Physiological And Molecular Responses Of Blueberry Anthocyanin Synthesis To Light Intensity

Blueberry,is a small berry fruit tree of the azalea family(Ericaceae)and cranberry(Vaccinium spp.).It is a perennial semi-evergreen shrub and is widely planted all over the world.Because of its peculiar flavor,soft taste,rich nutrition,it is a fine product among fresh fruits,and it is loved by consumers.Anthocyanin is the most important active ingredient in blueberry fruit and has strong antioxidant capacity.The blueberry fruit contains the highest content of anthocyanin in all fruits and vegetables,which has a wide range of development prospects and values.Anthocyanin synthesis and accumulation process are closely related to plant development and external environmental conditions.Among environmental factors,light intensity is one of the most important that affecting the biosynthesis of anthocyanin.So can the anthocyanin content in blueberry fruits be further increased by appropriately adjusting the light intensity during production? How to regulate?.Therefore,the analysis of light intensity emphasizes the molecular mechanism of controlling anthocyanin biosynthesis has theoretical value and practical significance.Previously,at the molecular level how light intensity induces the synthesis and accumulation of blueberry anthocyanin glycosides is not clear.At present,study on blueberry anthocyanin glycoside is mostly based on the phenotype and physiological level.This limits the technical research on artificially regulating the anthocyanin content of blueberry fruits under insufficient light conditions.In this paper,rabbit-eye blueberry ‘pink blue' is used as the research material,and 4 light intensities are set(light transmittance 25%,Q;light transmittance 50%,F;light transmittance 75%,H;light transmittance 100%,CK),respectively in three different development stages of the fruit(80d after flowering,S1;95d after flowering;S2 and 110 d after flowering,S3).Studies have been carried out on the anthocyanin content of blueberry fruits and the activity change characteristics of related synthase under different light intensities,combined with transcriptome sequencing and metabonomic analysis,and unearthed the key gene to the anthocyanin synthesis by light intensity in blueberry,and perform cloning and functional verification of the CDS region of key regulatory genes.The main results obtained in this study are as follows:(1)Insufficient light intensity significantly reduces fruit anthocyanin content and related physiological enzyme activities.Insufficient light intensity significantly delayed the color change time of blueberry fruits and reduced the anthocyanin content in the blueberry fruit.The anthocyanin content in the fruit decreased with the decrease in light transmittance at the three developmental stages,which were 19.26%,58.53% and 38.34% of the control at the lowest value,respectively.The highest accumulation of anthocyanin glycosides occurred at the middle stage of fruit development(95 days after anthesis).At the same time.Insufficient light intensity significantly reduced the anthocyanin content and enzyme activity of the anthocyanin synthesis pathway(CHS,ANS,F3'H,F3'5'H,PAL,F3 H,DFR,UFGT)in the fruit.The related enzyme activity in shading treatment is only 7.75%-58.91% of CK.At 15 days of re-lighting,the content of anthocyanin in the fruit and the key enzymes in the anthocyanin synthesis pathway(CHS,F3 H,F3'5'H,F3'H,UFGT,DFR,ANS)activity increased significantly,returned to normal levels,and remained basically unchanged after 30 days of rejuvenation.Correlation analysis found that the flavonoid 3',5' hydroxylase(F3'5'H)in the anthocyanin synthesis pathway was extremely significantly positively correlated with the anthocyanin content.(2)Insufficient light intensity reduces the types of fruit anthocyanins.Insufficient light intensity was reduced the types of anthocyanin in the fruit.30 and 13 anthocyanin components were detected in CK and Q treatment fruits,respectively.The content of Delphinidin-3-O-arabinoside,Peonidin-3-O-arabino-side,Petunidin-3-O-arabinoside increases with the increase of light intensity,but missing during Q treatment,especially delphinidin-3-O-Arabinosides account for 46% of the total anthocyanin content,which is a typical representative of light-inducible anthocyanin glycosides.(3)Light intensity can affect the accumulation of anthocyanin in blueberry fruits by regulating the expression level of related genes.Transcriptome sequencing analysis of fruits was performed based on different light intensities.It was found that the number of CK-vs-Q up-regulated and down-regulated genes were 9,711 and 13,598,respectively.The number of CK-vs-F up-regulated and down-regulated genes were 8,433 and 12,259,respectively.CK-vs-H up-regulated genes were 7824,and down-regulated were 11015 genes.While CK-vs-H,CK-vs-F and CK-vs-Q jointly up-regulated were 5599 genes.Moreover,16 gene families were screened using GO and KEGG enrichment analysis of DEGs that expressed anthocyanin synthesis structural genes.Among them,VcF3'5'H4,which encoded F3'5'H enzyme that may play a key role in the regulation of blueberry fruit anthocyanin synthesis by light intensity.Finally,the key regulatory genes of blueberry anthocyanin biosynthesis were identified,including VcbHLH004,VcERF061,VcNAC072 and VcMYB1.(4)VcF3'5'H family genes may be involved in light intensity regulation on blueberry anthocyanin synthesis.In this study,a total of 17 blueberry VcF3'5'H family genes were identified,and the 17 genes were analyzed by bioinformatics and gene expression level.It is found that the genes of this family are basically hydrophilic proteins,and the amino acid sequences are relatively conserved,and the subcellular localization is mainly in the endoplasmic reticulum.This family of genes contains typical light-responsive cis-acting elements such as ACE,G-box,AE-box and MYB binding sites,and the gene expression levels are higher under 100% light transmittance(CK)treatment,especially VcF3'5'H4.(5)VcMYB1 and VcbHLH004 are positive regulators of blueberry anthocyanin glycoside synthesis by light.VcF3'5'H4,VcMYB1 and VcbHLH004 were cloned and the overexpression vector was successfully constructed.Tobacco subcellular localization found that VcMYB1 and VcbHLH004 were located in the nucleus,while VcF3'5'H4 were located in the endoplasmic reticulum.Agrobacterium-mediated transformation of VcMYB1 and VcbHLH004 genes were performed in tomato.The color of leaves and fruits of the overexpression transgenic lines were darkened.The total anthocyanin content in the fruits were increased,and the increase rate reaches 45.12%-64.63%,which is significantly different from wild seedlings(P<0.05).Real-time fluorescent quantitative PCR detection found that the expression levels of VcMYB1 and VcbHLH004 genes in the fruits of the transgenic plants were high,while the expression levels of the target genes in the wild seedlings were basically not detected.The results of this study not only provide a theoretical basis for artificially regulating the anthocyanin content of blueberry fruits under the condition of insufficient light,but also provide new understanding of the theoretical mechanism of light intensity regulating blueberry fruit anthocyanin synthesis.