Abstract Hydathodes are specialized leaf tissues that mediate guttation, the release of liquid water droplets from vascular plants. They consist of xylem endings, water pores, and the epithem. The epithem comprises small cells whose cell types and functional properties remain poorly understood. While molecular genetic information on hydathodes has accumulated—particularly in the model plant Arabidopsis thaliana through recent RNA-seq analyses—high-resolution anatomical insights into their spatial organization remain limited. In this study, we aimed to characterize the specialized anatomy of Arabidopsis hydathodes using complementary imaging approaches. A fluorescent dye taken up from roots stained hydathodes, consistent with a connection between hydathodes and the vasculature. Microfocus–X-ray computed tomography revealed that the hydathodes protrude from the abaxial leaf surface and are more densely packed internally than surrounding tissues. Light microscopy showed that the epithem was heterogeneous, with elongated cells proximally and rounded cells distally. We found that the epithem was surrounded by cells (hereafter referred to as “boundary cells”) with plastids that resembled those of mesophyll cells. Transmission electron microscopy showed that the organelles in epithem cells were immature, and the apoplast contained electron-dense material. Finally, using transgenic plants expressing a secreted fluorescent marker protein, we detected GFP in guttation droplets, indicating that the droplets can contain apoplastic material. Together, these findings reveal that Arabidopsis hydathodes possess a dense and spatially heterogeneous epithem associated with the release of apoplastic fluid.

Alternative pre-mRNA splicing (AS) is a crucial regulatory layer of gene expression in eukaryotes. AS patterns can change in response to abiotic and biotic stress, allowing cellular functions to adapt to environmental conditions. Here, we examined the effects of cellular stress-inducing chemicals on AS-mediated gene regulation in Arabidopsis thaliana by investigating the alternatively spliced forms of SERINE-ARGININE PROTEIN30 (SRp30) and U1-70 K, encoding splicing factors, as well as ASCORBATE PEROXIDASE3 (APX3) and FOLYLPOLYGLUTAMATE SYNTHASE3 (FPGS3), encoding enzymes important for stress responses. Disrupting key cellular activities, including nitric oxide metabolism, ATPase activity, plastid function, and genome stability, affected AS patterns in Arabidopsis. Stress treatment altered the abundance of uridine-rich small nuclear RNAs (UsnRNAs), especially U1 snRNAs, which are essential non-coding RNA components of U1 small nuclear ribonucleoproteins (U1 snRNPs), suggesting that abnormalities in AS are partially mediated by changes in U1 snRNA levels. The shoot redifferentiation defectice2-1 (srd2-1) mutant defective for snRNA transcription was hypersensitive for stress treatment, since it showed changes in AS patterns at lower concentrations of stress inducers to compare with the wild type. Together, our data suggest that cellular stress can influence gene expression in plants by regulating AS, which is partially regulated by UsnRNA levels through the SRD2-mediated snRNA transcription.

The formation of secondary cell walls, which provide mechanical strength to the plant body, depends on numerous factors. Studies on rice brittle culm (bc) mutants allow us to identify these factors and gain insights into the mechanisms of secondary cell wall formation. Rice bc4 is a recessive bc mutant with fragile culms and leaves, similar to other bc mutants. We found that the bc4 mutant exhibited reduced cellulose content in the culm cell walls compared to the japonica cultivar Taichung 65 and the indica cultivar Kasalath, while hemicellulose content remained unchanged. Transmission electron microscopy revealed reduced cell wall thickness in the sclerenchyma cells of the bc4 culm, indicating that BC4 contributes to normal cellulose synthesis or deposition in secondary cell walls. Positional cloning and subsequent genome sequencing revealed that the BC4 gene encodes a four α-helical transmembrane protein with 205 amino acids, and that the bc4 mutation results in a premature termination codon in this gene. Four bc4 mutants generated from the japonica cultivar Nipponbare, using genome editing with the CRISPR/Cas9 system, exhibited reduced cellulose content along with bc phenotypes. Gene clustering analysis based on expression patterns and metabolomic analysis suggested that BC4 functions independently from secondary cell wall cellulose synthase catalytic subunits and COBRA-like protein. These results suggest that the BC4 protein is a newly identified factor involved in cellulose synthesis or deposition in the secondary cell walls of rice.

In vascular plants, xylem vessels transport water and contribute to structural integrity. As part of vessel formation, xylem cells deposit secondary cell walls (SCWs), which are composed of cellulose, hemicellulose, and lignin polymers. Under environmental challenges such as pathogen attack, a growth-defense trade-off limits xylem vessel development. Understanding the mechanism regulating this trade-off has implications for understanding of plant strategy to utilize their carbon resources because SCWs contain large amounts of densely packed high-carbon compounds. Here, we investigated the effect of pathogen responses induced by the peptide defense elicitor flagellin22 (flg22) on VASCULAR-RELATED NAC-DOMAIN7 (VND7)-dependent xylem vessel formation in Arabidopsis (Arabidopsis thaliana). Treatment with flg22 decreased xylem transport and delayed ectopic xylem vessel differentiation, including SCW deposition, in plants with a dexamethasone-inducible VND7 system, suggesting that the plant prioritized defense responses over xylem formation. Consistent with this, transcriptome analysis revealed that flg22 treatment suppressed SCW-related genes and activated immune-related genes. Salicylic acid (SA) treatment had a similar inhibitory effect on VND7-dependent xylem vessel formation, and the sid2-2 and npr1-1 mutants, which are defective in SA biosynthesis and signaling, respectively, did not exhibit flg22-induced inhibition of xylem transport activity; these highlight the role of SA, which is biosynthesized from phenylalanine during immune responses, as a key mediator of the competition between xylem vessel formation and immune responses. These findings provide insights into how plants flexibly regulate xylem development under biotic stress to optimize their development.

Xylem vessels function in the long-distance conduction of water in land plants. The NAC transcription factor VASCULAR-RELATED NAC-DOMAIN7 (VND7) is a master regulator of xylem vessel cell differentiation in Arabidopsis (Arabidopsis thaliana). We previously isolated suppressor of ectopic xylem vessel cell differentiation induced by VND7 (seiv) mutants. Here, we report that the responsible genes for seiv3, seiv4, seiv6, and seiv9 are protein ubiquitination-related genes encoding PLANT U-BOX46 (PUB46), an uncharacterized F-BOX protein (FBX), PUB36, and UBIQUITIN-SPECIFIC PROTEASE1 (UBP1), respectively. We also found decreased expression of genes downstream of VND7 and abnormal xylem transport activity in the seiv mutants. Upon VND7 induction, ubiquitination levels from 492 and 180 protein groups were upregulated and downregulated, respectively. VND7 induction resulted in the ubiquitination of proteins for cell wall biosynthesis and protein transport, whereas such active protein ubiquitination did not occur in the seiv mutants. We detected the ubiquitination of three lysine residues in VND7: K94, K105, and K260. Substituting K94 with arginine significantly decreased the transactivation activity of VND7, suggesting that the ubiquitination of K94 is crucial for regulating VND7 activity. Our findings highlight the crucial roles of target protein ubiquitination in regulating xylem vessel activity.

In plants, variations in seed size and number are outcomes of different reproductive strategies. Both traits are often environmentally influenced, suggesting that a mechanism exists to coordinate these phenotypes in response to available maternal resources. Yet, how maternal resources are sensed and influence seed size and number is largely unknown. Here, we report a mechanism that senses maternal resources and coordinates grain size and number in the wild rice Oryza rufipogon, a wild progenitor of Asian cultivated rice. We showed that FT-like 9 (FTL9) regulates both grain size and number and that maternal photosynthetic assimilates induce FTL9 expression in leaves to act as a long-range signal that increases grain number and reduces size. Our findings highlight a strategy that benefits wild plants to survive in a fluctuating environment. In this strategy, when maternal resources are sufficient, wild plants increase their offspring number while preventing an increase in offspring size by the action of FTL9, which helps expand their habitats. In addition, we found that a loss-of-function allele (ftl9) is prevalent among wild and cultivated populations, offering a new scenario in the history of rice domestication.

Parasitic plants that infect crops are devastating to agriculture throughout the world. These parasites develop a unique inducible organ called the haustorium that connects the vascular systems of the parasite and host to establish a flow of water and nutrients. Upon contact with the host, the haustorial epidermal cells at the interface with the host differentiate into specific cells called intrusive cells that grow endophytically toward the host vasculature. Following this, some of the intrusive cells re-differentiate to form a xylem bridge (XB) that connects the vasculatures of the parasite and host. Despite the prominent role of intrusive cells in host infection, the molecular mechanisms mediating parasitism in the intrusive cells remain poorly understood. In this study, we investigated differential gene expression in the intrusive cells of the facultative parasite Phtheirospermum japonicum in the family Orobanchaceae by RNA-sequencing of laser-microdissected haustoria. We then used promoter analyses to identify genes that are specifically induced in intrusive cells, and promoter fusions with genes encoding fluorescent proteins to develop intrusive cell-specific markers. Four of the identified intrusive cell-specific genes encode subtilisin-like serine proteases (SBTs), whose biological functions in parasitic plants are unknown. Expression of SBT inhibitors in intrusive cells inhibited both intrusive cell and XB development and reduced auxin response levels adjacent to the area of XB development. Therefore, we propose that subtilase activity plays an important role in haustorium development in P. japonicum.

Plant cell walls are typically composed of polysaccharide polymers and cell wall proteins (CWPs). CWPs account for approximately 10% of the plant cell wall structure and perform a wide range of functions. Previous studies have identified approximately 1000 CWPs in the model plant Arabidopsis thaliana; however, the analyses mainly targeted primary cell walls, which are generated at cell division. In contrast, little is known about CWPs in secondary cell walls (SCWs), which are rigid and contain the phenolic polymer lignin. Here, we performed a cell wall proteome analysis to obtain novel insights into CWPs in SCWs. To this end, we tested multiple methods for cell wall extraction with cultured Arabidopsis cells carrying the VND7-VP16-GR system, with which cells can be transdifferentiated into xylem-vessel-like cells with lignified SCWs by dexamethasone treatment. We then subjected the protein samples to in-gel trypsin digestion followed by LC-MS/MS analysis. The different extraction methods resulted in the detection of different cell wall fraction proteins (CWFPs). In particular, centrifugation conditions had a strong impact on the extracted CWFP species, resulting in the increased number of identified CWFPs. We successfully identified 896 proteins as CWFPs in total, including proteases, expansins, purple phosphatase, well-known lignin-related enzymes (laccase and peroxidase), and 683 of 896 proteins were newly identified CWFPs. These results demonstrate the usefulness of our CWP analysis method. Further analyses of SCW-related CWPs could be expected to produce information useful for understanding the roles of CWPs in plant cell functions.

Key message The homologs of VASCULAR RELATED NAC-DOMAIN in the peat moss Sphagnum palustre were identified and these transcriptional activity as the VNS family was conserved. In angiosperms, xylem vessel element differentiation is governed by the master regulators VASCULAR RELATED NAC-DOMAIN6 (VND6) and VND7, encoding plant-specific NAC transcription factors. Although vessel elements have not been found in bryophytes, differentiation of the water-conducting hydroid cells in the moss Physcomitrella patens is regulated by VND homologs termed VND-NST-SOMBRERO (VNS) genes. VNS genes are conserved in the land plant lineage, but their functions have not been elucidated outside of angiosperms and P. patens. The peat moss Sphagnum palustre, of class Sphagnopsida in the phylum Bryophyta, does not have hydroids and instead uses hyaline cells with thickened, helical-patterned cell walls and pores to store water in the leaves. Here, we performed whole-transcriptome analysis and de novo assembly using next generation sequencing in S. palustre, obtaining sequences for 68,305 genes. Among them, we identified seven VNS-like genes, SpVNS1-A, SpVNS1-B, SpVNS2-A, SpVNS2-B, SpVNS3-A, SpVNS3-B, and SpVNS4-A. Transient expression of these VNS-like genes, with the exception of SpVNS2-A, in Nicotiana benthamiana leaf cells resulted in ectopic thickening of secondary walls. This result suggests that the transcriptional activity observed in other VNS family members is functionally conserved in the VNS homologs of S. palustre.

KNOX and BELL transcription factors regulate distinct steps of diploid development in the green lineages. In the green alga Chlamydomonas reinhardtii, KNOX and BELL proteins are inherited by gametes of the opposite mating types, and heterodimerize in zygotes to activate diploid development. By contrast, in land plants such as Physcomitrella and Arabidopsis, KNOX and BELL proteins function in meristem maintenance and organogenesis during the later stages of diploid development. However, whether the contrasting functions of KNOX and BELL were acquired independently in algae and land plants is currently unknown. Here we show that in the basal land plant species Marchantia polymorpha , gamete-expressed KNOX and BELL are required to initiate zygotic development by promoting nuclear fusion in a manner strikingly similar to that of C. reinhardtii . Our results indicate that zygote activation is the ancestral role of KNOX/BELL transcription factors, which shifted toward meristem maintenance as land plants evolved.

Growth of biomass for lignocellulosic biofuels and biomaterials may take place on land unsuitable for foods, meaning the biomass plants are exposed to increased abiotic stresses. Thus, the understanding how this affects biomass composition and quality is important for downstream bioprocessing. Here, we analyzed the effect of drought and salt stress on cell wall biosynthesis in young shoots and xylem tissues of Populus trichocarpa using transcriptomic and biochemical methods. Following exposure to abiotic stress, stem tissues reduced vessel sizes, and young shoots increased xylem formation. Compositional analyses revealed a reduction in the total amount of cell wall polysaccharides. In contrast, the total lignin amount was unchanged, while the ratio of S/G lignin was significantly decreased in young shoots. Consistent with these observations, transcriptome analyses show that the expression of a subset of cell wall-related genes is tightly regulated by drought and salt stresses. In particular, the expression of a part of genes encoding key enzymes for S-lignin biosynthesis, caffeic acid O-methyltransferase and ferulate 5-hydroxylase, was decreased, suggesting the lower S/G ratio could be partly attributed to the down-regulation of these genes. Together, our data identifies a transcriptional abiotic stress response strategy in poplar, which results in adaptive changes to the plant cell wall.

The genus Cuscuta is stem parasitic angiosperms that parasitize a wide range of vascular plants via de novo formation of a distinctive parasitic organ called a haustorium. In the developing haustorium, meristematic cells, which are initiated from the stem cortical tissue, differentiate into haustorial parenchyma cells, which elongate, penetrate into the host tissues, and finally connect with the host vasculature. This interspecific vasculature connection allows the parasite to uptake water and nutrients from the host plant. Although histological aspects of haustorium development have been studied extensively, the molecular mechanisms underlying vasculature development and the interspecific connection with the host vasculature remain largely unknown. To gain insights into the interspecific cell-to-cell interactions involved in haustorium development, we established an in vitro haustorium induction system for Cuscuta campestris using Arabidopsis thaliana rosette leaves as the host plant tissue. The in vitro induction system was used to show that interaction with host tissue was required for the differentiation of parasite haustorial cells into xylem vessel cells. To further characterize the molecular events occurring during host-dependent xylem vessel cell differentiation in C. campestris, we performed a transcriptome analysis using samples from the in vitro induction system. The results showed that orthologs of genes involved in development and proliferation of vascular stem cells were up-regulated even in the absence of host tissue, whereas orthologs of genes required for xylem vessel cell differentiation were up-regulated only after some haustorial cells had elongated and contacted the host xylem. Consistent results were obtained by another transcriptome analysis of the haustorium development in C. campestris undergoing parasitization of an intact host plant. These findings suggest the involvement of host-derived signals in the regulation of non-autonomous xylem vessel differentiation and suggest that its connection to the host xylem during the haustorium development activates a set of key genes for differentiation into xylem vessel cells.

KEY MESSAGE:Plant-specific Dof transcription factors VDOF1 and VDOF2 are novel regulators of vascular cell differentiation through the course of a lifetime in Arabidopsis, with shifting their transcriptional target genes. Vascular system is one of critical tissues for vascular plants to transport low-molecular compounds, such as water, minerals, and the photosynthetic product, sucrose. Here, we report the involvement of two Dof transcription factors, named VASCULAR-RELATED DOF1 (VDOF1)/VDOF4.6 and VDOF2/VDOF1.8, in vascular cell differentiation and lignin biosynthesis in Arabidopsis. VDOF genes were expressed in vascular tissues, but the detailed expression sites were partly different between VDOF1 and VDOF2. Vein patterning and lignin analysis of VDOF overexpressors and double mutant vdof1 vdof2 suggested that VDOF1 and VDOF2 would function as negative regulators of vein formation in seedlings, and lignin deposition in inflorescence stems. Interestingly, effects of VDOF overexpression in lignin deposition were different by developmental stages of inflorescence stems, and total lignin contents were increased and decreased in VDOF1 and VDOF2 overexpressors, respectively. RNA-seq analysis of inducible VDOF overexpressors demonstrated that the genes for cell wall biosynthesis, including lignin biosynthetic genes, and the transcription factor genes related to stress response and brassinosteroid signaling were commonly affected by VDOF1 and VDOF2 overexpression. Taken together, we concluded that VDOF1 and VDOF2 are novel regulators of vascular cell differentiation through the course of a lifetime, with shifting their transcriptional target genes: in seedlings, the VDOF genes negatively regulate vein formation, while at reproductive stages, the VDOF proteins target lignin biosynthesis.

Next-generation sequencing technologies have made it possible to carry out transcriptome analysis at the single-cell level. Single-cell RNA-sequencing (scRNA-seq) data provide insights into cellular dynamics, including intercellular heterogeneity as well as inter- and intra-cellular fluctuations in gene expression that cannot be studied using populations of cells. The utilization of scRNA-seq is, however, restricted to cell types that can be isolated from their original tissues, and it can be difficult to obtain precise positional information for these cells in situ. Here, we established single cell-digital gene expression (1cell-DGE), a method of scRNA-seq that uses micromanipulation to extract the contents of individual living cells in intact tissue while recording their positional information. With 1cell-DGE, we could detect differentially expressed genes (DEGs) during the reprogramming of leaf cells of the moss Physcomitrella patens, identifying 6382 DEGs between cells at 0 and 24 h after excision. Furthermore, we identified a subpopulation of reprogramming cells based on their pseudotimes, which were calculated using transcriptome profiles at 24 h. 1cell-DGE with microcapillary manipulation can be used to analyze the gene expression of individual cells without detaching them from their tightly associated tissues, enabling us to retain positional information and investigate cell-cell interactions.

Root hairs protruding from epidermal cells increase the surface area for water absorption and nutrient uptake. Various environmental factors including light, oxygen concentration, carbon dioxide concentration, calcium and mycorrhizal associations promote root hair formation in Arabidopsis thaliana. Light regulates the expression of a large number of genes at the transcriptional and post-transcriptional levels; however, there is little information linking the light response to root hair development. In this study, we describe a novel mutant, light-sensitive root-hair development 1 (lrh1), that displays enhanced root hair development in response to light. Hypocotyl and root elongation was inhibited in the lrh1 mutant, which had a late flowering phenotype. We identified the gene encoding the p14 protein, a putative component of the splicing factor 3b complex essential for pre-mRNA splicing, as being responsible for the lrh1 phenotype. Indeed, regulation of alternative splicing was affected in lrh1 mutants and treatment with a splicing inhibitor mimicked the lrh1 phenotype. Genome-wide alterations in pre-mRNA splicing patterns including differential splicing events of light signaling- and circadian clock-related genes were found in lrh1 as well as a difference in transcriptional regulation of multiple genes including upregulation of essential genes for root hair development. These results suggest that pre-mRNA splicing is the key mechanism regulating root hair development in response to light signals.

Gene homology helps us understand gene function and speciation. The number of plant 70 genes and species registered in public data-banks is continuously increasing. It is useful 71 to associate homologous genes of various plants to better understand plant speciation. We designed the Gcorn plant database for the retrieval of information on homology and 73 evolution of a plant gene of interest. Amino acid sequences of 73 species (62 land plants 74 and 11 green algae), containing 2,682,261 sequences, were obtained from the RefSeq 75 database. Based on BLAST searches between these sequences, homologous genes were 76 grouped at various thresholds of homology indices devised by the authors. To show 77 functional and evolutionary traits of a gene of interest, a phylogenetic tree connecting 78 genes with high homology indices, and line charts of the numbers of genes with various 79 homology indices are depicted. In addition, such indices are projected on a network 80 graph in which species studied are connected based on the ratios of homologous genes, 81 and on a phylogenetic tree for species based on NCBI Taxonomy. Gcorn plant provides 82 information on homologous genes at various virtual time points along with speciation in 83 plants. Gcorn plant is an open-access database available for academic use at 84

Gene homology helps us understand gene function and speciation. The number of plant genes and species registered in public databanks is continuously increasing. It is useful to associate homologous genes of various plants to better understand plant speciation. We designed the Gcorn plant database for the retrieval of information on homology and evolution of a plant gene of interest. Amino acid sequences of 73 species (62 land plants and 11 green algae), containing 2,682,261 sequences, were obtained from the National Center for Biotechnology Information (NCBI) Reference Sequence database. Based on NCBI BLAST searches between these sequences, homologous genes were grouped at various thresholds of homology indices devised by the authors. To show functional and evolutionary traits of a gene of interest, a phylogenetic tree, connecting genes with high homology indices, and line charts of the numbers of genes with various homology indices, are depicted. In addition, such indices are projected on a network graph in which species studied are connected based on the ratios of homologous genes, and on a phylogenetic tree for species based on NCBI Taxonomy. Gcorn plant provides information on homologous genes at various virtual time points along with speciation in plants.

Plants generally possess a strong ability to regenerate organs; for example, in tissue culture, shoots can regenerate from callus, a clump of actively proliferating, undifferentiated cells. Processing of pre-mRNA and ribosomal RNAs is important for callus formation and shoot regeneration. However, our knowledge of the roles of RNA quality control via the nonsense-mediated mRNA decay (NMD) pathway in shoot regeneration is limited. Here, we examined the shoot regeneration phenotypes of the low-beta-amylase1 (lba1)/upstream frame shift1-1 (upf1-1) and upf3-1 mutants, in which the core NMD components UPF1 and UPF3 are defective. These mutants formed callus from hypocotyl explants normally, but this callus behaved abnormally during shoot regeneration: the mutant callus generated numerous adventitious root structures instead of adventitious shoots in an auxin-dependent manner. Quantitative RT-PCR and microarray analyses showed that the upf mutations had widespread effects during culture on shoot-induction medium. In particular, the expression patterns of early auxin response genes, including those encoding AUXIN/INDOLE ACETIC ACID (AUX/IAA) family members, were significantly affected in the upf mutants. Also, the upregulation of shoot apical meristem-related transcription factor genes, such as CUP-SHAPED COTYLEDON1 (CUC1) and CUC2, was inhibited in the mutants. Taken together, these results indicate that NMD-mediated transcriptomic regulation modulates the auxin response in plants and thus plays crucial roles in the early stages of shoot regeneration.

Vascular plants have two types of water-conducting cells, xylem vessel cells (in angiosperms) and tracheid cells (in ferns and gymnosperms). These cells are commonly characterized by secondary cell wall (SCW) formation and programmed cell death (PCD), which increase the efficiency of water conduction. The differentiation of xylem vessel cells is regulated by a set of NAC (NAM, ATAF1/2 and CUC2) transcription factors, called the VASCULAR-RELATED NAC-DOMAIN (VND) family, in Arabidopsis thaliana Linne. The VNDs regulate the transcriptional induction of genes required for SCW formation and PCD. However, information on the transcriptional regulation of tracheid cell differentiation is still limited. Here, we performed functional analysis of loblolly pine (Pinus taeda Linne) VND homologs (PtaVNS, for VND, NST/SND, SMB-related protein). We identified five PtaVNS genes in the loblolly pine genome, and four of these PtaVNS genes were highly expressed in tissues with tracheid cells, such as shoot apices and developing xylem. Transient overexpression of PtaVNS genes induced xylem vessel cell-like patterning of SCW deposition in tobacco (Nicotiana benthamiana Domin) leaves, and up-regulated the promoter activities of loblolly pine genes homologous to SCW-related MYB transcription factor genes and cellulose synthase genes, as well as to cysteine protease genes for PCD. Collectively, our data indicated that PtaVNS proteins possess transcriptional activity to induce the molecular programs required for tracheid formation, i.e., SCW formation and PCD. Moreover, these findings suggest that the VNS-MYB-based transcriptional network regulating water-conducting cell differentiation in angiosperm and moss plants is conserved in gymnosperms.