Dormancy is a crucial trait that allows land plants to withstand harsh terrestrial environments by pausing growth until favorable conditions return. The liverwort Marchantia polymorpha produces dormant propagules, gemmae, for asexual reproduction. The phytohormone ABA is known to regulate dormancy in the seeds of flowering plants and the gemmae of M. polymorpha. Here, we identify a basic helix-loop-helix protein, MpHYPNOS (MpHYP), as a central regulator of gemma dormancy. MpHYP expression induces germination arrest in gemmae. Transcriptome analysis revealed that MpHYP represses a D-type cyclin gene MpCYCD;1 and induces genes involved in ABA biosynthesis. Repression of MpCYCD;1 expression and germination arrest are both maintained even in an ABA-receptor mutant. By contrast, Mphyp and ABA-related mutants lack the desiccation tolerance observed in wild-type gemmae. We propose that MpHYP regulates gemma dormancy through two parallel pathways: an ABA-independent pathway that suppresses germination through cell cycle repression, and an ABA-dependent pathway that enhances desiccation tolerance via ABA biosynthesis. These findings highlight an integrated regulatory mechanism of dormancy via phytohormonal and cell cycle control in bryophytes.

ABSTRACT Colletotrichum higginsianum ( Ch ) is a hemibiotrophic fungal pathogen that infects Brassicaceae plants, including Arabidopsis thaliana . The molecular mechanisms underlying the Ch - A. thaliana interaction are not fully understood. Particularly, the susceptibility factor against Ch infection remains to be determined. Here, we report that A. thaliana ACTIN DEPOLYMERIZING FACTORs (ADFs), ancient proteins that regulate the organization and dynamics of actin filaments (AFs), function as susceptibility factors during Ch infection. Among 11 ADF s encoded in A. thaliana genome, subclass I ADF s that include ADF1 , -2 , -3 , and -4 , express throughout the plant. We found that knockout mutant of ADF4 and transgenic plants in which the expression of all of subclass I members is suppressed ( ADF1-4Ri ) exhibited increased resistance to Ch . Cytological analyses revealed that both Ch penetration and secondary hyphae formation were suppressed in adf4 and ADF1-4Ri . This enhanced resistance was associated with suppression of Ch -induced AF fragmentation. In addition, we found that PENETRATION 2 (PEN2) plays a critical role in the Ch resistance in adf4 and ADF1-4Ri . Our findings suggest that subclass I ADFs promote AF fragmentation during Ch infection, thereby suppressing PEN2-associated mitochondria accumulation at Ch entry sites. Together, these results raise the possibility that Ch exploits host ADF-dependent actin regulation to facilitate successful infection.

OBJECTIVE:Acid-sensing ion channel 3 (ASIC3), a proton-gated cation channel predominantly expressed in primary afferent nociceptors, is an acidosis-related pain generator. Previous experiments suggested that ASIC3 is also involved in the generation of itch. However, mechanistic links between ASIC3 and itch, including the expression of ASIC3 in itch-mediating primary sensory neurons, remain unclear. We examined ASIC3 expression in these sensory neurons and then investigated whether mild acid stimulation could induce ASIC3-dependent itch without overt pain in mice. METHODS:Immunohistochemical analyses were performed using ASIC3-FLAG-enhanced green fluorescent protein-FLAG (FEF) expressing mice. Citric acid was applied with a brush to shaved skin of the nape of the neck or cheek in wild-type and ASIC3 knockout (ASIC3 -/- ) mice. Hindlimb scratching and, in the cheek model, forelimb facial wiping were recorded. RESULTS:ASIC3-expressing neurons and plexin C1-positive/tachykinin 1-negative itch-mediating neurons essentially belonged to distinct subpopulations in dorsal root and trigeminal ganglia. Application of 0.2 M citric acid to the nape induced hindlimb scratching directed toward the citric acid-applied area in wild-type mice, and this response was significantly attenuated in ASIC3 -/- mice. Application of 0.5 M citric acid to the cheek induced ASIC3-dependent itch behavior (hindlimb scratching), accompanied by minimal or no pain behavior (forelimb wiping). CONCLUSION:Given the absence of ASIC3 in typical itch-mediating primary sensory neurons, citric acid-induced ASIC3 activation in nociceptive skin afferents primarily involved in pain likely underlies the observed itch behavior. As 0.5 M citric acid likely represents a weak noxious stimulus, weak activation of these pain-mediating afferents can evoke itch, supporting the intensity theory of itch.

The SABATH family enzymes are a group of plant-specific methyltransferases that catalyze the methylation of many small molecules, including several plant hormones. While this family originated before the evolution of land plants from streptophyte algae, little is known about its biological function in plant lineages other than angiosperms. Here, we identified 12 SABATH family genes from the liverwort Marchantia polymorpha and found that MpSABATH2 plays a critical role in development. Mpsabath2 mutants were severely inhibited in thallus growth and gemma cup formation, while they spontaneously formed sexual branches under noninductive conditions. These phenotypes resembled the developmental responses to far-red light, which was also supported by transcriptome analysis. Further genetic analysis connected this phenomenon with gibberellin (GA)-related metabolism. Blocking GA biosynthesis partially rescued Mpsabath2 phenotypes, which were restored by treatment with the GA precursor, ent-kaurenoic acid. Given that MpSABATH2 belongs to a phylogenetic clade distinct from previously reported phytohormone-methylating SABATH enzymes, our findings suggest that SABATH family enzymes independently acquired roles in developmental regulation through parallel evolution in land plants.

Actin filaments are essential for plant cellular functions, and their dynamics are altered in response to fungal infection, contributing to defense responses. However, the mechanisms by which pathogens suppress actin-mediated immunity remain largely unknown. Here, we report that infection by the adapted fungus Colletotrichum higginsianum induces actin fragmentation in Arabidopsis thaliana leaf cells. Imaging analyses revealed that actin fragmentation with increased apparent actin thickness occurred following C. higginsianum inoculation. Actin fragmentation was observed 2 days after inoculation, preceding hyphal invasion on Day 3. Rhodamine-phalloidin staining confirmed that endogenous actin filaments were disrupted in infected cells. Pretreatment with latrunculin B, which induces actin fragmentation, significantly increased the rate of C. higginsianum hyphal invasion, suggesting that fragmented actin filaments may facilitate fungal entry. Notably, actin fragmentation occurred even in pattern recognition receptor mutants, indicating that it is not triggered by pathogen-associated molecular pattern perception. No obvious changes in microtubule organization were observed. Inoculation with the nonadapted Colletotrichum tropicale did not induce actin fragmentation in A. thaliana. In contrast, C. higginsianum induced actin fragmentation in cucumber and Brassica crops, whereas Colletotrichum orbiculare did not, despite being pathogenic to cucumber. These findings suggest that C. higginsianum employs a distinctive infection strategy involving actin fragmentation that is independent of canonical immune recognition.

Flagella and cilia are conserved eukaryotic organelles whose formation depends on membrane trafficking, but how this is coupled to axonemal architecture and functional diversification remains unclear. RAB23, a small GTPase known for its role in Sonic hedgehog signaling, has an elusive function in ciliogenesis because its loss is embryonic lethal in animals. Here we analyze RAB23 in the liverwort Marchantia polymorpha, where motile flagella are confined to spermatozoids. Loss of MpRAB23 causes severe defects in spermatozoid morphology, motility, and 9 + 2 axonemal organization, while leaving vegetative growth unaffected. During spermiogenesis, MpRAB23 localizes to elongating flagella and is required for flagellar membrane organization, including polarized localization of MpPOFCAP, a calcium pump confined to the posterior flagellum. Disruption of MpPOFCAP reduces spermatozoid motility and fertility without affecting axonemal structure. These findings identify RAB23 as a regulator of flagellar biogenesis and uncover a specific mechanism by which asymmetric membrane protein targeting differentiates structurally similar flagella within a single cell.

Bioactive specialized metabolites (SMs) are synthesized and sequestered in specific cellular compartments or organelles as a self-defense strategy against their intrinsic toxicity. Liverwort-specific oil bodies accumulate large amounts of SMs and contribute to chemical defense; however, the molecular mechanisms underlying SM sequestration in oil bodies remain largely unknown. Therefore, in this study, we focused on MpABCG1 and MpABCG36, which are ATP-binding cassette (ABC) protein family members localized to the oil bodies of liverwort Marchantia polymorpha. Sesquiterpene (thujopsene, chamigrene, and himachalene) accumulation was reduced in the Mpabcg1 and Mpabcg36 loss-of-function mutants. Notably, levels of the bisbibenzyls, marchantins C and A, were predominantly reduced in Mpabcg1, but not in Mpabcg36. Although the Mpabcg1 mutant formed a number of oil bodies labeled with mCitrine-MpSYP12B (an oil body membrane marker) comparable to that of the wild type, the number of oil bodies stained with BODIPY 493/503, which has an affinity for lipophilic SMs, was reduced. This finding suggests that MpABCG1 and MpABCG36 mutations affect SM accumulation in the oil body but have little impact on oil body formation. Overall, our results highlight the involvement of MpABCG1 and MpABCG36 in the accumulation of SMs and/or their precursors in liverwort oil bodies.

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.

Autophagy, a critical process for the vacuolar degradation of proteins and organelles, is governed by multiple conserved autophagy-related (ATG) proteins. The central component of the ATG machinery is the ubiquitin-like protein ATG8, which is essential for multiple steps of the autophagy process, including phagophore expansion, autophagosome closure, trafficking and fusion with the lysosome/vacuole, and selective cargo recruitment. Currently, our understanding of the roles of ATG8 in plant autophagy and the functional specialization of ATG8 family members is limited due to genetic redundancy. To assess the roles of ATG8 genes in plant autophagy, here we used CRISPR/Cas9 technology to systematically knockout the Arabidopsis ATG8 genes. By analyzing the atg8 mutants, we found that in contrast to mammalian ATG8s, in which the LC3s and GABARAP subfamilies play distinct roles in the autophagic process, Arabidopsis ATG8s perform an overlapping function in controlling autophagic flux. Combinatorial mutations of Clade I and Clade II ATG8s resulted in severely impaired autophagy under nutrient-starved conditions. Furthermore, we found that RABG3 proteins, members of the RAB7/RABG GTPase family, interact with ATG8s through AIM-LDS interfaces, and that such interaction is essential for the association of RABG3 proteins with the autophagosomal membrane and probably for the fusion of autophagosome with the vacuole, but is not required for endosomal trafficking. With the collection of multiple high-order atg8 mutants generated in this study, we now provide a venue to study the roles of ATG8 genes in canonical autophagy and non-canonical autophagy in Arabidopsis. ### Competing Interest Statement The authors have declared no competing interest.

Autophagy is a catabolic process that degrades cytoplasmic components under cellular stress conditions such as nutrient deprivation, reactive oxygen species (ROS) accumulation, and pathogen infection. This process involves the formation of autophagosomes, which sequester cytoplasmic materials before fusing with lysosomes (or vacuoles in plant cells) for degradation. Historically, autophagy has been considered primarily as a stress adaptation mechanism, but emerging evidence indicates its involvement in programmed cell death (PCD), termed "autophagic cell death" (ACD). This dual role suggests that autophagy can promote either cell survival or cell death, depending on the biological context. While significant research has focused on autophagy's protective functions, its contribution to ACD remains less understood. Here, we review the physiological functions of autophagy, with particular emphasis on recently identified mechanisms underlying ACD in plants. We discuss how ACD contributes to various plant stress responses and developmental processes, providing insights for future research.

The ubiquitin-like protein ATG8 is a central component of the autophagy process and is required at multiple steps during both bulk and selective autophagy. Currently, our understanding of the roles of ATG8 in plants and the possible functional specialization of its family members is limited by genetic redundancy. Here, we employed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas)9 targeting technology to systematically inactivate all nine Arabidopsis thaliana ATG8 loci. Subsequent analyses of the resulting mutants revealed that, unlike mammalian ATG8 family members, which have distinct roles, Arabidopsis isoforms largely overlap in their functions controlling autophagic flux. Notably, combinatorial mutations have similarly impaired autophagy and misregulated proteomes much like other autophagy mutants. We further examined the functional redundancy of Arabidopsis ATG8s in late autophagy stages by investigating their interactions with Rab GTPase (RABG)3/RAB7 proteins. We found that all ATG8 representatives could interact with RABG3 proteins via ATG8-interacting motif-LC3-interacting region-docking site interfaces. Such interactions are crucial for RABG3 binding to the autophagosome membrane and probably for the fusion of autophagosomes with the vacuole. However, they are not necessary for endosomal trafficking. With this collection of multiple high-order atg8 mutants, we provide a venue to selectively study the roles of individual ATG8 isoforms during both canonical and noncanonical autophagy in Arabidopsis.

In recent years, deep learning has been used to analyze plant and cell images. One protein, MpPICALM-K, is localized at the base of flagella in Marchantia polymorpha (M.polymorpha) spermatozoids. MpPICALM-K is considered to be involved in the motility of the flagella that enable spermatozoids to swim. For the analysis of MpPICALM-K, video classification and visualization are used, and improving their performance can provide deeper biological insights. In this study, we propose a module called Dimensional-Fortes to improve Video Vision Transformer (ViViT). Dimensional-Fortes improved 6.15% in classification accuracy compared with the previous method (Integration-Net). Incorporating temporal information caused variations in the heatmaps, confirming its integration into the spatial visualization. These advancements are expected to provide stronger evidence that MpPICALM-K is involved in spermatozoid motility.

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. Feng et al. uncover a retrograde trafficking route from the plant vacuole, showing that sorting nexins retrieve the plant-specific SNARE VAMP727 and revealing distinct pathways from the core retromer system.

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.

Macroautophagy/autophagy is a highly conserved pathway responsible for the bulk degradation of cytoplasmic material through the formation of a double-membrane structure known as the autophagosome. However, the precise mechanisms governing the transport of autophagosomes to the vacuole for degradation in plants remain largely elusive. There exists an ongoing debate about whether RAB7, a key regulatory protein, is involved in the plant autophagy pathway. In this study, we demonstrate that upon autophagy induction by BTH treatment, RABG3e, a member of the RAB7 family, exhibits a partial localization with late-stage autophagosomes in Arabidopsis root cells, and its dysfunction leads to the accumulation of enlarged multilayered autophagosomes and a significant reduction in autophagic flux. We also showed that RABG3e is recruited to autophagosomes by its guanine nucleotide exchange factor (GEF) complex, MON1-CCZ1, which is targeted through the interaction between CCZ1 and SH3P2 (SH3 DOMAIN-CONTAINING PROTEIN 2), a plant-specific autophagy regulator. Subsequently, RABG3e recruits downstream effectors such as VPS39, which in turn promotes the recruitment of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) proteins, including SYP21, VTI12, SYP51, and VAMP711, that are essential for the fusion process. Arabidopsis mutants with dysfunction in the autophagosome-vacuole fusion process exhibit accelerated senescence and increased sensitivity to nitrogen starvation. Collectively, our findings provide new insights into the regulation of autophagosome-vacuole fusion in Arabidopsis, highlighting the essential roles of SH3P2-dependent targeting of the CCZ1-MON1-RABG3e module to late-stage autophagosomes as well as RABG3e effectors and a unique SNARE complex.

Gravitropism is a plant response to gravity that directs organ growth and development, playing a key role in the adaptation of land plants. While its molecular basis has been extensively studied in flowering plants, much less is known about this process in other plant lineages. Here, we investigated the gravitropic response of the liverwort Marchantia polymorpha, a model for early land plant evolution. In darkness, the thallus tips extended upward, forming several straight, narrow structures whose growth direction was consistently opposite to gravity and disrupted by clinostat treatment. These structures contained amyloplasts in parenchymatous cells, and their sedimentation preceded gravitropic curvature, suggesting a role as statoliths. Amyloplast sedimentation started near the tip and slowed with distance, and in more distal regions, both the size and the number of amyloplasts decreased. In starchless mutants (Mppgm1 and Mpaps1), the narrow structures displayed abnormal growth directions, although they still tended to elongate upward. These results indicate that while amyloplasts are required for proper gravitropism, M. polymorpha retains the ability to sense gravity even without well-developed amyloplasts. Our findings suggest that land plants use amyloplasts as statoliths but also possess amyloplast-independent mechanisms for gravitropic sensing.

Each eukaryotic cell possesses a specialized membrane trafficking system that emerged through paralogous expansion followed by the neofunctionalization of trafficking machinery components, including soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins, during evolution. We discovered that the acquisition of an acidic insertion in the polypeptide converted the secretory R-SNARE vesicle-associated membrane protein (VAMP)72 into a major component of plant vacuolar transport. The moderately acidic insertion, originating from alternative splicing in the common ancestor of zygnematophytes and embryophytes, conferred binding ability to the clathrin adapter protein complex-4 (AP-4) at the trans-Golgi network (TGN), partially redirecting the VAMP72 protein from the secretory to the vacuolar transport pathway. Increased acidity of the insertion in angiosperms further reinforced the interaction with AP-4, leading VAMP727 to discrete zoning during sorting at the TGN and a definitive conversion to endosomal localization. This stepwise neofunctionalization of VAMP72 provided an option for the development of the intricate and complex vacuolar transport system in extant angiosperms.

Gametogenesis, which is essential to the sexual reproductive system, has drastically changed during plant evolution. Bryophytes, lycophytes and ferns develop reproductive organs called gametangia-antheridia and archegonia for sperm and egg production, respectively. However, the molecular mechanism of early gametangium development remains unclear. Here we identified a 'non-canonical' type of BZR/BES transcription factor, MpBZR3, as a regulator of gametangium development in a model bryophyte, Marchantia polymorpha. Interestingly, overexpression of MpBZR3 induced ectopic gametangia. Genetic analysis revealed that MpBZR3 promotes the early phase of antheridium development in male plants. By contrast, MpBZR3 is required for the late phase of archegonium development in female plants. We demonstrate that MpBZR3 is necessary for the successful development of both antheridia and archegonia but functions in a different manner between the two sexes. Together, the functional specialization of this 'non-canonical' type of BZR/BES member may have contributed to the evolution of reproductive systems.

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.