AbstractsBiology & Animal Science

Transcriptional regulation of chloroplast ascorbate peroxidases in Arabidopsis thaliana

by Wenguo Cai

Institution: Freie Universität Berlin
Department: FB Biologie, Chemie, Pharmazie
Degree: PhD
Year: 2015
Record ID: 1113466
Full text PDF: http://edocs.fu-berlin.de/diss/receive/FUDISS_thesis_000000098507


In this project, the promoter regions of sAPX (At4g08390) and tAPX (At1g77490) were cloned to pHGWFS 7.0 vector and transform to Arabidopsis thaliana. The constructs allow the analysis of the promoter activities by measuring GFP and GUS. Three independent transformation lines were studied for each promoter in order to study the regulation by a spectrum of environmental stimulus. The promoter of tAPX gene was found to be controlled by chloroplastic H2O2. Application of chemicals blocking photosynthestic electron transport upstream of photosystem I suppressed the tAPX promoter activity. However, methyl viologen causing chloroplast reactive oxygen species production increased the tAPX promoter driven reporter expression. mRNA levels of tAPX in 2cpa, 2cpb, and 2cpa2cpb mutants, which have higher chloroplastic H2O2, were higher than in wild type plants. These results suggest a role of chloroplastic H2O2 in regulating tAPX gene. tAPX promoter activity was also shown to be elevated mechanic wounding. Although wounding caused cytosolic H2O2 production, wounding induction of of tAPX promoter activity was not primarily mediated by H2O2, since wounding in old leaves increased H2O2 but did not change tAPX promoter activity. Other factors, such as cold, drought, very strong high light, jasmonic acid or salicylic acid application, and exogenous feeding of H2O2, etc. made plants accumulate H2O2 in the cytosol, but did not activate the tAPX promoter. Taken these results together, chloroplatic but not cytosolic H2O2 regulate tAPX promoter. tAPX promoter was also shown to be regulated by light in a spectrum specific manner. Long term exposure to dark completely suppressed the activity of the tAPX expression. Exposure of etiolated plants to 8 h white light, blue light, and far red light, but not red light recovered the tAPX promoter activity. These results suggest that chloroplast H2O2 and photoreceptor mediated signals together regulate the tAPX promoter. Promoter truncation assays localized the important regulating region of tAPX promoter to -1295 to -734 bp upstream of translation start site. In silico prediction suggested that ARR2, ARR10, LEC2, SPL3, and SPL8 are putative transcription factors binding and regulating tAPX. Except LEC2, whose mutation caused a lethal phenotype, plants with T-DNA insertion within these genes are analyzed. mRNA levels of tAPX in spl3 and spl8 were shown higher than in wild type plants. Therefore, spl3 and spl8 are likely transcription factors negatively regulating tAPX. sAPX was found less intensively expressed in leaf mesophyll cells. Greater promoter activitywas observed in leaf veins. The promoter activity of sAPX in mesophyll cells and vasculature cells are differently controlled. Promoter was shown up-regulated by light in mesophyll cells, while down-regulated in leaf vasculature. Generally, stresses like cold, drought increases sAPX promoter activity. Block of photosynthetic electron transport suppresses sAPX promoter. The photosynthesis regulation of sAPX promoter was related to…