The Study Of Arabidopsis Flavanone-3-hydroxylase In Regulating Abiotic Stress Tolerance

Plants have to undergo a variety of environmental stresses during their life cycle, such as drought, high salinity, high temperature, pathogen attack and mechanical wounding. Although the stresses are divergent, they unanimously disturb the redox balance of plant cells, which result in the accumulation of reactive oxygen species (ROS) and subsequent oxidative stress.By testing different concentrations of methyl viologen (MV), we employed0.1μM MV as the final selection condition. After high throughput screening of EMS mutated M2plants, we deteced several mutants and one of them was analyzed further. In addition to the increased sensitivity to MV, this mutant also displayed transparent testa. We found that the mutation occurred at At3g51240through map-based cloing. This was a new allele of known TT6(Transparent Testa6) and we named the mutant as tt6-5. Both the complementation and hybridization experiment confirmed the reliability of the map-based cloing result.The responses of tt6-5to various abiotic stresses were examined and apparent phenotypes under oxidative stress, high salinity and ABA treatment conditions were observed. Under MV and high salinity conditions, the germination and growth of the tt6-5mutants were all suppressed obviously. When it was treated with ABA, the germination of tt6-5was faster than that of WT while the growth status was worse than WT. The complemented lines can recover all the phenotypes of the mutant while overexpression of TT6resulted in co-suppression and transgenic plants displayed mutant phenotypes.qRT-PCR and GUS staining results revealed that TT6was ubiqitously expressed in seedlings. During the reproductive stage, TT6was mainly detected in the cauline leaf and flower and relative lower expression in rosette leaf. However, TT6could not be detected in the lateral root. Subcellular location analysis suggested that TT6mainly located in the nucleus and cytomembrane.Flavonoids, an important category of secondary metabolites, were implicated in diverse cellular processes, such as antioxidation, affecting plant growth and responsing to biotic and abiotic stresses. F3H (flavanone-3-hydroxylase) catalyzes the third step of flavonoid biosynthesis pathway, which convert naringenin into dihydrokaempferol. HPLC analysis result showed that tt6-5accumulated higher naringenin and lower dihydrokaempferol. The total flavonoid content was also markedly reduced in tt6-5mutant. Accordingly, the tt6-5plants displayed lower total antioxiant capacity and accumulated more H2O2and MDA (malondialdehyde) especially under stressed conditions. Moreover, the expression levels of oxidative stress responsive genes were higher than that of WT plants. These results suggested that flavonoids play important roles in ROS scavengeing and abiotic stress tolerance.