surface receptors perceive signals from the environment and transfer them to the interior of the cell. The Arabidopsis thaliana PR5 receptor-like kinase (AtPR5K) subfamily consists of three members with extracellular domains that share sequence similarity with the PR5 proteins. In this study, we characterized the role of AtPR5K2 in plant drought-stress signaling. AtPR5K2 is predominantly expressed in leaves and localized to the plasma membrane. The atpr5k2-1 mutant showed tolerance to dehydration stress, while AtPR5K2-overexpressing plants was hypersensitive to drought. Bimolecular fluorescence complementation assays showed that AtPR5K2 physically interacted with the type 2C protein phosphatases ABA-insensitive 1 (ABI1) and ABI2 and the SNF1-related protein kinase 2 (SnRK2.6) proteins, all of which are involved in the initiation of abscisic acid (ABA) signaling; however, these interactions were inhibited by treatments of exogenous ABA. Moreover, AtPR5K2 was found to phosphorylate ABI1 and ABI2, but not SnRK2.6. Taken together, these results suggest that AtPR5K2 participates in ABA-dependent drought-stress signaling through the phosphorylation of ABI1 and ABI2.

Rheostatic Control of ABA Signaling through HOS15-Mediated OST1 Degradation.

Dehydrating stresses trigger the accumulation of abscisic acid (ABA), a key plant stress-signaling hormone that activates Snf1-Related Kinases (SnRK2s) to mount adaptive responses. However, the regulatory circuits that terminate the SnRK2s signal relay after acclimation or post-stress conditions remain to be defined. Here, we show that the desensitization of the ABA signal is achieved by the regulation of OST1 (SnRK2.6) protein stability via the E3-ubiquitin ligase HOS15. Upon ABA signal, HOS15-induced degradation of OST1 is inhibited and stabilized OST1 promotes the stress response. When the ABA signal terminates, protein phosphatases ABI1/2 promote rapid degradation of OST1 via HOS15. Notably, we found that even in the presence of ABA, OST1 levels are also depleted within hours of ABA signal onset. The unexpected dynamics of OST1 abundance are then resolved by systematic mathematical modeling, demonstrating a desensitizing feedback loop by which OST1-induced upregulation of ABI1/2 leads to the degradation of OST1. This model illustrates the complex rheostat dynamics underlying the ABA-induced stress response and desensitization.

Role and Functional Differences of HKT1-Type Transporters in Plants under Salt Stress.

Abiotic stresses generally cause a series of morphological, biochemical and molecular changes that unfavorably affect plant growth and productivity. Among these stresses, soil salinity is a major threat that can seriously impair crop yield. To cope with the effects of high salinity on plants, it is important to understand the mechanisms that plants use to deal with it, including those activated in response to disturbed Na⁺ and K⁺ homeostasis at cellular and molecular levels. HKT1-type transporters are key determinants of Na⁺ and K⁺ homeostasis under salt stress and they contribute to reduce Na⁺-specific toxicity in plants. In this review, we provide a brief overview of the function of HKT1-type transporters and their importance in different plant species under salt stress. Comparison between HKT1 homologs in different plant species will shed light on different approaches plants may use to cope with salinity.

Metabolic Adjustment of Arabidopsis Root Suspension Cells During Adaptation to Salt Stress and Mitotic Stress Memory.

Sessile plants reprogram their metabolic and developmental processes during adaptation to prolonged environmental stresses. To understand the molecular mechanisms underlying adaptation of plant cells to saline stress, we established callus suspension cell cultures from Arabidopsis roots adapted to high salt for an extended period of time. Adapted cells exhibit enhanced salt tolerance compared with control cells. Moreover, acquired salt tolerance is maintained even after the stress is relieved, indicating the existence of a memory of acquired salt tolerance during mitotic cell divisions, known as mitotic stress memory. Metabolite profiling using 1H-nuclear magnetic resonance (NMR) spectroscopy revealed metabolic discrimination between control, salt-adapted and stress-memory cells. Compared with control cells, salt-adapted cells accumulated higher levels of sugars, amino acids and intermediary metabolites in the shikimate pathway, such as coniferin. Moreover, adapted cells acquired thicker cell walls with higher lignin contents, suggesting the importance of adjustments of physical properties during adaptation to elevated saline conditions. When stress-memory cells were reverted to normal growth conditions, the levels of metabolites again readjusted. Whereas most of the metabolic changes reverted to levels intermediate between salt-adapted and control cells, the amounts of sugars, alanine, γ-aminobutyric acid and acetate further increased in stress-memory cells, supporting a view of their roles in mitotic stress memory. Our results provide insights into the metabolic adjustment of plant root cells during adaptation to saline conditions as well as pointing to the function of mitotic memory in acquired salt tolerance.

Arabidopsis AGDP1 links H3K9me2 to DNA methylation in heterochromatin.

Heterochromatin is a tightly packed form of chromatin that is associated with DNA methylation and histone 3 lysine 9 methylation (H3K9me). Here, we identify an H3K9me2-binding protein, Agenet domain (AGD)-containing p1 (AGDP1), in Arabidopsis thaliana. Here we find that AGDP1 can specifically recognize the H3K9me2 mark by its three pairs of tandem AGDs. We determine the crystal structure of the Agenet domain 1 and 2 cassette (AGD12) of Raphanus sativus AGDP1 in complex with an H3K9me2 peptide. In the complex, the histone peptide adopts a unique helical conformation. AGD12 specifically recognizes the H3K4me0 and H3K9me2 marks by hydrogen bonding and hydrophobic interactions. In addition, we find that AGDP1 is required for transcriptional silencing, non-CG DNA methylation, and H3K9 dimethylation at some loci. ChIP-seq data show that AGDP1 preferentially occupies long transposons and is associated with heterochromatin marks. Our findings suggest that, as a heterochromatin-binding protein, AGDP1 links H3K9me2 to DNA methylation in heterochromatin regions.

It's Hard to Avoid Avoidance: Uncoupling the Evolutionary Connection between Plant Growth, Productivity and Stress "Tolerance".

In the last 100 years, agricultural developments have favoured selection for highly productive crops, a fact that has been commonly associated with loss of key traits for environmental stress tolerance. We argue here that this is not exactly the case. We reason that high yield under near optimal environments came along with hypersensitization of plant stress perception and consequently early activation of stress avoidance mechanisms, such as slow growth, which were originally needed for survival over long evolutionary time periods. Therefore, mechanisms employed by plants to cope with a stressful environment during evolution were overwhelmingly geared to avoid detrimental effects so as to ensure survival and that plant stress "tolerance" is fundamentally and evolutionarily based on "avoidance" of injury and death which may be referred to as evolutionary avoidance (EVOL-Avoidance). As a consequence, slow growth results from being exposed to stress because genes and genetic programs to adjust growth rates to external circumstances have evolved as a survival but not productivity strategy that has allowed extant plants to avoid extinction. To improve productivity under moderate stressful conditions, the evolution-oriented plant stress response circuits must be changed from a survival mode to a continued productivity mode or to avoid the evolutionary avoidance response, as it were. This may be referred to as Agricultural (AGRI-Avoidance). Clearly, highly productive crops have kept the slow, reduced growth response to stress that they evolved to ensure survival. Breeding programs and genetic engineering have not succeeded to genetically remove these responses because they are polygenic and redundantly programmed. From the beginning of modern plant breeding, we have not fully appreciated that our crop plants react overly-cautiously to stress conditions. They over-reduce growth to be able to survive stresses for a period of time much longer than a cropping season. If we are able to remove this polygenic redundant survival safety net we may improve yield in moderately stressful environments, yet we will face the requirement to replace it with either an emergency slow or no growth (dormancy) response to extreme stress or use resource management to rescue crops under extreme stress (or both).

Arabidopsis Duodecuple Mutant of PYL ABA Receptors Reveals PYL Repression of ABA-Independent SnRK2 Activity.

Abscisic acid (ABA) is an important phytohormone controlling responses to abiotic stresses and is sensed by proteins from the PYR/PYL/RCAR family. To explore the genetic contribution of PYLs toward ABA-dependent and ABA-independent processes, we generated and characterized high-order Arabidopsis mutants with mutations in the PYL family. We obtained a pyl quattuordecuple mutant and found that it was severely impaired in growth and failed to produce seeds. Thus, we carried out a detailed characterization of a pyl duodecuple mutant, pyr1pyl1/2/3/4/5/7/8/9/10/11/12. The duodecuple mutant was extremely insensitive to ABA effects on seed germination, seedling growth, stomatal closure, leaf senescence, and gene expression. The activation of SnRK2 protein kinases by ABA was blocked in the duodecuple mutant, but, unexpectedly, osmotic stress activation of SnRK2s was enhanced. Our results demonstrate an important role of basal ABA signaling in growth, senescence, and abscission and reveal that PYLs antagonize ABA-independent activation of SnRK2s by osmotic stress.

Mutations in a subfamily of abscisic acid receptor genes promote rice growth and productivity.

Abscisic acid (ABA) is a key phytohormone that controls plant growth and stress responses. It is sensed by the pyrabactin resistance 1 (PYR1)/PYR1-like (PYL)/regulatory components of the ABA receptor (RCAR) family of proteins. Here, we utilized CRISPR/Cas9 technology to edit group I (PYL1-PYL6 and PYL12) and group II (PYL7-PYL11 and PYL13) PYL genes in rice. Characterization of the combinatorial mutants suggested that genes in group I have more important roles in stomatal movement, seed dormancy, and growth regulation than those in group II. Among all of the single pyl mutants, only pyl1 and pyl12 exhibited significant defects in seed dormancy. Interestingly, high-order group I mutants, but not any group II mutants, displayed enhanced growth. Among group I mutants, pyl1/4/6 exhibited the best growth and improved grain productivity in natural paddy field conditions, while maintaining nearly normal seed dormancy. Our results suggest that a subfamily of rice PYLs has evolved to have particularly important roles in regulating plant growth and reveal a genetic strategy to improve rice productivity.

Epigenetic switch from repressive to permissive chromatin in response to cold stress.

Switching from repressed to active status in chromatin regulation is part of the critical responses that plants deploy to survive in an ever-changing environment. We previously reported that HOS15, a WD40-repeat protein, is involved in histone deacetylation and cold tolerance in Arabidopsis However, it remained unknown how HOS15 regulates cold responsive genes to affect cold tolerance. Here, we show that HOS15 interacts with histone deacetylase 2C (HD2C) and both proteins together associate with the promoters of cold-responsive COR genes, COR15A and COR47 Cold induced HD2C degradation is mediated by the CULLIN4 (CUL4)-based E3 ubiquitin ligase complex in which HOS15 acts as a substrate receptor. Interference with the association of HD2C and the COR gene promoters by HOS15 correlates with increased acetylation levels of histone H3. HOS15 also interacts with CBF transcription factors to modulate cold-induced binding to the COR gene promoters. Our results here demonstrate that cold induces HOS15-mediated chromatin modifications by degrading HD2C. This switches the chromatin structure status and facilitates recruitment of CBFs to the COR gene promoters. This is an apparent requirement to acquire cold tolerance.

Control of Plant Water Use by ABA Induction of Senescence and Dormancy: An Overlooked Lesson from Evolution.

Drought stress is a condition that in specific climate contexts results in insufficient water availability and often limits plant productivity through perturbing development and reducing plant growth and survival. Plants use senescence of old leaves and dormancy of buds and seeds to survive extreme environmental conditions. The plant hormone ABA accumulates after drought stress, and increases plant survival by inducing quick responses such as stomatal closure, and long-term responses such as extended growth inhibition, osmotic regulation, accumulation of cuticular wax, senescence, abscission and dormancy. Here we focus on how the long-term ABA responses contribute to plant survival during severe drought stress. Leaf senescence and abscission of older leaves reduce total plant transpirational water loss and increase the transfer of nutrients to meristems and to some storage tissues. Osmotic regulation favors water consumption in sink tissues, and accumulation of cuticular wax helps to seal the plant surface and limits non-stomatal water loss.

The miR165/166 Mediated Regulatory Module Plays Critical Roles in ABA Homeostasis and Response in Arabidopsis thaliana.

The function of miR165/166 in plant growth and development has been extensively studied, however, its roles in abiotic stress responses remain largely unknown. Here, we report that reduction in the expression of miR165/166 conferred a drought and cold resistance phenotype and hypersensitivity to ABA during seed germination and post-germination seedling development. We further show that the ABA hypersensitive phenotype is associated with a changed transcript abundance of ABA-responsive genes and a higher expression level of ABI4, which can be directly regulated by a miR165/166 target. Additionally, we found that reduction in miR165/166 expression leads to elevated ABA levels, which occurs at least partially through the increased expression of BG1, a gene that is directly regulated by a miR165/166 target. Taken together, our results uncover a novel role for miR165/166 in the regulation of ABA and abiotic stress responses and control of ABA homeostasis.

A Single Amino-Acid Substitution in the Sodium Transporter HKT1 Associated with Plant Salt Tolerance.

A crucial prerequisite for plant growth and survival is the maintenance of potassium uptake, especially when high sodium surrounds the root zone. The Arabidopsis HIGH-AFFINITY K(+) TRANSPORTER1 (HKT1), and its homologs in other salt-sensitive dicots, contributes to salinity tolerance by removing Na(+) from the transpiration stream. However, TsHKT1;2, one of three HKT1 copies in Thellungiella salsuginea, a halophytic Arabidopsis relative, acts as a K(+) transporter in the presence of Na(+) in yeast (Saccharomyces cerevisiae). Amino-acid sequence comparisons indicated differences between TsHKT1;2 and most other published HKT1 sequences with respect to an Asp residue (D207) in the second pore-loop domain. Two additional T salsuginea and most other HKT1 sequences contain Asn (n) in this position. Wild-type TsHKT1;2 and altered AtHKT1 (AtHKT1(N-D)) complemented K(+)-uptake deficiency of yeast cells. Mutant hkt1-1 plants complemented with both AtHKT1(N) (-) (D) and TsHKT1;2 showed higher tolerance to salt stress than lines complemented by the wild-type AtHKT1 Electrophysiological analysis in Xenopus laevis oocytes confirmed the functional properties of these transporters and the differential selectivity for Na(+) and K(+) based on the n/d variance in the pore region. This change also dictated inward-rectification for Na(+) transport. Thus, the introduction of Asp, replacing Asn, in HKT1-type transporters established altered cation selectivity and uptake dynamics. We describe one way, based on a single change in a crucial protein that enabled some crucifer species to acquire improved salt tolerance, which over evolutionary time may have resulted in further changes that ultimately facilitated colonization of saline habitats.

ABA receptor PYL9 promotes drought resistance and leaf senescence.

Drought stress is an important environmental factor limiting plant productivity. In this study, we screened drought-resistant transgenic plants from 65 promoter-pyrabactin resistance 1-like (PYL) abscisic acid (ABA) receptor gene combinations and discovered that pRD29A::PYL9 transgenic lines showed dramatically increased drought resistance and drought-induced leaf senescence in both Arabidopsis and rice. Previous studies suggested that ABA promotes senescence by causing ethylene production. However, we found that ABA promotes leaf senescence in an ethylene-independent manner by activating sucrose nonfermenting 1-related protein kinase 2s (SnRK2s), which subsequently phosphorylate ABA-responsive element-binding factors (ABFs) and Related to ABA-Insensitive 3/VP1 (RAV1) transcription factors. The phosphorylated ABFs and RAV1 up-regulate the expression of senescence-associated genes, partly by up-regulating the expression of Oresara 1. The pyl9 and ABA-insensitive 1-1 single mutants, pyl8-1pyl9 double mutant, and snrk2.2/3/6 triple mutant showed reduced ABA-induced leaf senescence relative to the WT, whereas pRD29A::PYL9 transgenic plants showed enhanced ABA-induced leaf senescence. We found that leaf senescence may benefit drought resistance by helping to generate an osmotic potential gradient, which is increased in pRD29A::PYL9 transgenic plants and causes water to preferentially flow to developing tissues. Our results uncover the molecular mechanism of ABA-induced leaf senescence and suggest an important role of PYL9 and leaf senescence in promoting resistance to extreme drought stress.

A novel thiol-reductase activity of Arabidopsis YUC6 confers drought tolerance independently of auxin biosynthesis.

YUCCA (YUC) proteins constitute a family of flavin monooxygenases (FMOs), with an important role in auxin (IAA) biosynthesis. Here we report that Arabidopsis plants overexpressing YUC6 display enhanced IAA-related phenotypes and exhibit improved drought stress tolerance, low rate of water loss and controlled ROS accumulation under drought and oxidative stresses. Co-overexpression of an IAA-conjugating enzyme reduces IAA levels but drought stress tolerance is unaffected, indicating that the stress-related phenotype is not based on IAA overproduction. YUC6 contains a previously unrecognized FAD- and NADPH-dependent thiol-reductase activity (TR) that overlaps with the FMO domain involved in IAA biosynthesis. Mutation of a conserved cysteine residue (Cys-85) preserves FMO but suppresses TR activity and stress tolerance, whereas mutating the FAD- and NADPH-binding sites, that are common to TR and FMO domains, abolishes all outputs. We provide a paradigm for a single protein playing a dual role, regulating plant development and conveying stress defence responses.