Local accumulation of the plant growth regulator auxin mediates pattern formation in Arabidopsis roots and influences outgrowth and development of lateral root- and shoot-derived primordia. However, it has remained unclear how auxin can simultaneously regulate patterning and organ outgrowth and how its distribution is stabilized in a primordium-specific manner. Here we show that five PIN genes collectively control auxin distribution to regulate cell division and cell expansion in the primary root. Furthermore, the joint action of these genes has an important role in pattern formation by focusing the auxin maximum and restricting the expression domain of PLETHORA (PLT) genes, major determinants for root stem cell specification. In turn, PLT genes are required for PIN gene transcription to stabilize the auxin maximum at the distal root tip. Our data reveal an interaction network of auxin transport facilitators and root fate determinants that control patterning and growth of the root primordium.

A small organizing center, the quiescent center (QC), maintains stem cells in the Arabidopsis root and defines the stem cell niche. The phytohormone auxin influences the position of this niche by an unknown mechanism. Here, we identify the PLETHORA1 (PLT1) and PLT2 genes encoding AP2 class putative transcription factors, which are essential for QC specification and stem cell activity. The PLT genes are transcribed in response to auxin accumulation and are dependent on auxin response transcription factors. Distal PLT transcript accumulation creates an overlap with the radial expression domains of SHORT-ROOT and SCARECROW, providing positional information for the stem cell niche. Furthermore, the PLT genes are activated in the basal embryo region that gives rise to hypocotyl, root, and root stem cells and, when ectopically expressed, transform apical regions to these identities. Thus, the PLT genes are key effectors for establishment of the stem cell niche during embryonic pattern formation.

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.

Factors with a graded distribution can program fields of cells in a dose-dependent manner, but no evidence has hitherto surfaced for such mechanisms in plants. In the Arabidopsis thaliana root, two PLETHORA (PLT) genes encoding AP2-domain transcription factors have been shown to maintain the activity of stem cells. Here we show that a clade of four PLT homologues is necessary for root formation. Promoter activity and protein fusions of PLT homologues display gradient distributions with maxima in the stem cell area. PLT activities are largely additive and dosage dependent. High levels of PLT activity promote stem cell identity and maintenance; lower levels promote mitotic activity of stem cell daughters; and further reduction in levels is required for cell differentiation. Our findings indicate that PLT protein dosage is translated into distinct cellular responses.

Stem cells self-renew and produce daughter cells that differentiate. How stem cells are specified and maintained is a central question in developmental biology. Plant stem cells occupy a small region or niche in larger zones of mitotic activity called meristems. Here we provide molecular evidence that in the Arabidopsis root meristem, the stem cell population depends on a central group of cells, the quiescent center (QC), which positions the stem cell niche. We show that the putative transcription factor SCARECROW (SCR), first identified by its role in radial patterning, is required cell-autonomously for distal specification of the QC, which in turn regulates stem cell fate of immediately surrounding cells. Received October 21, 2002; revised version accepted November 29, 2002. Stem cell identity in various organisms is maintained in microenvironments called niches (Spradling et al. 2000). Their maintenance depends on local signaling events emanating from nearby cells that can be considered part of the niche (Spradling et al. 2000). Cap cells (Xie and Spradling 1998, 2000) and somatic hub cells (Kiger et al. 2001;Tulina and Matunis 2001) in Drosophila, distal tip cells in Caenorhabditis elegans (Kimble and White 1981), and cells expressing the homeodomain transcription factor WUSCHEL in the Arabidopsis shoot apical meristem (Mayer et al. 1998;Schoof et al. 2000) are examples of cells locally required for the maintenance of stem cells. In these cases, it remains to be established how the signaling cells themselves are specified to define stem cell location.In the Arabidopsis root meristem, the "initial cells" are the stem cells that give rise to all cell types of the root; they surround a small group of mitotically less active cells, the quiescent center (QC), and can be unequivocally identified (Fig. 1a;Dolan et al. 1993). Laser ablation experiments suggested that the QC is a source of cell nonautonomous signals, which prevent differentiation and hence maintain the surrounding stem cells (van den Berg et al. 1997). Hence, the QC and the surrounding cells that contact it can be considered a stem cell niche. The SCARECROW (SCR) gene encodes a putative transcription factor (Di Laurenzio et al. 1996) that is first expressed in QC precursor cells during embryogenesis, after which it extends to the initial cells for the ground tissue (cortex and endodermis) and the endodermis ; this expression pattern persists in the postembryonic root ( Fig. 1a; Di Laurenzio et al. 1996). In scr-1 mutants, the asymmetric cell division of the daughter of the cortex/endodermis initial does not occur, resulting in a single cell layer with mixed identity ( Fig. 1d; Di Laurenzio et al. 1996). Importantly, cells in the src-1 QC region are aberrant in shape and roots ultimately cease growth (Scheres et al. 1995;Di Laurenzio et al. 1996). Here, we provide evidence that these effects are not caused by the cortex/endodermis defect but rather reflect a direct requirement for SCR activity in QC cells for their specification and maintenance ...

Plant postembryonic development takes place in the meristems, where stem cells self-renew and produce daughter cells that differentiate and give rise to different organ structures. For the maintenance of meristems, the rate of differentiation of daughter cells must equal the generation of new cells: How this is achieved is a central question in plant development. In the Arabidopsis root meristem, stem cells surround a small group of organizing cells, the quiescent center. Together they form a stem cell niche [1, 2], whose position and activity depends on the combinatorial action of two sets of genes - PLETHORA1 (PLT1) and PLETHORA2 (PLT2)[3, 4] and SCARECROW (SCR) and SHORTROOT (SHR)[2] - as well as on polar auxin transport. In contrast, the mechanisms controlling meristematic cell differentiation remain unclear. Here, we report that cytokinins control the rate of meristematic cell differentiation and thus determine root-meristem size via a two-component receptor histidine kinase-transcription factor signaling pathway. Analysis of the root meristems of cytokinin mutants, spatial cytokinin depletion, and exogenous cytokinin application indicates that cytokinins act in a restricted region of the root meristem, where they antagonize a non-cell-autonomous cell-division signal, and we provide evidence that this signal is auxin.

During plant growth, dividing cells in meristems must coordinate transitions from division to expansion and differentiation, thus generating three distinct developmental zones: the meristem, elongation zone and differentiation zone 1 . Simultaneously, plants display tropisms, rapid adjustments of their direction of growth to adapt to environmental conditions. It is unclear how stable zonation is maintained during transient adjustments in growth direction. In Arabidopsis roots, many aspects of zonation are controlled by the phytohormone auxin and auxin-induced PLETHORA (PLT) transcription factors, both of which display a graded distribution with a maximum near the root tip 2-12 . In addition, auxin is also pivotal for tropic responses 13,14 . Here, using an iterative experimental and computational approach, we show how an interplay between auxin and PLTs controls zonation and gravitropism. We find that the PLT gradient is not a direct, proportionate readout of the auxin gradient. Rather, prolonged high auxin levels generate a narrow PLT transcription domain from which a gradient of PLT protein is subsequently generated through slow growth dilution and cell-to-cell movement. The resulting PLT levels define the location of developmental zones. In addition to slowly promoting PLT transcription, auxin also rapidly influences division, expansion and differentiation rates. We demonstrate how this specific regulatory design in which auxin cooperates with PLTs through different mechanisms and on Reprints and permissions information is available at www.nature.com/reprints.

SignificanceFixed nitrogen is essential for plant growth. Some plants, such as legumes, can host nitrogen-fixing bacteria within cells in root organs called nodules. Nodules are considered to have evolved in parallel in different lineages, but the genetic changes underlying this evolution remain unknown. Based on gene expression in the nitrogen-fixing nonlegume Parasponia andersonii and the legume Medicago truncatula, we find that nodules in these different lineages may share a single origin. Comparison of the genomes of Parasponia with those of related nonnodulating plants reveals evidence of parallel loss of genes that, in legumes, are essential for nodulation. Taken together, this raises the possibility that nodulation originated only once and was subsequently lost in many descendant lineages.