Search

Metabolic processes in prokaryotic and eukaryotic organisms are often modulated by kinases which are in turn, dependent on Ca 2+ 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., Competing Interests: Declaration of competing interest The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses or interpretation of the data; in the writing of the manuscript or in the decision to publish the results., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The cleavage of intracellular domains of receptor-like kinases (RLKs) has an important functional role in the transduction of signals from the cell surface to the nucleus in many organisms. However, the peptidases that catalyze protein cleavage during signal transduction remain poorly understood despite their crucial roles in diverse signaling processes. Here, we report in the flowering plant Arabidopsis thaliana that members of the DA1 family of ubiquitin-regulated Zn metallopeptidases cleave the cytoplasmic kinase domain of transmembrane kinase 1 (TMK1), releasing it for nuclear localization where it represses auxin-responsive cell growth during apical hook formation by phosphorylation and stabilization of the transcriptional repressors IAA32 and IAA34. Mutations in DA1 family members exhibited reduced apical hook formation, and DA1 family-mediated cleavage of TMK1 was promoted by auxin treatment. Expression of the DA1 family-generated intracellular kinase domain of TMK1 by an auxin-responsive promoter fully restored apical hook formation in a tmk1 mutant, establishing the function of DA1 family peptidase activities in TMK1-mediated differential cell growth and apical hook formation. DA1 family peptidase activity therefore modulates TMK1 kinase activity between a membrane location where it stimulates acid cell growth and initiates an auxin-dependent kinase cascade controlling cell proliferation in lateral roots and a nuclear localization where it represses auxin-mediated gene expression and growth.

SNF1-related Kinase 1 (SnRK1) is an evolutionarily conserved protein kinase with key functions in energy management during stress responses in plants. To address a potential role of SnRK1 under favorable conditions, we performed a metabolomic and transcriptomic characterization of rosettes of 20-d-old Arabidopsis (Arabidopsis thaliana) plants of SnRK1 gain- and loss-of-function mutants during the regular diel cycle. Our results show that SnRK1 manipulation alters the sucrose and trehalose 6-phosphate (Tre6P) relationship, influencing how the sucrose content is translated into Tre6P accumulation and modulating the flux of carbon to the tricarboxylic acid cycle downstream of Tre6P signaling. On the other hand, daily cycles of Tre6P accumulation were accompanied by changes in SnRK1 signaling, leading to a maximum in the expression of SnRK1-induced genes at the end of the night, when Tre6P levels are lowest, and to a minimum at the end of the day, when Tre6P levels peak. The expression of SnRK1-induced genes was strongly reduced by transient Tre6P accumulation in an inducible Tre6P synthase (otsA) line, further suggesting the involvement of Tre6P in the diel oscillations in SnRK1 signaling. Transcriptional profiling of wild-type plants and SnRK1 mutants also uncovered defects that are suggestive of an iron sufficiency response and of a matching induction of sulfur acquisition and assimilation when SnRK1 is depleted. In conclusion, under favorable growth conditions, SnRK1 plays a role in sucrose homeostasis and transcriptome remodeling in autotrophic tissues and its activity is influenced by diel fluctuations in Tre6P levels., (© The Author(s) 2021. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

The phytohormone auxin controls many processes in plants, at least in part through its regulation of cell expansion 1 . The acid growth hypothesis has been proposed to explain auxin-stimulated cell expansion for five decades, but the mechanism that underlies auxin-induced cell-wall acidification is poorly characterized. Auxin induces the phosphorylation and activation of the plasma membrane H + -ATPase that pumps protons into the apoplast 2 , yet how auxin activates its phosphorylation remains unclear. Here we show that the transmembrane kinase (TMK) auxin-signalling proteins interact with plasma membrane H + -ATPases, inducing their phosphorylation, and thereby promoting cell-wall acidification and hypocotyl cell elongation in Arabidopsis. Auxin induced interactions between TMKs and H + -ATPases in the plasma membrane within seconds, as well as TMK-dependent phosphorylation of the penultimate threonine residue on the H+-ATPases. Our genetic, biochemical and molecular evidence demonstrates that TMKs directly phosphorylate plasma membrane H + -ATPase and are required for auxin-induced H + -ATPase activation, apoplastic acidification and cell expansion. Thus, our findings reveal a crucial connection between auxin and plasma membrane H + -ATPase activation in regulating apoplastic pH changes and cell expansion through TMK-based cell surface auxin signalling., (© 2021. The Author(s).)

In Arabidopsis, ERECTA (ER) subfamily of leucine-rich repeat (LRR) receptor kinases (LRR-RKs) play important roles in cell division and cell elongation. However, the functions of OsER genes in rice are still very much unknown. In this study, sixty-seven TILLING and four gene-edited mutants were identified for one of the three OsERs, OsERL, and used for functional analyses. Results showed that mutations in OsERL led to striking defects in anther development. Compete male sterility and reduced numbers of anther lobes, more severe than knockout mutants, were observed in mutants with amino acid substitutions in the kinase domain. Among alleles with amino acid changes in LRRs, only one mutation in the 16 th LRR showed evident phenotype, suggesting a role of the LRR in ligand sensing. OsERL is expressed in shoot apcies, internodes and anthers, and within the anther OsERL is expressed in sporophytic and tapetal cells. Cell biological analyses revealed that mutations in OsERL led to defected periclinal division in archesporial cells in anthers, suggesting a critical role of OsERL in rice anther development., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The shoot apical meristem (SAM) and root apical meristem (RAM) act as pools of stem cells that give rise to aboveground and underground tissues and organs in higher plants, respectively. The CLAVATA3 (CLV3)-WUSCHEL (WUS) negative-feedback loop acts as a core pathway controlling SAM homeostasis, while CLV3/EMBRYO SURROUNDING REGION (ESR) 40 (CLE40) and WUSCHEL-RELATED HOMEOBOX5 (WOX5), homologs of CLV3 and WUS, direct columella stem cell fate. Moreover, CLV3 INSENSITIVE KINASES (CIKs) have been shown to be essential for maintaining SAM homeostasis, whereas whether they regulate the distal root meristem remains to be elucidated. Here, we report that CIKs are indispensable for transducing the CLE40 signal to maintain homeostasis of the distal root meristem. We found that the cik mutant roots displayed disrupted quiescent center and delayed columella stem cell (CSC) differentiation. Biochemical assays demonstrated that CIKs interact with ARABIDOPSIS CRINKLY4 (ACR4) in a ligand-independent manner and can be phosphorylated by ACR4 in vitro. In addition, the phosphorylation of CIKs can be rapidly induced by CLE40, which partially depends on ACR4. Although CIKs act as conserved and redundant regulators in the SAM and RAM, our results demonstrated that they exhibit differentiated functions in these meristems., (Copyright © 2021 The Author. Published by Elsevier Inc. All rights reserved.)

Pattern-triggered immunity (PTI) is typically initiated in plants by recognition of pathogen- or damage-associated molecular patterns (PAMP/DAMPs) by cell surface-localized pattern recognition receptors (PRRs). Here, we investigated the role in PTI of Arabidopsis thaliana brassinosteroid-signalling kinases 7 and 8 (BSK7 and BSK8), which are members of the receptor-like cytoplasmic kinase subfamily XII. BSK7 and BSK8 localized to the plant cell periphery and interacted in yeast and in planta with FLS2, but not with other PRRs. Consistent with a role in FLS2 signalling, bsk7 and bsk8 single and bsk7,8 double mutant plants were impaired in several immune responses induced by flg22, but not by other PAMP/DAMPs. These included resistance to Pseudomonas syringae and Botrytis cinerea, reactive oxygen species accumulation, callose deposition at the cell wall, and expression of the defence-related gene PR1, but not activation of MAP kinases and expression of the FRK1 and WRKY29 genes. bsk7, bsk8, and bsk7,8 plants also displayed enhanced susceptibility to P. syringae and B. cinerea. Finally, BSK7 and BSK8 variants mutated in their myristoylation site or in the ATP-binding site failed to complement defective phenotypes of the corresponding mutants, suggesting that localization to the cell periphery and kinase activity are critical for BSK7 and BSK8 functions. Together, these findings demonstrate that BSK7 and BSK8 play a role in PTI initiated by recognition of flg22 by interacting with the FLS2 immune receptor., (© 2021 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

In response to changing light quantity and quality, photosynthetic organisms perform state transitions, a process which optimizes photosynthetic yield and mitigates photo-damage. The serine/threonine-protein kinase STN7 phosphorylates the light-harvesting complex of photosystem II (PSII; light-harvesting complex II), which then migrates from PSII to photosystem I (PSI), thereby rebalancing the light excitation energy between the photosystems and restoring the redox poise of the photosynthetic electron transport chain. Two conserved cysteines forming intra- or intermolecular disulfide bonds in the lumenal domain (LD) of STN7 are essential for the kinase activity although it is still unknown how activation of the kinase is regulated. In this study, we show lumen thiol oxidoreductase 1 (LTO1) is co-expressed with STN7 in Arabidopsis (Arabidopsis thaliana) and interacts with the LD of STN7 in vitro and in vivo. LTO1 contains thioredoxin (TRX)-like and vitamin K epoxide reductase domains which are related to the disulfide-bond formation system in bacteria. We further show that the TRX-like domain of LTO1 is able to oxidize the conserved lumenal cysteines of STN7 in vitro. In addition, loss of LTO1 affects the kinase activity of STN7 in Arabidopsis. Based on these results, we propose that LTO1 helps to maintain STN7 in an oxidized active state in state 2 through redox interactions between the lumenal cysteines of STN7 and LTO1., (© The Author(s) 2021. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

LEAFY COTYLEDON1 (LEC1), a NUCLEAR FACTOR-Y (NF-Y) family member, plays a critical role in embryogenesis and seed development in Arabidopsis. Previous studies have shown that rice OsNF-YB9 and OsNF-YB7 are homologous to Arabidopsis LEC1. However, the functions of LEC1-like genes in rice remain unclear. Here we report that OsNF-YB9 and OsNF-YB7 display sub-functionalization in rice. We demonstrate that OsNF-YB7 is expressed mainly in the embryo, whereas OsNF-YB9 is preferentially expressed in the developing endosperm. Heterologous expression of either OsNF-YB9 or OsNF-YB7 in Arabidopsis lec1-1 was able to complement the lec1-1 defects. We failed to generate osnf-yb7 homozygous mutants due to lethality caused by OsNF-YB7 defects. Loss of OsNF-YB9 function caused abnormal seed development: seeds were longer, narrower and thinner and exhibited a higher chalkiness ratio. Furthermore, the expression of genes related to starch synthesis was deregulated in osnf-yb9. OsNF-YB9 could interact with SPK, a sucrose synthase protein kinase that is predominantly expressed in rice endosperm. Knockout of SPK resulted in chalky seeds similar to those observed in the osnf-yb9 mutants. Ectopic expression of OsNF-YB9 in both rice and Arabidopsis resulted in unhealthy plants with small seeds. Taken together, these results suggest a critical role for OsNF-YB9 in rice seed development., (© 2021 Society for Experimental Biology and John Wiley & Sons Ltd.)

Receptor-like kinases (RLKs) constitute a large group of cell surface receptors that play crucial roles in multiple biological processes. However, the function of most RLKs in plants has not been extensively explored, and much less for the class of cell wall associated kinases (WAKs) and WAK-like kinases (WAKLs). In this study, analyses of developmental expression patterns uncovered a putative role of AtWAKL10 in modulating leaf senescence, which was further investigated at physiological and molecular levels. The expression level of AtWAKL10 increased with the developmental progression and was rapidly upregulated in senescing leaf tissues. The promoter of AtWAKL10 contains various defense and hormone responsive elements, and its expression could be significantly induced by exogenous ABA, JA and SA. Moreover, the loss-of-function atwakl10 mutant showed earlier senescence along the course of natural development and accelerated leaf senescence under darkness and hormonal stresses, while plants overexpressing AtWAKL10 showed an opposite trend. Additionally, some defense and senescence related WRKY transcription factors could bind to the promoter of AtWAKL10 . In addition, deletion and overexpression of AtWAKL10 caused several specific transcriptional alterations, including genes involved in cell extension, cell wall modification, defense response and senescence related WRKYs, which may be implicated in regulatory mechanisms adopted by AtWAKL10 in controlling leaf senescence. Taken together, these results revealed that AtWAKL10 negatively regulated leaf senescence.

Polarity is a feature of life. In higher plants, non-autonomous polarity is largely directed by auxin, the morphogen that drives its own polarized flow, Polar Auxin Transport (PAT), to guide patterning events such as phyllotaxis and tropism. The plasma membrane-localized PIN-FORMED (PIN) auxin efflux carriers are rate-limiting factors in PAT. In yeasts and metazoans, the STE20 kinases are key players in cell polarity. We had previously characterized SIK1 as a STE20/Hippo orthologue in Arabidopsis and confirmed its function in mitotic exit and organ growth. Here we explore the possible link between SIK1, auxin, PIN, and polarity. Abnormal phyllotaxis and gravitropism were observed in sik1. sik1 was more sensitive to exogenous auxin in primary root elongation and lateral root emergence. RNA-Seq revealed reduced expression in auxin biosynthesis genes and induced expression of auxin flux carriers in sik1. However, normal tissue- and sub-cellular localization patterns of PIN1 and PIN2 were observed in sik1. The dark-induced vacuolar degradation of PIN2 also appeared normal in sik1. An additive phenotype was observed in the sik1 pin1 double mutant, indicating that SIK1 does not directly regulate PIN1. The polarity defects of sik1 are hence unlikely mediated by PINs and await future exploration., Competing Interests: Declaration of competing interest We declare no conflict of interest., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Invasive or penetrative growth is critical for developmental and reproductive processes (e.g., pollen tube penetration of pistils) and disease progression (e.g., cancer metastasis and fungal hyphae invasion). The invading or penetrating cells experience drastic changes in mechanical pressure from the surroundings and must balance growth with cell integrity. Here, we show that Arabidopsis pollen tubes sense and/or respond to mechanical changes via a cell-surface receptor kinase Buddha's Paper Seal 1 (BUPS1) while emerging from compressing female tissues. BUPS1-defective pollen tubes fail to maintain cell integrity after emergence from these tissues. The mechano-transduction function of BUPS1 is established by using a microfluidic channel device mimicking the mechanical features of the in vivo growth path. BUPS1-based mechano-transduction activates Rho-like GTPase from Plant 1 (ROP1) GTPase to promote exocytosis that facilitates secretion of BUPS1's ligands for mechanical signal amplification and cell wall rigidification in pollen tubes. These findings uncover a membrane receptor-based mechano-transduction system for cells to cope with the physical challenges during invasive or penetrative growth., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Sucrose non-fermenting-1-related protein kinase 2 s (SnRK2 s) are important stress-related plant protein kinases in plants. The interaction partners and phosphorylation substrates of group II and III SnRK2 s in Arabidopsis thaliana have been identified, but similar data for group I SnRK2 s are very limited. Here, we used a yeast two-hybrid (Y2H) screen to find proteins that interact with Arabidopsis AtSnRK2.4, a group I SnRK2. The transcription factor AtMYB21 was identified as an AtSnRK2.4 interaction partner, and its interaction with AtSnRK2.4 was confirmed by an in vitro pull-down assay and a bimolecular fluorescence complementation (BiFC) assay. A subcellular localization assay demonstrated that AtSnRK2.4 and AtMYB21 were located in the cytoplasm and nucleus of onion epidermal cells. AtSnRK2.4 and AtMYB21 were expressed in many tissues and upregulated in response to NaCl stress. Transgenic plants that overexpressed AtSnRK2.4 or AtMYB21 gene exhibited enhanced tolerance to salt stress at germination and post-germination stages. Moreover, the expression of downstream stress-responsive genes was upregulated in salt-stressed AtSnRK2.4 and AtMYB21 transgenic Arabidopsis. These results suggest that AtSnRK2.4 may act synergistically with AtMYB21 to mediate the response to salt stress through the upregulation of downstream stress-responsive genes., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Disruption of the MEKK1-MKK1/MKK2-MPK4 kinase cascade leads to activation of immunity mediated by the nucleotide-binding leucine-rich repeat (NLR) immune receptor SUMM2, which monitors the phosphorylation status of CRCK3. Here we report that two receptor-like kinases (RLKs), MDS1, and MDS2, function redundantly to promote SUMM2-mediated immunity. Activation of SUMM2-mediated immunity is dependent on MDS1, and to a less extent on MDS2. MDS1 associates with CRCK3 in planta and can phosphorylate CRCK3 in vitro, suggesting that it may target CRCK3 to positively regulate SUMM2-mediated signaling. Our finding highlights a new defense mechanism where RLKs promote NLR-mediated immunity., (© 2020 Institute of Botany, Chinese Academy of Sciences.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Unc-51-like kinase 4 (ULK4) is a pseudokinase that has been linked to the development of several diseases. Even though sequence motifs required for ATP binding in kinases are lacking, ULK4 still tightly binds ATP and the presence of the co-factor is required for structural stability of ULK4. Here, we present a high-resolution structure of a ULK4-ATPγS complex revealing a highly unusual ATP binding mode in which the lack of the canonical VAIK motif lysine is compensated by K39, located N-terminal to αC. Evolutionary analysis suggests that degradation of active site motifs in metazoan ULK4 has co-occurred with an ULK4-specific activation loop, which stabilizes the C helix. In addition, cellular interaction studies using BioID and biochemical validation data revealed high confidence interactors of the pseudokinase and armadillo repeat domains. Many of the identified ULK4 interaction partners were centrosomal and tubulin-associated proteins and several active kinases suggesting interesting regulatory roles for ULK4., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Many stresses induce the accumulation of unfolded and misfolded proteins in the endoplasmic reticulum, a phenomenon known as ER stress. In response to ER stress, cells initiate a protective response, known as unfolded protein response (UPR), to maintain cellular homeostasis. The UPR sensor, inositol-requiring enzyme 1 (IRE1), catalyzes the cytoplasmic splicing of bZIP transcription factor-encoding mRNAs to activate the UPR signaling pathway. Recently, we reported that pretreatment of Arabidopsis thaliana plants with tunicamycin, an ER stress inducer, increased their susceptibility to bacterial pathogens; on the other hand, IRE1 deficient mutants were susceptible to Pseudomonas syringae pv. maculicola (Psm) and failed to induce salicylic acid (SA)-mediated systemic acquired resistance. However, the functional relationship of IRE1 with the pathogen and TM treatment remains unknown. In the present study, we showed that bacterial pathogen-associated molecular patterns (PAMPs) induced IRE1 expression; however, PAMP-triggered immunity (PTI) response such as callose deposition, PR1 protein accumulation, or Pst DC3000 hrcC growth was not altered in ire1 mutants. We observed that IRE1 enhanced plant immunity against the bacterial pathogen P. syringae pv. tomato DC3000 (Pst DC3000) under ER stress. Moreover, TM-pretreated ire1 mutants were more susceptible to the avirulent strain Pst DC3000 (AvrRpt2) and showed greater cell death than wild-type plants during effector-triggered immunity (ETI). Additionally, Pst DC3000 (AvrRpt2)-mediated RIN4 degradation was reduced in ire1 mutants under TM-induced ER stress. Collectively, our results reveal that IRE1 plays a pivotal role in the immune signaling pathway to activate plant immunity against virulent and avirulent bacterial strains under ER stress., (Copyright © 2020. Published by Elsevier Masson SAS.)

Adverse environmental conditions trigger responses in plants that promote stress tolerance and survival at the expense of growth 1 . However, little is known of how stress signalling pathways interact with each other and with growth regulatory components to balance growth and stress responses. Here, we show that plant growth is largely regulated by the interplay between the evolutionarily conserved energy-sensing SNF1-related protein kinase 1 (SnRK1) protein kinase and the abscisic acid (ABA) phytohormone pathway. While SnRK2 kinases are main drivers of ABA-triggered stress responses, we uncover an unexpected growth-promoting function of these kinases in the absence of ABA as repressors of SnRK1. Sequestration of SnRK1 by SnRK2-containing complexes inhibits SnRK1 signalling, thereby allowing target of rapamycin (TOR) activity and growth under optimal conditions. On the other hand, these complexes are essential for releasing and activating SnRK1 in response to ABA, leading to the inhibition of TOR and growth under stress. This dual regulation of SnRK1 by SnRK2 kinases couples growth control with environmental factors typical for the terrestrial habitat and is likely to have been critical for the water-to-land transition of plants.

The plant-specific receptor-like cytoplasmic kinases (RLCKs) form a large, poorly characterized family. Members of the RLCK VI_A class of dicots have a unique characteristic: their activity is regulated by Rho-of-plants (ROP) GTPases. The biological function of one of these kinases was investigated using a T-DNA insertion mutant and RNA interference. Loss of RLCK VI_A2 function resulted in restricted cell expansion and seedling growth. Although these phenotypes could be rescued by exogenous gibberellin, the mutant did not exhibit lower levels of active gibberellins nor decreased gibberellin sensitivity. Transcriptome analysis confirmed that gibberellin is not the direct target of the kinase; its absence rather affected the metabolism and signalling of other hormones such as auxin. It is hypothesized that gibberellins and the RLCK VI_A2 kinase act in parallel to regulate cell expansion and plant growth. Gene expression studies also indicated that the kinase might have an overlapping role with the transcription factor circuit (PIF4-BZR1-ARF6) controlling skotomorphogenesis-related hypocotyl/cotyledon elongation. Furthermore, the transcriptomic changes revealed that the loss of RLCK VI_A2 function alters cellular processes that are associated with cell membranes, take place at the cell periphery or in the apoplast, and are related to cellular transport and/or cell wall reorganisation., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Phosphate (Pi) deficiency severely restricts plant growth and development, as Pi is an essential macronutrient. Calcium (Ca 2+ ) is a ubiquitous second messenger in plants; calcineurin B-like proteins (CBL) and CBL-interacting protein kinases (CIPK) are signalling pathways that act as an important Ca 2+ signalling network which integrates plants to fine tune the response to stress; however, whether CIPK are involved in Pi deficiency stress remains largely unknown. In this study, we carried out a reverse genetic strategy to screen T-DNA insertion mutants of CIPK isoforms under Pi deficiency in Arabidopsis thaliana. Then Pi content, transcription of phosphate starvation-induced (PSI) genes, acid phosphatase activity and hydrogen peroxide were determined in the wild-type (WT) and cipk1 mutant, respectively. The phenotype of CIPK1 complementation lines was analysed. The cipk1 mutant had a more sensitive phenotype, with lower root elongation and root length, and decreased Pi content compared with the WT under Pi deficiency. Moreover, CIPK1 mutation caused phosphate starvation-induced (PSI) genes to be significantly induced under Pi deficiency. Histological staining demonstrated that the cipk1 mutant had increased acid phosphatase activity and hydrogen peroxide concentration under Pi deficiency. By using the yeast two-hybrid system, we further demonstrated the interaction between CIPK1 and the WRKY transcription factors, WRKY6 and WRKY42. Overall, we demonstrate that CIPK1 is involved in the Pi deficiency signalling pathway in A. thaliana, revealing the important role of Ca 2+ in the Pi nutrition signalling pathway, and potentially providing a theoretical foundation for molecular breeding of crops with better Pi utilization efficiency., (© 2020 German Society for Plant Sciences and The Royal Botanical Society of the Netherlands.)

Transmembrane kinases (TMKs) are members of the plant receptor-like kinase (RLK) family. TMKs are characterized by an extracellular leucine-rich-repeat (LRR) domain, a single transmembrane region and a cytoplasmic kinase domain. TMKs have been shown to act as critical modulators of cell expansion and cell proliferation. Here, the crystal structure of the extracellular domain of TMK3 (TMK3-ECD) was determined to a resolution of 2.06 Å, with an R work of 17.69% and an R free of 20.58%. Similar to the extracellular domain of TMK1, the TMK3-ECD structure contains two solenoids with 13 LRRs and a non-LRR region (316-364) between the tenth and 11th LRRs. A comparison of TMK3-ECD with other LRR-RLKs that contain a non-LRR region indicates that the non-LRR region plays a critical role in structural integrity and may contribute to ligand interactions. The non-LRR region of TMK3-ECD is characterized by two disulfide bonds that may have critical biological implications.

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., (© 2020 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

In animals, extracellular ATP is a well-studied signaling molecule that is recognized by plasma membrane-localized P2-type purinergic receptors. However, in contrast, much less is known about purinergic signaling in plants. P2 receptors play critical roles in a variety of animal biological processes, including immune system regulation. The first plant purinergic receptor, Arabidopsis ( Arabidopsis thaliana ) P2K1 (L-type lectin receptor kinase-I.9), was shown to contribute to plant defense against bacterial, oomycete, and fungal pathogens. Here, we demonstrate the isolation of a second purinergic receptor, P2K2, by complementation of an Arabidopsis p2k1 mutant. P2K2 (LecRK-I.5) has 74% amino acid similarity to P2K1. The P2K2 extracellular lectin domain binds to ATP with higher affinity than P2K1 (dissociation constant [ K d ] = 44.47 ± 15.73 nm). Interestingly, p2k2 and p2k1 p2k2 mutant plants showed increased susceptibility to the pathogen Pseudomonas syringae, with the double mutant showing a stronger phenotype. In vitro and in planta studies demonstrate that P2K2 and P2K1 interact and cross-phosphorylate upon extracellular ATP treatment. Thus, similar to animals, plants possess multiple purinergic receptors., (© 2020 American Society of Plant Biologists. All Rights Reserved.)

Recognition of microbe-associated molecular patterns (MAMPs) by pattern recognition receptors (PRRs) triggers the first line of inducible defence against invading pathogens 1-3 . Receptor-like cytoplasmic kinases (RLCKs) are convergent regulators that associate with multiple PRRs in plants 4 . The mechanisms that underlie the activation of RLCKs are unclear. Here we show that when MAMPs are detected, the RLCK BOTRYTIS-INDUCED KINASE 1 (BIK1) is monoubiquitinated following phosphorylation, then released from the flagellin receptor FLAGELLIN SENSING 2 (FLS2)-BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1) complex, and internalized dynamically into endocytic compartments. The Arabidopsis E3 ubiquitin ligases RING-H2 FINGER A3A (RHA3A) and RHA3B mediate the monoubiquitination of BIK1, which is essential for the subsequent release of BIK1 from the FLS2-BAK1 complex and activation of immune signalling. Ligand-induced monoubiquitination and endosomal puncta of BIK1 exhibit spatial and temporal dynamics that are distinct from those of the PRR FLS2. Our study reveals the intertwined regulation of PRR-RLCK complex activation by protein phosphorylation and ubiquitination, and shows that ligand-induced monoubiquitination contributes to the release of BIK1 family RLCKs from the PRR complex and activation of PRR signalling.

Starch is a key energy-storage molecule in plants that requires controlled synthesis and breakdown for effective plant growth. β-Amylases (BAMs) hydrolyze starch into maltose to help to meet the metabolic needs of the plant. In the model plant Arabidopsis thaliana there are nine BAMs, which have apparently distinct functional and domain structures, although the functions of only a few of the BAMs are known and there are no 3D structures of BAMs from this organism. Recently, AtBAM2 was proposed to form a tetramer based on chromatography and activity assays of mutants; however, there was no direct observation of this tetramer. Here, small-angle X-ray scattering data were collected from AtBAM2 and its N-terminal truncations to describe the structure and assembly of the tetramer. Comparison of the scattering of the AtBAM2 tetramer with data collected from sweet potato (Ipomoea batatas) BAM5, which is also reported to form a tetramer, showed there were differences in the overall assembly. Analysis of the N-terminal truncations of AtBAM2 identified a loop sequence found only in BAM2 orthologs that appears to be critical for AtBAM2 tetramer assembly as well as for activity.

Yeast Snf1 (Sucrose non-fermenting1), mammalian AMPK (5' AMP-activated protein kinase) and plant SnRK1 (Snf1-Related Kinase1) are conserved heterotrimeric kinase complexes that re-establish energy homeostasis following stress. The hormone abscisic acid (ABA) plays a crucial role in plant stress response. Activation of SnRK1 or ABA signaling results in overlapping transcriptional changes, suggesting these stress pathways share common targets. To investigate how SnRK1 and ABA interact during stress response in Arabidopsis thaliana, we screened the SnRK1 complex by yeast two-hybrid against a library of proteins encoded by 258 ABA-regulated genes. Here, we identify 125 SnRK1- interacting proteins (SnIPs). Network analysis indicates that a subset of SnIPs form signaling modules in response to abiotic stress. Functional studies show the involvement of SnRK1 and select SnIPs in abiotic stress responses. This targeted study uncovers the largest set of SnRK1 interactors, which can be used to further characterize SnRK1 role in plant survival under stress.

Hydrogen peroxide (H 2 O 2 ) is a major reactive oxygen species in unicellular and multicellular organisms, and is produced extracellularly in response to external stresses and internal cues 1-4 . H 2 O 2 enters cells through aquaporin membrane proteins and covalently modifies cytoplasmic proteins to regulate signalling and cellular processes. However, whether sensors for H 2 O 2 also exist on the cell surface remains unknown. In plant cells, H 2 O 2 triggers an influx of Ca 2+ ions, which is thought to be involved in H 2 O 2 sensing and signalling. Here, by using forward genetic screens based on Ca 2+ imaging, we isolated hydrogen-peroxide-induced Ca 2+ increases (hpca) mutants in Arabidopsis, and identified HPCA1 as a leucine-rich-repeat receptor kinase belonging to a previously uncharacterized subfamily that features two extra pairs of cysteine residues in the extracellular domain. HPCA1 is localized to the plasma membrane and is activated by H 2 O 2 via covalent modification of extracellular cysteine residues, which leads to autophosphorylation of HPCA1. HPCA1 mediates H 2 O 2 -induced activation of Ca 2+ channels in guard cells and is required for stomatal closure. Our findings help to identify how the perception of extracellular H 2 O 2 is integrated with responses to various external stresses and internal cues in plants, and have implications for the design of crops with enhanced fitness.

SNF1-related protein kinases 2 (SnRK2s) are key signaling elements regulating abscisic acid-dependent plant development and responses to environmental stresses. Our previous data showed that the SnRK2-interacting Calcium Sensor (SCS) inhibits SnRK2 activity. Use of alternative transcription start sites located within the Arabidopsis ( Arabidopsis thaliana ) AtSCS gene results in two in-frame transcripts and subsequently two proteins, that differ only by the sequence position of the N terminus. We previously described the longer AtSCS-A, and now describe the shorter AtSCS-B and compare the two isoforms. The two isoforms differ substantially in their expression profiles in plant organs and in response to environmental stresses, in their calcium binding properties, and in their conformational dynamics in the presence and absence of Ca 2+ Only AtSCS-A has the features of a calcium sensor. Both forms inhibit SnRK2 activity, but while AtSCS-A requires calcium for inhibition, AtSCS-B does not. Analysis of Arabidopsis plants stably expressing 35S :: AtSCS - A - c - myc or 35S :: AtSCS - B - c - myc in the scs - 1 knockout mutant background revealed that, in planta, both forms are negative regulators of abscisic acid-induced SnRK2 activity and regulate plant resistance against water deficit. Moreover, the data highlight biochemical, biophysical, and functional properties of EF-hand-like motifs in plant proteins., (© 2020 American Society of Plant Biologists. All Rights Reserved.)

Osmoregulation is important for plant growth, development and response to environmental changes. SNF1-related protein kinase 2s (SnRK2s) are quickly activated by osmotic stress and are central components in osmotic stress and abscisic acid (ABA) signaling pathways; however, the upstream components required for SnRK2 activation and early osmotic stress signaling are still unknown. Here, we report a critical role for B2, B3 and B4 subfamilies of Raf-like kinases (RAFs) in early osmotic stress as well as ABA signaling in Arabidopsis thaliana. B2, B3 and B4 RAFs are quickly activated by osmotic stress and are required for phosphorylation and activation of SnRK2s. Analyses of high-order mutants of RAFs reveal critical roles of the RAFs in osmotic stress tolerance and ABA responses as well as in growth and development. Our findings uncover a kinase cascade mediating osmoregulation in higher plants.

Galls induced by plant-parasitic nematodes involve a hyperactivation of the plant mitotic and endocycle machinery for their profit. Dedifferentiation of host root cells includes drastic cellular and molecular readjustments. In such a background, potential DNA damage in the genome of gall cells is evident. We investigated whether DNA damage checkpoint activation followed by DNA repair occurred, or was eventually circumvented, in nematode-induced galls. Galls display transcriptional activation of the DNA damage checkpoint kinase WEE1, correlated with its protein localization in the nuclei. The promoter of the stress marker gene SMR7 was evaluated under the WEE1-knockout background. Drugs inducing DNA damage and a marker for DNA repair, PARP1, were used to understand the mechanisms for coping with DNA damage in galls. Our functional study revealed that gall cells lacking WEE1 conceivably entered mitosis prematurely, disturbing the cell cycle despite the loss of genome integrity. The disrupted nuclei phenotype in giant cells hinted at the accumulation of mitotic defects. In addition, WEE1-knockout in Arabidopsis and downregulation in tomato repressed infection and reproduction of root-knot nematodes. Together with data on DNA-damaging drugs, we suggest a conserved function for WEE1 in controlling G1/S cell cycle arrest in response to a replication defect in galls., (© 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.)

Appropriate timing of seed germination is crucial for the survival and propagation of plants, and for crop yield, especially in environments prone to salinity or drought. However, the exact mechanisms by which seeds perceive changes in soil conditions and integrate them to trigger germination remain elusive, especially once the seeds are non-dormant. In this study, we determined that the Arabidopsis ERECTA (ER), ERECTA-LIKE1 (ERL1), and ERECTA-LIKE2 (ERL2) leucine-rich-repeat receptor-like kinases regulate seed germination and its sensitivity to changes in salt and osmotic stress levels. Loss of ER alone, or in combination with ERL1 and/or ERL2, slows down the initiation of germination and its progression to completion, or arrests it altogether under saline conditions, until better conditions return. This function is maternally controlled via the tissues surrounding the embryo, with a primary role being played by the properties of the seed coat and its mucilage. These relate to both seed-coat expansion and subsequent differentiation and to salinity-dependent interactions between the mucilage, subtending seed coat layers and seed interior in the germinating seed. Salt-hypersensitive er105, er105 erl1.2, er105 erl2.1 and triple-mutant seeds also exhibit increased sensitivity to exogenous ABA during germination, and under salinity show an enhanced up-regulation of the germination repressors and inducers of dormancy ABA-insensitive-3, ABA-insensitive-5, DELLA-encoding RGL2, and Delay-Of-Germination-1. These findings reveal a novel role of the ERECTA receptor-kinases in the sensing of conditions at the seed surface and the integration of developmental, dormancy and stress signalling pathways in seeds. They also open novel avenues for the genetic improvement of plant adaptation to changing drought and salinity patterns., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Cell division cycle 5 (Cdc5) is a highly conserved nucleic acid binding protein among eukaryotes and plays critical roles in development. Cdc5 can simultaneously bind to DNA and RNA by its N-terminal DNA-binding domain (DBD), but molecular mechanisms describing its nucleic acid recognition and the regulation of development through its nucleic acid binding remain unclear. Herein, we present a crystal structure of the N-terminal DBD of MoCdc5 (MoCdc5-DBD) from the rice blast fungus Magnaporthe oryzae. Residue K100 of MoCdc5 is on the periphery of a positively charged groove that is formed by K42, K45, R47, and N92 and is evolutionally conserved. Mutation of K100 significantly reduces the affinity of MoCdc5-DBD to a Cdc5-binding element but not to a conventional myeloblastosis (Myb) domain-binding element, suggesting that K100 is a key residue of the high binding affinity to Cdc5-binding element. Another conserved residue (R31) is located close to the U6 RNA in the structure of the spliceosome, and its mutation dramatically reduces the binding capacity of MoCdc5-DBD for U6 RNA. Importantly, mutations in these key residues, including R31, K42, and K100 in AtCDC5, an Arabidopsis thaliana ortholog of MoCdc5, greatly impair the functions of AtCDC5, resulting in pleiotropic development defects and reduced levels of primary microRNA transcripts. Taken together, our findings suggest that Cdc5-DBD binds nucleic acids with two distinct binding surfaces, one for DNA and another for RNA, which together contribute to establishing the regulation mechanism of Cdc5 on development through nucleic acid binding., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

De novo phosphatidylcholine (PC) biosynthesis via the Kennedy pathway involves highly endergonic biochemical reactions that must be fine-tuned with energy homeostasis. Previous studies have shown that CTP:phosphocholine cytidylyltransferase (CCT) is an important regulatory enzyme in this pathway and that its activity can be controlled at both transcriptional and posttranslational levels. Here we identified an important additional mechanism regulating plant CCT1 activity. Comparative analysis revealed that Arabidopsis CCT1 (AtCCT1) contains catalytic and membrane-binding domains that are homologous to those of rat CCT1. In contrast, the C-terminal phosphorylation domain important for stringent regulation of rat CCT1 was apparently missing in AtCCT1. Instead, we found that AtCCT1 contains a putative consensus site (Ser-187) for modification by sucrose nonfermenting 1-related protein kinase 1 (SnRK1 or KIN10/SnRK1.1), involved in energy homeostasis. Phos-tag SDS-PAGE coupled with MS analysis disclosed that SnRK1 indeed phosphorylates AtCCT1 at Ser-187, and we found that AtCCT1 phosphorylation substantially reduces its activity by as much as 70%. An S187A variant exhibited decreased activity, indicating the importance of Ser-187 in catalysis, and this variant was less susceptible to SnRK1-mediated inhibition. Protein truncation and liposome binding studies indicated that SnRK1-mediated AtCCT1 phosphorylation directly affects the catalytic domain rather than interfering with phosphatidate-mediated AtCCT1 activation. Overexpression of the AtCCT1 catalytic domain in Nicotiana benthamiana leaves increased PC content, and SnRK1 co-expression reduced this effect. Taken together, our results suggest that SnRK1 mediates the phosphorylation and concomitant inhibition of AtCCT1, revealing an additional mode of regulation for this key enzyme in plant PC biosynthesis., (© 2019 Caldo et al.)

Regulation of protein synthesis is critical for maintaining cellular homeostasis. In mammalian systems, translational regulatory networks have been elucidated in considerable detail. In plants, however, regulation occurs through different mechanisms that remain largely elusive. In this study, we present evidence that the Arabidopsis thaliana energy sensing kinase SnRK1, a homologue of mammalian AMP-activated kinase and yeast sucrose non-fermenting 1 (SNF1), inhibits translation by phosphorylating the cap binding proteins eIF4E and eIFiso4E. We establish that eIF4E and eIFiso4E contain two deeply conserved SnRK1 consensus target sites and that both interact with SnRK1 in vivo. We then demonstrate that SnRK1 phosphorylation inhibits the ability of Arabidopsis eIF4E and eIFiso4E to complement a yeast strain lacking endogenous eIF4E, and that inhibition correlates with repression of polysome formation. Finally, we show that SnRK1 over-expression in Nicotiana benthamiana plants reduces polysome formation, and that this effect can be counteracted by transient expression of eIF4E or mutant eIF4E containing non-phosphorylatable SnRK1 target residues, but not by a phosphomimic eIF4E. Together, these studies elucidate a novel and direct pathway for translational control in plant cells. In light of previous findings that SnRK1 conditions an innate antiviral defense and is inhibited by geminivirus pathogenicity factors, we speculate that phosphorylation of cap binding proteins may be a component of the resistance mechanism., (© 2019 Federation of European Biochemical Societies.)

The SnRK1 (SNF1 related protein kinase 1), a plant homologue of SNF1 (Sucrose non-fermenting 1)/AMPK (AMP-activated protein kinase), is an important metabolic sensor involving in catabolic and anabolic processes. SnRK1 is essential for plant metabolism regulation and response to environmental stresses. The plant SnRK1 consists of one catalytic (α1/α2 subunit) and two regulatory subunits (β1/β2/β3 and γ/βγ subunits), and functions as a heterotrimeric complex. Here we took advantage of a tricistronic expression vector and successfully purified the holoenzyme containing three subunits of SnRK1 from the E.coli. Using advantages of the E.coli system, we would be able to purify SnRK1 complex with high yield and high purity. Moreover, the complex is stable with high homogeneity. Using the purified complex, we confirmed that the βγ rather than the γ subunit of the plant SnRK1 acts as the canonical regulatory subunit. Besides, some basic characters of the SnRK1 holoenzyme was studied. Together, our results provide a convenient way for purify the plant SnRK1 complexes, and this would be helpful for follow-up study on SnRK1's structure and mechanism., (Copyright © 2019. Published by Elsevier Inc.)

AGC protein kinases play important roles in plant growth and development. Several AGC kinases in Arabidopsis have been functionally characterized. However, the "AGC Other" subfamily, including IRE, IREH1, IRE3 and IRE4, has not been well understood. Here, we reported that ireh1 mutants displayed a root skewing phenotype, which can be enhanced by ire3 mutation. IREH1 and IRE3 were expressed in roots, consistent with their function in controlling root skewing. The fluorescence intensities of the microtubule marker KNpro:EGFP-MBD were decreased in ireh1, ire3 and ireh1 ire3 mutants compared to wild type. The microtubule arrangements in ireh1 and ireh1 ire3 mutants were also altered. IREH1 physically interacted with IRE3 in vitro and in planta. Thus, our findings demonstrate that IREH1 and IRE3 protein kinases play important roles in controlling root skewing, and maintaining microtubule network in Arabidopsis., (Copyright © 2019 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.)

Plant-unique membrane receptor kinases with leucine-rich repeat (LRR) extracellular domains are key regulators of development and immune responses. Here, the 1.55 Å resolution crystal structure of the immune receptor kinase SOBIR1 from Arabidopsis is presented. The ectodomain structure reveals the presence of five LRRs sandwiched between noncanonical capping domains. The disulfide-bond-stabilized N-terminal cap harbours an unusual β-hairpin structure. The C-terminal cap features a highly positively charged linear motif which was found to be largely disordered in this structure. Size-exclusion chromatography and right-angle light-scattering experiments suggest that SOBIR1 is a monomer in solution. The protruding β-hairpin, a set of highly conserved basic residues at the inner surface of the SOBIR LRR domain and the presence of a genetic missense allele in LRR2 together suggest that the SOBIR1 ectodomain may mediate protein-protein interaction in plant immune signalling., (open access.)

Nucleotide-binding, leucine-rich repeat receptors (NLRs) perceive pathogen effectors to trigger plant immunity. Biochemical mechanisms underlying plant NLR activation have until now remained poorly understood. We reconstituted an active complex containing the Arabidopsis coiled-coil NLR ZAR1, the pseudokinase RKS1, uridylated protein kinase PBL2, and 2'-deoxyadenosine 5'-triphosphate (dATP), demonstrating the oligomerization of the complex during immune activation. The cryo-electron microscopy structure reveals a wheel-like pentameric ZAR1 resistosome. Besides the nucleotide-binding domain, the coiled-coil domain of ZAR1 also contributes to resistosome pentamerization by forming an α-helical barrel that interacts with the leucine-rich repeat and winged-helix domains. Structural remodeling and fold switching during activation release the very N-terminal amphipathic α helix of ZAR1 to form a funnel-shaped structure that is required for the plasma membrane association, cell death triggering, and disease resistance, offering clues to the biochemical function of a plant resistosome., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Pathogen recognition by nucleotide-binding (NB), leucine-rich repeat (LRR) receptors (NLRs) plays roles in plant immunity. The Xanthomonas campestris pv. campestris effector AvrAC uridylylates the Arabidopsis PBL2 kinase, and the latter (PBL2 UMP ) acts as a ligand to activate the NLR ZAR1 precomplexed with the RKS1 pseudokinase. Here we report the cryo-electron microscopy structures of ZAR1-RKS1 and ZAR1-RKS1-PBL2 UMP in an inactive and intermediate state, respectively. The ZAR1 LRR domain, compared with animal NLR LRR domains, is differently positioned to sequester ZAR1 in an inactive state. Recognition of PBL2 UMP is exclusively through RKS1, which interacts with ZAR1 LRR PBL2 UMP binding stabilizes the RKS1 activation segment, which sterically blocks ZAR1 adenosine diphosphate (ADP) binding. This engenders a more flexible NB domain without conformational changes in the other ZAR1 domains. Our study provides a structural template for understanding plant NLRs., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Chitin, a major component of fungal cell walls, is a well-known pathogen-associated molecular pattern (PAMP) that triggers defense responses in several mammal and plant species. Here, we show that two chitooligosaccharides, chitin and chitosan, act as PAMPs in grapevine (Vitis vinifera) as they elicit immune signalling events, defense gene expression and resistance against fungal diseases. To identify their cognate receptors, the grapevine family of LysM receptor kinases (LysM-RKs) was annotated and their gene expression profiles were characterized. Phylogenetic analysis clearly distinguished three V. vinifera LysM-RKs (VvLYKs) located in the same clade as the Arabidopsis CHITIN ELICITOR RECEPTOR KINASE1 (AtCERK1), which mediates chitin-induced immune responses. The Arabidopsis mutant Atcerk1, impaired in chitin perception, was transformed with these three putative orthologous genes encoding VvLYK1-1, -2, or -3 to determine if they would complement the loss of AtCERK1 function. Our results provide evidence that VvLYK1-1 and VvLYK1-2, but not VvLYK1-3, functionally complement the Atcerk1 mutant by restoring chitooligosaccharide-induced MAPK activation and immune gene expression. Moreover, expression of VvLYK1-1 in Atcerk1 restored penetration resistance to the non-adapted grapevine powdery mildew (Erysiphe necator). On the whole, our results indicate that the grapevine VvLYK1-1 and VvLYK1-2 participate in chitin- and chitosan-triggered immunity and that VvLYK1-1 plays an important role in basal resistance against E. necator., (© 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Abscisic acid (ABA) regulates growth and developmental processes in response to limiting water conditions. ABA functions through a core signaling pathway consisting of PYR1/PYL/RCAR ABA receptors, type 2C protein phosphatases (PP2Cs), and SnRK2-type protein kinases. Other signaling modules might converge with ABA signals through the modulation of core ABA signaling components. We have investigated the role of the protein kinase WNK8 in ABA signaling. WNK8 interacted with PP2CA and PYR1, phosphorylated PYR1 in vitro, and was dephosphorylated by PP2CA. A hypermorphic wnk8-ct Arabidopsis mutant allele suppressed ABA and glucose hypersensitivities of pp2ca-1 mutants during young seedling development, and WNK8 expression in protoplasts suppressed ABA-induced reporter gene expression. We conclude that WNK8 functions as a negative modulator of ABA signaling., (© 2018 Federation of European Biochemical Societies.)

Plants employ receptor-like kinases (RLKs) and receptor-like proteins for rapid recognition of invading pathogens, and RLKs then transmit signals to receptor-like cytoplasmic kinases (RLCKs) to activate immune responses. RLKs are under fine regulation mediated by subcellular trafficking, which contributes to proper activation of plant immunity. In this study, we show that Arabidopsis thaliana RECEPTOR-LIKE KINASE 902 (RLK902) plays important roles in resistance to the bacterial pathogen Pseudomonas syringae, but not to the fungal powdery mildew pathogen Golovinomyces cichoracearum. RLK902 localizes at the plasma membrane and associates with ENHANCED DISEASE RESISTANCE 4 (EDR4), a protein involved in clathrin-mediated trafficking pathways. EDR4 and CLATHRIN HEAVY CHAIN 2 (CHC2) regulate the subcellular trafficking and accumulation of RLK902 protein. Furthermore, we found that RLK902 directly associates with the RLCK BRASSINOSTEROID-SIGNALING KINASE1 (BSK1), a key component of plant immunity, but not with other members of the FLAGELLIN SENSING 2 immune complex. RLK902 phosphorylates BSK1, and its Ser-230 is a key phosphorylation site critical for RLK902-mediated defense signaling. Taken together, our data indicate that EDR4 regulates plant immunity by modulating the subcellular trafficking and protein accumulation of RLK902, and that RLK902 transmits immune signals by phosphorylating BSK1., (Copyright © 2018 The Author. Published by Elsevier Inc. All rights reserved.)

The Aurora kinases are serine/threonine kinases with conserved functions in mitotic cell division in eukaryotes. In Arabidopsis, Aurora kinases play important roles in primary meristem maintenance, but their functions in vascular development are still elusive. We report a dominant xdi-d mutant showing the xylem development inhibition (XDI) phenotype. Gene identification and transgenic overexpression experiments indicated that the activation of the Arabidopsis Aurora 2 (AtAUR2) gene is responsible for the XDI phenotype. In contrast, the aur1-2 aur2-2 double mutant plants showed enhanced differentiation of phloem and xylem cells, indicating that the Aurora kinases negatively affect xylem differentiation. The transcript levels of key regulatory genes in vascular cell differentiation, i.e. ALTERED PHLOEM DEVELOPMENT (APL), VASCULAR-RELATED NAC-DOMAIN 6 (VND6) and VND7, were higher in the aur1-2 aur2-2 double mutant and lower in xdi-d mutants compared with the wild-type plants, further supporting the functions of α-Aurora kinases in vascular development. Gene mutagenesis and transgenic studies showed that protein phosphorylation and substrate binding, but not protein dimerization and ubiquitination, are critical for the biological function of AtAUR2. These results indicate that α-Aurora kinases play key roles in vascular cell differentiation in Arabidopsis.

Key Message: Here, we used a hxk1 mutant in the Col-0 background. We demonstrated that HXK1 regulates cell proliferation and expansion early during leaf development, and that HXK1 is involved in sucrose-induced leaf growth stimulation independent of GPT2. Furthermore, we identified KINγ as a novel HXK1-interacting protein. In the last decade, extensive efforts have been made to unravel the underlying mechanisms of plant growth control through sugar availability. Signaling by the conserved glucose sensor HEXOKINASE1 (HXK1) has been shown to exert both growth-promoting and growth-inhibitory effects depending on the sugar levels, the environmental conditions and the plant species. Here, we used a hxk1 mutant in the Col-0 background to investigate the role of HXK1 during leaf growth in more detail and show that it is affected in both cell proliferation and cell expansion early during leaf development. Furthermore, the hxk1 mutant is less sensitive to sucrose-induced cell proliferation with no significant increase in final leaf growth after transfer to sucrose. Early during leaf development, transfer to sucrose stimulates expression of GLUCOSE-6-PHOSPHATE/PHOSPHATE TRANSPORTER2 (GPT2) and represses chloroplast differentiation. However, in the hxk1 mutant GPT2 expression was still upregulated by transfer to sucrose although chloroplast differentiation was not affected, suggesting that GPT2 is not involved in HXK1-dependent regulation of leaf growth. Finally, using tandem affinity purification of protein complexes from cell cultures, we identified KINγ, a protein containing four cystathionine β-synthase domains, as an interacting protein of HXK1.

Microbial patterns are recognized by cell-surface receptors to initiate pattern-triggered immunity (PTI) in plants. Receptor-like cytoplasmic kinases (RLCKs), such as BIK1, and calcium-dependent protein kinases (CPKs) are engaged during PTI to activate the NADPH oxidase RBOHD for reactive oxygen species (ROS) production. It is unknown whether protein kinases besides CPKs and RLCKs participate in RBOHD regulation. We screened mutants in all ten Arabidopsis MAP4 kinases (MAP4Ks) and identified the conserved MAP4K SIK1 as a positive regulator of PTI. sik1 mutants were compromised in their ability to elicit the ROS burst in response to microbial features and exhibited compromised PTI to bacterial infection. SIK1 directly interacts with, phosphorylates, and stabilizes BIK1 in a kinase activity-dependent manner. Furthermore, SIK1 directly interacts with and phosphorylates RBOHD upon flagellin perception. Thus, SIK1 positively regulates immunity by stabilizing BIK1 and activating RBOHD to promote the extracellular ROS burst., (Copyright © 2018 Elsevier Inc. All rights reserved.)

In flowering plants, the interaction of pollen tubes with female tissues is important for the accomplishment of double fertilization. Little information is known about the mechanisms that underlie signalling between pollen tubes and female tissues. In this study, two Arabidopsis pollen tube-expressed CrRLK1L protein kinases, Buddha's Paper Seal 1 (BUPS1) and BUPS2, were identified as being required for normal tip growth of pollen tubes in the pistil. They are expressed prolifically in pollen and pollen tubes and are localized on the plasma membrane of the pollen tube tip region. Mutations in BUPS1 drastically reduced seed set. Most of the bups1 mutant pollen tubes growing in the pistil exhibited a swollen pollen tube tip, leading to failure of fertilization. The bups2 pollen tubes had a slightly abnormal morphology but could still accomplish double fertilization. The bups1 bups2 double mutant exhibited a slightly enhanced phenotype compared to the single bups1 mutants. The BUPS1 proteins could form homomers and heteromers with BUPS2, whereas BUPS2 could only form heteromers with BUPS1. The BUPS proteins could interact with the Arabidopsis pollen-expressed RopGEFs in the yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays. The results indicated that the BUPSs may mediate normal polar growth of pollen tubes in the pistil., (© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.)

When seeds are exposed to drought and salinity during germination, newly germinated embryos stop growth and enter a quiescent state called postgerminative growth (PGG) arrest. PGG arrest protects embryos from the stress, but it is not known how PGG is restored from the arrest when stress is eased. In this study, we show that under stress- or abscisic acid-induced PGG arrest conditions, Arabidopsis thaliana Raf-type protein kinase 22 (AtRaf22) overexpression accelerated photoautotrophic seedling establishment, whereas atraf22 knockout mutations enhanced the arrest. Furthermore, when the stress intensity was reduced subsequently, AtRaf22 overexpression plants resumed growth and accomplished photoautotrophic transition much faster than the knockout or wild-type plants. These results suggest that AtRaf22 activity is important for maintaining growth capacity during stress-induced PGG arrest, which is most likely critical for competitive growth when the stress subsides and plants resume growth. Such a factor has not been reported before., (© 2018 John Wiley & Sons Ltd.)

Highlight: An increasing number of reports question conclusions based on loss-of-function lines that have unexpected genetic backgrounds. In this opinion paper, we urge researchers to meticulously (re)investigate phenotypes retrieved from various genetic backgrounds and be critical regarding some previously drawn conclusions. As an example, we provide new evidence that acr4-2 mutant phenotypes with respect to columella stem cells are due to the lack of ACR4 and not - at least not as a major contributor - to a mutation in QRT1. In addition, we take the opportunity to alert the scientific community about the qrt1-2 background of a large number of Syngenta Arabidopsis Insertion Library (SAIL) T-DNA lines, a feature that is not commonly recognized by Arabidopsis researchers. This qrt1-2 background might have an important impact on the interpretation of the results obtained using these research tools, now and in the past. In conclusion, as a community, we should continuously assess and - if necessary - correct our conclusions based on the large number of (genetic) tools our work is built on. In addition, the positive or negative results of this self-criticism should be made available to the scientific community.

CLAVATA3/EMBRYO SURROUNDING REGION (CLE) peptides are secreted endogenous plant ligands that are sensed by receptor kinases (RKs) to convey environmental and developmental inputs. Typically, this involves an RK with narrow ligand specificity that signals together with a more promiscuous co-receptor. For most CLEs, biologically relevant (co-)receptors are unknown. The dimer of the receptor-like protein CLAVATA 2 (CLV2) and the pseudokinase CORYNE (CRN) conditions perception of so-called root-active CLE peptides, the exogenous application of which suppresses root growth by preventing protophloem formation in the meristem. clv2 as well as crn null mutants are resistant to root-active CLE peptides, possibly because CLV2-CRN promotes expression of their cognate receptors. Here, we have identified the CLE-RESISTANT RECEPTOR KINASE ( CLERK ) gene, which is required for full sensing of root-active CLE peptides in early developing protophloem. CLERK protein can be replaced by its close homologs, SENESCENCE-ASSOCIATED RECEPTOR-LIKE KINASE (SARK) and NSP-INTERACTING KINASE 1 (NIK1). Yet neither CLERK nor NIK1 ectodomains interact biochemically with described CLE receptor ectodomains. Consistently, CLERK also acts genetically independently of CLV2-CRN We, thus, have discovered a novel hub for redundant CLE sensing in the root., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

With No Lysine (WNK) kinase belongs to ser/thr protein kinase group in which conserved catalytic lysine (K) residue of subdomain II is shifted to subdomain I. In this study, we cloned full-length coding region of WNK9 from Oryza sativa (OsWNK9) and performed in silico studies to confirm the presence of all kinase signature regulatory elements. The transcript analysis revealed that OsWNK9 was strongly down regulated under salinity, drought and ABA stress in shoots. Constitutive expression of OsWNK9 in Arabidopsis thaliana imparted increased tolerance to salt, drought, and ABA stress. Transgenic lines showed healthy phenotypes such as green leaves, achieved higher fresh weight and longer roots under salt, drought and ABA stress as compared to wild-type (WT). Transgenic plants showed better seed germination, higher chlorophyll retention and less water loss under salt and drought stress compared to WT. Promoter/gene expression studies revealed that OsWNK9 were expressed throughout plant tissues with higher expression in roots. Subcellular localization studies of OsWNK9 showed their presence in the nucleus. The transcript analysis of abiotic stress marker genes and ABA dependent genes showed they were highly expressed in transgenic lines compared to WT in response to salt and drought stress. The endogenous ABA level under salt and drought stress in transgenic lines was higher than WT. The results indicated that OsWNK9 may regulate salt and drought response in ABA dependent manner., (Copyright © 2018 Elsevier B.V. All rights reserved.)