SNF1-related Protein Kinase Research Articles

SnRK2 kinases sense molecular crowding and form condensates to disrupt ABI1 inhibition.

Plants sense and respond to hyperosmotic stress via quick activation of sucrose nonfermenting 1-related protein kinase 2 (SnRK2). Under unstressed conditions, the protein phosphatase type 2C (PP2C) in clade A interact with and inhibit SnRK2s in subgroup III, which are released from the PP2C inhibition via pyrabactin resistance 1-like (PYL) abscisic acid receptors. However, how SnRK2s are released under osmotic stress is unclear. Here, we outline how subgroup I SnRK2s sense molecular crowding to interrupt PP2C-mediated inhibition in plants. Severe hyperosmotic stress triggers condensate formation to activate the subgroup I SnRK2s, which requires their intrinsically disordered region. PP2Cs interact with and inhibit subgroup I SnRK2s, and this interaction is disrupted by phase separation of SnRK2s. The subgroup I SnRK2s are critical for severe osmotic stress responses. Our findings elucidate a mechanism for how macromolecular crowding is sensed in plants and demonstrate that physical separation of signaling molecules can segregate negative regulators to initiate signaling.

Identification of a Novel Stomatal Opening Chemical, PP242, That Inhibits Early Abscisic Acid Signal Transduction in Guard Cells.

Plants control their stomatal apertures to optimize carbon dioxide uptake and water loss. Stomata open in response to light through the phosphorylation of the penultimate residue, Thr, of plasma membrane (PM) H+-ATPase in guard cells. Stomata close in response to drought and the phytohormone abscisic acid (ABA), and ABA suppresses the light-induced activation of PM H+-ATPase. However, the signaling pathways that regulate the stomatal aperture remain unclear. Previously, we identified a target of rapamycin (TOR) inhibitor, temsirolimus, to induce stomatal opening through chemical screening. In the present study, we further investigated other TOR inhibitors and identified PP242 as a novel stomatal opening chemical. PP242 induced stomatal opening even in the dark, as well as phosphorylation of the penultimate Thr of PM H+-ATPase in guard cells. Interestingly, PP242 completely suppressed ABA-induced stomatal closure, and inhibited ABA-induced activation of SNF1-related protein kinase 2s (SnRK2s), which are essential kinases for ABA signal transduction in guard cells. In vitro biochemical analysis revealed that PP242 did not directly inhibit SnRK2 but rather inhibited upstream ABA signaling components, specifically B3 clade Raf-like kinases. A quadruple mutant of B3 clade Raf-like kinases exhibited an open stoma phenotype that resembled the effect of PP242. However, PP242 still induced stomatal opening in this mutant, suggesting that PP242 also targets other guard cell components. Together, these results reveal that PP242 induces stomatal opening partly by inhibiting steady-state ABA signal transduction.

Calcium-dependent protein kinases CPK3/4/6/11 and 27 respond to osmotic stress and activate SnRK2s in Arabidopsis.

Drought and salinity are significant environmental threats that cause hyperosmotic stress in plants, which respond with a transient elevation of cytosolic Ca2+ and activation of Snf1-related protein kinase 2s (SnRK2s) and downstream responses. The exact regulators decoding Ca2+ signals to activate downstream responses remained unclear. Here, we show that the calcium-dependent protein kinases CPK3/4/6/11 and 27 respond to moderate osmotic stress and dehydration to activate SnRK2 phosphorylation in Arabidopsis. Using quantitative phosphoproteomics in a higher-order mutant lacking 12 pyrabactin resistance 1-like (PYL) abscisic acid (ABA) receptors, we identified six CPKs that are phosphorylated under osmotic stress. CPK3/4/6/11/27 phosphorylate the SnRK2s on multiple phosphosites within the activation loop. The cpk3/4/6/11/27 mutant is defective in SnRK2 activation, seed germination, and seedling growth under mild osmotic stress. Our findings elucidate the critical roles of CPK3/4/6/11/27 in decoding Ca2+ signals to activate SnRK2s and demonstrate a CPK-SnRK2 kinase cascade controlling osmotic stress responses in plants.

Sucrose non-fermenting 1-related protein kinase 2–14 participating in lipid elevating efficacy and biodiesel upgrade by Coccomyxa subllipsoidea

Sucrose non-fermenting 1-related protein kinase 2–14 participating in lipid elevating efficacy and biodiesel upgrade by Coccomyxa subllipsoidea

Evolution and divergence of the role of plant ethylene receptor-related histidine kinases in abscisic acid signaling.

Plant responses to the water environment are mediated by ethylene (submergence response) and abscisic acid (ABA, drought response). Ethylene is perceived by a family of histidine kinase receptors (ETR-HKs), which regulate the activity of the downstream B3 Raf-like (RAF) kinase CONSTITUTIVE TRIPLE RESPONSE1 (CTR1) in an ethylene-dependent manner. We previously demonstrated in the moss Physcomitrium patens that SNF1-related protein kinase 2 (SnRK2), an essential kinase in osmostress responses in land plants, is activated by the B3-RAF kinase ARK, which is also regulated by ETR-HKs in an ABA- and osmostress-dependent manner. Whether this regulatory mechanism is evolutionarily conserved in land plants remains unknown. We demonstrate through a cross-species complementation assay that ETR-HKs from a terrestrial alga, bryophytes, and a lycophyte, but not those from angiosperms, retain the ability to activate ARK/SnRK2-mediated ABA signaling in the moss. This suggests that the role of ETR-HKs in ABA signaling was ancestral but lost in seed plants. The γ-loop in the C-terminal receiver domain is crucially involved in this specification of ETR-HK function.

A Systematic Review on the Role of SnRK2 Gene in <i>Arabidopsis thaliana</i> Growth Stages under Abiotic Stresses

This systematic review examines the role of SnRK2 (Sucrose non-fermenting 1-Related protein Kinase 2) genes in Arabidopsis thaliana growth and responses to abiotic stresses. SnRK2 protein kinases are key components of abscisic acid (ABA) signaling and osmotic stress responses in plants. The review synthesizes findings from numerous studies on how different SnRK2 genes regulate Arabidopsis growth, development, and stress tolerance at various life stages. Key topics covered include SnRK2 functions under environmental stresses like drought, salinity, cold, and nutrient deficiency; SnRK2 roles in seed germination and early seedling growth; and applications of SnRK2 genes in developing transgenic Arabidopsis with enhanced stress tolerance. The review highlights the complex regulatory networks involving SnRK2 kinases and their interactions with other signaling components like PP2C phosphatases and AREB/ABF transcription factors. Overall, this comprehensive analysis provides insights into the multifaceted roles of SnRK2 genes in modulating plant growth and stress adaptation, with potential applications for improving crop resilience. Further research directions are suggested to elucidate remaining questions about SnRK2 functions and regulatory mechanisms in plants.

SnRK1α1-mediated RBOH1 phosphorylation regulates reactive oxygen species to enhance tolerance to low nitrogen in tomato.

Nitrogen is essential for plant growth and development. SNF1-related protein kinase 1 (SnRK1) is an evolutionarily conserved protein kinase pivotal for regulating plant responses to nutrient deficiency. Here, we discovered that the expression and activity of the SnRK1 α-catalytic subunit (SnRK1α1) increased in response to low-nitrogen stress. SnRK1α1 overexpression enhanced seedling tolerance, nitrate uptake capacity, apoplastic reactive oxygen species (ROS) accumulation, and NADPH oxidase activity in tomato (Solanum lycopersicum L.) under low-nitrogen stress compared to wild type plants, while snrk1α1 mutants exhibited the opposite phenotypes. Mutation of the NADPH oxidase gene Respiratory burst oxidase homolog 1 (RBOH1) suppressed numerous nitrate uptake and metabolism genes during low-nitrogen stress. rboh1 mutants displayed lower NADPH oxidase activity, apoplastic ROS production, and seedling tolerance to low nitrogen. Silencing RBOH1 expression also compromised SnRK1α1-mediated seedling tolerance to low-nitrogen stress. SnRK1α1 interacts with and activates RBOH1 through phosphorylation of three N-terminal serine residues, leading to increased apoplastic ROS production and enhanced tolerance to low nitrogen conditions. Furthermore, RBOH1-dependent ROS oxidatively modified the transcription factor TGA4 at residue Cys-334, which increased NRT1.1 and NRT2.1 expression under low-nitrogen stress. These findings reveal a SnRK1α1-mediated signaling pathway and highlight the essential role of RBOH1-dependent ROS production in enhancing plant tolerance to low nitrogen.

Incomplete filling in the basal region of maize endosperm: timing of development of starch synthesis and cell vitality.

Starch synthesis in maize endosperm adheres to the basipetal sequence from the apex downwards. However, the mechanism underlying nonuniformity among regions of the endosperm in starch accumulation and its significance is poorly understood. Here, we examined the spatiotemporal transcriptomes and starch accumulation dynamics in apical (AE), middle (ME), and basal (BE) regions of endosperm throughout the filling stage. Results demonstrated that the BE had lower levels of gene transcripts and enzymes facilitating starch synthesis, corresponding to incomplete starch storage at maturity, compared with AE and ME. Contrarily, the BE showed abundant gene expression for genetic processing and slow progress in physiological development (quantified by an index calculated from the expression values of development progress marker genes), revealing a sustained cell vitality of the BE. Further analysis demonstrated a significant parabolic correlation between starch synthesis and physiological development. An in-depth examination showed that the BE had more active signaling pathways of IAA and ABA than the AE throughout the filling stage, while ethylene showed the opposite pattern. Besides, SNF1-related protein kinase1 (SnRK1) activity, a regulator for starch synthesis modulated by trehalose-6-phosphate (T6P) signaling, was kept at a lower level in the BE than the AE and ME, corresponding to the distinct gene expression in the T6P pathway in starch synthesis regulation. Collectively, the findings support an improved understanding of the timing of starch synthesis and cell vitality in regions of the endosperm during development, and potential regulation from hormone signaling and T6P/SnRK1 signaling.

Mitogen-activated protein kinase-mediated regulation of plant specialized metabolism.

Post-transcriptional and post-translational modification of transcription factors (TFs) and pathway enzymes significantly affect the stress-stimulated biosynthesis of specialized metabolites (SMs). Protein phosphorylation is one of the conserved and ancient mechanisms that critically influences many biological processes including specialized metabolism. The phosphorylation of TFs and enzymes by protein kinases (PKs), especially the mitogen-activated protein kinases (MAPKs), is well studied in plants. While the roles of MAPKs in plant growth and development, phytohormone signaling, and immunity are well elucidated, significant recent advances have also been made in understanding the involvement of MAPKs in specialized metabolism. However, a comprehensive review highlighting the significant progress in the past several years is notably missing. This review focuses on MAPK-mediated regulation of several important SMs, including phenylpropanoids (flavonoids and lignin), terpenoids (artemisinin and other terpenoids), alkaloids (terpenoid indole alkaloids and nicotine), and other nitrogen- and sulfur-containing SMs (camalexin and indole glucosinolates). In addition to MAPKs, other PKs also regulate SM biosynthesis. For comparison, we briefly discuss the regulation by other PKs, such as sucrose non-fermenting-1 (SNF)-related protein kinases (SnRKs) and calcium-dependent protein kinases (CPKs). Furthermore, we provide future perspectives in this active area of research.

The multifaceted role of different SnRK gene family members in regulating multiple abiotic stresses in plants.

Abiotic stresses are a major constraint for agricultural productivity and food security in today's era of climate change. Plants can experience different types of abiotic stresses, either individually or in combination. Sometimes, more than one stress event may occur simultaneously or one after another during the lifecycle of the plant. In general, key survival strategies for stress tolerance may differ from one stress to another. However, at the molecular level, evolutionarily conserved protein kinase SUCROSE NONFERMENTING 1 (SNF1)-related protein kinase (SnRK) gene family members, comprising SnRK1, SnRK2, and SnRK3 gene families, play a key role in different types of stress and adaptive responses. SnRK gene family members can act as master regulators and regulate the central metabolism of plants, which determines the energy distribution in either survival or growth/developmental processes. The key mechanism of SnRK-mediated regulation is associated with the phosphorylation of downstream genes, which either induces or dampens the function of target proteins. This may be crucial for maintaining differential morpho-physiological and biochemical processes in plants, including potassium signalling, ROS homeostasis, sugar signalling, and energy homeostasis. Furthermore, phosphorylation sites associated with different targets were also reviewed, which showed that SnRK-mediated phosphorylation of Serine and Threonine residues of the target protein is a site-specific event, where the target consists of specific amino acid sequences, including RXXS/T, Serine-threonine rich regions, or AMPK/SNF1 types. Here, we review different classes of SnRK gene family members and their multifaceted roles in understanding the commonality of SnRK-mediated responses to multiple abiotic stresses in plants.

Abscisic acid-induced H2O2 production positively regulates the activity of SAPK8/9/10 through oxidation of the type one protein phosphatase OsPP47.

Subclass III sucrose nonfermenting1-related protein kinase 2s (SnRK2s) are positive regulators of abscisic acid (ABA) signaling and abiotic stress responses. However, the underlying activation mechanisms of osmotic stress/ABA-activated protein kinase 8/9/10 (SAPK8/9/10) of rice (Oryza sativa) subclass III SnRK2s in ABA signaling remain to be elucidated. In this study, we employed biochemical, molecular biology, cell biology, and genetic approaches to identify the molecular mechanism by which OsPP47, a type one protein phosphatase in rice, regulates SAPK8/9/10 activity in ABA signaling. We found that OsPP47 not only physically interacted with SAPK8/9/10 but also interacted with ABA receptors PYLs. OsPP47 negatively regulated ABA sensitivity in seed germination and root growth. In the absence of ABA, OsPP47 directly inactivated SAPK8/9/10 by dephosphorylation. In the presence of ABA, ABA-bound OsPYL2 formed complexes with OsPP47 and inhibited its phosphatase activity, partially releasing the inhibition of SAPK8/9/10. SAPK8/9/10-mediated H2O2 production inhibited OsPP47 activity by oxidizing Cys-116 and Cys-256 to form OsPP47 oligomers, resulting in not only preventing the OsPP47-SAPK8/9/10 interaction but also blocking the inhibition of SAPK8/9/10 activity by OsPP47. Our results reveal novel pathways for the inhibition of SAPK8/9/10 in the basal state and for the activation of SAPK8/9/10 induced by ABA in rice.

Effects of blue-light irradiation on abscisic acid signaling and sugar translocation in Vitis labruscana L.H. Bailey grapevines

AbstractThe effects of blue-light irradiation on abscisic acid (ABA) signaling, sugar metabolism and translocation, and photoreceptors and gene expressions were investigated to clarify the mechanism by which blue-LED irradiation increases sugar concentrations in grape berries (Vitis labruscana L.). Blue light-emitting diode (LED) irradiation increased the portion of 13C-photosynthates in the grapevine clusters that were fed 13CO2; compared to the portion in the cluster in the untreated control. Fructose and glucose concentrations and the expressions of VvSWEET10, VvSUC11, and VvSUS4 in blue LED-irradiated berries were increased. The blue LED-irradiated berries’ sucrose concentrations were significantly lower than the untreated control at 14 days after treatment. We speculated that the blue LED-treated berries’ decreased sucrose was associated with the increased Sugars Will Eventually be Exported Transporter (VvSWEET10), sucrose transporter (VvSUC11), and sucrose synthase (VvSUS4) expressions and promoted the translocation of 13C-photosynthates from the leaves that were fed 13CO2. Blue-LED irradiation increased the expressions of SNF1-related protein kinases (VvSnRK2.6) and ABA responding element binding transcription factor (VvABF1), while decreasing the expression of protein phosphateses 2C9 (VvPP2C9) genes, which are related to ABA signaling. Blue-LED irradiation increased the expressions of cryptochrome (VvCRYa) and phototropin (VvPHOT2), which are photoreceptor genes. The application of the pyrabactin resistance-like (PYL)-PP2C ABA receptor interaction antagonist AS6 did not affect endogenous ABA concentrations in the grape berries, but it decreased sucrose concentrations at harvest. The application of ABA did not affect sucrose, glucose, or fructose concentrations or the expressions of VvSnRK2.6 and VvPP2C9. The application of nordihydroguaiaretic acid (NDGA, an inhibitor of 9-cis-epoxycarotenoid dioxygenase activity in ABA biosynthesis) did not affect sugar concentrations at harvest. These results suggest that upregulation of photoreceptor gene expressions and ABA signaling are associated with sugar concentrations in grape berries.

Senescence-Associated Sugar Transporter1 affects developmental master regulators and controls senescence in Arabidopsis.

Sugar transport across membranes is critical for plant development and yield. However, an analysis of the role of intracellular sugar transporters in senescence is lacking. Here, we characterized the role of Senescence-Associated Sugar Transporter1 (SAST1) during senescence in Arabidopsis (Arabidopsis thaliana). SAST1 expression was induced in leaves during senescence and after the application of abscisic acid (ABA). SAST1 is a vacuolar protein that pumps glucose out of the cytosol. sast1 mutants exhibited a stay-green phenotype during developmental senescence, after the darkening of single leaves, and after ABA feeding. To explain the stay-green phenotype of sast1 mutants, we analyzed the activity of the glucose-induced master regulator TOR (target of rapamycin), which is responsible for maintaining a high anabolic state. TOR activity was higher in sast1 mutants during senescence compared to wild types, whereas the activity of its antagonist, SNF1-related protein kinase 1 (SnRK1), was reduced in sast1 mutants under senescent conditions. This deregulation of TOR and SnRK1 activities correlated with high cytosolic glucose levels under senescent conditions in sast1 mutants. Although sast1 mutants displayed a functional stay-green phenotype, their seed yield was reduced. These analyses place the activity of SAST1 in the last phase of a leaf's existence in the molecular program required to complete its life cycle.

Genome-wide analysis of the PYL-PP2C-SnRK2s family in the ABA signaling pathway of pitaya reveals its expression profiles under canker disease stress

BackgroundAbscisic acid (ABA) plays a crucial role in seed dormancy, germination, and growth, as well as in regulating plant responses to environmental stresses during plant growth and development. However, detailed information about the PYL-PP2C-SnRK2s family, a central component of the ABA signaling pathway, is not known in pitaya.ResultsIn this study, we identified 19 pyrabactin resistance-likes (PYLs), 70 type 2 C protein phosphatases (PP2Cs), and 14 SNF1-related protein kinase 2s (SnRK2s) from pitaya. In pitaya, tandem duplication was the primary mechanism for amplifying the PYL-PP2C-SnRK2s family. Co-linearity analysis revealed more homologous PYL-PP2C-SnRK2s gene pairs located in collinear blocks between pitaya and Beta vulgaris L. than that between pitaya and Arabidopsis. Transcriptome analysis showed that the PYL-PP2C-SnRK2s gene family plays a role in pitaya’s response to infection by N. dimidiatum. By spraying ABA on pitaya and subsequently inoculating it with N. dimidiatum, we conducted qRT-PCR experiments to observe the response of the PYL-PP2C-SnRK2s gene family and disease resistance-related genes to ABA. These treatments significantly enhanced pitaya’s resistance to pitaya canker. Further protein interaction network analysis helped us identify five key PYLs genes that were upregulated during the interaction between pitaya and N. dimidiatum, and their expression patterns were verified by qRT-PCR. Subcellular localization analysis revealed that the PYL (Hp1879) gene is primarily distributed in the nucleus.ConclusionThis study enhances our understanding of the response of PYL-PP2C-SnRK2s to ABA and also offers a new perspective on pitaya disease resistance.

Genome-wide identification, phylogenetic, structural and functional evolution of thecore components of ABA signaling in plant species: a focus on rice.

A genome-wide analysis had identified 642 ABA core component genes from 20 plant species, which were further categorized into three distinct subfamilies. The gene structures and evolutionary relationships of these genes had been characterized. PP2C_1, PP2C_2, and SnRK2_1 had emerged as key players in mediating the ABA signaling transduction pathway, specifically in rice, in response to abiotic stresses. The plant hormone abscisic acid (ABA) is essential for growth, development, and stress response, relying on its core components, pyrabactin resistance, pyrabactin resistance-like, and the regulatory component of ABA receptor (PYR/PYL/RCAR), 2C protein phosphatase (PP2C), sucrose non-fermenting-1-related protein kinase 2 (SnRK2). However, there's a lack of research on their structural evolution and functional differentiation across plants. Our study analyzed the phylogenetic, gene structure, homology, and duplication evolution of this complex in 20 plant species. We found conserved patterns in copy number and homology across subfamilies. Segmental and tandem duplications drove the evolution of these genes, while whole-genome duplication (WGD) expanded PYR/PYL/RCAR and PP2C subfamilies, enhancing environmental adaptation. In rice and Arabidopsis, the PYR/PYL/RCAR, PP2C, and SnRK2 genes showed distinct tissue-specific expression and responded to various stresses. Notably, PP2C_1 and PP2C_2 interacted with SnRK2_1 and were crucial for ABA signaling in rice. These findings offered new insights into ABA signaling evolution, interactions, and integration in green plants, benefiting future research in agriculture, evolutionary biology, ecology, and environmental science.

Melatonin mitigates drought stress by increasing sucrose synthesis and suppressing abscisic acid biosynthesis in tomato seedlings.

The increasing prevalence of drought events poses a major challenge for upcoming crop production. Melatonin is a tiny indolic tonic substance with fascinating regulatory functions in plants. While plants can respond in several ways to alleviate drought stress, the processes underpinning stress sensing and signaling are poorly understood. Hereafter, the objectives of this investigation were to explore the putative functions of melatonin in the regulation of sugar metabolism and abscisic acid biosynthesis in drought-stressed tomato seedlings. Melatonin (100 μM) and/or water were foliar sprayed, followed by the plants being imposed to drought stress for 14 days. Drought stress significantly decreased biomass accumulation, inhibited photosynthetic activity, and stimulated senescence-associated gene 12 (SAG12) expression. Melatonin treatment effectively reversed drought-induced growth retardation as evidenced by increased leaf pigment and water balance and restricted abscisic acid (ABA) accumulation. Sugar accumulation, particularly sucrose content, was higher in drought-imposed seedlings, possibly owing to higher transcription levels of sucrose non-fermenting 1-related protein kinase 2 (SnKR2.2) and ABA-responsive element binding factors 2 (AREB2). Melatonin addition further uplifted the sucrose content, which coincided with increased activity of sucrose synthase (SS, 130%), sucrose phosphate synthase (SPS, 137%), starch degradation encoding enzyme β-amylase (BAM, 40%) and α-amylase (AMY, 59%) activity and upregulated their encoding BAM1(10.3 folds) and AMY3 (8.1 folds) genes expression at day 14 relative to the control. Under water deficit conditions, melatonin supplementation decreased the ABA content (24%) and its biosynthesis gene expressions. Additionally, sugar transporter subfamily genes SUT1 and SUT4 expression were upregulated by the addition of melatonin. Collectively, our findings illustrate that melatonin enhances drought tolerance in tomato seedlings by stimulating sugar metabolism and negatively regulating ABA synthesis.

Identification of SnRK2 family and functional study of PeSnRK2.2A and PeSnRK2.2B for drought resistance in Phyllostachys edulis

Bamboo is a typical fast-growing non-timber bioresource in tropical and subtropical forests, yet its vulnerability to drought is one of the facing difficulties in commercial cultivation. The mechanism strategy in response to drought is poorly understood in bamboo plants. This study identified Sucrose non-fermenting 1-related protein kinase 2 s (SnRK2s) in the moso bamboo (Phyllostachys edulis), a group of kinases essential for plant adaptation. The expansion of PeSnKR2 family was driven by gene duplication. 18 PeSnKR2s were categorized into 3 subgroups based on their evolutionary relationships. Cis-elements analysis and expression analysis revealed that PeSnRK2s might be involved in stress resistance through ABA-dependent or ABA-independent pathways. 9 ABA-dependent and 9 ABA-independent PeSnKR2s were defined according to their sensibility to ABA. Furthermore, we examined the function of two ABA-independent SnRK2s, PeSnRK2.2A and PeSnRK2.2B, which were relevantly conserved at protein, transcript, and genomic levels, while showing distinct expression patterns under drought conditions. A transgenic rice study revealed that overexpression of either PeSnRK2.2A or PeSnRK2.2B relieved the overwhelming ROS in leaves to improve rice resistance under drought. The ABA signaling pathway was activated in transgenic rice indicating that PeSnRK2.2A or PeSnRK2.2B regulated tolerance via a conserved ABA-regulated manner downstream. Therefore, we concluded that PeSnRK2.2B and PeSnRK2.2A have similar roles in regulating ABA signals, but respond differently to environmental signals in P. edulis. PeSnRK2.2s represent the potential targets for the improvement of bamboo agronomic traits in the future. This study improves the understanding of drought-resistance mechanisms in moso bamboo and benefits genetic improvement and maintenance of bamboo sustainability.

A SNF1-related protein kinase regulatory subunit functions as a molecular tuner

Metabolic processes in prokaryotic and eukaryotic organisms are often modulated by kinases which are in turn, dependent on Ca2+ and the cyclic mononucleotides cAMP and cGMP. It has been established that some proteins have both kinase and cyclase activities and that active cyclases can be embedded within the kinase domains. Here, we identified phosphodiesterase (PDE) sites, enzymes that hydrolyse cAMP and cGMP, to AMP and GMP, respectively, in some of these proteins in addition to their kinase/cyclase twin-architecture. As an example, we tested the Arabidopsis thaliana KINγ, a subunit of the SnRK2 kinase, to demonstrate that all three enzymatic centres, adenylate cyclase (AC), guanylate cyclase (GC) and PDE, are catalytically active, capable of generating and hydrolysing cAMP and cGMP. These data imply that the signal output of the KINγ subunit modulates SnRK2, consequently affecting the downstream kinome. Finally, we propose a model where a single protein subunit, KINγ, is capable of regulating cyclic mononucleotide homeostasis, thereby tuning stimulus specific signal output.

Molecular mechanism of trehalose 6-phosphate inhibition of the plant metabolic sensor kinase SnRK1.

SUCROSE-NON-FERMENTING1-RELATED PROTEIN KINASE1 (SnRK1), a central plant metabolic sensor kinase, phosphorylates its target proteins, triggering a global shift from anabolism to catabolism. Molecular modeling revealed that upon binding of KIN10 to GEMINIVIRUS REP-INTERACTING KINASE1 (GRIK1), KIN10's activation T-loop reorients into GRIK1's active site, enabling its phosphorylation and activation. Trehalose 6-phosphate (T6P) is a proxy for cellular sugar status and a potent inhibitor of SnRK1. T6P binds to KIN10, a SnRK1 catalytic subunit, weakening its affinity for GRIK1. Here, we investigate the molecular details of T6P inhibition of KIN10. Molecular dynamics simulations and in vitro phosphorylation assays identified and validated the T6P binding site on KIN10. Under high-sugar conditions, T6P binds to KIN10, blocking the reorientation of its activation loop and preventing its phosphorylation and activation by GRIK1. Under these conditions, SnRK1 maintains only basal activity levels, minimizing phosphorylation of its target proteins, thereby facilitating a general shift from catabolism to anabolism.

Integrating multi-omics data reveals energy and stress signaling activated by abscisic acid in Arabidopsis.

Phytohormones are essential signaling molecules regulating various processes in growth, development, and stress responses. Genetic and molecular studies, especially using Arabidopsis thaliana (Arabidopsis), have discovered many important players involved in hormone perception, signal transduction, transport, and metabolism. Phytohormone signaling pathways are extensively interconnected with other endogenous and environmental stimuli. However, our knowledge of the huge and complex molecular network governed by a hormone remains limited. Here we report a global overview of downstream events of an abscisic acid (ABA) receptor, REGULATORY COMPONENTS OF ABA RECEPTOR (RCAR) 6 (also known as PYRABACTIN RESISTANCE 1 [PYR1]-LIKE [PYL] 12), by integrating phosphoproteomic, proteomic and metabolite profiles. Our data suggest that the RCAR6 overexpression constitutively decreases the protein levels of its coreceptors, namely clade A protein phosphatases of type 2C, and activates sucrose non-fermenting-1 (SNF1)-related protein kinase 1 (SnRK1) and SnRK2, the central regulators of energy and ABA signaling pathways. Furthermore, several enzymes in sugar metabolism were differentially phosphorylated and expressed in the RCAR6 line, and the metabolite profile revealed altered accumulations of several organic acids and amino acids. These results indicate that energy- and water-saving mechanisms mediated by the SnRK1 and SnRK2 kinases, respectively, are under the control of the ABA receptor-coreceptor complexes.