Kazuo Ebine

Small GTPases of the Ras superfamily are critical regulators of diverse cellular processes. While crosstalk between their signalling pathways has been documented in animals, similar mechanisms remain unexplored in plants, where small GTPases have undergone unique evolutionary diversification. Here we identify REAP1/AtSWAP70 as an effector in Arabidopsis thaliana that interacts with the active forms of both canonical RAB5 and the plant-specific RAB5, ARA6. Remarkably, REAP1 also binds to active ROP7, a plant-unique Rac-type GTPase, via its Dbl homology domain. REAP1 localizes to endosomes and facilitates ROP7 recruitment from the plasma membrane, a process dependent on RAB5 activity. Genetic analyses reveal that the RAB5–REAP1–ROP7 signalling cascade is essential for gametogenesis, impacting pollen viability and development. This study provides evidence of functional crosstalk between RAB and ROP signalling in plants, unveiling a layer of regulatory complexity in plant GTPase signalling during plant reproduction.

Receptor kinases are pivotal for growth, development, and environmental response of plants. Yet, their regulatory mechanisms and spatial dynamics remain underexplored. The ERECTA-family receptor kinases coordinate diverse developmental processes, including stomatal development. To understand the proteomic landscape of the ERECTA-mediated signaling pathways, we report comparative analyses of the ERECTA interactome and proximitome by epitope-tagged affinity-purification (ET-AP) and TurboID-based proximity labeling (TbID-PL) mass spectrometry, respectively. While ET-AP recovered receptor complex components (e.g., TOO MANY MOUTHS), TbID-PL effectively captured transient associations with the components of endosomal trafficking, i.e., clathrin-mediated endocytosis machinery. We further identify that specific subfamily members of phosphatidylinositol-binding clathrin assembly proteins (PICALMs) interact with and synergistically regulate ERECTA internalization. Mutations in PICALMs impair ERECTA endocytosis and lead to excessive stomatal clustering by dampening the downstream signaling output. Together, we provide a proteomic atlas of the ERECTA signaling network and demonstrate that timely removal of receptor kinase by the endocytosis machinery is essential for active signal transduction enforcing stomatal patterning.

A distinctive protein and lipid composition underlies the distinct function of each organelle, regulated by balanced anterograde and retrograde membrane trafficking. The vacuole, the largest plant organelle, is pivotal in various plant functions, and its protein composition is tightly regulated by bidirectional trafficking. However, the existence of retrograde transport from the plant vacuole has remained unverified. Here we demonstrate retrograde trafficking from the vacuole in Arabidopsis. We observed the retrieval of VAMP727, a plant-unique vacuolar membrane fusion machinery, from the vacuolar membrane. VAMP727 retrieval is facilitated by sorting nexin proteins, which independently diversified between plant and non-plant systems. Furthermore, we show that the core retromer complex and sorting nexins act independently in distinct retrograde transport events with specific cargos. Plant cells have thus elaborated a unique retrieval mechanism from the vacuole, underpinning the neofunctionalization of VAMP727 during plant evolution.

Receptor kinases are pivotal for growth, development, and environmental response of plants. Yet, their regulatory mechanisms and spatial dynamics are still underexplored. The ERECTA-family receptor kinases coordinate diverse developmental processes, including stomatal development. To understand the proteomic landscape of the ERECTA-mediated signaling pathways, we here report comparative analyses of the ERECTA interactome and proximitome by epitope-tagged affinity-purification (ET-AP) and TurboID-based proximity labeling (TbID-PL) mass-spectrometry, respectively. While ET-AP successfully recovered receptor complex components (e.g., TOO MANY MOUTHS), TbID-PL effectively captured transient associations with the components of endosomal trafficking, i.e., clathrin-mediated endocytosis (CME) machinery. We further identify that specific subfamily members of phosphatidylinositol-binding clathrin assembly proteins (PICALMs) interact with and synergistically regulate ERECTA internalization. Mutations of these PICALMs impair ERECTA endocytosis and lead to excessive stomatal clustering by dampening the downstream signaling output. Taken together, our work provides a proteomic atlas of the ERECTA signaling network and suggests that timely removal of receptor kinase by the endocytosis machinery is essential for active signal transduction enforcing stomatal patterning.

Small GTPases of the Ras superfamily are critical regulators of diverse cellular processes. While cross-talk between their signaling pathways has been documented in animals, similar mechanisms remain unexplored in plants, where small GTPases have undergone unique evolutionary diversification. Here, we identify REAP1/AtSWAP70 as a novel effector in Arabidopsis thaliana that interacts with the active forms of both canonical RAB5 and the plant-specific RAB5, ARA6. Remarkably, REAP1 also binds to active ROP7, a plant-unique Rac-type GTPase, via its DH domain. REAP1 localizes to endosomes and facilitates ROP7 recruitment from the plasma membrane, a process dependent on RAB5 activity. Genetic analyses reveal that the RAB5-REAP1-ROP7 signaling cascade is essential for gametogenesis, impacting pollen viability and development. This study provides the evidence of functional cross-talk between RAB and ROP signaling in plants, unveiling a novel layer of regulatory complexity in plant GTPase signaling during plant reproduction.

An Arabidopsis sterol mutant, smt2 smt3, defective in sterolmethyltransferase2 (SMT2), exhibits severe growth abnormalities. The loss of C-24 ethyl sterols, maintaining the biosynthesis of C-24 methyl sterols and brassinosteroids, suggests specific roles of C-24 ethyl sterols. We characterized the subcellular localizations of fluorescent protein-fused sterol biosynthetic enzymes, such as SMT2-GFP, and found these enzymes in the endoplasmic reticulum during interphase and identified their movement to the division plane during cytokinesis. The mobilization of endoplasmic reticulum-localized SMT2-GFP was independent of the polarized transport of cytokinetic vesicles to the division plane. In smt2 smt3, SMT2-GFP moved to the abnormal division plane, and unclear cell plate ends were surrounded by hazy structures from SMT2-GFP fluorescent signals and unincorporated cellulose debris. Unusual cortical microtubule organization and impaired cytoskeletal function accompanied the failure to determine the cortical division site and division plane formation. These results indicated that both endoplasmic reticulum membrane remodeling and cytokinetic vesicle transport during cytokinesis were impaired, resulting in the defects of cell wall generation. The cell wall integrity was compromised in the daughter cells, preventing the correct determination of the subsequent cell division site. We discuss the possible roles of C-24 ethyl sterols in the interaction between the cytoskeletal network and the plasma membrane.

The extended tubular shape of root hairs is established by tip growth and concomitant hardening. Here, we demonstrate that a syntaxin of plants (SYP)123–vesicle-associated membrane protein (VAMP)727-dependent secretion system delivers secondary cell wall components for hardening the subapical zone and shank of Arabidopsis (Arabidopsis thaliana) root hairs. We found increased SYP123 localization at the plasma membrane (PM) of the subapical and shank zones compared with the tip region in elongating root hairs. Inhibition of phosphatidylinositol (PtdIns)(3,5)P₂ production impaired SYP123 localization at the PM and SYP123-mediated root hair shank hardening. Moreover, root hair elongation in the syp123 mutant was insensitive to a PtdIns(3,5)P₂ synthesis inhibitor. SYP123 interacts with both VAMP721 and VAMP727. syp123 and vamp727 mutants exhibited reduced shank cell wall stiffness due to impaired secondary cell wall component deposition. Based on these results, we conclude that SYP123 is involved in VAMP721-mediated conventional secretion for root hair elongation as well as in VAMP727-mediated secretory functions for the delivery of secondary cell wall components to maintain root hair tubular morphology.

Summary: Land plant spermatozoids commonly possess characteristic structures such as the spline, which consists of a microtubule array, the multilayered structure (MLS) in which the uppermost layer is a continuum of the spline, and multiple flagella. However, the molecular mechanisms underpinning spermatogenesis remain to be elucidated. We successfully identified candidate genes involved in spermatogenesis, deeply divergent BLD10s, by computational analyses combining multiple methods and omics data. We then examined the functions of BLD10s in the liverwort Marchantia polymorpha and the moss Physcomitrium patens. MpBLD10 and PpBLD10 are required for normal basal body (BB) and flagella formation. Mpbld10 mutants exhibited defects in remodeling of the cytoplasm and nucleus during spermatozoid formation, and thus MpBLD10 should be involved in chromatin reorganization and elimination of the cytoplasm during spermiogenesis. We identified orthologs of MpBLD10 and PpBLD10 in diverse Streptophyta and found that MpBLD10 and PpBLD10 are orthologous to BLD10/CEP135 family proteins, which function in BB assembly. However, BLD10s evolved especially quickly in land plants and MpBLD10 might have acquired additional functions in spermatozoid formation through rapid molecular evolution.

Gametogenesis is an essential event for sexual reproduction in various organisms. Bryophytes employ motile sperm (spermatozoids) as male gametes, which locomote to the egg cells to accomplish fertilization. The spermatozoids of bryophytes harbor distinctive morphological characteristics, including a cell body with a helical shape and two flagella. During spermiogenesis, the shape and cellular contents of the spermatids are dynamically reorganized. However, the reorganization patterns of each organelle remain obscure. In this study, we classified the developmental processes during spermiogenesis in the liverwort Marchantia polymorpha according to changes in cellular and nuclear shapes and flagellar development. We then examined the remodeling of microtubules and the reorganization of endomembrane organelles. The results indicated that the state of glutamylation of tubulin changes during formation of the flagella and spline. We also found that the plasma membrane and endomembrane organelles are drastically reorganized in a precisely regulated manner, which involves the functions of endosomal sorting complexes required for transport (ESCRT) machineries in endocytic and vacuolar transport. These findings are expected to provide useful indices to classify developmental and subcellular processes of spermiogenesis in bryophytes.

Oogamy is a form of sexual reproduction and evolved independently in animals, fungi, and plants. In streptophyte plants, Charophyceae, Coleochaetophyceae, bryophytes, lycophytes, ferns (monilophytes), and some gymnosperms (Cycads and Ginkgo) utilize spermatozoids as the male gamete. Plant spermatozoids commonly possess characteristic structures such as the spline, which consists of a microtubule array, the multilayered structure (MLS) in which the uppermost layer is continuum of the spline, and multiple flagella. However, the molecular mechanisms underpinning plant spermatogenesis remain to be elucidated. To identify the genes involved in plant spermatogenesis, we performed computational analyses and successfully found deeply divergent BLD10 s by combining multiple methods and omics-data. We then validated the functions of candidate genes in the liverwort Marchantia polymorpha and the moss Physcomitrium patens and found that Mp BLD10 and Pp BLD10 are required for normal basal body and flagella formation. Mp bld10 mutants exhibited defects in remodeling of the cytoplasm and nucleus during spermatozoid formation, thus Mp BLD10 should be involved in chromatin reorganization and elimination of the cytoplasm during spermiogenesis. Streptophyte BLD10s are orthologous to BLD10/CEP135 family proteins, which function in basal body assembly, but we found that BLD10s evolved especially fast in land plants and MpBLD10 might obtain additional functions in spermatozoid formation through the fast molecular evolution. This study provides a successful example of combinatorial study from evolutionary and molecular genetic perspectives that elucidated a function of the key protein of the basal body formation that fast evolved in land plants.