Biology subjects

Umezawa, T.

Publications and source records attributed to Umezawa, T..

10 recordsLinked to original sources

bioRxiv2026

Ultrastructural analysis of engineered rice lines reveals ferulate cross-linking as a key factor mediating lignocellulose supramolecular assembly in grass cell walls

Within the secondary cell walls of vascular plants, cellulose, hemicelluloses, and lignin associate via various covalent and non-covalent linkages to form an intricate supramolecular assembly. Although the chemical structures and cross-linking levels of the lignin- hemicellulose matrix exhibit substantial diversity in planta, precisely how these structural variations affect lignocellulose supramolecular assembly and macroscopic biomass properties remains largely elusive. Here, we conducted a comparative multi-scale ultrastructural analysis across engineered rice lines with targeted modifications in lignin aromatic composition and ferulate (FA)-mediated cell wall cross-linking levels. Combined solid-state nuclear magnetic resonance and wide-angle X-ray diffraction analyses revealed that depleting FA cross-linking disrupts cellulose crystalline structure and accelerates molecular mobility markedly more severely than altering the guaiacyl-to-syringyl (G/S) lignin ratio, generating a more loosened lignocellulose network. Small-angle X-ray scattering analysis further demonstrated that specific FA-depleted lines, but none of those with an altered G/S ratio, also exhibited disruptions in the nano- to mesoscale organization of cellulose microfibrils. Furthermore, FA- depleted lines generally displayed greater improvements in saccharification efficiency and more rapid thermal softening than G/S-lignin-altered lines, suggesting that disruptions in lignocellulose molecular assembly induced by FA depletion can broadly translate into macroscopic biomass properties. These findings establish a molecular basis for the pivotal role of FA cross-linking in dictating grass cell wall architecture, offering a promising structural target for advancing grass biomass utility and crop design.

plant biology↗

bioRxiv2026

Engineering eudicot-like lignin in rice via targeted disruption of grass-specific lignin modification pathways

O_LILignin composition exhibits substantial diversity across plant lineages. Unlike eudicot and gymnosperm lignins, grass lignin incorporates p-coumarate and tricin units alongside canonical monolignols. To elucidate their specific biochemical and physiological roles, we engineered rice (Oryza sativa) to produce a "eudicot-like" lignin devoid of both modifications. C_LIO_LIUsing CRISPR-Cas9, we generated mutants deficient in p-coumarate and tricin by simultaneously targeting their respective biosynthetic genes. The resulting mutant cell walls underwent structural and functional characterization via wet-chemical analyses, nuclear magnetic resonance, gel permeation chromatography, and antioxidant capacity assays. C_LIO_LIThe newly generated ospmt1/2 osfnsII and ospmt1/2 osa3'h/c5'h triple-knockout mutants reached maturity with minor growth penalties. Subsequent cell wall analyses demonstrated near-complete depletion of both p-coumarate and tricin units in lignins. This structural shift substantially altered overall lignin content, monomeric composition, linkage distributions, and molecular weight, highlighting the divergent and synergistic roles of these units in lignin assembly and polymerization. Functionally, the free-radical scavenging capacity of rice lignin is markedly enhanced by p-coumaroylation, but attenuated by tricin incorporation. C_LIO_LIThe successful synthesis of eudicot-like lignin within a grass system underscores the inherent plasticity of lignification. These engineered rice lines offer a valuable platform to investigate the physiological functions and biotechnological potential of grass lignin modifications. C_LI

plant biology↗

bioRxiv2026

Iron-dependent reprogramming of damage-associated peptide receptor signaling coordinates immunity and phosphate stress adaptation

Plant adaptation to inorganic phosphate (Pi) limitation entails extensive developmental and metabolic reprogramming, collectively termed the phosphate starvation response (PSR). How plants balance nutrient acquisition with immune competence remains unresolved. Here, we show that PSR selectively remodels pattern-recognition receptor (PRR) signaling in Arabidopsis thaliana. Membrane proteomics reveals increased abundance of the Pep receptor PEPR1 under Pi deficiency, whereas most receptor kinases, including the microbial PRRs FLS2 and CERK1, are reduced. Immunoblotting further shows increased PEPR2 abundance. Consistently, Pep-triggered responses are strongly potentiated under Pi depletion, while flagellin- and chitin-induced responses are not, indicating selective PEPR sensitization rather than global immune suppression. This sensitization requires the PSR-associated ferroxidases LPR1/LPR2, NADPH oxidases RBOHD/RBOHF, and transcription factor WRKY33, linking iron-dependent redox remodeling to immune amplification. Beyond inducible defense, PEPRs promote basal PSR transcriptional reprogramming and growth under Pi limitation. Loss of PEPR1/PEPR2 compromises bacterial resistance and alters root-associated microbiota assembly under nutrient deficiency. Together, Pi depletion selectively sensitizes specific PRR pathways. PEPRs exhibit context-dependent dual functionality, promoting either immunity or nutrient stress adaptation, potentially in response to distinct PROPEP-derived ligands, thereby supporting nutrient acquisition without compromising immune vigilance. These findings provide a framework for how nutritional status selectively remodels PRR signaling to coordinate immunity with nutrient stress adaptation.

plant biology↗

bioRxiv2025

Stacked mutations disrupting syringyl and p-coumaroylated lignin biosynthesis in rice result in lignin dominated by guaiacyl units: insights into grass-specific lignin monomer biosynthesis and polymerization mechanisms

O_LIThe aromatic composition of lignin significantly impacts the usability of lignocellulosic biomass. In eudicots, transgenic and mutant lines with elevated guaiacyl (G) or syringyl (S) lignin units have been successfully generated by manipulating the expression level of CONIFERALDEHYDE 5-HYDROXYLASE (CAld5H). However, this bioengineering approach has proven less effective in grasses, implicating the potential existence of a grass-specific alternative pathway for S lignin biosynthesis. C_LIO_LIThrough characterization of genome-edited rice mutants, we demonstrated that S lignin in rice can be virtually eliminated by disrupting genes encoding CAld5H along with p- COUMAROYL-COENZYME A:MONOLIGNOL TRANSFERASE (PMT), a grass-specific enzyme essential for the biosynthesis of monolignol p-coumarate conjugates. In contrast, individual mutations in either CAld5H or PMT genes resulted in incomplete elimination of S lignin. These findings provide strong evidence that rice possesses a CAld5H-independent pathway leading to the grass-specific monolignol p-coumarate conjugates. C_LIO_LIIn-depth structural characterizations of G-dominated lignins from rice and Arabidopsis mutants, natural gymnosperm pine, and G-type synthetic lignin revealed pronounced effects of lineage-dependent cell wall environments on the linkage patterns and molecular weight distributions of the resulting lignin polymers. C_LIO_LIOverall, our findings highlight previously overlooked lineage-specific lignin monomer biosynthesis and polymerization patterns in grasses. C_LI

plant biology↗

bioRxiv2024

Mitochondrial Dysfunction and ER Stress in CB1 Receptor Antagonist-Induced Apoptosis in Human Neuroblastoma SH-SY5Y Cells

Cannabinoid receptor type 1 (CB1R) is the key modulator of neuronal viability. In particular, CB1R antagonists provide neuroprotective effects on neurotoxicity caused by e.g. neuronal injury. However, the underlying mechanisms and potential limitations of CB1R antagonism remain unclear. Here we investigated the impact of environmental conditions on CB1R antagonist effects. Unexpectedly, we have found that cell-permeable CB1R antagonists, rimonabant and AM251, induced significant cell death in human neuroblastoma SH-SY5Y cells under serum-free conditions. Mitochondrial morphological analysis revealed mitochondrial swelling characterized by their network fragmentation and cristae reduction. Additionally, phosphoproteomics analysis showed an upregulation of phosphorylated EIF2AK3 (also known as PERK), leading to the activation of the eIF2/ATF4/CHOP pathway, indicative of endoplasmic reticulum (ER) stress. Rimonabant and AM251 treatment also triggered caspase-dependent apoptosis, evidenced by the detection of cleaved caspase 3 and cleaved PARP. These results suggest that cell-permeable CB1R antagonists promote apoptosis via mitochondrial dysfunction and ER stress under serum-free conditions in SH-SY5Y cells. Our findings indicate that while CB1R antagonists may be neuroprotective in certain conditions, they may also pose a neurotoxic risk in environments characterized by cellular stress or nutrient deprivation.

pharmacology and toxicology↗

bioRxiv2024

Essential Yet Dispensable: The Role of CINNAMATE 4-HYDROXYLASE in Rice Cell Wall Lignification

A comprehensive understanding of the intricate lignin biosynthesis in grasses could contribute to enhancing our ability to utilize grass biomass. CINNAMATE 4-HYDROXYLASE (C4H), in conjunction with PHENYLALANINE AMMONIA-LYASE (PAL), initiates the entry of phenylalanine into the cinnamate/monolignol pathway, leading to the production of diverse phenylpropanoids, including lignin monomers. Despite extensive research on C4H in eudicots, genetic studies of C4H in grasses remain considerably limited. Notably, the role of C4H in the presence of PHENYLALANINE/TYROSINE AMMONIA-LYASE (PTAL), a grass-specific ammonia-lyase that can bypass the conserved PAL-C4H pathway by recruiting tyrosine into the cinnamate/monolignol pathway, remains unclear. To address this gap, a set of genome-edited rice mutants harboring knockout mutations in rice C4H genes were generated and subjected to the analysis of growth phenotype and cell wall chemotype, alongside isotopic feeding and chemical inhibitor assays to test the contributions of the PAL-C4H and PTAL pathways. The phenotype and chemotype characterizations of C4H-knockout rice mutants demonstrated that class I (OsC4H1/CYP73A38) and class II (OsC4H2a/CYP73A39 and OsC4H2b/CYP73A40) C4Hs cooperatively contribute to lignin biosynthesis in rice. Nevertheless, the impacts of C4H-deficiency on plant development and lignin formation in rice appeared to be less prominent compared to those reported in eudicots. The 13C-labeled phenylalanine and tyrosine feeding experiments demonstrated that even with the phenylalanine-derived PAL-C4H pathway completely blocked, the C4H-knockout rice can still produce significant levels of lignin and maintain sound cell walls by utilizing the tyrosine-derived PTAL pathway. Overall, this study demonstrates the essential but dispensable role of C4H in grass cell wall lignification.

plant biology↗

bioRxiv2024

MAP4K1 and MAP4K2 regulate ABA-induced and Ca2+-mediated stomatal closure in Arabidopsis

Abscisic acid (ABA)-induced stomatal closure limits water loss from plants under drought stress. To investigate signaling pathways involved in stomatal closure, we performed a phosphoproteomic analysis of ABA-treated Arabidopsis guard cell protoplasts (GCPs). From this analysis, we discovered that MITOGEN-ACTIVATED PROTEIN 4 KINASE 1 (MAP4K1) is phosphorylated in response to ABA. Subsequent biochemical assays showed that Ser-479 of MAP4K1 is directly phosphorylated by SRK2E (OST1/SnRK2.6), a protein kinase that plays a central role in ABA-induced responses. Mutational analyses of MAP4K1, as well as closely related MAP4K2, revealed that both kinases positively regulate ABA-induced stomatal closure, and that Ser-479 of MAP4K1 was required for this phenotype. In map4k1map4k2, stomatal closure was induced by exogenous Ca2+ but not H2O2. Furthermore, electrophysiology experiments showed that MAP4K1/2 are required for ABA-dependent activation of Ca2+-permeable channels in GCPs. Together, our results indicate that SnRK2 and MAP4K1/2 function as a signaling module to regulate Ca2+-mediated stomatal closure.

plant biology↗

bioRxiv2023

Hyperosmolarity-induced suppression of group B1 Raf-like protein kinases modulates drought-growth trade-off in Arabidopsis

When plants are exposed to drought stress, there is a trade-off between plant growth and stress responses. Here, we identified a signaling mechanism for the initial steps of the drought-growth trade-off. Phosphoproteomic profiling revealed that Raf13, a B1 subgroup Raf-like kinase, is dephosphorylated under drought conditions. Raf13 and the related B1-Raf Raf15 are required for growth rather than the acquisition of osmotolerance. We also found that Raf13 interacts with B55-family regulatory subunits of protein phosphatase 2A (PP2A), which mediates hyperosmolarity-induced dephosphorylation of Raf13. In addition, Raf13 interacts with an AGC kinase INCOMPLETE ROOT HAIR ELONGATION HOMOLOG 1 (IREH1), and Raf13 and IREH1 have similar functions in regulating cellular responses that promote plant growth. Overall, our results support a model in which Raf13-IREH1 activity promotes growth under nonstressed conditions, whereas PP2A activity suppresses Raf13-IREH1 during osmotic stress to modulate the physiological "trade-off" between plant growth and stress responses.

plant biology↗

bioRxiv2020

Activation of SnRK2 by the Raf-like kinase ARK represents a common mechanism of ABA response in embryophytes

The Raf-like protein kinase ARK previously identified in the moss Physcomitrella patens acts as an upstream regulator of subgroup III SnRK2, the key regulator of abscisic acid (ABA) and abiotic stress responses. However, the mechanisms underlying activation of ARK by ABA and abiotic stress for the regulation of SnRK2 including the role of ABA receptor-associated group A PP2C (PP2C-A) are not understood. We identified Ser1029 as the phosphorylation site in the activation loop of ARK, which provided a possible mechanism for regulation of its activity. Analysis of transgenic ark lines expressing ARK-GFP with Ser1029-to-Ala mutation indicated that this replacement causes reductions in ABA-induced gene expression, stress tolerance and SnRK2 activity. Immunoblot analysis using an anti-phosphopeptide antibody indicated that ABA treatments rapidly stimulate Ser1029 phosphorylation in wild type. The phosphorylation profile of Ser1029 in ABA-hypersensitive ppabi1 lacking PP2C-A was similar to that in wild type, whereas little Ser1029 phosphorylation was observed in ABA-insensitive ark missense lines. Furthermore, newly isolated ppabi1 ark lines showed ABA-insensitive phenotypes similar to those of ark lines. These results indicate that ARK is a primary activator of SnRK2, preceding negative regulation by PP2C-A in bryophytes, which provides a prototypal mechanism for ABA and abiotic stress-responses in embryophytes. One sentence summaryPhysiological characterization of various moss mutants revealed a common mechanism for phytohormone responses under water deficit in all land plants.

plant biology↗

bioRxiv2020

SNF1-related protein kinase 2 directly regulate group C Raf-like protein kinases in abscisic acid signaling

ABSTRUCTA phytohormone abscisic acid (ABA) has a major role in abiotic stress responses in plants, and subclass III SNF1-related protein kinase 2 (SnRK2) mediates ABA signaling. In this study, we identified Raf36, a group C Raf-like protein kinase in Arabidopsis, as an interacting protein with SnRK2. A series of reverse genetic and biochemical analyses revealed that Raf36 negatively regulates ABA responses and is directly phosphorylated by SnRK2s. In addition, we found that Raf22, another C-type Raf-like kinase, functions partially redundantly with Raf36 to regulate ABA responses. Comparative phosphoproteomic analysis using Arabidopsis wild-type and raf22raf36-1 plants identified proteins that are phosphorylated downstream of Raf36 and Raf22 in planta. Together, these results reveal a novel subsection of ABA-responsive phosphosignaling pathways branching from SnRK2.

plant biology↗