The establishment of new cell lineages during development often requires a symmetry-breaking event. An asymmetric division in the epidermis of plants initiates a lineage that ultimately produces stomatal guard cells. Stomata are pores in the epidermis that serve as the main conduits for gas exchange between plants and the atmosphere; they are critical for photosynthesis and exert a major influence on global carbon and water cycles. Recent studies implicated intercellular signalling in preventing the inappropriate production of stomatal complexes. Genes required to make stomata, however, remained elusive. Here we report the identification of a gene, SPEECHLESS (SPCH), encoding a basic helix-loop-helix (bHLH) transcription factor that is necessary and sufficient for the asymmetric divisions that establish the stomatal lineage in Arabidopsis thaliana. We demonstrate that SPCH and two paralogues are successively required for the initiation, proliferation and terminal differentiation of cells in the stomatal lineage. The stomatal bHLHs define a molecular pathway sufficient to create one of the key cell types in plants. Similar molecules and regulatory mechanisms are used during muscle and neural development, highlighting a conserved use of closely related bHLHs for cell fate specification and differentiation.

Coordination between cell proliferation and differentiation is essential to create organized and functional tissues. Arabidopsis thaliana stomata are created through a stereotyped series of symmetric and asymmetric cell divisions whose frequency and orientation are informed by cell-cell interactions. Receptor-like proteins and a mitogen-activated protein kinase kinase kinase were previously identified as negative regulators of stomatal development; here, we present the characterization of a bona fide positive regulator. FAMA is a putative basic helix-loop-helix transcription factor whose activity is required to promote differentiation of stomatal guard cells and to halt proliferative divisions in their immediate precursors. Ectopic FAMA expression is also sufficient to confer stomatal character. Physical and genetic interaction studies combined with functional characterization of FAMA domains suggest that stomatal development relies on regulatory complexes distinct from those used to specify other plant epidermal cells. FAMA behavior provides insights into the control of differentiation in cells produced through the activity of self-renewing populations.

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 (HEVHypSGHypN-PISN; 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-cellautonomous 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.CLV3/ESR-related (CLE) ͉ leucine-rich repeat receptor-like kinase ͉ phloem ͉ procambium ͉ xylem

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.

*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.KEY WORDS: Stomata, Monocotyledon, Rice, Arabidopsis, Maize, bHLH Development 136, 2265Development 136, -2276Development 136, (2009 DEVELOPMENT 2266Stomatal development in grasses can be divided into five stages (Stebbins, 1960) (Fig. 1B). Here, stomatal development exhibits a strong spatiotemporal gradient with early stages taking place in the proximal portions of the leaf and guard cells differentiating later in distal regions. In stage one, cell files that are capable of forming stomata are determined (blue shading at leaf base, Fig. 1B). Asymmetric division of cells in these files (stage two, middle leaf section, Fig. 1B) generates GMCs as the smaller daughters. A second asymmetric division then occurs in the cells adjacent to the newly specified GMCs to produce a pair of subsidiary mother cells (SMCs), so at this third stage the guard cell complex consists of a GMC and two subsidiary cells. In the fourth stage, the GMC divides symmetrically into two box-shaped guard cells. During the final stage, guard cells undergo extensive elongation and morphogenetic changes to form the final pair of dumbbell-shaped cells with a central pore between them (red cells at tip of leaf, Fig. 1B) (Sack, 1994).Despite the differences in stomatal ontogeny, morphology and pattern between monocots and dicots, protein sequences of the key regulatory genes SPCH, MUTE and FAMA are highly conserved between representatives of these two angiosperm divisions. In this study, we identify likely orthologs of Arabidopsis SPCH, MUTE and FAMA in two grass species: rice (Oryza sativa) and maize (Zea mays). Through mutation, transgenics, and by monitoring gene expression in situ, we demonstrate that there is significant conservation of function of the FAMA gene between monocots and dicots. By contrast, although MUTE and the two SPCH genes maintain some common functions in grasses, they have diverged in their roles and domains of expression. MATERIALS AND METHODS Plant growth conditionsArabidopsis thaliana Columbia ecotype seeds were ste...

Higher organisms possess mechanisms to maintain stem cells' proliferative and pluripotent states in stem cell niches [1]. Plants possess two types of stem cell niches in the root and shoot apical meristems, where regulatory interactions exist between stem cells and organizing cells. Recent studies provided new insights into the molecular mechanism of stem cell maintenance [2-4]. However, earlier and more essential developmental events such as the acquisition of stem cell proliferative activity are still unknown. In vascular tissues, procambial cells function as stem cells and differentiate into xylem, phloem, and procambium. Procambial cell proliferation starts at root apical meristem (RAM) postembryonically; therefore, procambial cell development in RAM is a good model for investigating the regulation of stem cell proliferation. LONESOME HIGHWAY (LHW) and TARGET OF MONOPTEROS5 (TMO5), as well as its homolog, TMO5-LIKE1 (T5L1), encode bHLH proteins that function as heterodimers (LHW-TMO5 and LHW-T5L1) in vascular tissue organization [5-7]. LHW-T5L1 promotes vascular-cell-specific proliferation in RAM [7]. Here, we demonstrate that LHW-T5L1 promotes expression of key cytokinin production genes, including LONELY GUY3 (LOG3) and LOG4, in xylem precursor cells, resulting in elevated cytokinin levels in the surrounding cells. LHW-T5L1 can also promote expression of AHP6, which suppresses cytokinin signaling and then maintains xylem precursor cells at a nondividing state. Our results indicate that LHW-T5L1 establishes xylem precursor cells as a signal center that promotes procambial-cell-specific proliferation through cytokinin response.

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-loophelix (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.

HD-Zip III homeobox genes are known to be essential transcriptional factors for vascular development. To further understand the relation of HD-Zip III genes in vascular differentiation, we isolated a new member of the HD-Zip III genes, ZeHB-13, as a Zinnia homolog of ATHB-15, and then characterized the expression profile using a Zinnia xylogenic cell culture and Zinnia plants. We compared the accumulation pattern of transcripts for ZeHB-13 and other HD-Zip III genes and suggested that the expression of ZeHB-13 was restricted to the procambium and was not severely suppressed by brassinazole, an inhibitor of brassinosteroid biosynthesis, unlike other HD-Zip III genes. We also characterized its Arabidopsis counterpart, ATHB-15. A histochemical promoter analysis using ATHB-15::GUS transgenic Arabidopsis plants indicated that ATHB-15 was active specifically in the procambium. These results strongly suggest that ZeHB-13/ATHB-15 is a pivotal transcriptional regulator responsible for early vascular development. Based on these results, we will discuss the regulation of xylem development in light of the functions of HD-Zip III members and brassinosteroids.