The Arabidopsis thaliana hypocotyl is widely used to study the effects of light and plant growth factors on cell elongation. To provide a framework for the molecular-genetic analysis of cell elongation in this organ, here we describe, at the cellular level, its morphology and growth and identify a number of characteristic, developmental differences between light-grown and dark-grown hypocotyls. First, in the light epidermal cells show a characteristic differentiation that is not observed in the dark. Second, elongation growth of this organ does not involve significant cortical or epidermal cell divisions. However, endoreduplication occurs, as revealed by the presence of 4C and 8C nuclei. In addition, 16C nuclei were found specifically in dark-grown seedlings. Third, in the dark epidermal cells elongate along a steep, acropetal spatial and temporal gradient along the hypocotyl. In contrast, in the light all epidermal cells elongated continuously during the entire growth period. These morphological and physiological differences, in combination with previously reported genetic data (1. Desnos, V. Orbovic, C. Bellini, j. Kronenberger, M. Caboche, j. Traas, H. Hofte [19961 Development 122: 683-693), illustrate that light does not simply inhibit hypocotyl growth in a cell-autonomous fashion, but that the observed growth response to light is a part of an integrated developmental change throughout the elongating organ.

Anthocyanidin reductase encoded by the BANYULS ( BAN ) gene is the core enzyme in proanthocyanidin (PA) biosynthesis. Here, we analyzed the developmental mechanisms that regulate the spatiotemporal expression of BAN in the developing Arabidopsis seed coat. PA-accumulating cells were localized histochemically in the inner integument (seed body and micropyle) and pigment strand (chalaza). BAN promoter activity was detected specifically in these cells. Gain-of-function experiments showed that an 86-bp promoter fragment functioned as an enhancer specific for PA-accumulating cells. Mutations in regulatory genes of PA biosynthesis abolished BAN promoter activity ( transparent testa2 [ tt2 ], tt8 , and transparent testa glabra1 [ ttg1 ]), modified its spatial pattern ( tt1 and tt16 ), or had no influence ( ttg2 ), thus revealing complex regulatory interactions at several developmental levels. Genetic ablation of PA-accumulating cells targeted by the BAN promoter fused to BARNASE led to the formation of normal plants that produced viable yellow seeds. Importantly, these seeds had no obvious defects in endosperm and embryo development.

Currently, examination of the cellular structure of plant organs and the gene expression therein largely relies on the production of tissue sections. Here, we present a staining technique that can be used to image entire plant organs using confocal laser scanning microscopy. This technique produces high-resolution images that allow three-dimensional reconstruction of the cellular organization of plant organs. Importantly, three-dimensional domains of gene expression can be analyzed with single-cell precision. We used this technique for a detailed examination of phloem cells in the wild type and mutants. We were also able to recognize phloem sieve elements and their differentiation state in any tissue type and visualize the structure of sieve plates. We show that in the altered phloem development mutant, a hybrid cell type with phloem and xylem characteristics develops from initially normally differentiated protophloem cells. The simplicity of sieve element data collection allows for the statistical analysis of structural parameters of sieve plates, essential for the calculation of phloem conductivity. Taken together, this technique significantly improves the speed and accuracy of the investigation of plant growth and development.

In Arabidopsis ton 2 mutants, abnormalities of the cortical microtubular cytoskeleton, such as disorganization of the interphase microtubule array and lack of the preprophase band before mitosis, markedly affect cell shape and arrangement as well as overall plant morphology. We present the molecular isolation of the TON 2 gene, which is highly conserved in higher plants and has a vertebrate homolog of unknown function. It encodes a protein similar in its C-terminal part to B ؆ regulatory subunits of type 2A protein phosphatases (PP2As). We show that the TON2 protein interacts with an Arabidopsis type A subunit of PP2A in the yeast two-hybrid system and thus likely defines a novel subclass of PP2A subunits that are possibly involved in the control of cytoskeletal structures in plants. INTRODUCTIONPlants possess unique features in many aspects of development compared with animals. At the cellular level, plants are characterized by specific features such as the presence of a pecto-cellulosic cell wall, continuous cytoplasmic connections through the plasmodesmata, and the lack of cell motility during morphogenesis (Kaplan and Hagemann, 1991). Reflecting these unique characteristics, the highly dynamic cortical arrays of microtubules (MTs) and actin filaments have adopted various specialized arrangements in plants. The organization of cortical arrays is coordinated tightly with other cellular events, and the cortical cytoskeleton, plasmalemma, and cell wall operate as a continuum (Wyatt and Carpita, 1993). The cortical cytoskeleton reorganizes steadily during all stages of the plant cell life and plays a crucial role in governing the orientation of both cell division and expansion (Cyr and Palevitz, 1995). In contrast to animals, fungi, and protists, in land plants the establishment of division planes depends on (1) the positioning of the preprophase band (PPB), a transient cortical ring of MTs that precisely foretells the position of the cell division plane at the G2/prophase transition, and (2) guidance of the phragmoplast to this predetermined cortical site during cytokinesis. During interphase, arrays of parallel MTs encircle the cell at the cortex. The positioning of MTs within these cortical arrays appears to involve both MT dynamicity (nucleation, growth/shrinkage, stabilization, and severing) and translocation, but the relative roles of these events and the proteins involved are unknown.To understand the molecular mechanisms underlying MT arrangements, mutants impaired in MT functions are essential, and a number of proteins involved in MT organization or dynamics have been identified through a genetic approach (Azimzadeh et al., 2001). These include the Arabidopsis botero1 mutant (which also is allelic to the fra2 mutant) (Zhong et al., 2001), which displays incorrect orientation of interphase cortical MTs and defects in anisotropic growth in root tip cells (Bichet et al., 2001). The FRA2 gene encodes a protein with high similarity to the sea urchin p60 subunit of katanin, which is known to be involved in MT...

Glutamate (Glu) metabolism and amino acid translocation were investigated in the young and old leaves of tobacco (Nicotiana tabacum L. cv Xanthi) using [15N]ammonium and [2-15N]Glu tracers. Regardless of leaf age, [15N]ammonium assimilation occurred via glutamine synthetase (GS; EC 6.1.1.3) and Glu synthase (ferredoxin [Fd]-GOGAT; EC 1.4.7.1; NADH-GOGAT; EC 1.4.1.14), both in the light and darkness, and it did not depend on Glu dehydrogenase (GDH; EC 1.4.1.2). The [15N]ammonium and ammonium accumulation patterns support the role of GDH in the deamination of [2-15N]Glu to provide 2-oxoglutarate and [15N]ammonium. In the dark, excess [15N]ammonium was incorporated into asparagine that served as an additional detoxification molecule. The constant Glu levels in the phloem sap suggested that Glu was continuously synthesized and supplied into the phloem regardless of leaf age. Further study using transgenic tobacco lines, harboring the promoter of the GLU1 gene (encoding Arabidopsis [Arabidopsis thaliana] Fd-GOGAT) fused to a GUS reporter gene, revealed that the expression of Fd-GOGAT remained higher in young leaves compared to old leaves, and higher in the veins compared to the mesophyll. Confocal laser-scanning microscopy localized the Fd-GOGAT protein to the phloem companion cells-sieve element complex in the leaf veins. The results are consistent with a role of Fd-GOGAT in supplying Glu for the synthesis and transport of amino acids. Taken together, the data provide evidence that the GS-GOGAT pathway and GDH play distinct roles in the source-sink nitrogen cycle of tobacco leaves.