Study On The Functional Difference Of Flavonols And Anthocyanins In The Abiotic Tolerance In Plants

Wheat is one of the most important crops worldwide.But its productivity are adversely affected by abiotic stresses such as drought.A new somatic hybrid introgression line Shanrong No.3(SR3)was generated.Previous omics-study showed that a set of genes that are involved in flavonoid synthesis display differential expression patterns between SR3 and JN177.Based on this information,we compared the transcriptional patterns of genes in flavonoid synthesis,clone a wheat flavonol synthase gene TaFLS1 and a wheat anthocyanin synthase gene TaANS1.Flavonols and anthocyanins are synthesized with dihydroflavonols as precursor under the catalysis by FLS,and DFR/ANS,respectively.O?r previo?s works indicated that TaFLSl overexpression enhanced drought tolerance via reducing the water loss rate.In this work,we constructed TaANS1 overexpression Arabidopsis lines,and used TaFLS1 and TaANS1 overexpression lines to compare the difference in the roles of anthocyanins and flavonols in stress response.Part 1:TaANSl encodes an ANS with in vivo activity.First,we measured the contents of anthocyanins and total flavonoids in TaANS1 overexpression wheat lines,TaANS1 overexpression(OE)lines and AtANSl mutant(ans),and their corresponding witdtypes(WT).Compared with WT,the contents of anthocyanins and total flavonoids were increased significantly in the overexpression lines,but they were reduced in ans.Consistently,the OE seedlings had blacker hypocotyls and petiole,but ans brighter.These data indicate that TaANS1 have in vivo activity.We determined the contents of quercetin and kaempferol(two kinds of common flavonols in higher plants)by HPLC and HPLC-MS analysis.The results showed that kaempferol and quercetin content was reduced in TaANSl OE lines,but was improved in ans.Real-time PCR analysis showed that in TaANS1 OE lines,the expression of genes involved in anthocyanin synthesis branch were significantly down-regulated,but those involved in flavonol synthesis branch were up-regulated.In combination with the findings of TaFLS1 overexpression lines,we found that the genetic manipulation of the wheat key enzymes of flavonol and anthocyanin synthesis branches also change the dynamic balance,and flavonoid enrichment can positively feedback regulate their synthesis branches.2.The role of TaANSl in drought response.Water-withholding assay showed that TaANS1 overexpression reduced drought tolerance of wheat and Arabidopsis,and the water loss rate of detached leaves was higher in the OE lines than in WT,but lower in ans,indicating that the reduced drought tolerance by TaANSl overexpression was achieved via increasing water loss.Under the control condition,the OE lines had more closed stomata and smaller stomatal aperture than WT.After exposure to ABA,the aperture became smaller,but the reduction strength was weaker in the OE lines,resulting in their stomatal aperture larger than WT.The results showed that alike TaFLS1 overexpression,TaANS1 overexpression also dually regulated the stomatal movement,but the dual effect was opposite.The addition of cyanidin but not delphinidin mimicked the effect of TaANS1 overexpression on stomatal movement,suggesting cyanidin may play an important role in stomatal movement.Under the control condition,in comparison with WT,the OE lines accumulated more H2O2 in guard cells,but ans produced less.After exposure to ABA,the difference was opposite:when compared to WT,the OE lines accumulated less H2O2 in guard cells,but ans had more content.The results indicated that the dual effect of anthocyanin on stomatal movement is achieved via dual effect on H2O2 in guard cells.After drought treatment,Arabidopsis accumulated more flaovnols,but the increase extent was the largest in ans,and the smallest in TaANS OE lines,resulting in the content of flavonols was significantly lower in the OE lines than in WT.In flsl-1,the application of anthocyanin had no effect on stomatal movement and H2O2 in guard cells,but in ans,the application of flaonols performed the dual effect.In chs,the dual effect on stomatal movement and H2O2 in guard cells was replicated by the application of flavonols but not anthocyanin.The results showed that the role of anthocyanin in drought tolerance and stomatal movement was accomplished by the shift of the balance between flaovnol and anthocyanin branches.After MV treatment,the growth inhibition and leaf etiolation were more pronounced in the OE lines than in WT.The results showed that anthocyanin enrichment reduce abiotic stress tolerance largely owed to the decrease of flavonol content,and consequently the decrease of total anti-oxidation capacity.