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Plants initiate specific defense responses by recognizing conserved epitope peptides within the flagellin proteins derived from bacteria. Proteolytic cleavage of epitope peptides from flagellin by plant apoplastic proteases is thought to be crucial for the perception of the epitope by the plant receptor. However, the identity of the plant proteases involved in this process remains unknown. Here, we establish an efficient identification system for the target proteases in Arabidopsis apoplastic fluid; the method employs native two-dimensional electrophoresis followed by an in-gel proteolytic assay using a fluorescence-quenching peptide substrate. We designed a substrate to specifically detect proteolytic activity at the C-terminus of the flg22 epitope in flagellin and identified two plant subtilases, SBT5.2 and SBT1.7, as specific proteases responsible for the C-terminal cleavage of flg22. In the apoplastic fluid of Arabidopsis mutant plants deficient in these two proteases, we observe a decrease in the C-terminal cleavage of the flg22 domain from flagellin, leading to a decrease in the efficiency of flg22 epitope liberation. Consequently, defensive reactive oxygen species (ROS) production is delayed in sbt5.2 sbt1.7 double-mutant leaf disks compared to wild type following flagellin exposure., (© 2024. The Author(s).)

Stomatal pores in the plant epidermis open and close to regulate gas exchange between leaves and the atmosphere. Upon light stimulation, the plasma membrane (PM) H + -ATPase is phosphorylated and activated via an intracellular signal transduction pathway in stomatal guard cells, providing a primary driving force for the opening movement. To uncover and manipulate this stomatal opening pathway, we screened a chemical library and identified benzyl isothiocyanate (BITC), a Brassicales-specific metabolite, as a potent stomatal-opening inhibitor that suppresses PM H + -ATPase phosphorylation. We further developed BITC derivatives with multiple isothiocyanate groups (multi-ITCs), which demonstrate inhibitory activity on stomatal opening up to 66 times stronger, as well as a longer duration of the effect and negligible toxicity. The multi-ITC treatment inhibits plant leaf wilting in both short (1.5 h) and long-term (24 h) periods. Our research elucidates the biological function of BITC and its use as an agrochemical that confers drought tolerance on plants by suppressing stomatal opening., (© 2023. The Author(s).)

Perception of pathogen-derived ligands by corresponding host receptors is a pivotal strategy in eukaryotic innate immunity. In plants, this is complemented by circadian anticipation of infection timing, promoting basal resistance even in the absence of pathogen threat. Here, we report that trichomes, hair-like structures on the epidermis, directly sense external mechanical forces, including raindrops, to anticipate pathogen infections in Arabidopsis thaliana. Exposure of leaf surfaces to mechanical stimuli initiates the concentric propagation of intercellular calcium waves away from trichomes to induce defence-related genes. Propagating calcium waves enable effective immunity against pathogenic microbes through the CALMODULIN-BINDING TRANSCRIPTION ACTIVATOR 3 (CAMTA3) and mitogen-activated protein kinases. We propose an early layer of plant immunity in which trichomes function as mechanosensory cells that detect potential risks., (© 2022. The Author(s).)

Plants tailor immune responses to defend against pathogens with different lifestyles. In this process, antagonism between the immune hormones salicylic acid (SA) and jasmonic acid (JA) optimizes transcriptional signatures specifically to the attacker encountered. Antagonism is controlled by the transcription cofactor NPR1. The indispensable role of NPR1 in activating SA-responsive genes is well understood, but how it functions as a repressor of JA-responsive genes remains unclear. Here, we demonstrate that SA-induced NPR1 is recruited to JA-responsive promoter regions that are co-occupied by a JA-induced transcription complex consisting of the MYC2 activator and MED25 Mediator subunit. In the presence of SA, NPR1 physically associates with JA-induced MYC2 and inhibits transcriptional activation by disrupting its interaction with MED25. Importantly, NPR1-mediated inhibition of MYC2 is a major immune mechanism for suppressing pathogen virulence. Thus, NPR1 orchestrates the immune transcriptome not only by activating SA-responsive genes but also by acting as a corepressor of JA-responsive MYC2., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The phytohormone abscisic acid (ABA) plays a major role in abiotic stress responses in plants, and subclass III SNF1-related protein kinase 2 (SnRK2) kinases mediate ABA signaling. In this study, we identified Raf36, a group C Raf-like protein kinase in Arabidopsis , as a protein that interacts with multiple SnRK2s. A series of reverse genetic and biochemical analyses revealed that 1) Raf36 negatively regulates ABA responses during postgermination growth, 2) the N terminus of Raf36 is directly phosphorylated by SnRK2s, and 3) Raf36 degradation is enhanced in response to ABA. In addition, Raf22, another C-type Raf-like kinase, functions partially redundantly with Raf36 to regulate ABA responses. A comparative phosphoproteomic analysis of ABA-induced responses of wild-type and raf22raf36-1 plants identified proteins that are phosphorylated downstream of Raf36 and Raf22 in planta. Together, these results support a model in which Raf36/Raf22 function mainly under optimal conditions to suppress ABA responses, whereas in response to ABA, the SnRK2 module promotes Raf36 degradation as a means of alleviating Raf36-dependent inhibition and allowing for heightened ABA signaling to occur., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Malate efflux from roots, which is regulated by the transcription factor STOP1 (SENSITIVE-TO-PROTON-RHIZOTOXICITY1) and mediates aluminum-induced expression of ALUMINUM-ACTIVATED-MALATE-TRANSPORTER1 (AtALMT1), is critical for aluminum resistance in Arabidopsis thaliana. Several studies showed that AtALMT1 expression in roots is rapidly observed in response to aluminum; this early induction is an important mechanism to immediately protect roots from aluminum toxicity. Identifying the molecular mechanisms that underlie rapid aluminum resistance responses should lead to a better understanding of plant aluminum sensing and signal transduction mechanisms. In this study, we observed that GFP-tagged STOP1 proteins accumulated in the nucleus soon after aluminum treatment. The rapid aluminum-induced STOP1-nuclear localization and AtALMT1 induction were detected in the presence of a protein synthesis inhibitor, suggesting that post-translational regulation is involved in these events. STOP1 also regulated rapid aluminum-induced expression for other genes that carry a functional/high-affinity STOP1-binding site in their promoter, including STOP2, GLUTAMATE-DEHYDROGENASE1 and 2 (GDH1 and 2). However STOP1 did not regulate Al resistance genes which have no functional STOP1-binding site such as ALUMINUM-SENSITIVE3, suggesting that the binding of STOP1 in the promoter is essential for early induction. Finally, we report that GDH1 and 2 which are targets of STOP1, are novel aluminum-resistance genes in Arabidopsis., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Homologous recombination is a key process for maintaining genome integrity and diversity. In eukaryotes, the nucleosome structure of chromatin inhibits the progression of homologous recombination. The DNA repair and recombination protein RAD54 alters the chromatin structure via nucleosome sliding to enable homology searches. For homologous recombination to progress, appropriate recruitment and dissociation of RAD54 is required at the site of homologous recombination; however, little is known about the mechanism regulating RAD54 dynamics in chromatin. Here, we reveal that the histone demethylase LYSINE-SPECIFIC DEMETHYLASE1-LIKE 1 (LDL1) regulates the dissociation of RAD54 at damaged sites during homologous recombination repair in the somatic cells of Arabidopsis ( Arabidopsis thaliana ). Depletion of LDL1 leads to an overaccumulation of RAD54 at damaged sites with DNA double-strand breaks. Moreover, RAD54 accumulates at damaged sites by recognizing histone H3 Lys 4 di-methylation (H3K4me2); the frequency of the interaction between RAD54 and H3K4me2 increased in the ldl1 mutant with DNA double-strand breaks. We propose that LDL1 removes RAD54 at damaged sites by demethylating H3K4me2 during homologous recombination repair and thereby maintains genome stability in Arabidopsis., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

Arabidopsis ASYMMETRIC LEAVES2 (AS2) plays a critical role in leaf adaxial-abaxial partitioning by repressing expression of the abaxial-determining gene ETTIN/AUXIN RESPONSE FACTOR3 (ETT/ARF3). We previously reported that six CpG dinucleotides in its exon 6 are thoroughly methylated by METHYLTRASFERASE1, that CpG methylation levels are inversely correlated with ETT/ARF3 transcript levels and that methylation levels at three out of the six CpG dinucleotides are decreased in as2-1. All these imply that AS2 is involved in epigenetic repression of ETT/ARF3 by gene body DNA methylation. The mechanism of the epigenetic repression by AS2, however, is unknown. Here, we tested mutations of NUCLEOLIN1 (NUC1) and RNA HELICASE10 (RH10) encoding nucleolus-localized proteins for the methylation in exon 6 as these mutations enhance the level of ETT/ARF3 transcripts in as2-1. Methylation levels at three specific CpGs were decreased in rh10-1, and two of those three overlapped with those in as2-1. Methylation levels at two specific CpGs were decreased in nuc1-1, and one of those three overlapped with that in as2-1. No site was affected by both rh10-1 and nuc1-1. One specific CpG was unaffected by these mutations. These results imply that the way in which RH10, NUC1 and AS2 are involved in maintaining methylation at five CpGs in exon 6 might be through at least several independent pathways, which might interact with each other. Furthermore, we found that AS2 binds specifically the sequence containing CpGs in exon 1 of ETT/ARF3, and that the binding requires the zinc-finger-like motif in AS2 that is structurally similar to the zinc finger-CxxC domain in vertebrate DNA methyltransferase1.

Reactive oxygen species (ROS) are known to be important signal molecules that are involved in biotic and abiotic stress responses as well as in growth regulation. However, the molecular mechanisms by which ROS act as a growth regulator, as well as how ROS-dependent growth regulation relates to its roles in stress responses, are not well understood. We performed a time-course microarray analysis of Arabidopsis root tips upon treatment with hydrogen peroxide, which we named "ROS-map." Using the ROS-map, we identified an MYB transcription factor, MYB30, which showed a strong response to ROS treatment and is the key regulator of a gene network that leads to the hydrogen peroxide-dependent inhibition of root cell elongation. Intriguingly, this network contained multiple genes involved in very-long-chain fatty acid (VLCFA) transport. Finally, we showed that MYB30 is necessary for root growth regulation during defense responses, thus providing a molecular link between these two ROS-associated processes., Competing Interests: The authors declare no conflict of interest.

The phytohormone auxin indole-3-acetic acid (IAA) regulates nearly all aspects of plant growth and development. Despite substantial progress in our understanding of auxin biology, delineating specific auxin response remains a major challenge. Auxin regulates transcriptional response via its receptors, TIR1 and AFB F-box proteins. Here we report an engineered, orthogonal auxin-TIR1 receptor pair, developed through a bump-and-hole strategy, that triggers auxin signaling without interfering with endogenous auxin or TIR1/AFBs. A synthetic, convex IAA (cvxIAA) hijacked the downstream auxin signaling in vivo both at the transcriptomic level and in specific developmental contexts, only in the presence of a complementary, concave TIR1 (ccvTIR1) receptor. Harnessing the cvxIAA-ccvTIR1 system, we provide conclusive evidence for the role of the TIR1-mediated pathway in auxin-induced seedling acid growth. The cvxIAA-ccvTIR1 system serves as a powerful tool for solving outstanding questions in auxin biology and for precise manipulation of auxin-mediated processes as a controllable switch.

Cell-free protein synthesis (CFPS) systems largely retain the endogenous translation machinery of the host organism, making them highly applicable for proteomics analysis of diverse biological processes. However, laborious and time-consuming cloning procedures hinder progress with CFPS systems. Herein, we report the development of a rapid and efficient two-step polymerase chain reaction (PCR) method to prepare linear DNA templates for a wheat germ CFPS system. We developed a novel, effective short 3'-untranslated region (3'-UTR) sequence that facilitates translation. Application of the short 3'-UTR to two-step PCR enabled the generation of various transcription templates from the same plasmid, including fusion proteins with N- or C-terminal tags, and truncated proteins. Our method supports the cloning-free expression of target proteins using an mRNA pool from biological material. The established system is a highly versatile platform for in vitro protein synthesis using wheat germ CFPS., (© 2017 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

In our previous study, a methodology was established to predict transcriptional regulatory elements in promoter sequences using transcriptome data based on a frequency comparison of octamers. Some transcription factors, including the NAC family, cannot be covered by this method because their binding sequences have non-specific spacers in the middle of the two binding sites. In order to remove this blind spot in promoter prediction, we have extended our analysis by including bipartite octamers that are composed of '4 bases-a spacer with a flexible length-4 bases'. 8,044 pre-selected bipartite octamers, which had an overrepresentation of specific spacer lengths in promoter sequences and sequences related to core elements removed, were subjected to frequency comparison analysis. Prediction of ER stress-responsive elements in the BiP/BiPL promoter and an ANAC017 target sequence resulted in precise detection of true positives, judged by functional analyses of a reported article and our own in vitro protein-DNA binding assays. These results demonstrate that incorporation of bipartite octamers with continuous ones improves promoter prediction significantly., (© The Author 2017. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.)

Perception of microbe-associated molecular patterns by host cell surface pattern recognition receptors (PRRs) triggers the intracellular activation of mitogen-activated protein kinase (MAPK) cascades. However, it is not known how PRRs transmit immune signals to MAPK cascades in plants. Here, we identify a complete phospho-signaling transduction pathway from PRR-mediated pathogen recognition to MAPK activation in plants. We found that the receptor-like cytoplasmic kinase PBL27 connects the chitin receptor complex CERK1-LYK5 and a MAPK cascade. PBL27 interacts with both CERK1 and the MAPK kinase kinase MAPKKK5 at the plasma membrane. Knockout mutants of MAPKKK5 compromise chitin-induced MAPK activation and disease resistance to Alternaria brassicicola PBL27 phosphorylates MAPKKK5 in vitro, which is enhanced by phosphorylation of PBL27 by CERK1. The chitin perception induces disassociation between PBL27 and MAPKKK5 in vivo Furthermore, genetic evidence suggests that phosphorylation of MAPKKK5 by PBL27 is essential for chitin-induced MAPK activation in plants. These data indicate that PBL27 is the MAPKKK kinase that provides the missing link between the cell surface chitin receptor and the intracellular MAPK cascade in plants., (© 2016 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Heat shock protein 90 (HSP90) is a molecular chaperone that is required for the function of various substrate proteins, also known as client proteins. It is proposed that HSP90 buffers or hides phenotypic variations in animals and plants by masking mutations in some of its client proteins. However, none of the client proteins with cryptic mutations has been identified to date. Here, we identify the first client protein example by which HSP90 buffers a mutation: the auxin receptor transport inhibitor response 1 (TIR1). TIR1 interacts with HSP90 in the nucleus. An HSP90-specific inhibitor abolished the nuclear localization of TIR1 and the auxin-induced degradation of a TIR1-substrate, indicating that TIR1 is an HSP90 client protein. Plants with a null mutation in the TIR1 gene had a defect in auxin response, whereas plants with a point mutation in the TIR1 gene responded to auxin treatment in young seedlings, but a cryptic defect in its auxin response was exposed with HSP90 inhibitor treatment. These results demonstrate that HSP90 masks a point mutation in the auxin receptor TIR1 and thereby buffers auxin-responsive phenotypes., (© The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Mitogen-activated protein kinase (MPK) cascades are important to cellular signaling in eukaryotes. They regulate growth, development and the response to environmental challenges. MPK cascades function via reversible phosphorylation of cascade components, MEKK, MEK, and MPK, but also by MPK substrate phosphorylation. Using mass spectrometry, we previously identified many in vivo MPK3 and MPK6 substrates in Arabidopsis thaliana, and we disclosed their phosphorylation sites., Results: We verified phosphorylation of several of our previously identified MPK3/6 substrates using a nonradioactive in vitro labeling assay. We engineered MPK3, MPK4, and MPK6 to accept bio-orthogonal ATPγS analogs for thiophosphorylating their appropriate substrate proteins. Subsequent alkylation of the thiophosphorylated amino acid residue(s) allows immunodetection using thiophosphate ester-specific antibodies. Site-directed mutagenesis of amino acids confirmed the protein substrates' site-specific phosphorylation by MPK3 and MPK6. A combined assay with MPK3, MPK6, and MPK4 revealed substrate specificity of the individual kinases., Conclusion: Our work demonstrates that the in vitro-labeling assay represents an effective, specific and highly sensitive test for determining kinase-substrate relationships.

The Arabidopsis (Arabidopsis thaliana) Early Light-Induced Protein (ELIP) is thought to act as a photoprotectant, reducing the damaging effects of high light (HL). Expression of ELIP2 is activated by multiple environmental stresses related to photoinhibition. We have identified putative regulatory elements in an ELIP2 promoter using an octamer-based frequency comparison method, analyzed the role of these elements using synthetic promoters, and revealed a key transcriptional regulatory unit for ultraviolet B (UV-B) radiation, HL, and cold stress responses. The unit is composed of two elements, designated as Elements A (TACACACC) and B (GGCCACGCCA), and shows functionality only when paired. Our genome-wide correlation analysis between possession of these elements in the promoter region and expression profiles in response to UV-B, HL, and cold suggests that Element B receives and integrates these multiple stress signals. In vitro protein-DNA binding assays revealed that LONG HYPOCOTYL5 (HY5), a basic domain-Leucine zipper transcription factor, directly binds to Element B. In addition, mutant analysis of HY5 showed partial involvement in the UV-B and HL responses but not in the cold stress response. These results suggest that signals for UV-B, HL, and cold stress join at Element B, which recognizes the signals of multiple transcription factors, including HY5., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

NPR1, a master regulator of basal and systemic acquired resistance in plants, confers immunity through a transcriptional cascade, which includes transcription activators (e.g., TGA3) and repressors (e.g., WRKY70), leading to the massive induction of antimicrobial genes. How this single protein orchestrates genome-wide transcriptional reprogramming in response to immune stimulus remains a major question. Paradoxically, while NPR1 is essential for defense gene induction, its turnover appears to be required for this function, suggesting that NPR1 activity and degradation are dynamically regulated. Here we show that sumoylation of NPR1 by SUMO3 activates defense gene expression by switching NPR1's association with the WRKY transcription repressors to TGA transcription activators. Sumoylation also triggers NPR1 degradation, rendering the immune induction transient. SUMO modification of NPR1 is inhibited by phosphorylation at Ser55/Ser59, which keeps NPR1 stable and quiescent. Thus, posttranslational modifications enable dynamic but tight and precise control of plant immune responses., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Redox Responsive Transcription Factor1 (RRTF1) in Arabidopsis is rapidly and transiently upregulated by H2O2, as well as biotic- and abiotic-induced redox signals. RRTF1 is highly conserved in angiosperms, but its physiological role remains elusive. Here we show that inactivation of RRTF1 restricts and overexpression promotes reactive oxygen species (ROS) accumulation in response to stress. Transgenic lines overexpressing RRTF1 are impaired in root and shoot development, light sensitive, and susceptible to Alternaria brassicae infection. These symptoms are diminished by the beneficial root endophyte Piriformospora indica, which reduces ROS accumulation locally in roots and systemically in shoots, and by antioxidants and ROS inhibitors that scavenge ROS. More than 800 genes were detected in mature leaves and seedlings of transgenic lines overexpressing RRTF1; ∼ 40% of them have stress-, redox-, ROS-regulated-, ROS-scavenging-, defense-, cell death- and senescence-related functions. Bioinformatic analyses and in vitro DNA binding assays demonstrate that RRTF1 binds to GCC-box-like sequences in the promoter of RRTF1-responsive genes. Upregulation of RRTF1 by stress stimuli and H2O2 requires WRKY18/40/60. RRTF1 is co-regulated with the phylogenetically related RAP2.6, which contains a GCC-box-like sequence in its promoter, but transgenic lines overexpressing RAP2.6 do not accumulate higher ROS levels. RRTF1 also stimulates systemic ROS accumulation in distal non-stressed leaves. We conclude that the elevated levels of the highly conserved RRTF1 induce ROS accumulation in response to ROS and ROS-producing abiotic and biotic stress signals., (Copyright © 2015 The Author. Published by Elsevier Inc. All rights reserved.)

In Arabidopsis (Arabidopsis thaliana) the root apex is protected from aluminum (Al) rhizotoxicity by excretion of malate, an Al chelator, by ALUMINUM-ACTIVATED MALATE TRANSPORTER1 (AtALMT1). AtALMT1 expression is fundamentally regulated by the SENSITIVE TO PROTON RHIZOTOXICITY1 (STOP1) zinc finger protein, but other transcription factors have roles that enable Al-inducible expression with a broad dynamic range. In this study, we characterized multiple cis-elements in the AtALMT1 promoter that interact with transcription factors. In planta complementation assays of AtALMT1 driven by 5' truncated promoters of different lengths showed that the promoter region between -540 and 0 (the first ATG) restored the Al-sensitive phenotype of atalm1 and thus contains cis-elements essential for AtALMT1 expression for Al tolerance. Computation of overrepresented octamers showed that eight regions in this promoter region contained potential cis-elements involved in Al induction and STOP1 regulation. Mutation in a position around -297 from the first ATG completely inactivated AtALMT1 expression and Al response. In vitro binding assays showed that this region contained the STOP1 binding site, which accounted for the recognition by four zinc finger domains of the protein. Other positions were characterized as cis-elements that regulated expression by repressors and activators and a transcription factor that determines root tip expression of AtALMT1. From the consensus of known cis-elements, we identified CALMODULIN-BINDING TRANSCRIPTION ACTIVATOR2 to be an activator of AtALMT1 expression. Al-inducible expression of AtALMT1 changed transcription starting sites, which increased the abundance of transcripts with a shortened 5' untranslated region. The present analyses identified multiple mechanisms that regulate AtALMT1 expression., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

DELLA protein is a key negative regulator of gibberellin (GA) signaling. Although how DELLA regulates downstream gene expression remains unclear, DELLA has been proposed to function as a transcriptional activator. However, because DELLA lacks a DNA-binding domain, intermediate protein(s) mediating the DELLA/DNA interaction are believed to be necessary for activating DELLA target genes. Here, using yeast hybrid screenings, we identified five members of indeterminate domain (IDD) protein family which bind physically to both DELLA and the promoter sequence of the GA-positive regulator SCARECROW-LIKE 3 (SCL3), which previously was characterized as a DELLA direct target gene. Transient assays using Arabidopsis protoplasts demonstrated that a luciferase reporter controlled by the SCL3 promoter was additively transactivated by REPRESSOR of ga1-3 (RGA) and IDDs. Phenotypic analysis of transgenic plants expressing AtIDD3 (one of the 16 IDDs in the Arabidopsis genome) fused with the plant-specific repression domain (SRDX) supported the possibility that AtIDD3 is positively involved in GA signaling. In addition, we found that SCL3 protein also interacts with IDDs, resulting in the suppression of its target gene expression. In this context, DELLA and SCL3 interact competitively with IDD proteins to regulate downstream gene expression. These results suggest that the coregulators DELLA and SCL3, using IDDs as transcriptional scaffolds for DNA binding, antagonistically regulate the expression of their downstream targets to control the GA signaling pathway.