Group C MAP kinases phosphorylate MBD10 to regulate ABA-induced leaf senescence in Arabidopsis.

Abscisic acid (ABA) is a phytohormone that promotes leaf senescence in response to environmental stress. We previously identified methyl CpG-binding domain 10 (MBD10) as a phosphoprotein that becomes differentially phosphorylated after ABA treatment in Arabidopsis. ABA-induced leaf senescence was delayed in mbd10 knockout plants but accelerated in MBD10-overexpressing plants, suggesting that MBD10 positively regulates ABA-induced leaf senescence. ABA-induced phosphorylation of MBD10 occurs in planta on Thr-89, and our results demonstrated that Thr-89 phosphorylation is essential for MBD10's function in leaf senescence. The in vivo phosphorylation of Thr-89 in MBD10 was significantly downregulated in a quadruple mutant of group C MAPKs (mpk1/2/7/14), and group C MAPKs directly phosphorylated MBD10 in vitro. Furthermore, mpk1/2/7/14 showed a similar phenotype as seen in mbd10 for ABA-induced leaf senescence, suggesting that group C MAPKs are the cognate kinases of MBD10 for Thr-89. Because group C MAPKs have been reported to function downstream of SnRK2s, our results indicate that group C MAPKs and MBD10 constitute a regulatory pathway for ABA-induced leaf senescence.

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.

Burning questions for a warming and changing world: 15 unknowns in plant abiotic stress.

We present unresolved questions in plant abiotic stress biology as posed by 15 research groups with expertise spanning eco-physiology to cell and molecular biology. Common themes of these questions include the need to better understand how plants detect water availability, temperature, salinity, and rising carbon dioxide (CO2) levels; how environmental signals interface with endogenous signaling and development (e.g. circadian clock and flowering time); and how this integrated signaling controls downstream responses (e.g. stomatal regulation, proline metabolism, and growth versus defense balance). The plasma membrane comes up frequently as a site of key signaling and transport events (e.g. mechanosensing and lipid-derived signaling, aquaporins). Adaptation to water extremes and rising CO2 affects hydraulic architecture and transpiration, as well as root and shoot growth and morphology, in ways not fully understood. Environmental adaptation involves tradeoffs that limit ecological distribution and crop resilience in the face of changing and increasingly unpredictable environments. Exploration of plant diversity within and among species can help us know which of these tradeoffs represent fundamental limits and which ones can be circumvented by bringing new trait combinations together. Better defining what constitutes beneficial stress resistance in different contexts and making connections between genes and phenotypes, and between laboratory and field observations, are overarching challenges.

Phosphoproteomic Approaches to Evaluate ABA Signaling.

Abscisic acid (ABA) is a major phytohormone that regulates various processes in plants (e.g., seed dormancy/germination, abiotic/biotic stress responses). As protein phosphorylation is involved in the major pathways of ABA signaling, it is necessary to elucidate the phosphosignaling pathway involved in the ABA response. Phosphoproteomics enables determination of the proteins phosphorylated in vivo, and recent studies have applied a comparative phosphoproteomic approach to analyze ABA signaling in plants. For example, ABA-responsive phosphoproteins were identified in barley embryos. Furthermore, a phosphoproteomic approach is useful for screening protein kinase substrates by comparative analysis using kinase knockout mutants. Here, some technical points regarding phosphoproteomic analyses of ABA responses in plants are described.

Growth Promotion or Osmotic Stress Response: How SNF1-Related Protein Kinase 2 (SnRK2) Kinases Are Activated and Manage Intracellular Signaling in Plants.

Reversible phosphorylation is a major mechanism for regulating protein function and controls a wide range of cellular functions including responses to external stimuli. The plant-specific SNF1-related protein kinase 2s (SnRK2s) function as central regulators of plant growth and development, as well as tolerance to multiple abiotic stresses. Although the activity of SnRK2s is tightly regulated in a phytohormone abscisic acid (ABA)-dependent manner, recent investigations have revealed that SnRK2s can be activated by group B Raf-like protein kinases independently of ABA. Furthermore, evidence is accumulating that SnRK2s modulate plant growth through regulation of target of rapamycin (TOR) signaling. Here, we summarize recent advances in knowledge of how SnRK2s mediate plant growth and osmotic stress signaling and discuss future challenges in this research field.

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.

Activation of SnRK2 by Raf-like kinase ARK represents a primary mechanism of ABA and abiotic stress responses.

The Raf-like protein kinase abscisic acid (ABA) and abiotic stress-responsive Raf-like kinase (ARK) previously identified in the moss Physcomitrium (Physcomitrella) patens acts as an upstream regulator of subgroup III SNF1-related protein kinase2 (SnRK2), the key regulator of ABA and abiotic stress responses. However, the mechanisms underlying activation of ARK by ABA and abiotic stress for the regulation of SnRK2, including the role of ABA receptor-associated group A PP2C (PP2C-A), are not understood. We identified Ser1029 as the phosphorylation site in the activation loop of ARK, which provided a possible mechanism for regulation of its activity. Analysis of transgenic P. patens ark lines expressing ARK-GFP with Ser1029-to-Ala mutation indicated that this replacement causes reductions in ABA-induced gene expression, stress tolerance, and SnRK2 activity. Immunoblot analysis using an anti-phosphopeptide antibody indicated that ABA treatments rapidly stimulate Ser1029 phosphorylation in the wild type (WT). The phosphorylation profile of Ser1029 in ABA-hypersensitive ppabi1 lacking protein phosphatase 2C-A (PP2C-A) was similar to that in the WT, whereas little Ser1029 phosphorylation was observed in ABA-insensitive ark missense mutant lines. Furthermore, newly isolated ppabi1 ark lines showed ABA-insensitive phenotypes similar to those of ark lines. Therefore, ARK is a primary activator of SnRK2, preceding negative regulation by PP2C-A in bryophytes, which provides a prototype mechanism for ABA and abiotic stress responses in plants.

Identification of novel compounds that inhibit SnRK2 kinase activity by high-throughput screening.

Abscisic acid (ABA) is a major phytohormone that regulates abiotic stress responses and development. SNF1-rerated protein kinase 2 (SnRK2) is a key regulator of ABA signaling. To isolate compounds which directly affect SnRK2 activity, we optimized a fluorescence-based system for high-throughput screening (HTS) of SnRK2 kinase regulators. Using this system, we screened a chemical library consisting of 16,000 compounds and identified ten compounds (INH1-10) as potential SnRK2 inhibitors. Further characterization of these compounds by in vitro phosphorylation assays confirmed that three of the ten compounds were SnRK2-specific kinase inhibitors. In contrast, seven of ten compounds inhibited ABA-responsive gene expression in Arabidopsis cells. From these results, INH1 was identified as a SnRK2-specific inhibitor in vitro and in vivo. We propose that INH1 could be a lead compound of chemical tools for studying ABA responses in various plant species.

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.

Arabidopsis Raf-like kinases act as positive regulators of subclass III SnRK2 in osmostress signaling.

Given their sessile nature, land plants must use various mechanisms to manage dehydration under water-deficit conditions. Osmostress-induced activation of the SNF1-related protein kinase 2 (SnRK2) family elicits physiological responses such as stomatal closure to protect plants during drought conditions. With the plant hormone ABA receptors [PYR (pyrabactin resistance)/PYL (pyrabactin resistance-like)/RCAR (regulatory component of ABA receptors) proteins] and group A protein phosphatases, subclass III SnRK2 also constitutes a core signaling module for ABA, and osmostress triggers ABA accumulation. How SnRK2 is activated through ABA has been clarified, although its activation through osmostress remains unclear. Here, we show that Arabidopsis ABA and abiotic stress-responsive Raf-like kinases (AtARKs) of the B3 clade of the mitogen-activated kinase kinase kinase (MAPKKK) family are crucial in SnRK2-mediated osmostress responses. Disruption of AtARKs in Arabidopsis results in increased water loss from detached leaves because of impaired stomatal closure in response to osmostress. Our findings obtained in vitro and in planta have shown that AtARKs interact physically with SRK2E, a core factor for stomatal closure in response to drought. Furthermore, we show that AtARK phosphorylates S171 and S175 in the activation loop of SRK2E in vitro and that Atark mutants have defects in osmostress-induced subclass III SnRK2 activity. Our findings identify a specific type of B3-MAPKKKs as upstream kinases of subclass III SnRK2 in Arabidopsis. Taken together with earlier reports that ARK is an upstream kinase of SnRK2 in moss, an existing member of a basal land plant lineage, we propose that ARK/SnRK2 module is evolutionarily conserved across 400 million years of land plant evolution for conferring protection against drought.

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.

Erratum: Publisher Correction: SnRK2 protein kinases represent an ancient system in plants for adaptation to a terrestrial environment.

[This corrects the article DOI: 10.1038/s42003-019-0281-1.].

Comparative Phosphoproteomic Analysis of Barley Embryos with Different Dormancy during Imbibition.

Dormancy is the mechanism that allows seeds to become temporally quiescent in order to select the right time and place to germinate. Like in other species, in barley, grain dormancy is gradually reduced during after-ripening. Phosphosignaling networks in barley grains were investigated by a large-scale analysis of phosphoproteins to examine potential changes in response pathways to after-ripening. We used freshly harvested (FH) and after-ripened (AR) barley grains which showed different dormancy levels. The LC-MS/MS analysis identified 2346 phosphopeptides in barley embryos, with 269 and 97 of them being up- or downregulated during imbibition, respectively. A number of phosphopeptides were differentially regulated between FH and AR samples, suggesting that phosphoproteomic profiles were quite different between FH and AR grains. Motif analysis suggested multiple protein kinases including SnRK2 and MAPK could be involved in such a difference between FH and AR samples. Taken together, our results revealed phosphosignaling pathways in barley grains during the water imbibition process.

SnRK2 protein kinases represent an ancient system in plants for adaptation to a terrestrial environment.

The SNF1-related protein kinase 2 (SnRK2) family includes key regulators of osmostress and abscisic acid (ABA) responses in angiosperms and can be classified into three subclasses. Subclass III SnRK2s act in the ABA response while ABA-nonresponsive subclass I SnRK2s are regulated through osmostress. Here we report that an ancient subclass III SnRK2-based signalling module including ABA and an upstream Raf-like kinase (ARK) exclusively protects the moss Physcomitrella patens from drought. Subclass III SnRK2s from both Arabidopsis and from the semiterrestrial alga Klebsormidium nitens, which contains all the components of ABA signalling except ABA receptors, complement Physcomitrella snrk2 - mutants, whereas Arabidopsis subclass I SnRK2 cannot. We propose that the earliest land plants developed the ABA/ARK/subclass III SnRK2 signalling module by recruiting ABA to regulate a pre-existing dehydration response and that subsequently a novel subclass I SnRK2 system evolved in vascular plants conferring osmostress protection independently from the ancient system.

Archetypal Roles of an Abscisic Acid Receptor in Drought and Sugar Responses in Liverworts.

Abscisic acid (ABA) controls seed dormancy and stomatal closure through binding to the intracellular receptor Pyrabactin resistance1 (Pyr1)/Pyr1-like/regulatory components of ABA receptors (PYR/PYL/RCAR) in angiosperms. Genes encoding PYR/PYL/RCAR are thought to have arisen in the ancestor of embryophytes, but the roles of the genes in nonvascular plants have not been determined. In the liverwort Marchantia polymorpha, ABA reduces growth and enhances desiccation tolerance through increasing accumulation of intracellular sugars and various transcripts such as those of Late Embryogenesis Abundant (LEA)-like genes. In this study, we analyzed a gene designated MpPYL1, which is closely related to PYR/PYL/RCAR of angiosperms, in transgenic liverworts. Transgenic lines overexpressing MpPYL1-GFP showed ABA-hypersensitive growth with enhanced desiccation tolerance, whereas Mppyl1 generated by CRISPR-Cas9-mediated genome editing showed ABA-insensitive growth with reduced desiccation tolerance. Transcriptome analysis indicated that MpPYL1 is a major regulator of abiotic stress-associated genes, including all 35 ABA-induced LEA-like genes. Furthermore, these transgenic plants showed altered responses to extracellular Suc, suggesting that ABA and PYR/PYL/RCAR function in sugar responses. The results presented here reveal an important role of PYR/PYL/RCAR in the ABA response, which was likely acquired in the common ancestor of land plants. The results also indicate the archetypal role of ABA and its receptor in sugar response and accumulation processes for vegetative desiccation tolerance in bryophytes.

Phosphoproteomic profiling reveals ABA-responsive phosphosignaling pathways in Physcomitrella patens.

Abscisic acid (ABA) and its signaling system are important for land plants to survive in terrestrial conditions. Here, we took a phosphoproteomic approach to elucidate the ABA signaling network in Physcomitrella patens, a model species of basal land plants. Our phosphoproteomic analysis detected 4630 phosphopeptides from wild-type P. patens and two ABA-responsive mutants, a disruptant of group-A type-2C protein phosphatase (PP2C; ppabi1a/b) and AR7, a defective mutant in ARK, identified as an upstream regulator of SnRK2. Quantitative analysis detected 143 ABA-responsive phosphopeptides in P. patens. The analysis indicated that SnRK2-mediated phosphorylation and target motifs were partially conserved in bryophytes. Our data demonstrate that the PpSnRK2B and AREB/ABF-type transcription factors are phosphorylated in vivo in response to ABA under the control of ARK. On the other hand, our data also revealed the following: (i) the entire ABA-responsive phosphoproteome in P. patens is quite diverse; (ii) P. patens PP2C affects additional pathways other than the known ABA signaling pathway; and (iii) ARK is mainly involved in ABA signaling. Taken together, we propose that the core ABA signaling pathway is essential in all land plants; however, some ABA-responsive phosphosignaling uniquely developed in bryophytes during the evolutionary process.

Plant Raf-like kinase integrates abscisic acid and hyperosmotic stress signaling upstream of SNF1-related protein kinase2.

Plant response to drought and hyperosmosis is mediated by the phytohormone abscisic acid (ABA), a sesquiterpene compound widely distributed in various embryophyte groups. Exogenous ABA as well as hyperosmosis activates the sucrose nonfermenting 1 (SNF1)-related protein kinase2 (SnRK2), which plays a central role in cellular responses against drought and dehydration, although the details of the activation mechanism are not understood. Analysis of a mutant of the moss Physcomitrella patens with reduced ABA sensitivity and reduced hyperosmosis tolerance revealed that a protein kinase designated "ARK" (for "ABA and abiotic stress-responsive Raf-like kinase") plays an essential role in the activation of SnRK2. ARK encoded by a single gene in P. patens belongs to the family of group B3 Raf-like MAP kinase kinase kinases (B3-MAPKKKs) mediating ethylene, disease resistance, and salt and sugar responses in angiosperms. Our findings indicate that ARK, as a novel regulatory component integrating ABA and hyperosmosis signals, represents the ancestral B3-MAPKKKs, which multiplied, diversified, and came to have specific functions in angiosperms.

Novel Abscisic Acid Antagonists Identified with Chemical Array Screening.

Abscisic acid (ABA) signaling is involved in multiple processes in plants, such as water stress control and seed dormancy. Major regulators of ABA signaling are the PYR/PYL/RCAR family receptor proteins, group A protein phosphatases 2C (PP2Cs), and subclass III of SNF1-related protein kinase 2 (SnRK2). Novel ABA agonists and antagonists to modulate the functions of these proteins would not only contribute to clarification of the signaling mechanisms but might also be used to improve crop yields. To obtain small molecules that interact with Arabidopsis ABA receptor PYR1, we screened 24 275 compounds from a chemical library at the RIKEN Natural Products Depository by using a chemical array platform. Subsequent SnRK2 and PP2C assays narrowed down the candidates to two molecules. One antagonized ABA in a competitive manner and inhibited the formation of the PYR1-ABA-PP2C ternary complex. These compounds might have potential as bioprobes to analyze ABA signaling.

Screening of kinase substrates using kinase knockout mutants.

Protein kinases are widely known to be major regulators of various signaling processes, particularly in eukaryotes, including plants. To understand their role in signal transduction pathways, it is necessary to determine which proteins are phosphorylated by these enzymes. Recent studies have applied a comparative phosphoproteomic approach to identify protein kinase substrates in plants. The results demonstrated that kinase knockout mutants are useful for screening protein kinase substrates via such a comparative analysis. Here some technical points are described for the experimental design and comparative analysis using kinase knockout mutants.

Phosphorylation networks in the abscisic Acid signaling pathway.

Abscisic acid (ABA) is one of the major phytohormones and regulates various processes in the plant life cycle, for example, seed development and abiotic/biotic stress responses. Recent studies have made significant progress in elucidating ABA signaling and established a simple ABA signaling model consisting of three core components: PYR/PYL/RCAR receptors, 2C-type protein phosphatases, and SnRK2 protein kinases. This model highlights the importance of protein phosphorylation mediated by SnRK2, but the downstream substrates of SnRK2 remain to be determined to complete the model. Previous studies have identified several SnRK2 substrates involving transcription factors and ion channels. Recently, SnRK2 substrates have been further surveyed by a phosphoproteomic approach, giving new insights on the SnRK2 downstream pathway. Other protein kinases, e.g., Ca(2+)-dependent protein kinase (CDPK) and mitogen-activated protein kinase (MAPK), have been identified as ABA signaling factors. Some evidence suggests that the SnRK2 pathway partially interacts with CDPK or MAPK pathways. In this chapter, recent advances in ABA signaling study are summarized, primarily focusing on two major protein kinases, SnRK2 and MAPK. Challenges for further study of the ABA-dependent protein phosphorylation network are also discussed.