A florigen-expressing subpopulation of companion cells expresses other small proteins and reveals a nitrogen-sensitive FT repressor.

The precise onset of flowering is crucial to ensure successful plant reproduction. The gene FLOWERING LOCUS T (FT) encodes florigen, a mobile signal produced in leaves that initiates flowering at the shoot apical meristem. In response to seasonal changes, FT is induced in phloem companion cells located in distal leaf regions. Thus far, a detailed molecular characterization of the FT-expressing cells has been lacking. Here, we used bulk nuclei RNA-seq and single nuclei RNA (snRNA)-seq to investigate gene expression in FT-expressing cells and other phloem companion cells. Our bulk nuclei RNA-seq demonstrated that FT-expressing cells in cotyledons and in true leaves differed transcriptionally. Within the true leaves, our snRNA-seq analysis revealed that companion cells with high FT expression form a unique cluster in which many genes involved in ATP biosynthesis are highly upregulated. The cluster also expresses other genes encoding small proteins, including the flowering and stem growth inducer FPF1-LIKE PROTEIN 1 (FLP1) and the anti-florigen BROTHER OF FT AND TFL1 (BFT). In addition, we found that the promoters of FT and the genes co-expressed with FT in the cluster were enriched for the consensus binding motifs of NITRATE-INDUCIBLE GARP-TYPE TRANSCRIPTIONAL REPRESSOR 1 (NIGT1). Overexpression of the paralogous NIGT1.2 and NIGT1.4 repressed FT expression and significantly delayed flowering under nitrogen-rich conditions, consistent with NIGT1s acting as nitrogen-dependent FT repressors. Taken together, our results demonstrate that major FT-expressing cells show a distinct expression profile that suggests that these cells may produce multiple systemic signals to regulate plant growth and development.

CDPK5 and CDPK13 play key roles in acclimation to low oxygen through the control of RBOH-mediated ROS production in rice.

CALCIUM-DEPENDENT PROTEIN KINASE (CDPK) stimulates reactive oxygen species (ROS)-dependent signaling by activating RESPIRATORY BURST OXIDASE HOMOLOG (RBOH). The lysigenous aerenchyma is a gas space created by cortical cell death that facilitates oxygen diffusion from the shoot to the root tips. Previously, we showed that RBOHH is indispensable for the induction of aerenchyma formation in rice (Oryza sativa) roots under low-oxygen conditions. Here, we showed that CDPK5 and CDPK13 localize to the plasma membrane where RBOHH functions. Mutation analysis of the serine at residues 92 and 107 of RBOHH revealed that these residues are required for CDPK5- and CDPK13-mediated activation of ROS production. The requirement of Ca2+ for CDPK5 and CDPK13 function was confirmed using in vitro kinase assays. CRISPR/Cas9-based mutagenesis of CDPK5 and/or CDPK13 revealed that the double knockout almost completely suppressed inducible aerenchyma formation, whereas the effects were limited in the single knockout of either CDPK5 or CDPK13. Interestingly, the double knockout almost suppressed the induction of adventitious root formation, which is widely conserved in vascular plants, under low-oxygen conditions. Our results suggest that CDPKs are essential for the acclimation of rice to low-oxygen conditions, and also for many other plant species conserving CDPK-targeted phosphorylation sites in RBOH homologues.

Arabidopsis SBT5.2 and SBT1.7 subtilases mediate C-terminal cleavage of flg22 epitope from bacterial flagellin.

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.

Florigen-producing cells express FPF1-LIKE PROTEIN 1 that accelerates flowering and stem growth in long days with sunlight red/far-red ratio in Arabidopsis.

Seasonal changes in spring induce flowering by expressing the florigen, FLOWERING LOCUS T (FT), in Arabidopsis. FT is expressed in unique phloem companion cells with unknown characteristics. The question of which genes are co-expressed with FT and whether they have roles in flowering remains elusive. Through tissue-specific translatome analysis, we discovered that under long-day conditions with the natural sunlight red/far-red ratio, the FT-producing cells express a gene encoding FPF1-LIKE PROTEIN 1 (FLP1). The master FT regulator, CONSTANS (CO), controls FLP1 expression, suggesting FLP1's involvement in the photoperiod pathway. FLP1 promotes early flowering independently of FT, is active in the shoot apical meristem, and induces the expression of SEPALLATA 3 (SEP3), a key E-class homeotic gene. Unlike FT, FLP1 facilitates inflorescence stem elongation. Our cumulative evidence indicates that FLP1 may act as a mobile signal. Thus, FLP1 orchestrates floral initiation together with FT and promotes inflorescence stem elongation during reproductive transitions.

Arabidopsis ASYMMETRIC LEAVES2 and Nucleolar Factors Are Coordinately Involved in the Perinucleolar Patterning of AS2 Bodies and Leaf Development.

Arabidopsis ASYMMETRIC LEAVES2 (AS2) plays a key role in the formation of flat symmetric leaves. AS2 represses the expression of the abaxial gene ETTIN/AUXIN RESPONSE FACTOR3 (ETT/ARF3). AS2 interacts in vitro with the CGCCGC sequence in ETT/ARF3 exon 1. In cells of leaf primordia, AS2 localizes at peripheral regions of the nucleolus as two AS2 bodies, which are partially overlapped with chromocenters that contain condensed 45S ribosomal DNA repeats. AS2 contains the AS2/LOB domain, which consists of three sequences conserved in the AS2/LOB family: the zinc finger (ZF) motif, the ICG sequence including the conserved glycine residue, and the LZL motif. AS2 and the genes NUCLEOLIN1 (NUC1), RNA HELICASE10 (RH10), and ROOT INITIATION DEFECTIVE2 (RID2) that encode nucleolar proteins coordinately act as repressors against the expression of ETT/ARF3. Here, we examined the formation and patterning of AS2 bodies made from as2 mutants with amino acid substitutions in the ZF motif and the ICG sequence in cells of cotyledons and leaf primordia. Our results showed that the amino acid residues next to the cysteine residues in the ZF motif were essential for both the formation of AS2 bodies and the interaction with ETT/ARF3 DNA. The conserved glycine residue in the ICG sequence was required for the formation of AS2 bodies, but not for the DNA interaction. We also examined the effects of nuc1, rh10, and rid2 mutations, which alter the metabolism of rRNA intermediates and the morphology of the nucleolus, and showed that more than two AS2 bodies were observed in the nucleolus and at its periphery. These results suggested that the patterning of AS2 bodies is tightly linked to the morphology and functions of the nucleolus and the development of flat symmetric leaves in plants.

Identification and improvement of isothiocyanate-based inhibitors on stomatal opening to act as drought tolerance-conferring agrochemicals.

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.

Identification of a pluripotency-inducing small compound, PLU, that induces callus formation via Heat Shock Protein 90-mediated activation of auxin signaling.

Plants retain the ability to generate a pluripotent tissue called callus by dedifferentiating somatic cells. A pluripotent callus can also be artificially induced by culturing explants with hormone mixtures of auxin and cytokinin, and an entire body can then be regenerated from the callus. Here we identified a pluripotency-inducing small compound, PLU, that induces the formation of callus with tissue regeneration potency without the external application of either auxin or cytokinin. The PLU-induced callus expressed several marker genes related to pluripotency acquisition via lateral root initiation processes. PLU-induced callus formation required activation of the auxin signaling pathway though the amount of active auxin was reduced by PLU treatment. RNA-seq analysis and subsequent experiments revealed that Heat Shock Protein 90 (HSP90) mediates a significant part of the PLU-initiated early events. We also showed that HSP90-dependent induction of TRANSPORT INHIBITOR RESPONSE 1, an auxin receptor gene, is required for the callus formation by PLU. Collectively, this study provides a new tool for manipulating and investigating the induction of plant pluripotency from a different angle from the conventional method with the external application of hormone mixtures.

Proteasome-associated ubiquitin ligase relays target plant hormone-specific transcriptional activators.

The ubiquitin-proteasome system is vital to hormone-mediated developmental and stress responses in plants. Ubiquitin ligases target hormone-specific transcriptional activators (TAs) for degradation, but how TAs are processed by proteasomes remains unknown. We report that in Arabidopsis, the salicylic acid- and ethylene-responsive TAs, NPR1 and EIN3, are relayed from pathway-specific ubiquitin ligases to proteasome-associated HECT-type UPL3/4 ligases. Activity and stability of NPR1 were regulated by sequential action of three ubiquitin ligases, including UPL3/4, while proteasome processing of EIN3 required physical handover between ethylene-responsive SCFEBF2 and UPL3/4 ligases. Consequently, UPL3/4 controlled extensive hormone-induced developmental and stress-responsive transcriptional programs. Thus, our findings identify unknown ubiquitin ligase relays that terminate with proteasome-associated HECT-type ligases, which may be a universal mechanism for processive degradation of proteasome-targeted TAs and other substrates.

Mechanosensory trichome cells evoke a mechanical stimuli-induced immune response in Arabidopsis thaliana.

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.

CFI 25 Subunit of Cleavage Factor I is Important for Maintaining the Diversity of 3' UTR Lengths in Arabidopsis thaliana (L.) Heynh.

Cleavage and polyadenylation at the 3' end of the pre-mRNA is essential for mRNA function, by regulating its translatability, stability and translocation to the cytoplasm. Cleavage factor I (CFI) is a multi-subunit component of the pre-mRNA 3' end processing machinery in eukaryotes. Here, we report that plant CFI 25 subunit of CFI plays an important role in maintaining the diversity of the 3' ends of mRNA. The genome of Arabidopsis thaliana (L.) Heynh. contained four genes encoding three putative CFI subunits (AtCFI 25, AtCFI 59 and AtCFI 68), orthologous to the mammalian CFI subunits. There were two CFI 25 paralogs (AtCFI 25a and AtCFI 25b) that shared homology with human CFI 25. Two null alleles of AtCFI 25a displayed smaller rosette leaves, longer stigmatic papilla, smaller anther, earlier flowering and lower fertility compared to wild-type plants. Null alleles of AtCFI 25b, as well as, plants ectopically expressing full-length cDNA of AtCFI 25a, displayed no obvious morphological defects. AtCFI 25a was shown to interact with AtCFI 25b, AtCFI 68 and itself, suggesting various forms of CFI in plants. Furthermore, we show that AtCFI 25a function was essential for maintaining proper diversity of the 3' end lengths of transcripts coding for CFI subunits, suggesting a self-regulation of the CFI machinery in plants. AtCFI 25a was also important to maintain 3' ends for other genes to different extent. Collectively, AtCFI 25a, but not AtCFI 25b, seemed to play important roles during Arabidopsis development by maintaining proper diversity of the 3' UTR lengths.

Suppression of MYC transcription activators by the immune cofactor NPR1 fine-tunes plant immune responses.

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.

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

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

Different DNA-binding specificities of NLP and NIN transcription factors underlie nitrate-induced control of root nodulation.

Leguminous plants produce nodules for nitrogen fixation; however, nodule production incurs an energy cost. Therefore, as an adaptive strategy, leguminous plants halt root nodule development when sufficient amounts of nitrogen nutrients, such as nitrate, are present in the environment. Although legume NODULE INCEPTION (NIN)-LIKE PROTEIN (NLP) transcription factors have recently been identified, understanding how nodulation is controlled by nitrate, a fundamental question for nitrate-mediated transcriptional regulation of symbiotic genes, remains elusive. Here, we show that two Lotus japonicus NLPs, NITRATE UNRESPONSIVE SYMBIOSIS 1 (NRSYM1)/LjNLP4 and NRSYM2/LjNLP1, have overlapping functions in the nitrate-induced control of nodulation and act as master regulators for nitrate-dependent gene expression. We further identify candidate target genes of LjNLP4 by combining transcriptome analysis with a DNA affinity purification-seq approach. We then demonstrate that LjNLP4 and LjNIN, a key nodulation-specific regulator and paralog of LjNLP4, have different DNA-binding specificities. Moreover, LjNLP4-LjNIN dimerization underlies LjNLP4-mediated bifunctional transcriptional regulation. These data provide a basic principle for how nitrate controls nodulation through positive and negative regulation of symbiotic genes.

High affinity promoter binding of STOP1 is essential for early expression of novel aluminum-induced resistance genes GDH1 and GDH2 in Arabidopsis.

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.

Identification of Abscisic Acid-Dependent Phosphorylated Basic Helix-Loop-Helix Transcription Factors in Guard Cells of Vicia faba by Mass Spectrometry.

An unknown 61 kDa protein is phosphorylated by abscisic acid (ABA)-activated protein kinase in response to ABA and binds to 14-3-3 protein in a phosphorylation-dependent manner in guard-cell protoplasts (GCPs) from Vicia faba. Subsequently, ABA-dependent phosphorylated proteins were identified as basic helix-loop-helix transcription factors, named ABA-responsive kinase substrates (AKSs) in GCPs from Arabidopsis thaliana. However, whether the 61 kDa protein in Vicia GCPs is an AKS is unclear. We performed immunoprecipitation of ABA-treated Vicia GCPs using anti-14-3-3 protein antibodies and identified several AKS isoforms in V. faba (VfAKSs) by mass spectrometry. The 61 kDa protein was identified as VfAKS1. Phosphoproteomic analysis revealed that VfAKSs are phosphorylated at Ser residues, which are important for 14-3-3 protein binding and monomerisation, in response to ABA in GCPs. Orthologs of AtABCG40, an ABA importer in guard cells, and CHC1, a clathrin heavy chain and a regulator of stomatal movement, also co-immunoprecipitated with 14-3-3 protein from guard cells.

ANAC032 regulates root growth through the MYB30 gene regulatory network.

Reactive oxygen species (ROS) play important roles as root growth regulators. We previously reported a comprehensive transcriptomic atlas, which we named ROS-map, that revealed ROS-responsible genes in Arabidopsis root tips. By using ROS-map, we have characterised an early ROS response key transcription factor, MYB30, as a regulator of root cell elongation under ROS signals. However, there are other ROS-responsible transcription factors which have the potential to regulate root growth. In the present study, we characterised the function of another early ROS-responsible transcription factor, ANAC032, that was selected from ROS-map. Overexpression of ANAC032 fused with the transcriptional activation domain, VP16, inhibited root growth, especially decreasing cell elongation. By transcriptome analysis, we revealed that ANAC032 regulated many stress-responsible genes in the roots. Intriguingly, ANAC032 upregulated MYB30 and its target genes. The upregulation of MYB30 target genes was completely abolished in the ANAC032-VP16x2 OX and ANAC032 estradiol-inducible line in myb30-2 mutants. Moreover, root growth inhibition was alleviated in ANAC032-OX in myb30-2 mutants. Overall, we characterised an upstream transcription factor, ANAC032, of the MYB30 transcriptional cascade which is a key regulator for root cell elongation under ROS signalling.

LSD1-LIKE1-Mediated H3K4me2 Demethylation Is Required for Homologous Recombination Repair.

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.

Rapid and easy method for in vitro determination of transcription factor binding core motifs.

We introduce a rapid method for easily elucidating transcription factor (TF) cis-elements by adopting a highly efficient in vitro protein synthesis method and identifying protein-DNA interactions using PCR. We determined two cis-elements for plant TFs using this method, and the results confirmed our method as an easy and time-saving alternative for elucidating TF cis-elements using common laboratory procedures.

Transcriptome Analysis and Identification of a Transcriptional Regulatory Network in the Response to H2O2.

Hydrogen peroxide (H2O2) is a common signal molecule initiating transcriptional responses to all the known biotic and abiotic stresses of land plants. However, the degree of involvement of H2O2 in these stress responses has not yet been well studied. Here we identify time-dependent transcriptome profiles stimulated by H2O2 application in Arabidopsis (Arabidopsis thaliana) seedlings. Promoter prediction based on transcriptome data suggests strong crosstalk among high light, heat, and wounding stress responses in terms of environmental stresses and between the abscisic acid (ABA) and salicylic acid (SA) responses in terms of phytohormone signaling. Quantitative analysis revealed that ABA accumulation is induced by H2O2 but SA is not, suggesting that the implied crosstalk with ABA is achieved through ABA accumulation while the crosstalk with SA is different. We identified potential direct regulatory pairs between regulator transcription factor (TF) proteins and their regulated TF genes based on the time-course transcriptome analysis for the H2O2 response, in vivo regulation of the regulated TF by the regulator TF identified by expression analysis of mutants and overexpressors, and in vitro binding of the regulator TF protein to the target TF promoter. These analyses enabled the establishment of part of the transcriptional regulatory network for the H2O2 response composed of 15 regulatory pairs of TFs, including five pairs previously reported. This regulatory network is suggested to be involved in a wide range of biotic and abiotic stress responses in Arabidopsis.

Brassinosteroid Induces Phosphorylation of the Plasma Membrane H+-ATPase during Hypocotyl Elongation in Arabidopsis thaliana.

Brassinosteroids (BRs) are steroid phytohormones that regulate plant growth and development, and promote cell elongation at least in part via the acid-growth process. BRs have been suggested to induce cell elongation by the activating plasma membrane (PM) H+-ATPase. However, the mechanism by which BRs activate PM H+-ATPase has not been clarified. In this study, we investigated the effects of BR on hypocotyl elongation and the phosphorylation status of a penultimate residue, threonine, of PM H+-ATPase, which affects the activation, in the etiolated seedlings of Arabidopsis thaliana. Brassinolide (BL), an active endogenous BR, induced hypocotyl elongation, phosphorylation of the penultimate, threonine residue of PM H+-ATPase, and binding of the 14-3-3 protein to PM H+-ATPase in the endogenous BR-depleted seedlings. Changes in both BL-induced elongation and phosphorylation of PM H+-ATPase showed similar concentration dependency. BL did not induce phosphorylation of PM H+-ATPase in the BR receptor mutant bri1-6. In contrast, bikinin, a specific inhibitor of BIN2 that acts as a negative regulator of BR signaling, induced its phosphorylation. Furthermore, BL accumulated the transcripts of SMALL AUXIN UP RNA 9 (SAUR9) and SAUR19, which suppress dephosphorylation of the PM H+-ATPase penultimate residue by inhibiting D-clade type 2C protein phosphatase in the hypocotyls of etiolated seedlings. From these results, we conclude that BL-induced phosphorylation of PM H+-ATPase penultimate residue is mediated via the BRI1-BIN2 signaling pathway, together with the accumulation of SAURs during hypocotyl elongation.