Accumulation of Phosphorylated SnRK2 Substrate 1 Promotes Drought Escape in Arabidopsis.

Plants adopt optimal tolerance strategies depending on the intensity and duration of stress. Retaining water is a priority under short-term drought conditions, whereas maintaining growth and reproduction processes takes precedence over survival under conditions of prolonged drought. However, the mechanism underlying changes in the stress response depending on the degree of drought is unclear. Here, we report that SNF1-related protein kinase 2 (SnRK2) substrate 1 (SNS1) is involved in this growth regulation under conditions of drought stress. SNS1 is phosphorylated and stabilized by SnRK2 protein kinases reflecting drought conditions. It contributes to the maintenance of growth and promotion of flowering as drought escape by repressing stress-responsive genes and inducing FLOWERING LOCUS T (FT) expression, respectively. SNS1 interacts with the histone methylation reader proteins MORF-related gene 1 (MRG1) and MRG2, and the SNS1-MRG1/2 module cooperatively regulates abscisic acid response. Taken together, these observations suggest that the phosphorylation and accumulation of SNS1 in plants reflect the intensity and duration of stress and can serve as a molecular scale for maintaining growth and adopting optimal drought tolerance strategies under stress conditions.

Constitutively active B2 Raf-like kinases are required for drought-responsive gene expression upstream of ABA-activated SnRK2 kinases.

Osmotic stresses, such as drought and high salinity, adversely affect plant growth and productivity. The phytohormone abscisic acid (ABA) accumulates in response to osmotic stress and enhances stress tolerance in plants by triggering multiple physiological responses through ABA signaling. Subclass III SNF1-related protein kinases 2 (SnRK2s) are key regulators of ABA signaling. Although SnRK2s have long been considered to be self-activated by autophosphorylation after release from PP2C-mediated inhibition, they were recently revealed to be activated by two independent subfamilies of group B Raf-like kinases, B2-RAFs and B3-RAFs, under osmotic stress conditions. However, the relationship between SnRK2 phosphorylation by these RAFs and SnRK2 autophosphorylation and the individual physiological roles of each RAF subfamily remain unknown. In this study, we indicated that B2-RAFs are constantly active and activate SnRK2s when released from PP2C-mediated inhibition by ABA-binding ABA receptors, whereas B3-RAFs are activated only under stress conditions in an ABA-independent manner and enhance SnRK2 activity. Autophosphorylation of subclass III SnRK2s is not sufficient for ABA responses, and B2-RAFs are needed to activate SnRK2s in an ABA-dependent manner. Using plants grown in soil, we found that B2-RAFs regulate subclass III SnRK2s at the early stage of drought stress, whereas B3-RAFs regulate SnRK2s at the later stage. Thus, B2-RAFs are essential kinases for the activation of subclass III SnRK2s in response to ABA under mild osmotic stress conditions, and B3-RAFs function as enhancers of SnRK2 activity under severe stress conditions.

Peculiar properties of tuber starch in a potato mutant lacking the α-glucan water dikinase 1 gene GWD1 created by targeted mutagenesis using the CRISPR/dMac3-Cas9 system.

Glucose chains in starch are phosphorylated and contribute to structural stabilization. Phosphate groups contained in starch also play a role in retaining moisture. α-Glucan water dikinase 1 (GWD1) is involved in the phosphorylation of glucose chains in starch. In this study, we generated potato mutants of the GWD1 gene using the CRISPR/dMac3-Cas9 system. Observation of the phenotypes of the GWD1-deficient mutants revealed their physiological roles in tuber starch formation. The 4-allele mutants showed growth retardation and a delay in tuber formation. A significant decrease in phosphorus content was detected in the tuber starch of the gwd1 mutant. This mutant starch showed a higher amylose content than the wild-type starch, whereas its gelatinization temperature was slightly lower than that of the WT starch. The peak viscosity of the mutant starch was lower than that of the WT starch. These observations revealed that the starch of the gwd1 mutants had peculiar and unique properties compared to those of WT, sbe3 and gbss1 mutant starches. The amount of tissue-released water due to freeze-thawing treatment was determined on tubers of the gwd1 mutant and compared with those of WT and the other mutants. Significantly less water loss was found in the gwd1, sbe3 and gbss1 mutant tubers than in the WT tubers. Our results indicate that the GWD1 gene is not only important for potato growth, but also largely effective for the traits of tuber starch.

Use of Micrografting to Study the Role Played by Peptide Signals in ABA Biosynthesis.

Plants survive by deploying appropriate stress responses under rapidly changing environmental conditions. Vascular plants transmit environmental information among distant organs. Long-distance signaling plays a crucial role in plant adaptation and subsequent survival under severe environmental conditions. In model plants, the micrografting method has recently emerged as an important method to elucidate tissue-to-tissue communication via hormones, RNAs, and peptides. In this chapter, I describe a micrografting method for Arabidopsis thaliana to graft shoots onto roots of plants with different genotypes. This grafting method may facilitate further research investigating how vascular plants integrate environmental information among distant organs via long-distance signaling.

Affinity Purification Followed by Liquid Chromatography-Tandem Mass Spectrometry to Identify Proteins Interacting with ABA Signaling Components.

Abscisic acid (ABA) is a key phytohormone involved in plant development, seed germination and responses to osmotic stresses, such as drought and high salinity. SNF1-related protein kinases (SnRK2s) play important roles in ABA-dependent and ABA-independent osmotic stress signaling. SnRK2s phosphorylate transcription factors and ion channels in response to ABA or osmotic stress to induce the expression of stress-responsive genes and stomatal closure, respectively, to confer osmotic stress tolerance. The activity of SnRK2s is directly or indirectly regulated by several protein factors. Identification of downstream substrates or upstream regulators of SnRK2s is very useful for elucidating protein components that regulate ABA and osmotic stress signaling. Here, we describe the use of affinity purification by coimmunoprecipitation and liquid chromatography-tandem mass spectrometry to identify protein complexes involved in ABA and osmotic stress signaling in plants. We previously identified several protein factors that regulate ABA and osmotic stress signaling by using this method.

Arabidopsis TBP-ASSOCIATED FACTOR 12 ortholog NOBIRO6 controls root elongation with unfolded protein response cofactor activity.

Plant root growth is indeterminate but continuously responds to environmental changes. We previously reported on the severe root growth defect of a double mutant in bZIP17 and bZIP28 (bz1728) modulating the unfolded protein response (UPR). To elucidate the mechanism by which bz1728 seedlings develop a short root, we obtained a series of bz1728 suppressor mutants, called nobiro, for rescued root growth. We focused here on nobiro6, which is defective in the general transcription factor component TBP-ASSOCIATED FACTOR 12b (TAF12b). The expression of hundreds of genes, including the bZIP60-UPR regulon, was induced in the bz1728 mutant, but these inductions were markedly attenuated in the bz1728nobiro6 mutant. In view of this, we assigned transcriptional cofactor activity via physical interaction with bZIP60 to NOBIRO6/TAF12b. The single nobiro6/taf12b mutant also showed an altered sensitivity to endoplasmic reticulum stress for both UPR and root growth responses, demonstrating that NOBIRO6/TAF12b contributes to environment-responsive root growth control through UPR.

Inter-tissue and inter-organ signaling in drought stress response and phenotyping of drought tolerance.

Plant response to drought stress includes systems for intracellular regulation of gene expression and signaling, as well as inter-tissue and inter-organ signaling, which helps entire plants acquire stress resistance. Plants sense water-deficit conditions both via the stomata of leaves and roots, and transfer water-deficit signals from roots to shoots via inter-organ signaling. Abscisic acid is an important phytohormone involved in the drought stress response and adaptation, and is synthesized mainly in vascular tissues and guard cells of leaves. In leaves, stress-induced abscisic acid is distributed to various tissues by transporters, which activates stomatal closure and expression of stress-related genes to acquire drought stress resistance. Moreover, the stepwise stress response at the whole-plant level is important for proper understanding of the physiological response to drought conditions. Drought stress is sensed by multiple types of sensors as molecular patterns of abiotic stress signals, which are transmitted via separate parallel signaling networks to induce downstream responses, including stomatal closure and synthesis of stress-related proteins and metabolites. Peptide molecules play important roles in the inter-organ signaling of dehydration from roots to shoots, as well as signaling of osmotic changes and reactive oxygen species/Ca2+ . In this review, we have summarized recent advances in research on complex plant drought stress responses, focusing on inter-tissue signaling in leaves and inter-organ signaling from roots to shoots. We have discussed the mechanisms via which drought stress adaptations and resistance are acquired at the whole-plant level, and have proposed the importance of quantitative phenotyping for measuring plant growth under drought conditions.

Corrigendum: Drought Stress Responses and Resistance in Plants: From Cellular Responses to Long-Distance Intercellular Communication.

[This corrects the article DOI: 10.3389/fpls.2020.556972.].

Transcriptome Analysis of Chloris virgata, Which Shows the Fastest Germination and Growth in the Major Mongolian Grassland Plant.

Plants in Mongolian grasslands are exposed to short, dry summers and long, cold winters. These plants should be prepared for fast germination and growth activity in response to the limited summer rainfall. The wild plant species adapted to the Mongolian grassland environment may allow us to explore useful genes, as a source of unique genetic codes for crop improvement. Here, we identified the Chloris virgata Dornogovi accession as the fastest germinating plant in major Mongolian grassland plants. It germinated just 5 h after treatment for germination initiation and showed rapid growth, especially in its early and young development stages. This indicates its high growth potential compared to grass crops such as rice and wheat. By assessing growth recovery after animal bite treatment (mimicked by cutting the leaves with scissors), we found that C. virgata could rapidly regenerate leaves after being damaged, suggesting high regeneration potential against grazing. To analyze the regulatory mechanism involved in the high growth potential of C. virgata, we performed RNA-seq-based transcriptome analysis and illustrated a comprehensive gene expression map of the species. Through de novo transcriptome assembly with the RNA-seq reads from whole organ samples of C. virgata at the germination stage (2 days after germination, DAG), early young development stage (8 DAG), young development stage (17 DAG), and adult development stage (28 DAG), we identified 21,589 unified transcripts (contigs) and found that 19,346 and 18,156 protein-coding transcripts were homologous to those in rice and Arabidopsis, respectively. The best-aligned sequences were annotated with gene ontology groups. When comparing the transcriptomes across developmental stages, we found an over-representation of genes involved in growth regulation in the early development stage in C. virgata. Plant development is tightly regulated by phytohormones such as brassinosteroids, gibberellic acid, abscisic acid, and strigolactones. Moreover, our transcriptome map demonstrated the expression profiles of orthologs involved in the biosynthesis of these phytohormones and their signaling networks. We discuss the possibility that C. virgata phytohormone signaling and biosynthesis genes regulate early germination and growth advantages. Comprehensive transcriptome information will provide a useful resource for gene discovery and facilitate a deeper understanding of the diversity of the regulatory systems that have evolved in C. virgata while adapting to severe environmental conditions.

Arabidopsis group C Raf-like protein kinases negatively regulate abscisic acid signaling and are direct substrates of SnRK2.

The phytohormone abscisic acid (ABA) plays a major role in abiotic stress responses in plants, and subclass III SNF1-related protein kinase 2 (SnRK2) kinases mediate ABA signaling. In this study, we identified Raf36, a group C Raf-like protein kinase in Arabidopsis, as a protein that interacts with multiple SnRK2s. A series of reverse genetic and biochemical analyses revealed that 1) Raf36 negatively regulates ABA responses during postgermination growth, 2) the N terminus of Raf36 is directly phosphorylated by SnRK2s, and 3) Raf36 degradation is enhanced in response to ABA. In addition, Raf22, another C-type Raf-like kinase, functions partially redundantly with Raf36 to regulate ABA responses. A comparative phosphoproteomic analysis of ABA-induced responses of wild-type and raf22raf36-1 plants identified proteins that are phosphorylated downstream of Raf36 and Raf22 in planta. Together, these results support a model in which Raf36/Raf22 function mainly under optimal conditions to suppress ABA responses, whereas in response to ABA, the SnRK2 module promotes Raf36 degradation as a means of alleviating Raf36-dependent inhibition and allowing for heightened ABA signaling to occur.

Cellular Phosphorylation Signaling and Gene Expression in Drought Stress Responses: ABA-Dependent and ABA-Independent Regulatory Systems.

Drought is a severe and complex abiotic stress that negatively affects plant growth and crop yields. Numerous genes with various functions are induced in response to drought stress to acquire drought stress tolerance. The phytohormone abscisic acid (ABA) accumulates mainly in the leaves in response to drought stress and then activates subclass III SNF1-related protein kinases 2 (SnRK2s), which are key phosphoregulators of ABA signaling. ABA mediates a wide variety of gene expression processes through stress-responsive transcription factors, including ABA-RESPONSIVE ELEMENT BINDING PROTEINS (AREBs)/ABRE-BINDING FACTORS (ABFs) and several other transcription factors. Seed plants have another type of SnRK2s, ABA-unresponsive subclass I SnRK2s, that mediates the stability of gene expression through the mRNA decay pathway and plant growth under drought stress in an ABA-independent manner. Recent research has elucidated the upstream regulators of SnRK2s, RAF-like protein kinases, involved in early responses to drought stress. ABA-independent transcriptional regulatory systems and ABA-responsive regulation function in drought-responsive gene expression. DEHYDRATION RESPONSIVE ELEMENT (DRE) is an important cis-acting element in ABA-independent transcription, whereas ABA-RESPONSIVE ELEMENT (ABRE) cis-acting element functions in ABA-responsive transcription. In this review article, we summarize recent advances in research on cellular and molecular drought stress responses and focus on phosphorylation signaling and transcription networks in Arabidopsis and crops. We also highlight gene networks of transcriptional regulation through two major regulatory pathways, ABA-dependent and ABA-independent pathways, that ABA-responsive subclass III SnRK2s and ABA-unresponsive subclass I SnRK2s mediate, respectively. We also discuss crosstalk in these regulatory systems under drought stress.

Long-distance stress and developmental signals associated with abscisic acid signaling in environmental responses.

Flowering plants consist of highly differentiated organs, including roots, leaves, shoots and flowers, which have specific roles: root system for water and nutrient uptake, leaves for photosynthesis and gas exchange and reproductive organs for seed production. The communication between organs through the vascular system, by which water, nutrient and signaling molecules are transported, is essential for coordinated growth and development of the whole plant, particularly under adverse conditions. Here, we highlight recent progress in understanding how signaling pathways of plant hormones are associated with long-distance stress and developmental signals, with particular focus on environmental stress responses. In addition to the root-to-shoot peptide signal that induces abscisic acid accumulation in leaves under drought stress conditions, we summarize the diverse stress-responsive peptide signals reported to date to play a role in environmental responses.

Large-Scale Phosphoproteomic Study of Arabidopsis Membrane Proteins Reveals Early Signaling Events in Response to Cold.

Cold stress is one of the major factors limiting global crop production. For survival at low temperatures, plants need to sense temperature changes in the surrounding environment. How plants sense and respond to the earliest drop in temperature is still not clearly understood. The plasma membrane and its adjacent extracellular and cytoplasmic sites are the first checkpoints for sensing temperature changes and the subsequent events, such as signal generation and solute transport. To understand how plants respond to early cold exposure, we used a mass spectrometry-based phosphoproteomic method to study the temporal changes in protein phosphorylation events in Arabidopsis membranes during 5 to 60 min of cold exposure. The results revealed that brief cold exposures led to rapid phosphorylation changes in the proteins involved in cellular ion homeostasis, solute and protein transport, cytoskeleton organization, vesical trafficking, protein modification, and signal transduction processes. The phosphorylation motif and kinase-substrate network analysis also revealed that multiple protein kinases, including RLKs, MAPKs, CDPKs, and their substrates, could be involved in early cold signaling. Taken together, our results provide a first look at the cold-responsive phosphoproteome changes of Arabidopsis membrane proteins that can be a significant resource to understand how plants respond to an early temperature drop.

Drought Stress Responses and Resistance in Plants: From Cellular Responses to Long-Distance Intercellular Communication.

The drought stress responses of vascular plants are complex regulatory mechanisms because they include various physiological responses from signal perception under water deficit conditions to the acquisition of drought stress resistance at the whole-plant level. It is thought that plants first recognize water deficit conditions in roots and that several molecular signals then move from roots to shoots. Finally, a phytohormone, abscisic acid (ABA) is synthesized mainly in leaves. However, the detailed molecular mechanisms of stress sensors and the regulators that initiate ABA biosynthesis in response to drought stress conditions are still unclear. Another important issue is how plants adjust ABA propagation, stress-mediated gene expression and metabolite composition to acquire drought stress resistance in different tissues throughout the whole plant. In this review, we summarize recent advances in research on drought stress responses, focusing on long-distance signaling from roots to shoots, ABA synthesis and transport, and metabolic regulation in both cellular and whole-plant levels of Arabidopsis and crops. We also discuss coordinated mechanisms for acquiring drought stress adaptations and resistance via tissue-to-tissue communication and long-distance signaling.

Arabidopsis SMN2/HEN2, Encoding DEAD-Box RNA Helicase, Governs Proper Expression of the Resistance Gene SMN1/RPS6 and Is Involved in Dwarf, Autoimmune Phenotypes of mekk1 and mpk4 Mutants.

In Arabidopsis thaliana, a mitogen-activated protein kinase pathway, MEKK1-MKK1/MKK2-MPK4, is important for basal resistance and disruption of this pathway results in dwarf, autoimmune phenotypes. To elucidate the complex mechanisms activated by the disruption of this pathway, we have previously developed a mutant screening system based on a dwarf autoimmune line that overexpressed the N-terminal regulatory domain of MEKK1. Here, we report that the second group of mutants, smn2, had defects in the SMN2 gene, encoding a DEAD-box RNA helicase. SMN2 is identical to HEN2, whose function is vital for the nuclear RNA exosome because it provides non-ribosomal RNA specificity for RNA turnover, RNA quality control and RNA processing. Aberrant SMN1/RPS6 transcripts were detected in smn2 and hen2 mutants. Disease resistance against Pseudomonas syringae pv. tomato DC3000 (hopA1), which is conferred by SMN1/RPS6, was decreased in smn2 mutants, suggesting a functional connection between SMN1/RPS6 and SMN2/HEN2. We produced double mutants mekk1smn2 and mpk4smn2 to determine whether the smn2 mutations suppress the dwarf, autoimmune phenotypes of the mekk1 and mpk4 mutants, as the smn1 mutations do. As expected, the mekk1 and mpk4 phenotypes were suppressed by the smn2 mutations. These results suggested that SMN2 is involved in the proper function of SMN1/RPS6. The Gene Ontology enrichment analysis using RNA-seq data showed that defense genes were downregulated in smn2, suggesting a positive contribution of SMN2 to the genome-wide expression of defense genes. In conclusion, this study provides novel insight into plant immunity via SMN2/HEN2, an essential component of the nuclear RNA exosome.

Plant Raf-like kinases regulate the mRNA population upstream of ABA-unresponsive SnRK2 kinases under drought stress.

SNF1-related protein kinases 2 (SnRK2s) are key regulators governing the plant adaptive responses to osmotic stresses, such as drought and high salinity. Subclass III SnRK2s function as central regulators of abscisic acid (ABA) signalling and orchestrate ABA-regulated adaptive responses to osmotic stresses. Seed plants have acquired other types of osmotic stress-activated but ABA-unresponsive subclass I SnRK2s that regulate mRNA decay and promote plant growth under osmotic stresses. In contrast to subclass III SnRK2s, the regulatory mechanisms underlying the rapid activation of subclass I SnRK2s in response to osmotic stress remain elusive. Here, we report that three B4 Raf-like MAP kinase kinase kinases (MAPKKKs) phosphorylate and activate subclass I SnRK2s under osmotic stress. Transcriptome analyses reveal that genes downstream of these MAPKKKs largely overlap with subclass I SnRK2-regulated genes under osmotic stress, which indicates that these MAPKKKs are upstream factors of subclass I SnRK2 and are directly activated by osmotic stress.

Comparative Phosphoproteomic Analysis Reveals a Decay of ABA Signaling in Barley Embryos during After-Ripening.

Abscisic acid (ABA) is a phytohormone and a major determinant of seed dormancy in plants. Seed dormancy is gradually lost during dry storage, a process known as 'after-ripening', and this dormancy decay is related to a decline in ABA content and sensitivity in seeds after imbibition. In this study, we aimed at investigating the effect of after-ripening on ABA signaling in barley, our cereal model species. Phosphosignaling networks in barley grains were investigated by a large-scale analysis of phosphopeptides to examine potential changes in response pathways to after-ripening. We used freshly harvested (FH) and after-ripened (AR) barley grains which showed different ABA sensitivity. A total of 1,730 phosphopeptides were identified in barley embryos isolated from half-cut grains. A comparative analysis showed that 329 and 235 phosphopeptides were upregulated or downregulated, respectively after ABA treatment, and phosphopeptides profiles were quite different between FH and AR embryos. These results were supported by peptide motif analysis which suggested that different sets of protein kinases are active in FH and AR grains. Furthermore, in vitro phosphorylation assays confirmed that some phosphopeptides were phosphorylated by SnRK2s, which are major protein kinases involved in ABA signaling. Taken together, our results revealed very distinctive phosphosignaling networks in FH and AR embryos of barley, and suggested that the after-ripening of barley grains is associated with differential regulation of phosphosignaling pathways leading to a decay of ABA signaling.

Hormone-like peptides and small coding genes in plant stress signaling and development.

Recent works have shed light on the long-distance interorgan signaling by which hormone-like peptides precisely regulate physiological effects in a manner similar to phytohormones. Many such peptides have already been identified in the primary model plant, Arabidopsis thaliana. In addition, Arabidopsis genome reanalysis revealed over 7000 novel candidate small coding genes, some of which are likely to be associated with hormone-like peptides. Hormone-like peptides have also been reported to play critical roles in interorgan communications during morphogenesis and stress responses. In this review, we focus on the functional roles of hormone-like peptides and small coding genes in cell-to-cell and/or long-distance communications during plant stress signaling and development and discuss the evolutionary conservation of these peptides among plants.

NF-YB2 and NF-YB3 Have Functionally Diverged and Differentially Induce Drought and Heat Stress-Specific Genes.

Functional diversification of transcription factors allows the precise regulation of transcriptomic changes under different environmental conditions. The NUCLEAR FACTOR Y (NF-Y) transcription factor comprises three subunits, NF-YA, NF-YB, and NF-YC, and is broadly diversified in plant species, whereas Humans (Homo sapiens) have one protein for each subunit. However, there remains much to be learned about the diversified functions of each subunit in plants. Here, we found that NF-YB2 and NF-YB3, which have the greatest sequence similarity to each other among NF-YB family proteins in Arabidopsis (Arabidopsis thaliana), are functionally diversified and specifically activate dehydration-inducible and heat-inducible genes, according to environmental conditions. Overexpression of NF-YB2 and NF-YB3 specifically enhanced drought and heat stress tolerance, respectively, and each single knockout mutant showed adverse stress-sensitive phenotypes. Transcriptomic analyses confirmed that overexpression of NF-YB2 and NF-YB3 largely affected the transcriptomic changes under dehydration and heat stress conditions, respectively. The DNA-binding profiles of each protein in planta also suggested that dehydration and heat stress increased the DNA-binding activity of NF-YB2 and NF-YB3 to dehydration-inducible and heat stress-inducible target genes, respectively. Moreover, phylogenetic analysis suggested that the NF-YB proteins of angiosperm plants belong to divergent NF-YB2 and NF-YB3 subgroups. These results demonstrate the functional diversification of NF-Y through evolutionary processes and how plants adapt to various abiotic stresses under fluctuating environments.