Analysis Of Both The Function Of Athspr In Cell Expansion And Vascular-specific Expression Of Athspr Promoter In Arabidopsis

Cell expansion is an important step of tissue and organ morphogenesis in plants. Both vascular tissues and cytoskeleton play vital roles in tissue and organ development and morphogenesis. Besides, cytoskeleton is also critical for stress tolerance of plants. An Arabidopsis pleiotropic mutant named Athspr (Arabidopsis thaliana heat shock protein related) was isolated through the gene trap vector mediated by T-DNA in our laboratory. Compared to the wild-type (WT) C24, Athspr mutant exhibits a dwarf phenotype including the stable phenotype of shorten and broaden silique. Moreover, Athspr mutant is sensitive to NaCl stress. Furthermore, the GUS reporter gene was expressed widely in the vascular systems of various organs and tissues of Athspr mutant. According to the previous study of Athspr mutant, we conclude that the dwarf phenotype in Athspr mutant may be the change of cell morphology or the abnormal development of vascular tissues. Cytoskeleton is a ubiquitous structure in cells, and normal cytoskeleton is also necessary for the development of vascular tissues. Thus, histochemistry and fluorescence staining, vector construction and transgenic technology, combined with microexamination were used to study the functions of Athspr gene in the morphogenesis of cells and the development of vascular systems, and the main results are as follows:1. Scanning electron microscopy (SEM) and paraffin section showed that cells in hypocotyl, petiole and papilla were notably shorter in Athspr mutant; the epidermal valve cells were out of shape in the mature silique; the medial vascular bundle (mv) in replum was thinner; the ovules showed a delayed development in the ovary, compared with WT.2. Fluorescence staining showed that the MF organizationin of hypocotyl cells in Athspr mutant was different from that in WT. The ratio of cells with fine and well-organized MF network in Athspr mutant was lower than that in WT under normal growth condition, and the ratio of cells with transverse-directed-network in Athspr mutant was higher than that in WT. While the chaotic-organized-MF was more observed after 50mM NaCl treatment, and the ratio of cells with chaotic-organized-MF in Athspr mutant was 12.6% higher than that in WT.3. Expression pattern of GUS in Athspr mutant and 1.7k-proAthspr::GUS-transformed lines showed that Athspr promoter was widely expressed in vascular tissues of various tissues and organs, primarily in xylem, cambium and phloem of inflorescence stem, especially in the SAM, leaf vein, junction of hypocotyl and root, and branches of the root.4. The 4.3kb upstream sequence of Athspr gene was cloned and amplified to construct proAthspr::GUS expression vector. Transformation of wide type C24 was performed, and 17 transgenic T1 lines were generated. GUS staining showed that GUS was expressed in vascular tissues of all the tissues and organs in Arabidopsis, including vascular tissues in sepals, petals, filaments, as well as the style, replum, valve and septum of siliques.5.10-day-old seedlings of Athspr mutant and proAthspr::GUS-transgenic lines were treated with 50uM IAA, GA and 6-BA. And the results suggested that Athspr promoter-controlled GUS activity could be up-regulated by these phytohormones, especially after GA treatment.The results above indicate that Athspr gene may play an important role in the development and morphogenesis of organs that contain vascular tissues in Arabidopsis. Athspr gene may be involved in the maintenance of morphological structure of MF. Furthermore, Athspr gene may participate in the salt stress response through MF skeleton, and the expression and function of Athspr gene could be regulated by different phytohormone signals.