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Basic study of plant cell cycle

Analysis of endoreduplication was focused mostly on the role of the ccs52 protein family and its interactors. CCS52 is an activator of the anaphase promoting complex APC in all organisms including plants. CCS52 triggers the destruction of M phase specific cyclins, thus pushes cells into endoreduplication. Several genes (E2F/DP like proteins, ORCc, MCMs) that play a role in G1/S transition were investigated for their role in endoreduplication. Inze's group has shown that co-expression of E2Fa/Dp1a in transgenic Arabidopsis enhances endoreduplication levels in certain tissues. This important finding sheds light on the relationship between G1/S transition and endoreduplication and the control mechanisms that regulate both processes.

In synchronised cell cultures of Medicago truncatula expression of ccs52A was constitutive in the cell cycle, in contrast to that of ccs52B, which was observed only in G2-M. The protein level of ccs52B correlated with the transcript levels. For ccs52A, a decrease in the protein level was observed in the S-G2 phase. As a prototype for the ccs52 proteins, different point and deletion mutations were generated in the Mt/MsCcs52A protein. The effect of the mutations was studied in fission yeast where over-expression of the wild type protein elicited growth arrest, cell enlargement and endoreduplication. This analysis revealed that deletion of the two, conserved N-terminal oligopeptide motifs and the C-terminal IR residues are essential for the biological activity. In contrast, elimination of the CDK phosphorylation sites rendered the protein constitutively active.

In Arabidopsis, GUS expression patterns of the ccs52A and ccs52B genes was only partially overlapping, most often the two genes exhibited complementary activities (most strikingly during flower development). The expression was linked to development of certain cell types (e.g.trichome, hairs), cell layers (e.g. tapetum/A/, pollen tubes /B/), tissues (e.g.vascular tissues, meristems) or organs (e.g. primordia, different parts of the flower), indicating non-redundant functions of the ccs52A and ccs52B genes. Since no ccs52 mutants could be found in the mutant collection of INRA, France, CropDesign has made RNAi constructs for the ccs52A and ccs52B genes. Various overall growth phenotypes have been observed. Plants over-expressing the Arabidopsis CCS52A1 gene under the control of the strong constitutive promoter 35S showed smaller leaves, decreased flower and seed production; Over-expression of CCS52A1 under the control of a mild constitutive promoter led to much more positive phenotypes such as rounded leaves, increased stem diameter, altered trichome architecture, bigger seeds. Additional effects have been found at the microscopic level on cell size, cell number, and endoreduplication. Several contracts have been made by CropDesign with the Arabidopsis CCS52 genes using different promoters (p35S; pUBI; p2S2) and the different phenotypes have been compared. Over-expression under the control of p35S of the 3 Arabidopsis CCS52 genes conducted to the similar drastic growth reduction, accumulation of anthocyanins in the leaves and strong reduction of fertility and seed production.

The yeast two-hybrid screens were performed with both the ccs52A and B proteins from Arabidopsis. In the case of both the Arabidopsis, the strongest interaction was found with a CCA3 homologous protein. In addition, several new, predominantly Destruction box-containing interacting partners were identified including a putative homeodomain transcription factor, as well as a known one (ATHB-14), an unknown TRAF-homologue protein, a hypothetical, carotenoid biosynthesis regulator homologue, an autocrine motility factor receptor-homologue as well as an EREBP4-like protein. Several CCS52 interactors constructs have been produced by CropDesign. One known gene (Athb14) is involved in leaf symmetry and dorsoventrality, which could explain the effects of over-expression of CCS52 on leaf development.

The anaphase-promoting complex (APC/C) is an essential ubiquitin protein ligase that regulates mitotic progression and exit by controlling the stability of cell cycle regulatory proteins, such as the mitotic cyclins. In plants, the function, regulation, and substrates of the APC/C are poorly understood. The plant APC2 gene is able to partially complement a budding yeast apc2 ts mutant. By yeast two-hybrid assays, interaction of APC2 with two other APC/C subunits: APC11 and APC8/CDC23 has been shown and corroborates the function of this subunit in the APC complex. A reverse-genetic approach identified Arabidopsis plants carrying T-DNA insertions in the APC2 gene. Apc2 null mutants are impaired in female megagametogenesis. The APC2 gene is expressed in various plant organs and does not seem to be cell cycle regulated. This work suggests a conserved function of the APC/C in plants but a different mode of regulation. (Capron et al., 2003).

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