Diego Albani

The E2F family of transcription factors from Arabidopsis thaliana: Novel and conserved components of the retinoblastoma/E2F pathway in plants

The E2F transcription factors are key components of the cyclin D/retinoblastoma/E2F pathway. Here we demonstrate thatArabidopsis thaliana contains six functional AtE2F genes that are all expressed in cell suspension culture but show different patterns of expression during cell cycle progression. According to their structural and functional features, the six AtE2Fs can be divided into two distinct groups; although the three members of the first group, AtE2Fa, AtE2Fb and AtE2Fc, possess all the conserved domains found in other plant and animal E2Fs, the remaining AtE2Fs are novel proteins, which reveal a duplication of the DNA binding domain but lack any other conserved region. Furthermore, the AtE2Fs of the first group are functional transcription factors that in association with AtDP proteins can recognize specifically an E2F cis-element and can transactivate an E2F-responsive reporter gene in plant cells. In contrast, the AtE2Fs of the second group can bind specifically the E2F site without interacting with DP partners but cannot activate gene expression and, instead, are able to inhibit E2F-dependent activation of gene expression in Arabidopsis cells. These findings suggest distinctive roles for the plant E2F proteins and point to a complex concerted regulation of E2F-dependent gene expression in plant cells.

Cloning and characterization of a Brassica napus gene encoding a homologue of the B subunit of a heteromeric CCAAT-binding factor

The CCAAT motif present in the promoter of several genes is recognized in yeast and animals by a highly specific heteromeric factor (variously called HAP, CBF, CP1 or NF-Y) which is composed of a minimum of three subunits. A plant homologue of the CBF-B/HAP2 subunit is described for the first time in this report. Sequence comparison of the Brassica napus (Bn) CCAAT-binding factor (CBF) B subunit with the homologous yeast and animal proteins revealed that the critical amino-acid domains involved in DNA binding and subunit assembly are also conserved in plants. Interestingly, the Gln-rich regions found in the animal and yeast proteins, which may be involved in transcriptional activation, are absent in the Bn CBF-B subunit. The analysis of various cDNAs and of a genomic clone revealed the presence of alternatively spliced transcripts which could originate from different promoters.

The E2FD/DEL2 factor is a component of a regulatory network controlling cell proliferation and development in Arabidopsis

An emerging view of plant cell cycle regulators, including the E2F transcription factors, implicates them in the integration of cell proliferation and development. Arabidopsis encodes six E2F proteins that can act as activators or repressors of E2F-responsive genes. E2FA, E2FB and E2FC interact with the retinoblastoma-like RBR protein and bind to DNA together with their DP partners. In contrast, E2FD, E2FE and E2FF (also known as DEL2, DEL1 and DEL3) are atypical E2Fs that possess duplicated DNA binding regions, lack trans-activating and RBR-binding domains and are believed to act as transcriptional inhibitors/repressors. E2FE/DEL1 has been shown to inhibit the endocycle and E2FF/DEL3 appears to control cell expansion but the role of E2FD/DEL2 has not been reported so far. In this study, we investigated the expression of E2FD/DEL2 and analysed the accumulation of its product. These studies revealed that E2FD/DEL2 accumulation is subject to negative post-translational regulation mediated by the plant hormone auxin. Moreover, the analysis of mutant and transgenic plants characterized by altered expression of E2FD/DEL2 has revealed that this atypical E2F can affect plant growth by promoting cell proliferation and repressing cell elongation. Overexpression of E2FD/DEL2 increased the expression of E2FA, E2FB and E2FE/DEL1 whereas its inactivation led to the up-regulation of genes encoding repressors of cell division. These results suggest that E2FD/DEL2 is part of a regulatory network that controls the balance between cell proliferation and development in Arabidopsis.

Perturbation of cytokinin and ethylene-signalling pathways explain the strong rooting phenotype exhibited by Arabidopsis expressing the Schizosaccharomyces pombe mitotic inducer, cdc25

BackgroundEntry into mitosis is regulated by cyclin dependent kinases that in turn are phosphoregulated. In most eukaryotes, phosphoregulation is through WEE1 kinase and CDC25 phosphatase. In higher plants a homologous CDC25 gene is unconfirmed and hence the mitotic inducer Schizosaccharomyces pombe (Sp) cdc25 has been used as a tool in transgenic plants to probe cell cycle function. Expression of Spcdc25 in tobacco BY-2 cells accelerates entry into mitosis and depletes cytokinins; in whole plants it stimulates lateral root production. Here we show, for the first time, that alterations to cytokinin and ethylene signaling explain the rooting phenotype elicited by Spcdc25 expression in Arabidopsis.ResultsExpressing Spcdc25 in Arabidopsis results in increased formation of lateral and adventitious roots, a reduction of primary root width and more isodiametric cells in the root apical meristem (RAM) compared with wild type. Furthermore it stimulates root morphogenesis from hypocotyls when cultured on two way grids of increasing auxin and cytokinin concentrations. Microarray analysis of seedling roots expressing Spcdc25 reveals that expression of 167 genes is changed by > 2-fold. As well as genes related to stress responses and defence, these include 19 genes related to transcriptional regulation and signaling. Amongst these was the up-regulation of genes associated with ethylene synthesis and signaling. Seedlings expressing Spcdc25 produced 2-fold more ethylene than WT and exhibited a significant reduction in hypocotyl length both in darkness or when exposed to 10 ppm ethylene. Furthermore in Spcdc25 expressing plants, the cytokinin receptor AHK3 was down-regulated, and endogenous levels of iPA were reduced whereas endogeous IAA concentrations in the roots increased.ConclusionsWe suggest that the reduction in root width and change to a more isodiametric cell phenotype in the RAM in Spcdc25 expressing plants is a response to ethylene over-production. The increased rooting phenotype in Spcdc25 expressing plants is due to an increase in the ratio of endogenous auxin to cytokinin that is known to stimulate an increased rate of lateral root production. Overall, our data reveal important cross talk between cell division and plant growth regulators leading to developmental changes.

Characterization of genes expressed during the development ofBrassica napus pollen

SummaryThe study of the formation of pollen in plants has been the focus of extensive morphologic and cytologic observations. This complex developmental process requires the coordinated activity of both gametophytic and sporophytic tissues. The events that occur during microspore development represent a carefully orchestrated program of physiologic, biochemical, and genetic activities. Genes expressed specifically in pollen or in sporophytic tissues that support pollen development have only recently been identified and desribed. In the present paper we describe several genes expressed during pollen development in the important oil seed speciesBrassica napus (oil seed rape/canola). The characterization of three gene families expressed during microspore development is reviewed which provides a basis for comparison with other genes expressed during pollen maturation. The, potential value of these genes for the development of novel plant breeding strategies and hybrid seed production is discussed.

In tobacco BY-2 cells xyloglucan oligosaccharides alter the expression of genes involved in cell wall metabolism, signalling, stress responses, cell division and transcriptional control

Xyloglucan oligosaccharides (XGOs) are breakdown products of XGs, the most abundant hemicelluloses of the primary cell walls of non-Poalean species. Treatment of cell cultures or whole plants with XGOs results in accelerated cell elongation and cell division, changes in primary root growth, and a stimulation of defence responses. They may therefore act as signalling molecules regulating plant growth and development. Previous work suggests an interaction with auxins and effects on cell wall loosening, however their mode of action is not fully understood. The effect of an XGO extract from tamarind (Tamarindus indica) on global gene expression was therefore investigated in tobacco BY-2 cells using microarrays. Over 500 genes were differentially regulated with similar numbers and functional classes of genes up- and down-regulated, indicating a complex interaction with the cellular machinery. Up-regulation of a putative XG endotransglycosylase/hydrolase-related (XTH) gene supports the mechanism of XGO action through cell wall loosening. Differential expression of defence-related genes supports a role for XGOs as elicitors. Changes in the expression of genes related to mitotic control and differentiation also support previous work showing that XGOs are mitotic inducers. XGOs also affected expression of several receptor-like kinase genes and transcription factors. Hence, XGOs have significant effects on expression of genes related to cell wall metabolism, signalling, stress responses, cell division and transcriptional control.

Gene dosage effect of WEE1 on growth and morphogenesis from arabidopsis hypocotyl explants

BACKGROUND AND AIMS How plant cell-cycle genes interface with development is unclear. Preliminary evidence from our laboratory suggested that over-expression of the cell cycle checkpoint gene, WEE1, repressed growth and development. Here the hypothesis is tested that the level of WEE1 has a dosage effect on growth and development in Arabidospis thaliana. To do this, a comparison was made of the development of gain- and loss-of-function WEE1 arabidopsis lines both in vivo and in vitro. METHODS Hypocotyl explants from an over-expressing Arath;WEE1 line (WEE1(oe)), two T-DNA insertion lines (wee1-1 and wee1-4) and wild type (WT) were cultured on two-way combinations of kinetin and naphthyl acetic acid. Root growth and meristematic cell size were also examined. KEY RESULTS Quantitative data indicated a repressive effect in WEE1(oe) and a significant increase in morphogenetic capacity in the two T-DNA insertion lines compared with WT. Compared with WT, WEE1(oe) seedlings exhibited a slower cell-doubling time in the root apical meristem and a shortened primary root, with fewer laterals, whereas there were no consistent differences in the insertion lines compared with WT. However, significantly fewer adventitious roots were recorded for WEE1(oe) and significantly more for the insertion mutant wee1-1. Compared with WT there was a significant increase in meristem cell size in WEE1(oe) for all three ground tissues but for wee1-1 only cortical cell size was reduced. CONCLUSIONS There is a gene dosage effect of WEE1 on morphogenesis from hypocotyls both in vitro and in vivo.

Gene expression during Brassica napus pollen development.

The development of pollen is a complex process that requires the coordinate participation of various tissue types and specific gene expression patterns. Both gametophytic (pollen) and sporophytic (anther, tapetum) tissues participate in this process. Of the many nutrients and macromolecules that are involved in the ultimate production of pollen, some are made within the pollen grain while others are contributed by the tapetal tissue. Recently more attention has been paid to this process and molecular and cytological studies have been undertaken in different plant species. Our study focuses on pollen development in Brassica napus, an important oilseed crop. Here we describe some of the molecular events that occur during the formation of pollen grains in this plant species.