Functional Analysis Of TaRAN1, A Small GTP-binding Protein During Cell Cycle And Development In Plant

As important switches in cell signaling, the small GTPases are apparently conserved in all organisms. In spite of their small size, the interaction of small GTPase with various regulators and effectors generates functional diversity and creates novel functions in different phyla. Functional innovation of a conserved GTPase together with birth of new members of the superfamily endows plants with the capacity to use small GTPase as a key molecular switch for the modulation of many plant-specific signaling pathways and functions. It is one of the hot topics of developmental biology. The function of plant Ran family, however, has not been elucidated, although the research progresses of its interactional proteins are intriguing.TaRAN1 (AF488730) was cloned from wheat cold-induced cDNA library in our laboratory. The function of Ran proteins, however, is few known in plants. TaRANl was identified as a small GTP-binding protein in biochemical level. We also found TaRANl modulates diverse important processes, including cell cycle, auxin and stress responses signal transduction on molecular level. The results suggested that TaRANl protein might be involved in many fundamental regulational mechanism of cell development as a key molecular switch in plant signaling.The fusion protein of TaRanl from the recombined strain cells of E. coli was affinitily purified. The binding of [a-32P]-GTP was able to effectively compete when a non-hydrolyzable GTP analog was included in the buffer as a competitor. These results demonstrated that the binding site of this protein was thus specific for guanine nucleotides. The TaRANl transcript was expressed more abundant in young stems and flower buds, little in old leaves in wheat. It localized to the nucleus predominantly using transient expression assay in onion epidermal cells. But the typical nuclear localization sequence (NLS) does not appear in TaRAN1 sequence.Cell cycle is one of the most conspicuous features of life and is usually a hot topic in biological research field. Although recent data have strongly expanded our knowledge of the molecular events of cell cycle in animals, it is still poorly understood in plants. The fission yeast, Schizosaccharomyces pombe, is an excellent model to study cell morphogenesis and cell cycle progression. Fission yeast cells overexpressed TaRANl exhibited a number of novel cell biological phenotypes. These included elongated and multinucleated cell, G2 cell cycle delay, hypersensitivity to UV. Antisense expression of the TaRANl caused S. pombe cells to round up, with highly condensed nucleus, and suppressed the growth rates. The number of nucleomixis andanucleate cells were increased dramatically. Changes of the cell cycle phases of the transgenic yeast cells also monitored by a flow cytometry. The results suggested that TaRAN1 protein might be involved in regulational mechanism of cell division and development and highlighted a role in regulation of genome integrity via maintaining chromatin structures. We also analyzed microtubules in transgenic yeast cells using approach of indirect immunofluorescence. The overexpressed TaRANl cells had aberrant spindle structures, such as short "v" - shaped spindles, resulted in a subsequent loss of chromosome segregation fidelity. The spindles of the antisense TaRANl strain disrupted, and were not aligned along the long axis of the cell. It suggested that TaRANl plays a role in mitotic spindle assembly and microtubule integrity and stability. Finally, we also studied the function of TaRANl in transgenic Arabidopsis and rice plants. The abnormal phenomenon of cell cycle events also occurred in transgenic plant cells, including the increase of primodium development in stem apical, the changes of the mitotic index in root tip cells and the abnormal mitotic phases. These results further suggested that TaRAN1 has been presumed to be involved in regulating the G2/M transition of the cell cycle events in plant.Expression of TaRANl gene was specifically induced by auxin. The expression level on mRNA was enhanced with the high...