Shunsuke Miyashima

Conserved factors regulate signalling in Arabidopsis thaliana shoot and root stem cell organizers

Throughout the lifespan of a plant, which in some cases can last more than one thousand years, the stem cell niches in the root and shoot apical meristems provide cells for the formation of complete root and shoot systems, respectively. Both niches are superficially different and it has remained unclear whether common regulatory mechanisms exist. Here we address whether root and shoot meristems use related factors for stem cell maintenance. In the root niche the quiescent centre cells, surrounded by the stem cells, express the homeobox gene WOX5 (WUSCHEL-RELATED HOMEOBOX 5), a homologue of the WUSCHEL (WUS) gene that non-cell-autonomously maintains stem cells in the shoot meristem. Loss of WOX5 function in the root meristem stem cell niche causes terminal differentiation in distal stem cells and, redundantly with other regulators, also provokes differentiation of the proximal meristem. Conversely, gain of WOX5 function blocks differentiation of distal stem cell descendents that normally differentiate. Importantly, both WOX5 and WUS maintain stem cells in either a root or shoot context. Together, our data indicate that stem cell maintenance signalling in both meristems employs related regulators.

Callose Biosynthesis Regulates Symplastic Trafficking during Root Development

Plant cells are connected through plasmodesmata (PD), membrane-lined channels that allow symplastic movement of molecules between cells. However, little is known about the role of PD-mediated signaling during plant morphogenesis. Here, we describe an Arabidopsis gene, CALS3/GSL12. Gain-of-function mutations in CALS3 result in increased accumulation of callose (β-1,3-glucan) at the PD, a decrease in PD aperture, defects in root development, and reduced intercellular trafficking. Enhancement of CALS3 expression during phloem development suppressed loss-of-function mutations in the phloem abundant callose synthase, CALS7 indicating that CALS3 is a bona fide callose synthase. CALS3 alleles allowed us to spatially and temporally control the PD aperture between plant tissues. Using this tool, we are able to show that movement of the transcription factor SHORT-ROOT and microRNA165 between the stele and the endodermis is PD dependent. Taken together, we conclude that regulated callose biosynthesis at PD is essential for cell signaling.

Non-cell-autonomous microRNA165 acts in a dose-dependent manner to regulate multiple differentiation status in the Arabidopsis root

In the development of multicellular organisms, cell fate is usually determined by exchanging positional information. Animals employ a class of intercellular signaling molecules that specify different cell fates by their dosage, but the existence of an equivalent system has not been demonstrated in plants, except that the growth regulator auxin has been proposed to act in a similar manner in certain developmental contexts. Recently, it has been reported that, in the Arabidopsis root meristem, endodermis-derived microRNA (miR) 165/166 non-cell-autonomously suppress the expression of the Class III HD-ZIP transcription factor PHABULOSA (PHB) in the peripheral stele, thereby specifying xylem differentiation. Here, we show that the miR165/166-dependent suppression of PHB is required not only for xylem specification, but also for differentiation of the pericycle, as well as for ground tissue patterning. Furthermore, using a plant system that allows quantitative control of miR165 production in the ground tissue, we show that endodermis-derived miR165 acts in a dose-dependent manner to form a graded distribution of PHB transcripts across the stele. These results reveal a previously unidentified role of miR165 in the differentiation of a broad range of root cell types and suggest that endodermis-derived miR165 acts in a dose-dependent manner to control multiple differentiation status in the Arabidopsis root.

Stem cell function during plant vascular development.

The plant vascular system, composed of xylem and phloem, evolved to connect plant organs and transport various molecules between them. During the post‐embryonic growth, these conductive tissues constitutively form from cells that are derived from a lateral meristem, commonly called procambium and cambium. Procambium/cambium contains pluripotent stem cells and provides a microenvironment that maintains the stem cell population. Because vascular plants continue to form new tissues and organs throughout their life cycle, the formation and maintenance of stem cells are crucial for plant growth and development. In this decade, there has been considerable progress in understanding the molecular control of the organization and maintenance of stem cells in vascular plants. Noticeable advance has been made in elucidating the role of transcription factors and major plant hormones in stem cell maintenance and vascular tissue differentiation. These studies suggest the shared regulatory mechanisms among various types of plant stem cell pools. In this review, we focus on two aspects of stem cell function in the vascular cambium, cell proliferation and cell differentiation.

Symplastic intercellular connectivity regulates lateral root patterning

Cell-to-cell communication coordinates the behavior of individual cells to establish organ patterning and development. Although mobile signals are known to be important in lateral root development, the role of plasmodesmata (PD)-mediated transport in this process has not been investigated. Here, we show that changes in symplastic connectivity accompany and regulate lateral root organogenesis in Arabidopsis. This connectivity is dependent upon callose deposition around PD affecting molecular flux through the channel. Two plasmodesmal-localized β-1,3 glucanases (PdBGs) were identified that regulate callose accumulation and the number and distribution of lateral roots. The fundamental role of PD-associated callose in this process was illustrated by the induction of similar phenotypes in lines with altered callose turnover. Our results show that regulation of callose and cell-to-cell connectivity is critical in determining the pattern of lateral root formation, which influences root architecture and optimal plant performance.

Arabidopsis NAC45/86 direct sieve element morphogenesis culminating in enucleation

Removing the nucleus in sieve elements Although a cells nucleus performs critical command and control functions, some cell types, such as enucleated red blood cells, seem to do without. Sieve element cells in plants similarly carry out their function of transporting nutrients and signals from one end of the plant to the other without the guidance of a nucleus. Furuta et al. watched how the nucleus self-destructs during the development of sieve element cells (see the Perspective by Geldner). The process is regulated under the control of transcription factors, even as the entire nuclear edifice crumbles into nothingness. Science, this issue p. 933; see also p. 875 Cellular remodeling to develop phloem cells orchestrates degradation of the cell’s nucleus. [Also see Perspective by Geldner] Photoassimilates such as sugars are transported through phloem sieve element cells in plants. Adapted for effective transport, sieve elements develop as enucleated living cells. We used electron microscope imaging and three-dimensional reconstruction to follow sieve element morphogenesis in Arabidopsis. We show that sieve element differentiation involves enucleation, in which the nuclear contents are released and degraded in the cytoplasm at the same time as other organelles are rearranged and the cytosol is degraded. These cellular reorganizations are orchestrated by the genetically redundant NAC domain–containing transcription factors, NAC45 and NAC86 (NAC45/86). Among the NAC45/86 targets, we identified a family of genes required for enucleation that encode proteins with nuclease domains. Thus, sieve elements differentiate through a specialized autolysis mechanism.

A Comprehensive Expression Analysis of the Arabidopsis MICRORNA165/6 Gene Family during Embryogenesis Reveals a Conserved Role in Meristem Specification and a Non-cell-autonomous Function

One of the most fundamental events in plant ontogeny is the specification of the shoot and root apical meristem (SAM and RAM) in embryogenesis. In Arabidopsis, the restricted expression of class III homeodomain leucine zipper (HD-ZIP III) transcription factors (TFs) at the central-apical domain of early embryos is required for the correct specification of the SAM and RAM. Because the expression of HD-ZIP III TFs is suppressed by microRNA165/166 (miR165/6), elucidation of the sites of miR165/6 production and their activity range is a key to understanding the molecular basis of SAM and RAM specification in embryogenesis. Here, we present a comprehensive reporter analysis of all nine Arabidopsis MICRORNA165/166 (MIR165/6) genes during embryogenesis. We show that five MIR165/6 genes are transcribed in a largely conserved pattern in embryos, with their expression being preferentially focused at the basal-peripheral region of embryos. Our analysis also indicated that MIR165/6 transcription does not depend on SCARECROW (SCR) function in early embryos, in contrast to its requirement in post-embryonic roots. Furthermore, by observing the expression pattern of the miR-resistant PHBmu-GFP (green fluorescent protein) reporter, in either the presence or absence of the MIR165Amu transgene, which targets PHBmu-GFP, we obtained data that indicate a non-cell-autonomous function for miR165 in early embryos. These results suggest that miR165, and possibly miR166 as well, has the capacity to act as a positional cue from the basal-peripheral region of early embryos, and remotely controls SAM and RAM specification with their non-cell-autonomous function.

CHOLINE TRANSPORTER-LIKE1 is required for sieve plate development to mediate long-distance cell-to-cell communication

Phloem, a plant tissue responsible for long-distance molecular transport, harbours specific junctions, sieve areas, between the conducting cells. To date, little is known about the molecular framework related to the biogenesis of these sieve areas. Here we identify mutations at the CHER1/AtCTL1 locus of Arabidopsis thaliana. The mutations cause several phenotypic abnormalities, including reduced pore density and altered pore structure in the sieve areas associated with impaired phloem function. CHER1 encodes a member of a poorly characterized choline transporter-like protein family in plants and animals. We show that CHER1 facilitates choline transport, localizes to the trans-Golgi network, and during cytokinesis is associated with the phragmoplast. Consistent with its function in the elaboration of the sieve areas, CHER1 has a sustained, polar localization in the forming sieve plates. Our results indicate that the regulation of choline levels is crucial for phloem development and conductivity in plants.

Tryptophan-dependent auxin biosynthesis is required for HD-ZIP III-mediated xylem patterning

The development and growth of higher plants is highly dependent on the conduction of water and minerals throughout the plant by xylem vessels. In Arabidopsis roots the xylem is organized as an axis of cell files with two distinct cell fates: the central metaxylem and the peripheral protoxylem. During vascular development, high and low expression levels of the class III HD-ZIP transcription factors promote metaxylem and protoxylem identities, respectively. Protoxylem specification is determined by both mobile, ground tissue-emanating miRNA165/6 species, which downregulate, and auxin concentrated by polar transport, which promotes HD-ZIP III expression. However, the factors promoting high HD-ZIP III expression for metaxylem identity have remained elusive. We show here that auxin biosynthesis promotes HD-ZIP III expression and metaxylem specification. Several auxin biosynthesis genes are expressed in the outer layers surrounding the vascular tissue in Arabidopsis root and downregulation of HD-ZIP III expression accompanied by specific defects in metaxylem development is seen in auxin biosynthesis mutants, such as trp2-12, wei8 tar2 or a quintuple yucca mutant, and in plants treated with L-kynurenine, a pharmacological inhibitor of auxin biosynthesis. Some of the patterning defects can be suppressed by synthetically elevated HD-ZIP III expression. Taken together, our results indicate that polar auxin transport, which was earlier shown to be required for protoxylem formation, is not sufficient to establish a proper xylem axis but that root-based auxin biosynthesis is additionally required.

ARGONAUTE1 acts in Arabidopsis root radial pattern formation independently of the SHR/SCR pathway.

The formation of radially symmetric tissue patterns is one of the most basic processes in the development of vascular plants. In Arabidopsis thaliana, plant-specific GRAS-type transcription factors, SHORT-ROOT (SHR) and SCARECROW (SCR), are required for asymmetric cell divisions that separate two ground tissue cell layers, the endodermis and cortex, as well as for endodermal cell fate specification. While loss of SHR or SCR results in a single-layered ground tissue, radially symmetric cellular patterns are still maintained, suggesting that unknown regulatory mechanisms act independently of the SHR/SCR-dependent pathway. In this study, we identified a novel root radial pattern mutant and found that it is a new argonaute1 (ago1) allele. Multiple ago1 mutant alleles contained supernumerary ground tissue cell layers lacking a concentric organization, while expression patterns of SHR and SCR were not affected, revealing a previously unreported role for AGO1 in root ground tissue patterning. Analyses of ago1 scr double mutants demonstrated that the simultaneous loss of the two pathways caused a dramatic reduction in cellular organization and ground tissue identity as compared with the single mutants. Based on these results, we propose that highly symmetric root ground tissue patterns are maintained by the actions of two independent pathways, one using post-transcriptional regulation mediated by AGO1 and the other using the SHR/SCR transcriptional regulator.