Kyoko Ohashi-Ito

Non-cell-autonomous control of vascular stem cell fate by a CLE peptide/receptor system

Land plants evolved a long-distance transport system of water and nutrients composed of the xylem and phloem, both of which are generated from the procambium- and cambium-comprising vascular stem cells. However, little is known about the molecular mechanism of cell communication governing xylem–phloem patterning. Here, we show that a dodecapeptide (HEVHypSGHypNPISN; Hyp, 4-hydroxyproline), TDIF (tracheary element differentiation inhibitory factor), is secreted from the phloem and suppresses the differentiation of vascular stem cells into xylem cells through a leucine-rich repeat receptor-like kinase (LRR-RLK). TDIF binds in vitro specifically to the LRR-RLK, designated TDR (putative TDIF receptor), whose expression is restricted to procambial cells. However, the combined analysis of TDIF with a specific antibody and the expression profiles of the promoters of two genes encoding TDIF revealed that TDIF is synthesized mainly in, and secreted from, the phloem and its neighboring cells. The observation that TDIF is capable of promoting proliferation of procambial cells while suppressing xylem differentiation suggests that this small peptide functions as a phloem-derived, non-cell-autonomous signal that controls stem cell fate in the procambium. Our results indicate that we have discovered a cell communication system governing phloem–xylem cross-talk.

Arabidopsis VASCULAR-RELATED NAC-DOMAIN6 Directly Regulates the Genes That Govern Programmed Cell Death and Secondary Wall Formation during Xylem Differentiation

This article reports the identification of the downstream genes regulated by a master regulator of differentiation of tracheary elements, VND6, and of xylem fibers, SND1. VND6 was found to directly regulate several genes involved in programmed cell death and secondary cell wall formation through binding to a specific cis-element. Xylem consists of three types of cells: tracheary elements (TEs), parenchyma cells, and fiber cells. TE differentiation includes two essential processes, programmed cell death (PCD) and secondary cell wall formation. These two processes are tightly coupled. However, little is known about the molecular mechanisms underlying these processes. Here, we show that VASCULAR-RELATED NAC-DOMAIN6 (VND6), a master regulator of TEs, regulates some of the downstream genes involved in these processes in a coordinated manner. We first identified genes that are expressed downstream of VND6 but not downstream of SECONDARY WALL-ASSOCIATED NAC DOMAIN PROTEIN1 (SND1), a master regulator of xylem fiber cells, using transformed suspension culture cells in microarray experiments. We found that VND6 and SND1 governed distinct aspects of xylem formation, whereas they regulated a number of genes in common, specifically those related to secondary cell wall formation. Genes involved in TE-specific PCD were upregulated only by VND6. Moreover, we revealed that VND6 directly regulated genes that harbor a TE-specific cis-element, TERE, in their promoters. Thus, we found that VND6 is a direct regulator of genes related to PCD as well as to secondary wall formation.

Regulation of the Arabidopsis root vascular initial population by LONESOME HIGHWAY

Complex organisms consist of a multitude of cell types arranged in a precise spatial relation to each other. Arabidopsis roots generally exhibit radial tissue organization; however, within a tissue layer, cells are not identical. Specific vascular cell types are arranged in diametrically opposed longitudinal files that maximize the distance between them and create a bilaterally symmetric (diarch) root. Mutations in the LONESOME HIGHWAY (LHW) gene eliminate bilateral symmetry and reduce the number of cells in the center of the root, resulting in roots with only single xylem and phloem poles. LHW does not appear to be required for the creation of any specific cell type, but coordinately controls the number of all vascular cell types by regulating the size of the pool of cells from which they arise. We cloned LHW and found that it encodes a protein with weak sequence similarity to basic helix-loop-helix (bHLH)-domain proteins. LHW is a transcriptional activator in vitro. In plants, LHW is nuclear-localized and is expressed in the root meristems, where we hypothesize it acts independently of other known root-patterning genes to promote the production of stele cells, but might also indirectly feed into established regulatory networks for the maintenance of the root meristem.

Orthologs of Arabidopsis thaliana stomatal bHLH genes and regulation of stomatal development in grasses.

Stomata are adjustable pores in the plant epidermis that regulate gas exchange between the plant and atmosphere; they are present on the aerial portions of most higher plants. Genetic pathways controlling stomatal development and distribution have been described in some detail for one dicot species, Arabidopsis, in which three paralogous bHLH transcription factors, FAMA, MUTE and SPCH, control discrete sequential stages in stomatal development. Orthologs of FAMA, MUTE and SPCH are present in other flowering plants. This observation is of particular interest when considering the grasses, because both the morphology of guard cells and their tissue distributions differ substantially between Arabidopsis and this group. By examining gene expression patterns, insertional mutants and cross-species complementation studies, we find evidence that FAMA function is conserved between monocots and dicots, despite their different stomatal morphologies, whereas the roles of MUTE and two SPCH paralogs are somewhat divergent.

Transcriptional regulation of vascular cell fates.

In vascular development, uncommitted cells differentiate into different xylem cells through vascular stem cells, such as procambial cells, during vein formation as well as embryogenesis. Cascades of transcriptional regulation of genes play crucial roles in the progress of vascular development. Auxin, cytokinin, and brassinosteroids also function in procambial cell determination, procambial maintenance, and xylem cell differentiation from procambial cells, respectively, through transcriptional regulation. The positive feedback loop typically shown in auxin-flow-MONOPTEROS-(HD-ZIP IIIs)-PIN1-auxin-flow in procambial precursor cell determination and VND7-ASL/LBD-VND7 in xylem vessel cell determination, may be a crucial mechanism that determines vascular cell fates, which occurs in stages.

Differentiation of Arabidopsis guard cells: analysis of the networks incorporating the basic helix-loop-helix transcription factor, FAMA.

Nearly all extant land plants possess stomata, the epidermal structures that mediate gas exchange between the plant and the environment. The developmental pathways, cell division patterns, and molecules employed in the generation of these structures are simple examples of processes used in many developmental contexts. One specific module is a set of “master regulator” basic helix-loop-helix transcription factors that regulate individual consecutive steps in stomatal development. Here, we profile transcriptional changes in response to inducible expression of Arabidopsis (Arabidopsis thaliana) FAMA, a basic helix-loop-helix protein whose actions during the final stage in stomatal development regulate both cell division and cell fate. Genes identified by microarray and candidate approaches were then further analyzed to test specific hypothesis about the activity of FAMA, the shape of its regulatory network, and to create a new set of stomata-specific or stomata-enriched reporters.

A novel function of TDIF-related peptides: promotion of axillary bud formation.

Small peptides derived from the CLAVATA3/EMBRYO SURROUNDING REGION-related (CLE) gene family play a key role in various cell-cell communications in land plants. Among them, tracheary element differentiation inhibition factor (TDIF; CLE41/CLE44 peptide) and CLE42 peptide of Arabidopsis have almost identical amino acid sequences and act as inhibitors of tracheary element differentiation. In this study, we report a novel function of TDIF and CLE42. We found by the GUS (β-glucuronidase) reporter gene assay that while CLE41 and CLE44 are expressed preferentially in vascular bundles, CLE42 is expressed strongly in the shoot apical meristem (SAM) and axillary meristems. Overexpression of CLE42 and CLE41 enhanced axillary bud formation in the leaf and cotyledon axils. Before floral transition, the emergence of axillary buds in these plants occurred in an acropetal order. Exogenous supply of either TDIF or CLE42 peptide to the wild type induced similar excess bud emergence. In vascular bundles, the TDIF RECEPTOR (TDR) acts as the main receptor for TDIF. The axillary bud emergence of tdr mutants was little affected by either of the peptides. It was confirmed by scanning electron microscopy that peptide-treated wild-type plants form an axillary meristem-like structure earlier than non-treated plants. SHOOT MERISTEMLESS (STM), a marker gene for meristems, was up-regulated in peptide-treated plants before the axillary meristem becomes morphologically distinguishable. These results indicate that CLE42 peptide and TDIF have an activity to enhance axillary bud formation via the TDR. Judging from its expression pattern, CLE42 may play an important role in the regulation of secondary shoot development.

An atypical bHLH transcription factor regulates early xylem development downstream of auxin

The vascular system in plants, which comprises xylem, phloem and vascular stem cells, originates from provascular cells and forms a continuous network throughout the plant body. Although various aspects of vascular development have been extensively studied, the early process of vascular development remains largely unknown. LONESOME HIGHWAY (LHW), which encodes an atypical basic helix-loop-helix (bHLH) transcription factor, plays an essential role in establishing vascular cells. Here, we report the analysis of LHW homologs in relation to vascular development. Three LHW homologs, LONESOME HIGHWAY LIKE 1-3 (LHL1-LHL3), were preferentially expressed in the plant vasculature. Genetic analysis indicated that, although the LHL3 loss-of-function mutant showed no obvious phenotype, the lhw lhl3 double mutant displayed more severe phenotypic defects in the vasculature of the cotyledons and roots than the lhw single mutant. Only one xylem vessel was formed at the metaxylem position in lhw lhl3 roots, whereas the lhw root formed one protoxylem and one or two metaxylem vessels. Conversely, overexpression of LHL3 enhanced xylem development in the roots. Moreover, N-1-naphthylphthalamic acid caused ectopic LHL3 expression in accordance with induced auxin maximum. These results suggest that LHL3 plays a positive role in xylem differentiation downstream of auxin.

Auxin-associated initiation of vascular cell differentiation by LONESOME HIGHWAY

Plant vascular tissues are essential for the existence of land plants. Many studies of transcriptional regulation and cell-cell communication have revealed the process underlying the development of vascular tissues from vascular initial cells. However, the initiation of vascular cell differentiation is still a mystery. Here, we report that LONESOME HIGHWAY (LHW), which encodes a bHLH transcription factor, is expressed in pericycle-vascular mother cells at the globular embryo stage and is required for proper asymmetric cell division to generate vascular initial cells. In addition, ectopic expression of LHW elicits an ectopic auxin response. Moreover, LHW is required for the correct expression patterns of components related to auxin flow, such as PIN-FORMED 1 (PIN1), MONOPTEROS (MP) and ATHB-8, and ATHB-8 partially rescues the vascular defects of lhw. These results suggest that LHW functions as a key regulator to initiate vascular cell differentiation in association with auxin regulation.

A Negative Feedback Loop Controlling bHLH Complexes Is Involved in Vascular Cell Division and Differentiation in the Root Apical Meristem.

Controlling cell division and differentiation in meristems is essential for proper plant growth. Two bHLH heterodimers consisting of LONESOME HIGHWAY (LHW) and TARGET OF MONOPTEROS 5 (TMO5)/TMO5-LIKE1 (T5L1) regulate periclinal cell division in vascular cells in the root apical meristem (RAM). In this study, we further investigated the functions of LHW-T5L1, finding that in addition to controlling cell division, this complex regulates xylem differentiation in the RAM via a novel negative regulatory system. LHW-T5L1 upregulated the thermospermine synthase gene ACAULIS5 (ACL5), as well as SUPPRESSOR OF ACAULIS5 LIKE3 (SACL3), which encodes a bHLH protein, in the RAM. The SACL3 promoter sequence contains a conserved upstream open reading frame (uORF), which blocked translation of the main SACL3 ORF in the absence of thermospermine. Thermospermine eliminated the negative effect of uORF and enhanced SACL3 production. Further genetic and molecular biological analyses indicated that ACL5 and SACL3 suppress the function of LHW-T5L1 through a protein-protein interaction between LHW and SACL3. Finally, we showed that a negative feedback loop consisting of LHW-T5L1, ACL5, SACL3, and LHW-SACL3 contributes to maintain RAM size and proper root growth. These findings suggest that a negative feedback loop regulates the LHW-T5L1 output level to coordinate cell division and differentiation in a cell-autonomous manner.