Faculty Bibliography

Soil salinity affects large areas of cultivated land, causing significant reductions in crop yield globally. The Na+ toxicity of many crop plants is correlated with overaccumulation of Na+ in the shoot. We have previously suggested that the engineering of Na+ exclusion from the shoot could be achieved through an alteration of plasma membrane Na+ transport processes in the root, if these alterations were cell type specific. Here, it is shown that expression of the Na+ transporter HKT1;1 in the mature root stele of Arabidopsis thaliana decreases Na+ accumulation in the shoot by 37 to 64%. The expression of HKT1;1 specifically in the mature root stele is achieved using an enhancer trap expression system for specific and strong overexpression. The effect in the shoot is caused by the increased influx, mediated by HKT1;1, of Na+ into stelar root cells, which is demonstrated in planta and leads to a reduction of root-to-shoot transfer of Na+. Plants with reduced shoot Na+ also have increased salinity tolerance. By contrast, plants constitutively expressing HKT1;1 driven by the cauliflower mosaic virus 35S promoter accumulated high shoot Na+ and grew poorly. Our results demonstrate that the modification of a specific Na+ transport process in specific cell types can reduce shoot Na+ accumulation, an important component of salinity tolerance of many higher plants.

To facilitate the monitoring of guard cells during development and isolation, a population of 704 GAL4 GFP enhancer trap lines was screened and four single insert lines with guard cell GFP expression and one with developmentally-regulated guard cell GFP expression were identified. The location of the T-DNA inserts, the expression of the flanking genes, and the promoter activity of the genomic DNA upstream of the T-DNA were characterized. The results indicated that the GFP expression pattern in at least one of the lines was due to elements in the intergenic DNA immediately upstream of the T-DNA, rather than due to the activity of the promoters of genes flanking the insert, and provide evidence for the involvement of Dof elements in regulating guard cell gene expression. It is shown further that the GAL4 GFP lines can be used to track the contribution of guard cell material in vitro, and this method was used to assess the purity of guard cell samples obtained using two methods of guard cell isolation.

Because plant cells do not migrate, cell division planes are crucial determinants of plant cellular architecture. In Arabidopsis roots, stringent control of cell divisions leads to a virtually invariant division pattern, including those that create new tissue layers. However, the mechanisms that control oriented cell divisions are hitherto poorly understood. Here, we reveal one such mechanism in which FEZ and SOMBRERO (SMB), two plant-specific NAC-domain transcription factors, control the delicately tuned reorientation and timing of cell division in a subset of stem cells. FEZ is expressed in root cap stem cells, where it promotes periclinal, root cap-forming cell divisions. In contrast, SMB negatively regulates FEZ activity, repressing stem cell-like divisions in the root cap daughter cells. FEZ becomes expressed in predivision stem cells, induces oriented cell division, and activates expression of its negative regulator, SMB, thus generating a feedback loop for controlled switches in cell division plane.

BACKGROUND:Plant cell death is a normal process during plant development. Mutant plants may exhibit misregulation of this process, which can lead to severe growth defects. Simple ways of visualising cell death in living plant tissues can aid the study of plant development and physiology. RESULTS:Spectral variants of the fluorescent SYTOX dyes were tested for their usefulness for the detection of non-viable cells within plant embryos and roots using confocal laser-scanning microscopy. The dyes were selective for non-viable cells and showed very little background staining in living cells. Simultaneous detection of SYTOX dye and fluorescent protein (e.g. GFP) fluorescence was possible. CONCLUSION/CONCLUSIONS:The fluorescent SYTOX dyes are useful for an easy and quick first assay of plant cell viability in living plant samples using fluorescence and confocal laser-scanning microscopy.

BACKGROUND AND AIMS/OBJECTIVE:During the development of multicellular organisms, cells are capable of interacting with each other through a range of biological and physical mechanisms. A description of these networks of cell-cell interactions is essential for an understanding of how cellular activity is co-ordinated in regionalized functional entities such as tissues or organs. The difficulty of experimenting on living tissues has been a major limitation to describing such systems, and computer modelling appears particularly helpful to characterize the behaviour of multicellular systems. The experimental difficulties inherent to the multitude of parallel interactions that underlie cellular morphogenesis have led to the need for computer models. METHODS:A new generic model of plant cellular morphogenesis is described that expresses interactions amongst cellular entities explicitly: the plant is described as a multi-scale structure, and interactions between distinct entities is established through a topological neighbourhood. Tissues are represented as 2D biphasic systems where the cell wall responds to turgor pressure through a viscous yielding of the cell wall. KEY RESULTS/RESULTS:This principle was used in the development of the CellModeller software, a generic tool dedicated to the analysis and modelling of plant morphogenesis. The system was applied to three contrasting study cases illustrating genetic, hormonal and mechanical factors involved in plant morphogenesis. CONCLUSIONS:Plant morphogenesis is fundamentally a cellular process and the CellModeller software, through its underlying generic model, provides an advanced research tool to analyse coupled physical and biological morphogenetic mechanisms.

BACKGROUND:The Arabidopsis outer ovule integument is a simple two-cell layered structure that grows around the developing embryo and develops into the outer layer of the seed coat. As one of the functions of the seed coat is the protection of the plant embryo, the outer ovule integument is an example for a plant organ whose morphogenesis has to be precisely regulated. RESULTS:To better characterise outer ovule integument morphogenesis, we have isolated some marker lines that show GFP expression in this organ. We have used those lines to identify distinct cell types in the outer integument and to demonstrate similarities between leaves and the outer integument. Using confocal microscopy, we showed that cell sizes and shapes differ between the two cell layers of the outer integument. Expression of KNAT1 in the integuments leads to extra cell divisions specifically in the outer layer of the outer integument. This is being compensated for by a decrease of cell volume in this layer, thus showing that mechanisms exist to control proper ovule integument morphogenesis. CONCLUSION/CONCLUSIONS:The Arabidopsis outer ovule integument can be used as a good model system to study the basic principles of plant organ morphogenesis. This work provides new insights into its development and opens new possibilities for the identification of factors involved in the regulation of cell division and elongation during plant organ growth.

The outer tissues of dicotyledonous plant roots (i.e. epidermis, cortex, and endodermis) are clearly organized in distinct concentric layers in contrast to the diarch to polyarch vascular tissues of the central stele. Up to now, the outermost layer of the stele, the pericycle, has always been regarded, in accordance with the outer tissue layers, as one uniform concentric layer. However, considering its lateral root-forming competence, the pericycle is composed of two different cell types, with one subset of cells being associated with the xylem, showing strong competence to initiate cell division, whereas another group of cells, associated with the phloem, appears to remain quiescent. Here, we established, using detailed microscopy and specific Arabidopsis thaliana reporter lines, the existence of two distinct pericycle cell types. Analysis of two enhancer trap reporter lines further suggests that the specification between these two subsets takes place early during development, in relation with the determination of the vascular tissues. A genetic screen resulted in the isolation of mutants perturbed in pericycle differentiation. Detailed phenotypical analyses of two of these mutants, combined with observations made in known vascular mutants, revealed an intimate correlation between vascular organization, pericycle fate, and lateral root initiation potency, and illustrated the independence of pericycle differentiation and lateral root initiation from protoxylem differentiation. Taken together, our data show that the pericycle is a heterogeneous cell layer with two groups of cells set up in the root meristem by the same genetic pathway controlling the diarch organization of the vasculature.

Homeodomain proteins set up domains of gene expression during the development of animal and plant body plans. In plants, homeodomain proteins of the KNOX class I family have been shown to play a role in shoot apical meristem development. Recently, we have investigated the role of the Arabidopsis thaliana KNOX class II genes KNAT3, KNAT4 and KNAT5 in root development. These genes showed root domain and cell type specific expression patterns, and their expression was regulated by hormones that influence root growth. Moreover, sub-cellular localization of the KNAT proteins exhibited regulation, suggesting that post-transcriptional control contributes to KNOX class II protein activity. Our data provide a survey of KNAT gene expression in the root and indicate that the investigated KNAT genes might play distinct roles during root development.

We tested the hypothesis that the circadian clock modulates Ca(2+)-based signalling pathways, using low-temperature (LT)-induced Ca(2+) signals. We investigated the relationship between diurnal and circadian modulation of LT-induced increases in cytosolic-free calcium ([Ca(2+)](cyt)), and regulation of [Ca(2+)](cyt)-dependent outputs of the LT-signalling network (RD29A transcript abundance and stomatal closure). We measured [Ca(2+)](cyt) non-invasively using aequorin, and targeted aequorin to the guard cell using a guard cell-specific GAL4-green fluorescent protein enhancer trap line. LT caused transient increases in whole plant and guard cell [Ca(2+)](cyt). In guard cells, the LT-induced [Ca(2+)](cyt) elevation preceded stomatal closure. In whole plants, the magnitude of LT-induced [Ca(2+)](cyt) transients, measured from the entire plant or specifically the guard cell, varied with the time of day: LT-induced [Ca(2+)](cyt) transients were significantly higher during the mid-photoperiod than at the beginning or end. Diurnal variation in LT-induced guard cell [Ca(2+)](cyt) increases was not correlated to diurnal variation in LT-induced stomatal closure. There was circadian modulation of LT-induced whole plant [Ca(2+)](cyt) increases, which were correlated to the circadian pattern of RD29A induction. In order to understand the significance of LT-induced [Ca(2+)](cyt) increases, we used a computer simulation to demonstrate that, in guard cells, LT-induced [Ca(2+)](cyt) increases measured from a population of cells are likely to represent the summation of cold-induced single-cell [Ca(2+)](cyt) oscillations.

Armadillo/beta-catenin and related proteins have important functions during animal and Dictyostelium development, regulating cell differentiation, proliferation, and adhesion. Armadillo-repeat-containing proteins also exist in plants, but the majority have unknown roles. The Arabidopsis genes that show greatest sequence homology to Armadillo/beta-catenin are called ARABIDILLO-1 and -2. Here, we demonstrate that ARABIDILLO-1 and -2 promote lateral root development. arabidillo-1/-2 mutants form fewer lateral roots, and ARABIDILLO-1-overexpressing lines produce more lateral roots than wild-type seedlings. ARABIDILLO-yellow fluorescent protein fusions are nuclear. ARABIDILLO proteins contain an F-box motif, and thus may target other proteins for proteasomal degradation. Overexpression of ARABIDILLO-1 protein fragments, including F-box fragments, in wild-type seedlings reduces lateral root formation to the level of the arabidillo-1/-2 mutant. We have shown that plant beta-catenin-related proteins regulate root development. We suggest that ARABIDILLO proteins may target an inhibitor of lateral root development for degradation and propose that Arabidopsis beta-catenin-related proteins define a previously uncharacterized pathway that promotes root branching.