Yoshikatsu Matsubayashi

Summary: Meristem activity is controlled by the CLAVATA (CLV) signaling pathway, which involves a suite of leucine‐rich receptor (LRR) receptors, receptor‐like proteins, and CLV‐EMBRYO SURROUNDING REGION (CLE) peptide ligands. FASCIATED EAR 3 (FEA3) is a leucine‐rich receptor (LRR) receptor‐like protein important for meristem maintenance in maize and acts independently of canonical CLV receptors. To identify FEA3's interaction network, we used TurboID‐based proximity labeling in Zea mays meristems and identified a putative co‐receptor, BARELY ANY MERISTEM 1D (BAM1D). BAM1D and FEA3 proximity labeling proteomes shared over 40 proteins, including many signaling proteins, suggesting they feed into a common signaling pathway. fea3 was epistatic to bam1d in the control of inflorescence meristem (IM) size, supporting the idea that FEA3 and BAM1D interact physically. However, fea3 and bam1d act antagonistically because fea3 mutants had larger IMs, whereas bam1d mutants produced smaller IMs. This study demonstrates how in vivo TurboID‐based proximity labeling clarifies complex genetic interactions between CLV receptors and expands our knowledge of downstream signaling components of CLV signaling pathways, which are largely uncharacterized. Our findings support the notion that multiple, partially overlapping CLV receptor complexes coordinately control meristem maintenance.

In response to drought stress, land plants close their stomata to minimize transpiration. This action precedes a gradual accumulation of the stress hormone abscisic acid (ABA) that enhances plant drought tolerance. However, the molecular mechanisms that cause the time lag between the onset of stomatal closure and ABA accumulation and coordinate these two phases remain unexplained. Here, we found that Arabidopsis thaliana loss-of-function CLAVATA3/ENDOSPERM SURROUNDING REGION 5 (CLE5) mutants are less tolerant to drought. The CLE5 dodecapeptide (CLE5p) acts as a local signal to induce stomatal closure by binding to the LEUCINE-RICH REPEAT RECEPTOR-LIKE KINASE (LRR-RLK) receptor complex, BARELY ANY MERISTEM 1 (BAM1)–GUARD CELL HYDROGEN PEROXIDE-RESISTANT 1 (GHR1), in guard cells. The BAM1–GHR1–CLE5p module directly phosphorylates two SNF1-related protein kinases, OPEN STOMATA1 (SRK2E) and SRK2D, the central regulators of drought responses in plants, to regulate stomatal movement and drought-responsive gene expression without stimulating ABA biosynthesis or ROS accumulation. Our findings mark a critical step in understanding how plants promptly counteract environmental stresses. The CLEp–LRR-RLK signalling components are highly conserved across plant phyla, suggesting that peptide-mediated rapid stomatal closure is a widespread survival strategy and can be exploited to generate drought-resistant crops.

Meristem activity is controlled by the CLAVATA (CLV) signaling pathway, which involves a suite of leucine rich receptor (LRR) receptors, receptor-like proteins and CLV- EMBRYO SURROUNDING REGION (CLE) peptide ligands. FASCIATED EAR 3 (FEA3) is a leucine rich receptor (LRR) receptor-like protein important for meristem maintenance in maize, and acts independently of canonical CLV receptors. Weak alleles of fea3 can increase yield-related traits in maize, so understanding how FEA3 controls inflorescence development can maximize its potential as a crop improvement target. To identify FEA3’s interaction network, we used TurboID-based proximity labeling in maize meristems, and identified a putative co-receptor, BARELY ANY MERISTEM 1D (BAM1D). BAM1D and FEA3 proximity labeling proteomes shared over 100 proteins, including many signaling proteins, suggesting they feed into a common signaling pathway. fea3 was epistatic to bam1d in the control of IM size, supporting the idea that FEA3 and BAM1D interact physically. However, fea3 and bam1d act antagonistically, because fea3 mutants had larger inflorescence meristems (IMs), whereas bam1d mutants produced smaller IMs. Together, this study demonstrates how in vivo TurboID-based proximity labeling clarifies complex genetic interactions between CLV receptors and expands our knowledge of downstream signaling components of CLV signaling pathways, which are largely uncharacterized. Our findings support the notion that multiple, partially overlapping CLV receptor complexes coordinately control meristem maintenance.

SUMMARY: In the accumulation response, chloroplasts move toward weak blue light (BL) to maximize photosynthetic efficiency; in the avoidance response, they move away from strong BL to reduce photodamage. The BL receptor kinase phototropin (phot) mediates these chloroplast relocation responses, and the chloroplast relocation response requires phot kinase activity. Upon receiving BL, phot undergoes autophosphorylation; however, the molecular mechanisms that regulate chloroplast relocation through phot autophosphorylation remain unclear. In this study, we conducted biochemical experiments using phot in the liverwort Marchantia polymorpha and revealed that phot employs cis‐autophosphorylation under weak BL and both cis‐ and trans‐autophosphorylation under strong BL. Inhibiting trans‐autophosphorylation reduced phot autophosphorylation and suppressed the avoidance response, but not the accumulation response. These findings suggest that phot employs two modes of autophosphorylation to alternate between the accumulation and avoidance responses in plants.

Small interference RNA (siRNA) selectively accumulates and acts in RNA interference (RNAi). Although the components involved in siRNA production have long been the focus of studies to elucidate RNAi processes, the mechanism(s) for selectivity of siRNA (or RNAi effectivity) remains unclear. In a novel approach, we developed a progressive deep learning (DL) framework integrating Transformer and convolutional neural networks to predict the sequences of selectively accumulated siRNAs across various land plant species. These approaches achieved high-accuracy prediction of selectively accumulated 21-nt siRNAs and further identified their key signals, which are positionally and linguistically flexible sequences surrounding the target siRNA. We experimentally validated the contribution of these flexible key signal sequences to siRNA accumulation selectivity using virus-induced gene silencing (VIGS) in Nicotiana benthamiana , and identified RNA-binding proteins that directly recognize the key signal sequences to act for selective siRNA accumulation. These insights provide a novel framework for investigating RNAi mechanisms in plants. One sentence summary: We discovered a novel mechanism centered on RNA-protein interactions involving the selective accumulation of small-RNAs in plants by applying advanced deep learning frames.

Unlike plants in the field, which experience significant temporal fluctuations in environmental conditions, plants in the laboratory are typically grown in controlled, stable environments. Therefore, signaling pathways evolved for survival in fluctuating environments often remain functionally latent in laboratory settings. Here, we show that TGA1 and TGA4 act as hub transcription factors through which the expression of genes involved in high-affinity nitrate uptake are regulated in response to shoot-derived phloem mobile polypeptides, CEP DOWNSTREAM 1 (CEPD1), CEPD2 and CEPD-like 2 (CEPDL2) as nitrogen (N) deficiency signals, and Glutaredoxin S1 (GrxS1) to GrxS8 as N sufficiency signals. CEPD1/2/CEPDL2 and GrxS1-S8 competitively bind to TGA1/4 in roots, with the former acting as transcription coactivators that enhance the uptake of nitrate, while the latter function as corepressor complexes together with TOPLESS (TPL), TPL-related 1 (TPR1) and TPR4 to limit nitrate uptake. Arabidopsis plants deficient in TGA1/4 maintain basal nitrate uptake and exhibit growth similar to wild-type plants in a stable N environment, but are impaired in regulation of nitrate acquisition in response to shoot N demand, leading to defective growth under fluctuating N environments where rhizosphere nitrate ions switch periodically between deficient and sufficient states. TGA1/4 are crucial transcription factors that enable plants to survive under fluctuating and challenging N environmental conditions.

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.

Legumes control root nodule symbiosis (RNS) in response to environmental nitrogen availability. Despite the recent understanding of the molecular basis of external nitrate-mediated control of RNS, it remains mostly elusive how plants regulate physiological processes depending on internal nitrogen status. In addition, iron (Fe) acts as an essential element that enables symbiotic nitrogen fixation; however, the mechanism of Fe accumulation in nodules is poorly understood. Here, we focus on the transcriptome in response to internal nitrogen status during RNS in Lotus japonicus and identify that IRON MAN (IMA) peptide genes are expressed during symbiotic nitrogen fixation. We show that LjIMA1 and LjIMA2 expressed in the shoot and root play systemic and local roles in concentrating internal Fe to the nodule. Furthermore, IMA peptides have conserved roles in regulating nitrogen homeostasis by adjusting nitrogen-Fe balance in L. japonicus and Arabidopsis thaliana. These findings indicate that IMA-mediated Fe provision plays an essential role in regulating nitrogen-related physiological processes.

Binding assays provide ultimate proof that a particular peptide and receptor kinase (RK) do indeed function as a ligand-receptor pair. Among available binding assays, proximity-induced photoaffinity labeling is superior for confirming direct contact between the peptide ligand and the receptor. Our binding assay employs covalent photoaffinity labeling followed by immunoprecipitation to specifically evaluate the ligand binding activity of the target RKs. Here, we describe a protocol for the synthesis of photoactivatable peptide ligands and the UV-induced formation of covalent bonds between photoaffinity ligands and RKs. Key words Photoaffinity labeling Receptor kinase Peptide ligand Ligand-receptor interaction