Alteration of gibberellin response in transgenic tobacco plants which express a human Lewis fucosyltransferase.

In plants, Lewisa type N-glycans may be involved in cell-to-cell communication and recognition. N-glycoproteins harboring Lewisa glycotopes are mainly found in plasma membranes and cell walls. Some can be also involved in cell wall synthesis or the loosening process, and subsequently in cell elongation. In order to determine the potential role(s) of the alpha4-fucosylation during vegetative development, transgenic tobacco plants overexpressing a human Lewis fucosyltransferase (hFUT3), which transfers a fucose residue in a alpha(1,4)-linkage on complex glycans, have been developed. The heterologous enzyme hFUT3 was strongly expressed and fully functional in transgenic tobacco. Transgenic plants showed a delay in growth linked to a reduction of internode length. Furthermore, transgenic seedling roots were significantly shorter than wild-type roots and the length of their epidermis cells was reduced. Strikingly, root growth was completely and specifically restored following gibberellin treatment. Etiolated hypocotyls of hFUT3 overexpressors were also more sensitive to exogenous gibberellin. Furthermore, paclobutrazol, an inhibitor of gibberellin synthesis, induced a similar effect on control and transgenic dark-grown hypocotyls suggesting that gibberellin biosynthesis was probably not altered in seedlings overexpressing hFUT3. Thus, alpha4-fucosylation could act as a possible modulator of conformation and/or functioning of N-glycoproteins involved in the gibberellin-dependent elongation process.

Plant D-type cyclins and the control of G1 progression.

The basic pattern of controls that operate during the G1 phase of the plant cell cycle shows much closer similarity to animals than to the yeasts and other fungi. The activity of D-type cyclin (CycD) kinases is induced in response to stimulatory signals, and these phosphorylate the plant homologue of the retinoblastoma tumour susceptibility (Rb) protein. It is likely that Rb phosphorylation results in the activation of genes under the control of E2F transcription factors, including those required for S phase entry. As the initial triggers of the cascade, attention has focused on the CycDs, and a family of 10 genes is present in Arabidopsis, divided into three major and three minor groups. Analysis to date suggests that these groups are functionally distinct.

alpha4-Fucosyltransferase is regulated during flower development: increases in activity are targeted to pollen maturation and pollen tube elongation.

alpha4-Fucosylation represents a final step of protein N- glycosylation. alpha4-fucosylated N-glycans are thought to be involved in cell-to-cell communication and recognition in primates and plants. Nevertheless, in the plant life cycle, the function of alpha4-fucosylation remains largely unknown. To gain an insight into the role of alpha4-fucosylation during development, the study focused on tobacco flowers. It is shown that an increase in alpha(1,4)fucosyltransferase (Fuc-T) activity is only observed during anther development, whereas it remains at a constant but low level (around 20 pmol Fuc h(-1) mg(-1) protein) in the gynoecium and perianth. At least a 4-fold higher activity is detected in mature pollen grains. These data suggest that alpha(1,4)Fuc-T activity is regulated during anther development. Furthermore, alpha(1,4)Fuc-T activity could be required during pollen tube elongation where the activity level peaks at 350 pmol h(-1) mg(-1) protein. Based on enzyme profile and cycloheximide effects on pollen germination and activity, it is hypothesized that the gene encoding alpha4-Fuc-T could be regulated late during pollen development. A potential role of alpha4- fucosylation during pollen tube elongation is also discussed.

The CDK inhibitor NtKIS1a is involved in plant development, endoreduplication and restores normal development of cyclin D3; 1-overexpressing plants.

Plant development requires stringent controls between cell proliferation and cell differentiation. Proliferation is positively regulated by cyclin dependent kinases (CDKs). Acting in opposition to CDKs are CDK inhibitors (CKIs). The first tobacco CKI (NtKIS1a) identified was shown to inhibit in vitro the kinase activity of CDK/cyclin complexes and to interact with CDK and D-cyclins. However, these features, which are common to other plant and animal CKIs already characterised, did not provide information about the function of NtKIS1a in plants. Thus, to gain insight into the role of NtKIS1a and especially its involvement in cell proliferation during plant development, we generated transgenic Arabidopsis thaliana plants that overexpress NtKIS1a. These plants showed reduced growth with smaller organs that contained larger cells. Moreover, these plants displayed modifications in plant morphology. These results demonstrated that plant organ size and shape, as well as organ cell number and cell size, might be controlled by modulation of the single NtKIS1a gene activity. Since in mammals, D-cyclins control cell cycle progression in a CDK-dependent manner but also play a CDK independent role by sequestering the CKIs p27(Kip1) and p21(Cip1), we tested the significance of cyclin D-CKI interaction within a living plant. With this aim, NtKIS1a and AtCycD3;1 were overexpressed simultaneously in plants by two different methods. Our results demonstrated that overexpression of the CKI NtKIS1a restores essentially normal development in plants overexpressing AtCycD3;1, providing the first evidence of cyclin D-CKI co-operation within the context of a living plant.