Misato Ohtani

VASCULAR‐RELATED NAC‐DOMAIN 7 directly regulates the expression of a broad range of genes for xylem vessel formation

The Arabidopsis thaliana NAC domain transcription factor, VASCULAR-RELATED NAC-DOMAIN7 (VND7), acts as a key regulator of xylem vessel differentiation. In order to identify direct target genes of VND7, we performed global transcriptome analysis using Arabidopsis transgenic lines in which VND7 activity could be induced post-translationally. This analysis identified 63 putative direct target genes of VND7, which encode a broad range of proteins, such as transcription factors, IRREGULAR XYLEM proteins and proteolytic enzymes, known to be closely associated with xylem vessel formation. Recombinant VND7 protein binds to several promoter sequences present in candidate direct target genes: specifically, in the promoter of XYLEM CYSTEINE PEPTIDASE1, two distinct regions were demonstrated to be responsible for VND7 binding. We also found that expression of VND7 restores secondary cell wall formation in the fiber cells of inflorescence stems of nst1 nst3 double mutants, as well as expression of NAC SECONDARY WALL THICKENING PROMOTING FACTOR3 (NST3, however, the vessel-type secondary wall deposition was observed only as a result of VND7 expression. These findings indicated that VND7 upregulates, directly and/or indirectly, many genes involved in a wide range of processes in xylem vessel differentiation, and that its target genes are partially different from those of NSTs.

VND-INTERACTING2, a NAC Domain Transcription Factor, Negatively Regulates Xylem Vessel Formation in Arabidopsis

VND7 has been shown to regulate differentiation of xylem vessels. This work identifies VND-INTERACTING2 (VNI2), encoding a NAC domain transcription factor, as a protein that interacts with VND7 and demonstrates that VNI2 inhibits transcriptional activation activities of VND7 and negatively regulates xylem vessel differentiation. The Arabidopsis thaliana NAC domain transcription factor VASCULAR-RELATED NAC-DOMAIN7 (VND7) acts as a master regulator of xylem vessel differentiation. To understand the mechanism by which VND7 regulates xylem vessel differentiation, we used a yeast two-hybrid system to screen for proteins that interact with VND7 and identified cDNAs encoding two NAC domain proteins, VND-INTERACTING1 (VNI1) and VNI2. Binding assays demonstrated that VNI2 effectively interacts with VND7 and the VND family proteins, VND1-5, as well as with other NAC domain proteins at lower affinity. VNI2 is expressed in both xylem and phloem cells in roots and inflorescence stems. The expression of VNI2 overlaps with that of VND7 in elongating vessel precursors in roots. VNI2 contains a predicted PEST motif and a C-terminally truncated VNI2 protein, which lacks part of the PEST motif, is more stable than full-length VNI2. Transient reporter assays showed that VNI2 is a transcriptional repressor and can repress the expression of vessel-specific genes regulated by VND7. Expression of C-terminally truncated VNI2 under the control of the VND7 promoter inhibited the normal development of xylem vessels in roots and aerial organs. These data suggest that VNI2 regulates xylem cell specification as a transcriptional repressor that interacts with VND proteins and possibly also with other NAC domain proteins.

VASCULAR-RELATED NAC-DOMAIN6 and VASCULAR-RELATED NAC-DOMAIN7 Effectively Induce Transdifferentiation into Xylem Vessel Elements under Control of an Induction System

We previously showed that the VASCULAR-RELATED NAC-DOMAIN6 (VND6) and VND7 genes, which encode NAM/ATAF/CUC domain protein transcription factors, act as key regulators of xylem vessel differentiation. Here, we report a glucocorticoid-mediated posttranslational induction system of VND6 and VND7. In this system, VND6 or VND7 is expressed as a fused protein with the activation domain of the herpes virus VP16 protein and hormone-binding domain of the animal glucocorticoid receptor, and the proteins activity is induced by treatment with dexamethasone (DEX), a glucocorticoid derivative. Upon DEX treatment, transgenic Arabidopsis (Arabidopsis thaliana) plants carrying the chimeric gene exhibited transdifferentiation of various types of cells into xylem vessel elements, and the plants died. Many genes involved in xylem vessel differentiation, such as secondary wall biosynthesis and programmed cell death, were up-regulated in these plants after DEX treatment. Chemical analysis showed that xylan, a major hemicellulose component of the dicot secondary cell wall, was increased in the transgenic plants after DEX treatment. This induction system worked in poplar (Populus tremula × tremuloides) trees and in suspension cultures of cells from Arabidopsis and tobacco (Nicotiana tabacum); more than 90% of the tobacco BY-2 cells expressing VND7-VP16-GR transdifferentiated into xylem vessel elements after DEX treatment. These data demonstrate that the induction systems controlling VND6 and VND7 activities can be used as powerful tools for understanding xylem cell differentiation.

A NAC domain protein family contributing to the regulation of wood formation in poplar

Wood harvested from trees is one of the most widely utilized natural materials on our planet. Recent environmental issues have prompted an increase in the demand for wood, especially as a cost-effective and renewable resource for industry and energy, so it is important to understand the process of wood formation. In the present study, we focused on poplar (Populus trichocarpa) NAC domain protein genes which are homologous to well-known Arabidopsis transcription factors regulating the differentiation of xylem vessels and fiber cells. From phylogenetic analysis, we isolated 16 poplar NAC domain protein genes, and named them PtVNS (VND-, NST/SND- and SMB-related proteins) genes. Expression analysis revealed that 12 PtVNS (also called PtrWND) genes including both VND and NST groups were expressed in developing xylem tissue and phloem fiber, whereas in primary xylem vessels, only PtVNS/PtrWND genes of the VND group were expressed. By using the post-translational induction system of Arabidopsis VND7, a master regulator of xylem vessel element differentiation, many poplar genes functioning in xylem vessel differentiation downstream from NAC domain protein genes were identified. Transient expression assays showed the variation in PtVNS/PtrWND transactivation activity toward downstream genes, even between duplicate gene pairs. Furthermore, overexpression of PtVNS/PtrWND genes induced ectopic secondary wall thickening in poplar leaves as well as in Arabidopsis seedlings with different levels of induction efficiency according to the gene. These results suggest that wood formation in poplar is regulated by cooperative functions of the NAC domain proteins.

Mutations in MYB3R1 and MYB3R4 Cause Pleiotropic Developmental Defects and Preferential Down-Regulation of Multiple G2/M-Specific Genes in Arabidopsis

R1R2R3-Myb proteins represent an evolutionarily conserved class of Myb family proteins important for cell cycle regulation and differentiation in eukaryotic cells. In plants, this class of Myb proteins are believed to regulate the transcription of G2/M phase-specific genes by binding to common cis-elements, called mitosis-specific activator (MSA) elements. In Arabidopsis (Arabidopsis thaliana), MYB3R1 and MYB3R4 act as transcriptional activators and positively regulate cytokinesis by activating the transcription of KNOLLE, which encodes a cytokinesis-specific syntaxin. Here, we show that the double mutation myb3r1 myb3r4 causes pleiotropic developmental defects, some of which are due to deficiency of KNOLLE whereas other are not, suggesting that multiple target genes are involved. Consistently, microarray analysis of the double mutant revealed altered expression of many genes, among which G2/M-specific genes showed significant overrepresentation of the MSA motif and a strong tendency to be down-regulated by the double mutation. Our results demonstrate, on a genome-wide level, the importance of the MYB3R-MSA pathway for regulating G2/M-specific transcription. In addition, MYB3R1 and MYB3R4 may have diverse roles during plant development by regulating G2/M-specific genes with various functions as well as genes possibly unrelated to the cell cycle.

Arabidopsis ROOT INITIATION DEFECTIVE1, a DEAH-Box RNA Helicase Involved in Pre-mRNA Splicing, Is Essential for Plant Development

This article investigates the developmental roles of a pre-mRNA splicing factor by analyzing ROOT INITIATION DEFECTIVE1 (RID1), a DEAH-box RNA helicase. The results show that the requirement for RID1 is not constitutive, but changes dynamically throughout development, suggesting that robust levels of pre-mRNA splicing are critical for several specific aspects of plant development. Pre-mRNA splicing is a critical process in gene expression in eukaryotic cells. A multitude of proteins are known to be involved in pre-mRNA splicing in plants; however, the physiological roles of only some of these have been examined. Here, we investigated the developmental roles of a pre-mRNA splicing factor by analyzing root initiation defective1-1 (rid1-1), an Arabidopsis thaliana mutant previously shown to have severe defects in hypocotyl dedifferentiation and de novo meristem formation in tissue culture under high-temperature conditions. Phenotypic analysis in planta indicated that RID1 is differentially required during development and has roles in processes such as meristem maintenance, leaf morphogenesis, and root morphogenesis. RID1 was identified as encoding a DEAH-box RNA helicase implicated in pre-mRNA splicing. Transient expression analysis using intron-containing reporter genes showed that pre-mRNA splicing efficiency was affected by the rid1 mutation, which supported the presumed function of RID1 in pre-mRNA splicing. Our results collectively suggest that robust levels of pre-mRNA splicing are critical for several specific aspects of plant development.

Transcriptional repression by MYB3R proteins regulates plant organ growth

In multicellular organisms, temporal and spatial regulation of cell proliferation is central for generating organs with defined sizes and morphologies. For establishing and maintaining the post‐mitotic quiescent state during cell differentiation, it is important to repress genes with mitotic functions. We found that three of the Arabidopsis MYB3R transcription factors synergistically maintain G2/M‐specific genes repressed in post‐mitotic cells and restrict the time window of mitotic gene expression in proliferating cells. The combined mutants of the three repressor‐type MYB3R genes displayed long roots, enlarged leaves, embryos, and seeds. Genome‐wide chromatin immunoprecipitation revealed that MYB3R3 binds to the promoters of G2/M‐specific genes and to E2F target genes. MYB3R3 associates with the repressor‐type E2F, E2FC, and the RETINOBLASTOMA RELATED proteins. In contrast, the activator MYB3R4 was in complex with E2FB in proliferating cells. With mass spectrometry and pairwise interaction assays, we identified some of the other conserved components of the multiprotein complexes, known as DREAM/dREAM in human and flies. In plants, these repressor complexes are important for periodic expression during cell cycle and to establish a post‐mitotic quiescent state determining organ size.

Particular Significance of SRD2-Dependent snRNA Accumulation in Polarized Pattern Generation during Lateral Root Development of Arabidopsis

Lateral root primordia are initiated by anticlinal division of cells in the pericycle and are constructed through an ordered set of cell divisions. At the completion of the development of the primordium, cell division ceases, after which the lateral root meristem is activated. In Arabidopsis, this course of lateral root morphogenesis was found to be significantly susceptible to srd2-1, a temperature-sensitive mutation of the SRD2 gene encoding an activator of small nuclear RNA (snRNA) transcription. The srd2-1 mutation altered the organization of cells of the root primordium and, importantly, maintained primordial cell division for a long period, resulting in the formation of abnormal hemispherical laterals. Expression patterns of various reporter genes suggested that both the apical-basal and radial axes were not well established in the lateral root primordia of the srd2-1 mutant. In the early stages of development of the primordium, the srd2-1 mutation reduced the amount of the auxin efflux facilitator PIN-FORMED (PIN) and, probably by this means, interfered with the generation of an auxin gradient. Spliceosomal snRNAs accumulated at high levels throughout young root primordia and then decreased in association with the arrest of cell division, and finally increased again when the apical meristem became activated. The accumulation of snRNAs was severely suppressed by the srd2-1 mutation, and this was detectable before any morphological defect became evident. These findings suggest that high-level accumulation of snRNA involving the SRD2 function is particularly important for expression of PINs in polarized pattern generation during the development of lateral root primordia.

Differential requirement for the function of SRD2, an snRNA transcription activator, in various stages of plant development

Small nuclear RNA (snRNA) is a class of eukaryotic noncoding RNAs, which have essential roles in pre-mRNA splicing and rRNA processing. As these functions are fundamental to cell activities, the regulation of snRNA transcription should be a vital issue for all eukaryotes. Here we address developmental control of snRNA transcription and its significance through the analysis of the SRD2 gene of Arabidopsis (Arabidopsis thaliana), which encodes an activator of snRNA transcription. In young seedlings, a high level of SRD2 expression was observed in shoot and root apical meristems, leaf primordia, and root stele tissues, where a large amount of snRNA accumulated. In mature plants, SRD2 was highly expressed in developing leaves and flowers as well as apical meristems. Mutations in the SRD2 gene interfered with many, but not all, aspects of development in the regions that showed strong expression of SRD2. Of note, establishment of the fully active state of apical meristems in the seedling stage was very sensitive to the srd2-1 mutation, while maintenance of the established meristems was substantially insensitive. These results demonstrated differential requirement for the SRD2 function in various stages of plant development.

Evolution of plant conducting cells: perspectives from key regulators of vascular cell differentiation

One crucial problem that plants faced during their evolution, particularly during the transition to growth on land, was how to transport water, nutrients, metabolites, and small signaling molecules within a large, multicellular body. As a solution to this problem, land plants developed specific tissues for conducting molecules, called water-conducting cells (WCCs) and food-conducting cells (FCCs). The well-developed WCCs and FCCs in extant plants are the tracheary elements and sieve elements, respectively, which are found in vascular plants. Recent molecular genetic studies revealed that transcriptional networks regulate the differentiation of tracheary and sieve elements, and that the networks governing WCC differentiation are largely conserved among land plant species. In this review, we discuss the molecular evolution of plant conducting cells. By focusing on the evolution of the key transcription factors that regulate vascular cell differentiation, the NAC transcription factor VASCULAR-RELATED NAC-DOMAIN for WCCs and the MYB-coiled-coil (CC)-type transcription factor ALTERED PHLOEM DEVELOPMENT for sieve elements, we describe how land plants evolved molecular systems to produce the specialized cells that function as WCCs and FCCs.