Functional Analysis Of BR Regulatory Proteins PMRP In Arabidopsis Thaliana

START domain(the lipid/sterol-binding StAR-related lipid transfer protein domains) is a kind of steroid regulatory protein of30Kda with conserved domain, which combines and transports cholesterol to inner membrane of mitochondrion. It is defined as an amino acid sequence of200amino acids and is related to the combination with lipids and sterols. Arabidopsis contains35members of the protein family with START domain, and they can be further divided into7subfamilies according to the structure and size. Among the35members,21contain HD ZLZ START domain,5with PH START DUF1336domain, and9just contain START domain. The functions of9proteins containing START domain have been determined, including GL2, ANL2, PDF2, ATML1, FWA, PHV, PHB, ATHB-8and REV. All of the proteins above with known functions belong to the protein subfamily with HD ZLZ START domain, regulating such growth processes as cortex development (ATML1), floral organ formation (PDF2), anthocyanin accumulation (ANL2、FWA), epidermal hair growth (GL2), adaxial and abaxialpolarity (PHV, PHB, REV) and vascular bundle build up (ATHB-8). However, no report has yet presented the functions of other proteins containing START domain. In this study, we selected PMRP, a conserved protein with only START domain as subject to further analyze the functions of START domain. The function analysis of PMRP gene was analyzed in this study to demonstrate the function of PMRP in the process of plant growth and development. The results are as follows.1. PMRP is induced by Brassinosteroids (BRs). In this study, Col-0Arabidopsis of4-week-growth was treated with100μM of2,4-epibrassinolide (eBL) of3,6,9,12,18,24h respectively. The result of RT-PCR showed that PMRPexpressed quantity increased gradually with the extension of time, indicating that BR induces PMRP expression.2. Expression pattern analysis of PMRP(1) The tissue localization carrier was established with PMRP promoter connecting GUS reporter gene to analyze the tissue localization of PMRP. The result showed that PMRP was expressed in the whole seedling with the most expression quantity in cotyledon and hypocotyl. In true leaf, PMRP expression mainly localized in the genesis areas of leaf vein and epidermal hair. The expression in root was also obvious.(2) Preliminary analysis of the subcellular localization of PMRP was performed through the establishment of PMRP-GFP fusion expression vector and protoplast transformation. The result showed that PMRP was mainly localized on cell membrane and some expression in cytoplasm could also be observed.(3) Real-time PCR was adopted to analyze the temporal and spatial expression of PMRP in Arabidopsis. The result showed that the most expression quantity of PMRP was found in stem leaf, with the second expression quantity in the rosette leaf and the third in stem, and the expression in the flower and seed was relatively low. Further study demonstrated that PMRP expression quantity increases along with time in rosette leaf, stem leaf and stem, but remains steady in root and the whole part above the earth.3. Biological function analysis of PMRPBiological function was identified by establishing the genetically modified Arabidopsis with PMRP function gaining and PMRP function lost and phenotype analysis.(1) Arabidopsis with PMRP overexpression showed enlarged leaf and lengthened root. T-DNA insertion mutant showed short and small size. The bolting number of mutant was fewer than that of wild type, and the bough of the mutant often extended out from one side. This indicated that PMRP is significant in the growth and polarity formation of Arabidopsis.(2) The morphology observation of leaf epidermal cells showed that the abaxialcells in T-DNA insertion mutant were more compact than those in the wild type, and manifested adaxial features. The adaxial cells of Arabidopsis leaf with PMRP overexpression manifested the morphological characteristicsof abaxial cells. This further verified the regulatory effect of PMRP in polarity establishment.(3) Anthocyanin content analysis was performed in the leaf of genetically modified Arabidopsis with PMRP overexpression. The result showed that the anthocyanin content was significantly higher than that of the wild type, indicating the regulatory effect of PMRP on anthocyanin.4. Mechanism study of PMRP in regulating the growth and development of Arabidopsis(1) Mechanism analysis of PMRP in regulating the polarity establishment of ArabidopsisIn order to identify the regulatory mechanism of PMRP in polarity establishment, RT-PCR was adopted in this study to quantify the expression of the polarity-related genes in the gain of function mutant of PMRP. The result showed that the expression of the genes related to leaf growth increased, including WOX1and PRS. The expression of REV, which controls vascular bundle differentiation also increased, while the expression of the genePHB related to adaxial differentiation decreased. Other polarity-related genes (ARF3,AS1,KANAD1,AE7and YABBY2) showed no significant changes. This indicated that PMRP may control the growth of Arabidopsis through regulating the polarity-related genes above.(2) The influence of BR on the expression of polarity-related genes ofArabidopsisIn this study, Col-0Arabidopsis of4-week-growth was treated with100μM of2,4-epibrassinolide (eBL) with treating time of3,6,9,12,18,24h respectively. Results of RT-PCR showed that the expression quantity of YABBY2, FIL, AS1and WOX1dropped and then increased, reaching the lowest point in about the12th hour. The expression of PHB and PHV genes were constantly down regulated. No significant changes were noted in WOX2and AE7genes. The expression of PRS and ARF3genes showed constant increase. The expression changes of PRS and PHB were in accordance with those in the gain-of function mutant of PMRP.