Study On Accumulation Of Anthocyanin And Its Mechanism In Response To Mechanical Damage In Foxtail Millet

Foxtail millet,which plays an important role in dry farming,is an important cereal crop and forage crop in north of China.It has the characteristics of drought tolerance,barren tolerance,short growth period and long suitable sowing period.In the growth process of foxtail millet,it endures impacts from various adverse environmental,which affect its physiology and metabolism leading to slow growth and yield decline.Anthocyanin,which belongs to flavonoid,is important secondary metabolite in plant and can participate in the resistance and adaptation to biotic and abiotic stress.By now,there are a large number of reports about anthocyanin biosynthesis and metabolism in plant,but there are few studies on anthocyanin accumulation induced by mechanical damage.In this study,B104 and YG were selected as the experimental materials.The anthocyanin,photosynthetic pigment,ROS,MDA contents and antioxidant capacity in mature leaves of two varieties were measured and analyzed at different time points after mechanical damage treatment.The mature leaves of B104 were sampled at different time points after mechanical damage treatment for transcriptome sequencing analysis,in oder to select DEGs(differentially expressed genes)related to stress response and anthocyanin synthesis.Finally,the expression patterns of these DEGs were verified by q RT-PCR.In this study,the molecular mechanism of anthocyanin accumulation induced by mechanical damage was preliminarily explored,which provided theoretical basis for breeding resistant foxtail millet varieties rich in anthocyanin.The main results were as follows:(1)Physiological index analysis of mature leaves in foxtail millet at different time points after mechanical damage treatmentAfter mechanical damage treatment,the leaves veins of B104 below the folding gradually turned purple at 36 h,and the whole leaf turned purple at 42 h.By contrast,the leaves phenotype of YG did not change significantly and were still green.Compared with YG,the damage treatment had more obvious effects on the physiological indexes related to stress resistance in B104 leaves.After mechanical damage treatment,the anthocyanin,SOD and POD contents of B104 leaves increased significantly.In addition,ROS showed a trend of increasing firstly and then decreasing,while the contents of chlorophyll and carotenoid firstly decreased and then increased.The contents of soluble sugar and proline in B104 leaves increased after mechanical damage treatment,and significantly increased(18 h,24 h)before the phenotype change of leaves.Therefore,over accumulation of soluble sugar may be one of the reasons for anthocyanin accumulation in B104 leaves under the mechanical damage treatment.(2)Transcriptome analysis of mature leaves in B104 at different time points after mechanical damage treatmentAfter mechanical damage treatment of B104 leaves,10516 DEGs were obtained from the transcriptome analysis.The functional annotation analysis of DEGs in 12 h vs 0 h,18 h vs 0 h,24 h vs 0 h,36 h vs 0 h,42 h vs 0 h showed that COG,GO,KEGG databases enriched in stimulus response,biological regulation,antioxidation,signal transduction,biological defense,flavonoid biosynthesis,proline metabolism and other processes.According to the enrichment and expression pattern analysis,there were22 genes responding to stress and anthocyanin biosynthetic pathway.The genes of Si KCS5,Si POD,Si SFR2,Si UVR8,Si OXS3 were related to stress response;Si PIP1 and Si TIP1 were related to aquaporin synthesis;Si CIPK1,Si CIPK4 and Si CIPK6 were related to plant protein kinase synthesis;Si ATHB7 and Si UGT74E2 regulated the synthesis of plant hormones.The structural genes(Si CYP450,Si PAL1,Si PAL2,Si UFGT,Si DFR,Si GT,Si4CL)and transcription factors(Sib HLH,Si NAC,Si MYB)were involved in the synthesis of flavonoids and anthocyanins.(3)Analysis of expression patterns of candidate genes related to anthocyanin synthesis and response to mechanical damage treatmentThe expression patterns of candidate genes were analyzed at different time points after mechanical damage by q RT-PCR.The expression trends of these genes were basically consistent with the transcriptome results.Compared with the control,gene responding to adversity stress(Si OXS3),the transcription factors regulated to anthocyanin synthesis(Si NAC,Si MYB)and the gene(Si UGT74E2)regulated to auxin were significantly down-regulated,and the other genes were significantly up-regulated after mechanical damage treatment.The expression of genes related to anthocyanin synthesis increased significantly at 18 h,and the expression trends of Si DFR and Sib HLH were consistent.Therefore,the Sib HLH transcription factor may increase the expression of Si DFR,which in turn promoted the accumulation of anthocyanins in B104 leaves,leading to its color gradually turn red after mechanical damage.In conclusion,the anthocyanin accumulation in leaves of B104 after mechanical damage treatment was mainly caused by the the over accumulation of soluble sugar and the up-regulation of Sib HLH transcription factors and they can enhance the antioxidant capacity of of B104,thus improving the tolerance and adaptability of B104 foxtail millet to mechanical damage.