Insights into Land Plant Evolution Garnered from the Marchantia polymorpha Genome.

The evolution of land flora transformed the terrestrial environment. Land plants evolved from an ancestral charophycean alga from which they inherited developmental, biochemical, and cell biological attributes. Additional biochemical and physiological adaptations to land, and a life cycle with an alternation between multicellular haploid and diploid generations that facilitated efficient dispersal of desiccation tolerant spores, evolved in the ancestral land plant. We analyzed the genome of the liverwort Marchantia polymorpha, a member of a basal land plant lineage. Relative to charophycean algae, land plant genomes are characterized by genes encoding novel biochemical pathways, new phytohormone signaling pathways (notably auxin), expanded repertoires of signaling pathways, and increased diversity in some transcription factor families. Compared with other sequenced land plants, M. polymorpha exhibits low genetic redundancy in most regulatory pathways, with this portion of its genome resembling that predicted for the ancestral land plant. PAPERCLIP.

Active protein ubiquitination regulates xylem vessel functionality.

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.

A leaf-emanated signal orchestrates grain size and number in response to maternal resources.

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.

VND Genes Redundantly Regulate Cell Wall Thickening during Parasitic Nematode Infection.

Plant parasitic root-knot nematodes are major agricultural pests worldwide, as they infect plant roots and cause substantial damages to crop plants. Root-knot nematodes induce specialized feeding cells known as giant cells (GCs) in the root vasculature, which serve as nutrient reservoirs for the infecting nematodes. Here, we show that the cell walls of GCs thicken to form pitted patterns that superficially resemble metaxylem cells. Interestingly, VASCULAR-RELATED NAC-DOMAIN1 (VND1) was found to be upregulated, while the xylem-type programmed cell death marker XYLEM CYSTEINE PEPTIDASE 1 was downregulated upon nematode infection. The vnd2 and vnd3 mutants showed reduced secondary cell wall pore size, while the vnd1 vnd2 vnd3 triple mutant produced significantly fewer nematode egg masses when compared with the wild type. These results suggest that the GC development pathway likely shares common signaling modules with the metaxylem differentiation pathway and VND1, VND2, and VND3 redundantly regulate plant-nematode interaction through secondary cell wall formation.

Pulvinar slits: Cellulose-deficient and de-methyl-esterified pectin-rich structures in a legume motor cell.

The cortical motor cells (CMCs) in a legume pulvinus execute the reversible deformation in leaf movement that is driven by changes in turgor pressure. In contrast to the underlying osmotic regulation property, the cell wall structure of CMCs that contributes to the movement has yet to be characterized in detail. Here, we report that the cell wall of CMCs has circumferential slits with low levels of cellulose deposition, which are widely conserved among legume species. This structure is unique and distinct from that of any other primary cell walls reported so far; thus, we named them "pulvinar slits." Notably, we predominantly detected de-methyl-esterified homogalacturonan inside pulvinar slits, with a low deposition of highly methyl-esterified homogalacturonan, as with cellulose. In addition, Fourier transform infrared spectroscopy analysis indicated that the cell wall composition of pulvini is different from that of other axial organs, such as petioles or stems. Moreover, monosaccharide analysis showed that pulvini are pectin-rich organs like developing stems and that the amount of galacturonic acid in pulvini is greater than in developing stems. Computer modeling suggested that pulvinar slits facilitate anisotropic extension in the direction perpendicular to the slits in the presence of turgor pressure. When tissue slices of CMCs were transferred to different extracellular osmotic conditions, pulvinar slits altered their opening width, indicating their deformability. In this study, we thus characterized a distinctive cell wall structure of CMCs, adding to our knowledge of repetitive and reversible organ deformation as well as the structural diversity and function of the plant cell wall.

Regulatory mechanisms for specification and patterning of plant vascular tissues.

Plant vascular tissues, the conduits of water, nutrients, and small molecules, play important roles in plant growth and development. Vascular tissues have allowed plants to successfully adapt to various environmental conditions since they evolved 450 Mya. The majority of plant biomass, an important source of renewable energy, comes from the xylem of the vascular tissues. Efforts have been made to identify the underlying mechanisms of cell specification and patterning of plant vascular tissues and their proliferation. The formation of the plant vascular system is a complex process that integrates signaling and gene regulation at transcriptional and posttranscriptional levels. Recently, a wealth of molecular genetic studies and the advent of cell biology and genomic tools have enabled important progress toward understanding its underlying mechanisms. Here, we provide a comprehensive review of the cell and developmental processes of plant vascular tissue and resources recently available for studying them that will enable the discovery of new ways to develop sustainable energy using plant biomass.

Simultaneous analysis of shape and internal structure of a curved Hibiscus cannabinus pulvinus: X-ray microtomography and semi-automated quantification.

In the Malvaceae family, dynamic solar tracking by leaves is actuated by the deformation of the pulvinus, a thickened region at the leaf blade-petiole junction. While the internal structure is believed to play a crucial role in this process, experimental verification has been challenging due to technical limitations. To address this gap, we developed a semi-automated workflow, which integrates data analysis and image processing to simultaneously analyze the shape and internal structure of a Malvaceae pulvinus using X-ray microtomography. Firstly, we found that kenaf (Hibiscus cannabinus L.), a Malvaceae species with curved pulvini, exhibited solar-tracking leaf movement and selected it as a model system. We employed diffusible iodine-based contrast-enhanced computed tomography to visualize the internal structure of the kenaf pulvinus. Analysis of the pulvini's shape revealed variations in pulvinus morphology, yet plausible prediction of the centerline was accomplished using polar polynomial regression. Upon slicing the pulvini perpendicular to the centerline, we observed distinct gray value gradients along the proximo-distal and adaxial-abaxial axes, challenging threshold-based tissue segmentation. This workflow successfully generated three modified 3D images and derived quantitative parameters. Using these quantitative parameters, we conducted network analysis and found the linkage between the size-normalized cortex cross-sectional area and curvature. Polynomial least absolute shrinkage and selection operator (LASSO) regression revealed the relationship between the size-normalized cortex cross-sectional area and curvature commonly in all three tested samples. This workflow enables simultaneous analysis of the shape and internal structure, significantly improving the reproducibility of Malvaceae leaf pulvinus characterization.

The Carbon Flow Shifts from Primary to Secondary Metabolism during Xylem Vessel Cell Differentiation in Arabidopsis thaliana.

Xylem vessel cell differentiation is characterized by the deposition of a secondary cell wall (SCW) containing cellulose, hemicellulose and lignin. VASCULAR-RELATED NAC-DOMAIN7 (VND7), a plant-specific NAC (NAM, ATAF1/2, and CUC2) transcription factor, is a master regulator of xylem vessel cell differentiation in Arabidopsis (Arabidopsis thaliana). Previous metabolome analysis using the VND7-inducible system in tobacco BY-2 cells successfully revealed significant quantitative changes in primary metabolites during xylem vessel cell differentiation. However, the flow of primary metabolites is not yet well understood. Here, we performed a metabolomic analysis of VND7-inducible Arabidopsis T87 suspension cells. Capillary electrophoresis-time-of-flight mass spectrometry quantified 57 metabolites, and subsequent data analysis highlighted active changes in the levels of UDP-glucose and phenylalanine, which are building blocks of cellulose and lignin, respectively. In a metabolic flow analysis using stable carbon 13 (13C) isotope, the 13C-labeling ratio specifically increased in 3-phosphoglycerate after 12 h of VND7 induction, followed by an increase in shikimate after 24 h of induction, while the inflow of 13C into lactate from pyruvate was significantly inhibited, indicating an active shift of carbon flow from glycolysis to the shikimate pathway during xylem vessel cell differentiation. In support of this notion, most glycolytic genes involved in the downstream of glyceraldehyde 3-phosphate were downregulated following the induction of xylem vessel cell differentiation, whereas genes for the shikimate pathway and phenylalanine biosynthesis were upregulated. These findings provide evidence for the active shift of carbon flow from primary metabolic pathways to the SCW polymer biosynthetic pathway at specific points during xylem vessel cell differentiation.

Subtilase activity in intrusive cells mediates haustorium maturation in parasitic plants.

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 secondary cell wall proteome analysis with an inducible system for xylem vessel cell differentiation.

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.

Transgenic poplar trees overexpressing AtGolS2, a stress-responsive galactinol synthase gene derived from Arabidopsis thaliana, improved drought tolerance in a confined field.

Drought is an abiotic stress that limits plant growth and productivity, and the development of trees with improved drought tolerance is expected to expand potential plantation areas and to promote sustainable development. Previously we reported that transgenic poplars (Populus tremula × P. tremuloides, T89) harboring the stress-responsive galactinol synthase gene, AtGolS2, derived from Arabidopsis thaliana were developed and showed improved drought stress tolerance in laboratory conditions. Herein we report a field trial evaluation of the AtGolS2-transgenic poplars. The rainfall-restricted treatments on the poplars started in late May 2020, 18 months after transplanting to the field, and were performed for 100 days. During these treatments, the leaf injury levels were observed by measuring photosynthetic quantum yields twice a week. Observed leaf injury levels varied in response to soil moisture fluctuation and showed a large difference between transgenic and non-transgenic poplars during the last month. Comparison of the leaf injury levels against three stress classes clustered by the machine learning approach revealed that the transgenic poplars exhibited significant alleviation of leaf injuries in the most severe stress class. The transgenes and transcript levels were stable in the transgenic poplars cultivated in the field conditions. These results indicated that the overexpression of AtGolS2 significantly improved the drought stress tolerance of transgenic poplars not only in the laboratory but also in the field. In future studies, molecular breeding using AtGolS2 will be an effective method for developing practical drought-tolerant forest trees.

Feature selection for RNA cleavage efficiency at specific sites using the LASSO regression model in Arabidopsis thaliana.

BACKGROUND: RNA degradation is important for the regulation of gene expression. Despite the identification of proteins and sequences related to deadenylation-dependent RNA degradation in plants, endonucleolytic cleavage-dependent RNA degradation has not been studied in detail. Here, we developed truncated RNA end sequencing in Arabidopsis thaliana to identify cleavage sites and evaluate the efficiency of cleavage at each site. Although several features are related to RNA cleavage efficiency, the effect of each feature on cleavage efficiency has not been evaluated by considering multiple putative determinants in A. thaliana. RESULTS: Cleavage site information was acquired from a previous study, and cleavage efficiency at the site level (CSsite value), which indicates the number of reads at each cleavage site normalized to RNA abundance, was calculated. To identify features related to cleavage efficiency at the site level, multiple putative determinants (features) were used to perform feature selection using the Least Absolute Shrinkage and Selection Operator (LASSO) regression model. The results indicated that whole RNA features were important for the CSsite value, in addition to features around cleavage sites. Whole RNA features related to the translation process and nucleotide frequency around cleavage sites were major determinants of cleavage efficiency. The results were verified in a model constructed using only sequence features, which showed that the prediction accuracy was similar to that determined using all features including the translation process, suggesting that cleavage efficiency can be predicted using only sequence information. The LASSO regression model was validated in exogenous genes, which showed that the model constructed using only sequence information can predict cleavage efficiency in both endogenous and exogenous genes. CONCLUSIONS: Feature selection using the LASSO regression model in A. thaliana identified 155 features. Correlation coefficients revealed that whole RNA features are important for determining cleavage efficiency in addition to features around the cleavage sites. The LASSO regression model can predict cleavage efficiency in endogenous and exogenous genes using only sequence information. The model revealed the significance of the effect of multiple determinants on cleavage efficiency, suggesting that sequence features are important for RNA degradation mechanisms in A. thaliana.

Expression of peat moss VASCULAR RELATED NAC-DOMAIN homologs in Nicotiana benthamiana leaf cells induces ectopic secondary wall formation.

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.

Functional Analysis of Poplar Sombrero-Type NAC Transcription Factors Yields a Strategy to Modify Woody Cell Wall Properties.

Woody cells generate lignocellulosic biomass, which is a promising sustainable bioresource for wide industrial applications. Woody cell differentiation in vascular plants, including the model plant poplar (Populus trichocarpa), is regulated by a set of NAC family transcription factors, the VASCULAR-RELATED NAC-DOMAIN (VND), NAC SECONDARY CELL WALL THICKENING PROMOTING FACTOR (NST)/SND, and SOMBRERO (SMB) (VNS)-related proteins, but the precise contributions of each VNS protein to wood quality are unknown. Here, we performed a detailed functional analysis of the poplar SMB-type VNS proteins PtVNS13-PtVNS16. PtVNS13-PtVNS16 were preferentially expressed in the roots of young poplar plantlets, similar to the Arabidopsis thalianaSMB gene. PtVNS13 and PtVNS14, as well as the NST-type PtVNS11, suppressed the abnormal root cap phenotype of the Arabidopsis sombrero-3 mutant, whereas the VND-type PtVNS07 gene did not, suggesting a functional gap between SMB- or NST-type VNS proteins and VND-type VNS proteins. Overexpressing PtVNS13-PtVNS16 in Arabidopsis seedlings and poplar leaves induced ectopic xylem-vessel-like cells with secondary wall deposition, and a transient expression assay showed that PtVNS13-16 transactivated woody-cell-related genes. Interestingly, although any VNS protein rescued the pendant stem phenotype of the Arabidopsis nst1-1 nst3-1 mutant, the resulting inflorescence stems exhibited distinct cell wall properties: poplar VNS genes generated woody cell walls with higher enzymatic saccharification efficiencies compared with Arabidopsis VNS genes. Together, our data reveal clear functional diversity among VNS proteins in woody cell differentiation and demonstrate a novel VNS-based strategy for modifying woody cell wall properties toward enhanced utilization of woody biomass.

Changes in mRNA Degradation Efficiencies under Varying Conditions Are Regulated by Multiple Determinants in Arabidopsis thaliana.

Multiple mechanisms are involved in gene expression, with mRNA degradation being critical for the control of mRNA accumulation. In plants, although some trans-acting factors and motif sequences have been identified in deadenylation-dependent mRNA degradation, endonucleolytic cleavage-dependent mRNA degradation has not been studied in detail. Previously, we developed truncated RNA-end sequencing (TREseq) in Arabidopsis thaliana and detected G-rich sequence motifs around 5' degradation intermediates. However, it remained to be elucidated whether degradation efficiencies of 5' degradation intermediates in A. thaliana vary among growth conditions and developmental stages. To address this issue, we conducted TREseq of cultured cells under heat stress and at three developmental stages (seedlings, expanding leaves and expanded leaves) and compared 5' degradation intermediates data among the samples. Although some 5' degradation intermediates had almost identical degradation efficiencies, others differed among conditions. We focused on the genes and sites whose degradation efficiencies differed. Changes in degradation efficiencies at the gene and site levels revealed an effect on mRNA accumulation in all comparisons. These changes in degradation efficiencies involved multiple determinants, including mRNA length and translation efficiency. These results suggest that several determinants govern the efficiency of mRNA degradation in plants, helping the organism to adapt to varying conditions by controlling mRNA accumulation.

Patterned Deposition of Xylan and Lignin is Independent from that of the Secondary Wall Cellulose of Arabidopsis Xylem Vessels.

The secondary cell wall (SCW) of xylem vessel cells provides rigidity and strength that enables efficient water conduction throughout the plant. To gain insight into SCW deposition, we mutagenized Arabidopsis thaliana VASCULAR-RELATED NAC-DOMAIN7-inducible plant lines, in which ectopic protoxylem vessel cell differentiation is synchronously induced. The baculites mutant was isolated based on the absence of helical SCW patterns in ectopically-induced protoxylem vessel cells, and mature baculites plants exhibited an irregular xylem (irx) mutant phenotype in mature plants. A single nucleic acid substitution in the CELLULOSE SYNTHASE SUBUNIT 7 (CESA7) gene in baculites was identified: while the mutation was predicted to produce a C-terminal truncated protein, immunoblot analysis revealed that cesa7bac mutation results in loss of production of CESA7 proteins, indicating that baculites is a novel cesa7 loss-of-function mutant. In cesa7bac , despite a lack of patterned cellulose deposition, the helically-patterned deposition of other SCW components, such as the hemicellulose xylan and the phenolic polymer lignin, was not affected. Similar phenotypes were found in another point mutation mutant cesa7mur10-2 , and an established knock-out mutant, cesa7irx3-4 Taken together, we propose that the spatio-temporal deposition of different SCW components, such as xylan and lignin, is not dependent on cellulose patterning.

Single-cell transcriptome analysis of Physcomitrella leaf cells during reprogramming using microcapillary manipulation.

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.

Affinity-based high-resolution analysis of DNA binding by VASCULAR-RELATED NAC-DOMAIN7 via fluorescence correlation spectroscopy.

VASCULAR-RELATED NAC-DOMAIN7 (VND7) is the master transcription factor for vessel element differentiation in Arabidopsis thaliana. To identify the cis-acting sequence(s) bound by VND7, we employed fluorescence correlation spectroscopy (FCS) to find VND7-DNA interactions quantitatively. This identified an 18-bp sequence from the promoter of XYLEM CYSTEINE PEPTIDASE1 (XCP1), a direct target of VND7. A quantitative assay for binding affinity between VND7 and the 18-bp sequence revealed the core nucleotides contributing to specific binding between VND7 and the 18-bp sequence. Moreover, by combining the systematic evolution of ligands by exponential enrichment (SELEX) technique with known consensus sequences, we defined a motif termed the Ideal Core Structure for binding by VND7 (ICSV). We also used FCS to search for VND7 binding sequences in the promoter regions of other direct targets. Taking these data together, we proposed that VND7 preferentially binds to the ICSV sequence. Additionally, we found that substitutions among the core nucleotides affected transcriptional regulation by VND7 in vivo, indicating that the core nucleotides contribute to vessel-element-specific gene expression. Furthermore, our results demonstrate that FCS is a powerful tool for unveiling the DNA-binding properties of transcription factors.

Plant-specific Dof transcription factors VASCULAR-RELATED DOF1 and VASCULAR-RELATED DOF2 regulate vascular cell differentiation and lignin biosynthesis in Arabidopsis.

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.

Arabidopsis FLYING SAUCER 2 Functions Redundantly with FLY1 to Establish Normal Seed Coat Mucilage.

Following exposure to water, mature Arabidopsis seeds are surrounded by a gelatinous capsule, termed mucilage. The mucilage consists of pectin-rich polysaccharides, which are produced in epidermal cells of the seed coat. Although pectin is a major component of plant cell walls, its biosynthesis and biological functions are not fully understood. Previously, we reported that a transmembrane RING E3 ubiquitin ligase, FLYING SAUCER 1 (FLY1) regulates the degree of pectin methyl esterification for mucilage capsule formation. The Arabidopsis thaliana genome has a single FLY1 homolog, FLY2. In this study, we show that the FLY2 protein functions in mucilage modification together with FLY1. FLY2 was expressed in seed coat epidermal cells during mucilage synthesis, but its expression level was much lower than that of FLY1. While fly2 showed no obvious difference in mucilage capsule formation from wild type, the fly1 fly2 double mutants showed more severe defects in mucilage than fly1 alone. FLY2-EYFP that was expressed under the control of the FLY1 promoter rescued fly1 mucilage, showing that FLY2 has the same molecular function as FLY1. FLY2-EYFP colocalized with marker proteins of Golgi apparatus (sialyltransferase-mRFP) and late endosome (mRFP-ARA7), indicating that as FLY1, FLY2 controls pectin modification by functioning in these endomembrane organelles. Furthermore, phylogenetic analysis suggests that FLY1 and FLY2 originated from a common ancestral gene by gene duplication prior to the emergence of Brassicaceae. Taken together, our findings suggest that FLY2 functions in the Golgi apparatus and/or the late endosome of seed coat epidermal cells in a manner similar to FLY1.