RESEARCH

Tracing back from molecules to mechanisms

Peptide hormones and receptors in plants

In biology, the general methodology is to first focus on a specific biological phenomenon, then to identify the key molecules involved, and finally to explain the mechanism. However, if we first focus on a specific molecule, analyze its function, and finally aim to discover new mechanisms and phenomena, a new biology may be born. We have been searching for novel peptide hormone-receptor pairs by using the following strategies: (1) in silico identification of genes encoding small secreted peptide hormones, (2) determination of their mature peptide structures by mass spectrometry, and (3) identification of their receptors by binding experiments using an expression library of receptor candidates. This approach has led to the discovery of peptide hormones involved in growth and environmental responses, including RGF (Science 2010), which regulates activity and patterning of root apical meristem; CEP (Science 2014), which mediates systemic regulation of nitrogen acquisition; CIF (Science 2017), which is involved in Casparian strip development (Science 2017), and PSY (Science 2022), which regulates the trade-off between growth and stress responses.

Post-translational modifications

Post-translational modifications of peptides also provide important clues for tracing back from molecules to mechanisms. Because post-translational modifications generally have a critical effect on peptide function, phenotypic analysis of a mutant deficient in a post-translational modification enzyme provides an overall picture of the function of a group of post-translationally modified peptides. For example, mutants deficient in tyrosine sulfotransferase have shorter roots and dwarf shoots (PNAS 2009), a phenotype that was a major clue to the discovery of peptide hormones such as RGF and PSY. In mutants deficient in enzymes involved in extracellular protein glycosylation such as Hyp O-arabinosylation (Nature Chem. Biol. 2013) and Hyp O-galactosylation (Plant J. 2015), various tissue defects have also been observed, suggesting the existence of as-yet-unknown extracellular signaling mechanisms. In particular, Hyp O-galactosylation is an essential modification of arabinogalactan protein (AGP) function. Mutants deficient in this modification are unable to generate functional AGPs, which allows us to overcome the gene redundancy problem and gain a better understanding of the functions of AGPs.

  • RESEARCH
  • RESEARCH

Phosphorylation/dephosphorylation-mediated signaling

When a peptide hormone binds to a receptor on the cell membrane, the intracellular signal transduction system through protein phosphorylation and dephosphorylation is activated. Tracking this phosphorylation and dephosphorylation process tells us how cell signaling works. In addition, there are still many protein phosphatases and kinases in plants whose functions remain unknown, and the search for their substrates is also an attractive research target that will lead to the elucidation of new mechanisms. Using our improved 15N metabolic labeled quantitative phosphoproteomics system, we can identify the substrates of the protein phosphatases. For example, PP2C family phosphatase, CEPH, dephosphorylates the Ser501 residue of NRT2.1, a major nitrate transporter in roots, which turns on nitrate uptake activity (Nature Plants 2021).

Phloem-mobile non-secreted peptides

Our recent studies have revealed that non-secreted peptides such as CEPD (Nature Plants 2017) and CEPDL2 (Nature Commun. 2020), which are induced in leaves in a nitrogen deficiency-dependent manner, translocate to roots through phloem and regulate the expression of nitrate uptake transporters. Phloem is a vascular tissue comprised of sieve-tube element cells, with numerous pores, called sieve pores, at the cell-cell interface, allowing the cytoplasm inside to migrate between cells. Therefore, non-secreted peptides in the phloem can migrate long distances between organs. This is a mechanism unique to plants that is not found in animals, and there are still many unknowns. Analysis of a group of non-secreted peptides that exhibit similar phloem-mobile nature may reveal dynamic communication between plant organs.

PUBLICATIONS

  • Integration of shoot-derived signals by root TGA

    Integration of shoot-derived signals by root TGA Nature Commun (2024)

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    Integration of shoot-derived polypeptide signals by root TGA transcription factors is essential for survival under fluctuating nitrogen environments

    Ryutaro Kobayashi, Yuri Ohkubo, Mai Izumi, Ryosuke Ota, Keiko Yamada, Yoko Hayashi, Yasuko Yamashita, Saki Noda, Mari Ogawa-Ohnishi and Yoshikatsu Matsubayashi*

    Nature Commun. 15, 6903 (2024)

    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, CEPD1, CEPD2 and CEPDL2 as nitrogen (N) deficiency signals, and 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, 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 were 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.

    https://www.nature.com/articles/s41467-024-48108-4
  • Subtilases mediating flg22 cleavage from flagellin

    Subtilases mediating flg22 cleavage from flagellin Nature Commun (2024)

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

    Sayaka Matsui, Saki Noda, Keiko Kuwata, Mika Nomoto, Yasuomi Tada, Hidefumi Shinohara and Yoshikatsu Matsubayashi*

    Nature Commun. 15, 3762 (2024)

    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 perception of the epitope by the plant receptor. However, the identity of the plant proteases involved in this process remains unknown. Here, we established 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 observed 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 was delayed in sbt5.2 sbt1.7 double-mutant leaf disks compared to wild type following flagellin exposure.

    https://www.nature.com/articles/s41467-024-48108-4
  • AGP glycans contribute plasmodesmata biogenesis

    AGP glycans contribute plasmodesmata biogenesis Plant J (2023)

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    AGP polysaccharide chains are required for normal biogenesis of plasmodesmata

    Ryoya Okawa, Yoko Hayashi, Yasuko Yamashita, Yoshikatsu Matsubayashi and Mari Ogawa-Ohnishi*

    Plant J. 113, 493-503 (2023)

    Arabinogalactan proteins (AGPs) are a plant-specific family of extracellular proteoglycans characterized by large and complex galactose-rich polysaccharide chains. Functional elucidation of AGPs, however, has been hindered by the high degree of redundancy of AGP genes. To uncover as yet unexplored roles of AGPs in Arabidopsis, a mutant of Hyp O-galactosyltransferase (HPGT), a critical enzyme that catalyzes the common initial step of Hyp-linked arabinogalactan (AG) chain biosynthesis, was used. Here we show, using the hpgt1,2,3 triple mutant, that a reduction in functional AGPs leads to a stomatal patterning defect in which two or more stomata are clustered together. This defect is attributed to increased and dysregulated symplastic transport following changes in plasmodesmata structure, such that highly permeable complex branched plasmodesmata with cavities in branching parts increased in the mutant. We also found that the hpgt1,2,3 mutation causes a reduction of cellulose in the cell wall and accumulation of pectin, which controls cell wall porosity. Our results highlight the importance of AGPs in the correct biogenesis of plasmodesmata, possibly acting through the regulation of cell wall properties surrounding the plasmodesmata.

    https://onlinelibrary.wiley.com/doi/abs/10.1111/tpj.16061
  • Peptide mediates growth-stress response trade-off

    A peptide mediating growth-stress response trade-off Science (2022)

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    Peptide ligand-mediated trade-off between plant growth and stress response

    Mari Ogawa-Ohnishi, Tomohide Yamashita, Mitsuru Kakita, Takuya Nakayama, Yuri Ohkubo, Yoko Hayashi, Yasuko Yamashita, Taizo Nomura, Saki Noda, Hidefumi Shinohara and Yoshikatsu Matsubayashi*

    Science 378, 175-180 (2022)

    The decision of whether to grow or to divert energy to stress response is a key physiological trade-off for plants seeking to survive under fluctuating environments. However, the cell-to-cell communication underlying this decision is largely unknown. We showed that three orphan leucine-rich repeat receptor kinases (LRR-RKs) act as direct ligand-perceiving receptors for PSY-family peptides and play a key role in local switching between two opposing pathways. In contrast to the known LRR-RKs which activate signaling upon ligand binding, PSY receptor (PSYR) signaling, which intrinsically induces a stress response, usually is repressed by ligands to permit growth. Conversely, PSYRs activate the expression of various genes encoding stress response transcription factors upon depletion of the ligands. Loss of PSYRs resulted in defects in the plant’s tolerance to both biotic and abiotic stresses. This ligand-deprivation-dependent activation system enables plants to exert exquisitely tuned regulation of stress responses in the tissues proximal to metabolically dysfunctional damaged sites where ligand production is impaired.

    https://www.science.org/doi/10.1126/science.abq5735
  • A PP2C phosphatase that activates NRT2.1

    A PP2C phosphatase that activates NRT2.1 Nature Plants (2021)

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    A type 2C protein phosphatase activates high-affinity nitrate uptake by dephosphorylating NRT2.1

    Yuri Ohkubo, Keiko Kuwata and Yoshikatsu Matsubayashi*

    Nature Plants 7, 310 (2021)

    Nitrate transporter NRT2.1, which plays a central role in high-affinity nitrate uptake in roots, is activated at the post-translational level in response to nitrogen (N) starvation. However, critical enzymes required for post-translational activation of NRT2.1 remain to be identified. We found that a type 2C protein phosphatase, designated CEPD-induced phosphatase (CEPH), activates high-affinity nitrate uptake by directly dephosphorylating Ser501 of NRT2.1, a residue that functions as a negative phospho-switch in Arabidopsis. CEPH is predominantly expressed in epidermal and cortex cells in roots and up-regulated by N starvation via a CEPDL2/CEPD1/2-mediated long-distance signaling from shoots. Loss of CEPH leads to a marked decrease in high-affinity nitrate uptake, tissue nitrate content, and plant biomass. Collectively, our results identify CEPH as a crucial enzyme in N starvation-dependent activation of NRT2.1, providing molecular and mechanistic insights into how plants regulate high-affinity nitrate uptake at the post-translational level in response to the N environment.

    https://www.nature.com/articles/s41477-021-00870-9
  • On-demand nitrate uptake by shoot-derived peptide

    On-demand nitrate uptake by shoot-derived peptide Nature Commun (2020)

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    Shoot-to-root mobile CEPD-like 2 integrates shoot nitrogen status to systemically regulate nitrate uptake in Arabidopsis

    Ryosuke Ota, Yuri Ohkubo, Yasuko Yamashita, Mari Ogawa-Ohnishi and Yoshikatsu Matsubayashi*

    Nature Commun. 11, 641 (2020)

    Plants modulate the efficiency of root nitrogen (N) acquisition in response to shoot N demand. However, molecular components directly involved in this shoot-to-root communication remain to be identified. We uncovered that phloem-mobile CEPD-like 2 (CEPDL2) polypeptide is upregulated in the leaf vasculature in response to decreased shoot N status and, after translocation to the roots, promotes high-affinity uptake and root-to-shoot transport of nitrate. Loss of CEPDL2 leads to a reduction in shoot nitrate content and plant biomass. CEPDL2 contributes to N acquisition cooperatively with CEPD1 and CEPD2 that mediate root N status, and the complete loss of all three proteins severely impairs N homeostasis in plants. Reciprocal grafting analysis provided conclusive evidence that the shoot CEPDL2/CEPD genotype defines the high-affinity nitrate uptake activity in root. Our results indicate that plants integrate shoot N status and root N status in leaves and systemically regulate the efficiency of root N acquisition.

    https://www.nature.com/articles/s41467-020-14440-8
  • Non-peptide antagonist for the peptide hormone receptor

    Non-peptide antagonist for the peptide hormone receptor Commun Biol (2019)

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    Screening and identification of a non-peptide antagonist for the peptide hormone receptor in Arabidopsis

    Hidefumi Shinohara, Naoko Yasue, Tetsuo Onuki, Yasumitsu Kondoh, Minoru Yoshida and Yoshikatsu Matsubayashi*

    Commun Biol. 2, 61 (2019)

    Intercellular signaling mediated by peptide hormones and membrane-localized receptor kinases plays crucial roles in plant developmental processes. Because of their diverse functions, agonistic or antagonistic modulation of peptide signaling holds enormous promise for agricultural applications. We established a high-throughput screening system using a bead-immobilized receptor kinase and fluorescent-labeled peptide ligand to identify small molecules that bind peptide hormone receptors in competition with natural ligands. We used the Arabidopsis CLE9-BAM1 ligand-receptor pair to screen a library of ≈30,000 chemicals and identified NPD12704 as an antagonist for BAM1. NPD12704 also inhibited CLV3 binding to BAM1 but only minimally interfered with CLV3 binding to CLV1, the closest homolog of BAM1, demonstrating preferential receptor specificity. Treatment of clv1-101 mutant seedlings with NPD12704 enhanced the enlarged shoot apical meristem phenotype. Our results provide a technological framework enabling high-throughput identification of small non-peptide chemicals that specifically control receptor kinase–mediated peptide hormone signaling in plants.

    https://www.nature.com/articles/s42003-019-0307-8
  • Shoot-to-root mobile peptide regulates nitrate uptake

    Shoot-to-root mobile peptide regulates nitrate uptake Nature Plants (2017)

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    Shoot-to-root mobile polypeptides involved in systemic regulation of nitrogen acquisition

    Yuri Ohkubo, Mina Tanaka, Ryo Tabata, Mari Ogawa-Ohnishi and Yoshikatsu Matsubayashi*

    Nature Plants 3, 17029 (2017)

    Plants uptake nitrogen (N) from the soil mainly in the form of nitrate. However, nitrate is often distributed heterogeneously in natural soil. Plants, therefore, have a systemic long-distance signaling mechanism by which N-starvation on one side of the root leads to a compensatory N uptake on the other N-rich side. This systemic N acquisition response is triggered by a root-to-shoot mobile peptide hormone, C-terminally Encoded Peptide (CEP), originating from the N-starved roots, but the molecular nature of the descending shoot-to-root signal remains elusive. We showed that phloem-specific polypeptides that are induced in leaves upon perception of root-derived CEP act as descending long-distance mobile signals translocated to each root. These shoot-derived polypeptides, which we named CEP Downstream 1 (CEPD1) and CEPD2, upregulate the expression of the nitrate transporter gene NRT2.1 in roots specifically when nitrate is present in the rhizosphere. Arabidopsis plants deficient in this pathway show impaired systemic N-acquisition response accompanied with N-deficiency symptoms. These fundamental mechanistic insights should provide a conceptual framework for understanding systemic nutrient acquisition responses in plants.

    https://www.nature.com/articles/nplants201729
  • A peptide required for Casparian strip formation

    A peptide required for Casparian strip formation Science (2017)

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    A peptide hormone required for Casparian strip diffusion-barrier formation in Arabidopsis roots

    Takuya Nakayama, Hidefumi Shinohara, Mina Tanaka, Koki Baba, Mari Ogawa-Ohnishi and Yoshikatsu Matsubayashi*

    Science 355, 284-286 (2017)

    Plants achieve mineral ion homeostasis by restricting lateral diffusion of ions between the root vascular bundles and the soil by a hydrophobic barrier on endodermal cells called the Casparian strip. We identified a family of sulfated peptides required for contiguous Casparian strip formation in Arabidopsis roots. These peptide hormones, which we named Casparian strip integrity factor 1 (CIF1) and CIF2, are expressed in the root stele and specifically bind endodermis-expressed leucine-rich-repeat receptor kinase, GASSHO1 (GSO1)/SCHENGEN3, and its homolog, GSO2. A mutant devoid of CIF peptides is defective in ion homeostasis in the xylem. CIF genes are environmentally responsive. Casparian strip regulation is not just a passive process driven by root developmental cues but also serves as an active strategy to cope with adverse soil conditions.

    https://www.science.org/doi/10.1126/science.aai9057
  • Receptors for peptide hormone RGF

    Receptors for peptide hormone RGF PNAS (2016)

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    Identification of three LRR-RKs involved in perception of root meristem growth factor in Arabidopsis

    Hidefumi Shinohara, Ayaka Mori, Naoko Yasue, Kumiko Sumida and Yoshikatsu Matsubayashi*

    PNAS 113, 3897-3902 (2016)

    A peptide hormone, root meristem growth factor (RGF), regulates root meristem development through the PLETHORA (PLT) stem cell transcription factor pathway, but it remains to be uncovered how extracellular RGF signals are transduced to the nucleus. We identified, using a combination of a custom-made receptor kinase (RK) expression library and exhaustive photoaffinity labeling, three leucine-rich repeat RKs (LRR-RKs) that directly interact with RGF peptides in Arabidopsis. These three LRR-RKs, which we named RGFR1, RGFR2 and RGFR3, are expressed in root tissues including the proximal meristem, the elongation zone and the differentiation zone. The triple rgfr mutant was insensitive to externally applied RGF peptide and displayed a short root phenotype accompanied by a considerable decrease in meristematic cell number. In addition, PLT1 and PLT2 protein gradients, observed as a gradual gradient decreasing toward the elongation zone from the stem cell area in wild type, steeply declined at the root tip in the triple mutant. Because RGF peptides have been shown to create a diffusion-based concentration gradient extending from the stem cell area, our results strongly suggest that RGFRs mediate the transformation of an RGF peptide gradient into a PLT protein gradient in the proximal meristem, thereby acting as key regulators of root meristem development.

    https://www.pnas.org/doi/10.1073/pnas.1522639113
  • A galactosyltransferase critical for AGP biosynthesis

    A galactosyltransferase critical for AGP biosynthesis Plant J (2015)

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    Identification of three potent hydroxyproline O-galactosyltransferases in Arabidopsis

    Mari Ogawa-Ohnishi and Yoshikatsu Matsubayashi*

    Plant J. 81, 736-746 (2015)

    Arabinogalactan proteins (AGPs) are plant-specific extracellular glycoproteins implicated in a variety of processes during growth and development. AGP biosynthesis involves O-galactosylation of hydroxyproline (Hyp) residues followed by a stepwise elongation of the complex sugar chains. However, functionally dominant Hyp O-galactosyltransferases, such that their disruption produces phenocopies of AGP-deficient mutants, remain to be identified. We purified and identified three potent Hyp O-galactosyltransferases, HPGT1, HPGT2 and HPGT3, from Arabidopsis microsomal fractions. Loss-of-function analysis indicated that approximately 90% of the endogenous Hyp O-galactosylation activity is attributable to these three enzymes. Loss-of-function mutant plants exhibited a pleiotropic phenotype of longer lateral roots, longer root hairs, radial expansion of the cells in the root tip, small leaves, shorter inflorescence stems, reduced fertility and shorter siliques. Our findings provide genetic evidence that Hyp-linked arabinogalactan polysaccharide chains are critical for AGP function and clues to how arabinogalactan moieties of AGPs contribute to cell-to-cell communication during plant growth and development.

    https://onlinelibrary.wiley.com/doi/10.1111/tpj.12764
  • A peptide hormone mediating systemic nitrogen signaling

    A peptide hormone mediating systemic nitrogen signaling Science (2014)

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    Perception of root-derived peptides by shoot LRR-RKs mediates systemic N-demand signaling

    Ryo Tabata, Kumiko Sumida, Tomoaki Yoshii, Kentaro Ohyama, Hidefumi Shinohara and Yoshikatsu Matsubayashi*

    Science 346, 343-346 (2014)

    Nitrogen (N) is a critical nutrient for plants but is often distributed unevenly in the soil. Plants therefore have evolved a systemic mechanism by which N starvation on one side of the root system leads to a compensatory upregulation of nitrate uptake on the other side. However, the molecular components underlying this long-distance signaling, called N-demand signaling, remain elusive. We showed that perception of N starvation-induced small secreted peptides derived from roots by two specific leucine-rich repeat receptor kinases (LRR-RKs) in shoots constitutes the initial step in systemic N-demand signaling. Arabidopsis plants deficient in this pathway show growth retardation accompanied with N deficiency symptoms. Our findings provide new insights into the molecular basis of plant adaptation to a dynamically fluctuating N environment.

    https://www.science.org/doi/10.1126/science.1257800
  • An enzyme catalyzing peptide hormone arabinosylation

    An enzyme catalyzing peptide hormone arabinosylation Nature Chem Biol (2013)

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    Identification of three hydroxyproline O-arabinosyltransferases in Arabidopsis thaliana

    Mari Ogawa-Ohnishi, Wataru Matsushita and Yoshikatsu Matsubayashi*

    Nature Chem. Biol. 9, 726 (2013)

    Hydroxyproline (Hyp) O-arabinosylation is a posttranslational modification that is prominent in extracellular glycoproteins in plants. Hyp O-arabinosylation is generally found in these glycoproteins in the form of linear oligoarabinoside chains and plays a key role in their function by contributing to conformational stability. However, Hyp O-arabinosyltransferase (HPAT), a key enzyme that catalyzes the transfer of the L-arabinose to the hydroxyl group of Hyp residues, has remained undiscovered. We purified and identified Arabidopsis HPAT as a Golgi-localized transmembrane protein that is structurally similar to the glycosyltransferase GT8 family. Loss-of-function mutations in HPAT genes cause pleiotropic phenotypes that include enhanced hypocotyl elongation, defects in cell wall thickening, early flowering, early senescence, and impaired pollen tube growth. Our results indicate essential roles of Hyp O-arabinosylation in both vegetative and reproductive growth in plants.

    https://www.nature.com/articles/nchembio.1351
  • CLE glycopeptides control nodulation

    CLE glycopeptides control nodulation Nature Commun (2013)

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    Root-derived CLE glycopeptides control nodulation by direct binding to HAR1 receptor kinase

    Satoru Okamoto, Hidefumi Shinohara, Tomoko Mori, Yoshikatsu Matsubayashi* and Masayoshi Kawaguchi*

    Nature Commun 4, 2191 (2013)

    Leguminous plants establish a symbiosis with rhizobia to enable nitrogen fixation in root nodules under the control of the presumed root-to-shoot-to-root negative feedback called autoregulation of nodulation. In Lotus japonicus, autoregulation is mediated by CLE-RS genes that are specifically expressed in the root, and the receptor kinase HAR1 that functions in the shoot. However, the mature functional structures of CLE-RS gene products and the molecular nature of CLE-RS/HAR1 signaling governed by these spatially distant components remain elusive. We showed that CLE-RS2 is a posttranslationally arabinosylated glycopeptide derived from the CLE domain. Chemically synthesized CLE-RS glycopeptides cause significant suppression of nodulation and directly bind HAR1 in an arabinose-chain and sequence-dependent manner. In addition, CLE-RS2 glycopeptide specifically produced in the root is found in xylem sap collected from the shoot. We propose that CLE-RS glycopeptides are the long sought mobile signals responsible for the initial step of autoregulation of nodulation.

    https://www.nature.com/articles/ncomms3191
  • Ligand-binding domain of LRR-RK BAM1

    Ligand-binding domain of LRR-RK BAM1 Plant J (2012)

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    Biochemical mapping of a ligand-binding domain within Arabidopsis BAM1 reveals diversified ligand recognition mechanisms of plant LRR-RKs

    Hidefumi Shinohara, Yuji Moriyama, Kentaro Ohyama and Yoshikatsu Matsubayashi*

    Plant J. 70, 845-854 (2012)

    Leucine-rich repeat receptor kinases (LRR-RKs) comprise the largest subfamily of the transmembrane receptor kinases in plants. In several LRR-RKs, a loop-out region called an “island domain” that intercepts the extracellular tandem LRRs at a position near the transmembrane domain constitutes the ligand-binding pocket, but the absence of the island domain in numerous LRR-RKs raises questions about which domain specifically recognizes the corresponding ligands in non-island domain-carrying LRR-RKs. We determined by photoaffinity labeling followed by chemical and enzymatic digestion that BAM1 directly interacts with the small peptide ligand CLE9 at the LRR6-8 region that is relatively distal from the transmembrane domain. Multiple sequence alignment and homology modeling revealed that the inner concave side of LRR6-8 of the CLV1/BAM family LRR-RKs is slightly deviatory from the LRR consensus. Our results indicate that ligand recognition mechanisms of plant LRR-RKs are more complex and diversified than anticipated.

    https://onlinelibrary.wiley.com/doi/full/10.1111/j.1365-313X.2012.04934.x
  • A peptide hormone regulating root meristem development

    A peptide hormone regulating root meristem development Science (2010)

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    Secreted peptide signals required for maintenance of root stem cell niche in Arabidopsis

    Yo Matsuzaki, Mari Ogawa-Ohnishi, Ayaka Mori and Yoshikatsu Matsubayashi*

    Science 329, 1065-1067 (2010)

    Stem cells are maintained in the niche by intercellular interactions and signaling networks. We searched for extracellular signals required for maintenance of the root stem cell niche in higher plants. We identified a family of functionally redundant homologous peptides that are secreted, tyrosine-sulfated, and expressed mainly in the stem cell area and the innermost layer of central columella cells. We named these peptides root meristem growth factors (RGF). RGFs are required for maintenance of the root stem cell niche and transit amplifying cell proliferation in Arabidopsis. RGF defines expression levels and patterns of the stem cell transcription factor PLETHORA mainly at the post-transcriptional level. The RGFs function independently of the auxin pathway. These peptide signals play a crucial role in post-embryonic root development.

    https://www.science.org/doi/10.1126/science.1191132
  • An enzyme catalyzing peptide hormone tyrosine sulfation

    An enzyme catalyzing peptide hormone tyrosine sulfation PNAS (2009)

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    Identification of tyrosylprotein sulfotransferase in Arabidopsis

    Ryota Komori, Yukari Amano, Mari Ogawa-Ohnishi and Yoshikatsu Matsubayashi*

    PNAS 106, 15067-15072 (2009)

    Tyrosine sulfation is a posttranslational modification common in peptides and proteins synthesized by the secretory pathway in most eukaryotes. In plants, this modification is critical for the biological activities of a subset of peptide hormones such as PSK and PSY. In animals, tyrosine sulfation is catalyzed by Golgi-localized type II transmembrane protein called tyrosylprotein sulfotransferase (TPST). However, no orthologs of animal TPST genes have been found in plants, suggesting that plants have evolved plant-specific TPSTs structurally distinct from their animal counterparts. To investigate the mechanisms of tyrosine sulfation in plants, we purified TPST activity from microsomal fractions of Arabidopsis cells and identified a protein that specifically interacts with the sulfation motif of PSY precursor peptide. This protein is a type I transmembrane protein that shows no sequence similarity to animal TPSTs. A loss-of-function mutant of AtTPST displayed a marked dwarf phenotype accompanied by stunted roots, pale green leaves, reduction in higher order veins, early senescence and a reduced number of flowers and siliques. Our results indicate that plants and animals independently acquired tyrosine sulfation enzymes through convergent evolution.

    https://www.pnas.org/doi/10.1073/pnas.0902801106
  • CLV3 is an arabinosylated glycopeptide

    CLV3 is an arabinosylated glycopeptide Nature Chem Biol (2009)

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    A glycopeptide regulating stem cell fate in Arabidopsis

    Kentaro Ohyama, Hidefumi Shinohara, Mari Ogawa-Ohnishi and Yoshikatsu Matsubayashi*

    Nature Chem. Biol. 5, 578-580 (2009)

    A secreted peptide gene CLAVATA3 (CLV3) regulates stem cell fate in the shoot apical meristem in Arabidopsis plants, but the molecular structure of the active mature CLV3 peptide is controversial. We showed, by nano-LC-MS/MS analysis of apoplastic peptides of Arabidopsis plants overexpressing CLV3, that CLV3 is a 13-amino-acid arabinosylated glycopeptide. Post-translational arabinosylation of CLV3 is critical for its biological activity and high-affinity binding to its receptor CLV1.

    https://www.nature.com/articles/nchembio.182
  • In silico gene screening of peptide hormone candidates

    In silico gene screening of peptide hormone candidates Plant J (2008)

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    Identification of a biologically active small secreted peptide in Arabidopsis by in silico gene screening followed by LC-MS-based structure analysis

    Kentaro Ohyama, Mari Ogawa and Yoshikatsu Matsubayashi*

    Plant J. 55, 152-160 (2008)

    Peptidomics is a challenging field to create a link between genomic information and biological function through biochemical analysis of expressed peptides, including precise identification of post-translational modifications and proteolytic processing. We found that secreted peptides in Arabidopsis plants diffuse into the medium of whole-plant submerged culture and can be effectively identified by o-chlorophenol extraction followed by LC-MS analysis. Using this system, we first confirmed that 12-amino-acid mature CLE44 peptide accumulated at a considerable level in the culture medium of transgenic plants overexpressing CLE44. Next, using an in silico approach, we identified a novel gene family encoding small secreted peptides that exhibit significant sequence similarity within the C-terminal short conserved domain. We determined that the mature peptide encoded by At1g47485, a member of this gene family, is a 15-amino-acid peptide containing two hydroxyproline residues derived from the conserved domain. This peptide, which we have named CEP1, is mainly expressed in the lateral root primordia and, when overexpressed or externally applied, significantly arrests root growth. CEP1 is a candidate for a novel peptide plant hormone.

    https://onlinelibrary.wiley.com/doi/full/10.1111/j.1365-313X.2008.03464.x
  • CLV3 peptide directly binds CLV1 extracellular domain

    CLV3 peptide directly binds CLV1 extracellular domain Science (2008)

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    Arabidopsis CLV3 peptide directly binds CLV1 ectodomain

    Mari Ogawa, Hidefumi Shinohara, Youji Sakagami and Yoshikatsu Matsubayashi*

    Science 319, 294 (2008)

    CLV1, which encodes a leucine-rich-repeat receptor kinase, and CLV3, which encodes a secreted peptide, function in the same genetic pathway to maintain stem cell populations in the Arabidopsis shoot apical meristem. We showed biochemical evidence, by ligand binding assay and photoaffinity labeling, that CLV3 peptide directly binds the CLV1 ectodomain with a dissociation constant of 17.5 nM. The CLV1 ectodomain also interacts with the structurally related CLE peptides, with distinct affinities depending on the specific amino acid sequence. Our results provide definitive evidence that CLV3 and CLV1 function as a ligand-receptor pair involved in stem cell maintenance.

    https://www.science.org/doi/10.1126/science.1150083
  • A tyrosine-sulfated peptide promoting cell proliferation

    A tyrosine-sulfated peptide promoting cell proliferation PNAS (2007)

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    A tyrosine-sulfated glycopeptide involved in cellular proliferation and expansion in Arabidopsis

    Yukari Amano, Hiroko Tsubouchi, Hidefumi Shinohara, Mari Ogawa and Yoshikatsu Matsubayashi*

    PNAS 104, 18333-18338 (2007)

    Post-translational modification can confer special functions to peptides. Based on exhaustive LC-MS analysis targeting tyrosine-sulfated peptides, we identified a novel 18-amino-acid tyrosine-sulfated glycopeptide in Arabidopsis cell suspension culture medium. This peptide, which we have named PSY1, significantly promotes cellular proliferation and expansion at nanomolar concentrations. PSY1 is widely expressed in various Arabidopsis tissues, including shoot apical meristem, and is highly upregulated by wounding. Perception of PSY1 is dependent on At1g72300, which is a novel leucine-rich repeat receptor kinase (LRR-RK) whose two paralogs are involved in perception of PSK.
    (Note added in 2022)
    We note that PSY1 showed no direct interaction with At1g72300, which is often called a PSY1 receptor (PSY1R), indicating that At1g72300 is not a direct ligand-perceiving receptor for PSY peptides. In 2022, we report that three LRR-RKs (At1g17230, At2g33170 and At5g63930) act as direct ligand-perceiving receptors for PSY-family peptides and mediate the trade-off between plant growth and the stress response.

    https://www.pnas.org/doi/10.1073/pnas.0706403104
  • Ligand fishing using immobilized receptor kinase

    Ligand fishing using immobilized receptor kinase Plant J (2007)

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    Functional immobilization of plant receptor-like kinase onto microbeads toward receptor array construction and receptor-based ligand fishing

    Hidefumi Shinohara and Yoshikatsu Matsubayashi*

    Plant J. 52, 175-184 (2007)

    Despite the large number of receptor-like kinases (RLKs) in plants, few of them have known specific ligands. Ligand fishing is one of the most challenging post-genomic technologies. We established a strategy for functional immobilization of plant RLKs on microbeads via covalent linkage. Due to the high density of immobilized RLK, ligand-receptor interaction can be visualized at high sensitivity using fluorescent-labeled ligands under the confocal laser scanning microscope. Moreover, using receptor-based affinity chromatography system with RLK microbeads, the ligand of the receptor was directly retrieved at high purity from complex natural samples. Our RLK microbeads and receptor-based ligand fishing approach is a feasible alternative to conventional forward genetics and bioassay-based biochemical purification for identification of novel ligand-receptor pairs in plants.

    https://onlinelibrary.wiley.com/doi/10.1111/j.1365-313X.2007.03204.x
PUBLICATION LIST

PEOPLE

PEOPLE
  • Yoshikatsu Matsubayashi

    ProfessorYoshikatsu Matsubayashi

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  • Assistant Professor

    Assistant ProfessorMari Ogawa-Ohnishi

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  • Yuri Ohkubo

    Assistant ProfessorYuri Ohkubo

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