Regulating Of Anthocyanin Biosynthesis Under Abiotic Stresses In Plant

Anthocyanins are a class of natural water-soluble pigments,which widely exist in all parts of plants.For plants,anthocyanins can not only attract insect pollination and help them spread seeds and reproduce,but also improve plant stress resistance to a variety of adverse stresses.For humans,anthocyanins have rich nutritional value and health care functions.Anthocyanins can antioxidant,anti-radiation,anti-tumor,sunscreen,protect vision,improve memory,etc.Therefore,the research on the regulation of anthocyanin synthesis under abiotic stress has important practical significance for cultivating high stress resistant crops as well as economic plants with high anthocyanin content.Although abiotic stress can promote anthocyanin accumulation,the underlying regulatory mechanisms remain largely unknown.MicroRNA(miRNA)is a kind of endogenous non-coding small molecular RNA with a length of about 19-24 nt.It plays important roles in regulating plant growth,development and resistance to various stresses.Our previous study has shown that miR858 a is a positive regulator of anthocyanin accumulation in plants.It can inhibit the translation of MYBL2 gene,a negative regulator in anthocyanin synthesis pathway,and MIR858 A promoter contains cis-acting elements potentially bound by multiple abiotic stress response factors.Therefore,it is speculated that abiotic stresses may regulate anthocyanin biosynthesis mainly through miR858a-MYBL2 module in plants.In current work,we selected three abiotic stress factors to analyze how they affect anthocyanin accumulation in plant,including cold stress(4 ℃),salt stress(100 m M Na Cl)and drought stress(100 m M mannitol).The anthocyanin contents of wild-type Arabidopsis seedlings were significantly increased after the above three abiotic stress treatments,meanwhile the results of reverse transcription real-time quantitative PCR(RT-q PCR)showed that miR858 a along with genes involved in anthocyanin biosynthesis were induced in varying degrees.Sequence analysis showed that the MIR858 A promoter region contains multiple Crepeat/DRE motifs in response to abiotic stresses,which may be directly bound by Crepeat/DRE binding factor 1(CBF1).In order to explore whether transcription factor CBF1 can interact with MIR858 A promoter and regulate MIR858 A expression,we constructed CBF1 prokaryotic expression vector to induce and purify CBF1 proteins.The EMSA experiment confirmed that CBF1 protein could bind to the MIR858 A promoter.Next,experiments such as transient transformation in protoplast or tobacco leaves indicated that CBF1 inhibited the transcriptional activity of MIR858 A.In order to further reveal the relationship between CBF1 and anthocyanin accumulation,we constructed CBF1 overexpression transgenic plants.Compared to the wild type,the anthocyanin contents in CBF1 overexpression plants were dramatically decreased while the anthocyanin contents in cbf1 mutant significantly increased.Thus,these results indicate that CBF1 is a negative regulator of anthocyanin synthesis,which is consistent with the result that CBF1 inhibits the positive regulator MIR858 A of anthocyanin synthesis.Stress treatment of the pro MYBL2::MYBL2-HA/WT transgenic plants showed that three abiotic stresses could inhibit the translation of MYBL2,a negative regulator of anthocyanin synthesis.Taken together,our results preliminarily clarify that CBF1-MIR858AMYBL2 pathway represents a regulatory mechanism for plants to promote anthocyanin synthesis in response to external abiotic stress,which provides an important reference basis for creating plant germplasm resources with high anthocyanin content.