Identification And Functional Analyses Of Key Genes Related To Betalain Biosynthesis In Pitaya

Pitaya（Hylocereus spp.）is a new,promising fruit crop in China.Pitaya is popular with growers and consumers due to its adaptability,attractive colors and shape,delicious taste as well as high nutritional value such as betalains.Betalains exhibit various biological activities in term of antioxidant,anti-inflammatory,hypoglycemic,antiproliferative,cardioactive,radioprotective,neuroprotective,diuretic and hypolipidemic.Current,pitaya is the only commercial cultivation of fruit containing abundant betalains for consumer.Therefore,elucidation of the metabolites and betalain biosynthesis in pitaya is crucial for improving fruit quality and cultivar breeding.Betalains are divided into betaxanthins（yellow-orange pigments）and betacyanins（redviolet pigments）.The main synthesis steps are as follows:L-tyrosine is hydroxylated and oxidated to L-DOPA and form cyclo-DOPA by both tyrosinase（TYR）or cytochrome P450 enzyme（CYP76ADs）.L-DOPA is then converted to betalamic acid by DOPA 4,5dioxygenase（DODA）.The diversity and stability of betalains are increased by two glycosylation processes.Betalain-related glucosyltransferases catalyze the 5-O or 6-O glucosylation of betanidin or the 5-O glucosylation of cyclo-DOPA.In addition,betaxanthins are produced by spontaneous condensation reaction between the betalamic acid and the amino group or amino acid.However,genes involved in pitaya betalain biosynthesis are not fully clarified yet.In this study,changes in sugar,organic acid,total phenol,total flavone and antioxidant activity were investigated during developmental stages of different pitaya fruit.The differentially expressed genes and proteins involved in betalain biosynthesis were screened using genome,RNA-Seq and iTRAQ.The functions of candidate genes involved in betalain biosynthesis were identified by virus induced gene silencing（VIGS）,tobacco transient expression,yeast recombinant expression and genetic transformation system.The main results were as follows:1.Changes of chlorophyll,betalains,phenols,flavonoids,total starch,sugars,organic acids and total antioxidant activity were investigated during fruit development of the three pitaya cultivars.Betalain contents were increasing while total chlorophyll contents were decreasing.Sugar contents in pulps increased rapidly while contents of the major organic acids decreased after the 25th DAP.Citramalic acid showed an increasing trend up to the 23rd DAP and higher than the other organic acids.Total antioxidant activity,phenol and flavonoid contents were increased during fruit development of the three pitaya cultivars which is consistent with changes of betalain contents.There was a strong positive correlation between betalain contents and total antioxidant activity,phenol and flavonoid contents in pitayas.Those results suggested that the accumulation of sugar,organic acid and citramalic acid are contributed to the production of pitaya betalains.2.Based on genome data,54 candidate genes i.e.ADH（2 genes）,CYP76AD（15 genes）,DODA（3 genes）,GTs（34 genes）related to betalain biosynthesis were obtained by alignment.Seven differentially expressed gene were further identified by RT-qPCR.The candidate genes were HU03G02979.1（ADHα）,HU03G00480.1（CYP76AD）,HU03G00481.1（CYP76AD）,HU03G01342.1（DODAα1）,HU08G01572.1（DODAαl）,HU01G00156.1（B5GT）and HU07G00240.1（CDOPA5GT）.3.RNA-Seq and iTRAQ-based proteomic analyses were used to screen key genes and proteins involved in betalain biosynthesis in pitaya fruits.8,871 differentially expressed genes（DEGs）including 4,107 up-regulated genes were identified using a threshold false discovery rate（FDR）of ≦1%and Fold change=2.GO and KEGG analysis showed that the DEGs significantly enriched in pigment process,flavone and flavonol biosynthesis and flavonoid biosynthesis.The DEGs involved in betalain biosynthesis were analyzed using RTqPCR.Seven DEGs of betalain biosynthesis were identified,such as DODA（comp30986_c0_seq1 and comp37692_c0_seq1）,CYP76AD（comp16058_c0_s eq1,comp35191_c2_seq2 and comp36993_c0_seq4）,GT（comp32889_c0_seq1）and DDC（comp37261_c1_seq14）.1,946 differentially expressed proteins including 936 up-regulated proteins were identified,using a threshold false discovery rate（FDR）of≦1%and Fold change=1.5.According to the combination analysis of RNA-Seq and iTRAQ,57 differentially expressed proteins including 36 up-regulated proteins and 21 down-regulated proteins were identified using a threshold of p-value<0.05,FC（pro）>1.5 and FC（RNA）>2.They belonged to "phenylpropanoid biosynthesis","tyrosine metabolism","flavonoid biosynthesis","ascorbate and aldarate metabolism","betalain biosynthesis" and"anthocyanin biosynthesis".In betalain biosynthesis pathway,several proteins such as CYP76AD3（comp 16058_c0_seq1₄）and 4,5-DOPA dioxygenase extradiol（comp37692_c0_seq1₇）were identified.4.Subcellular localization assay showed that HpDODAα1 and HpDODAα2 were localized in the nucleus and plasma membrane.Yeast heterologous expression analysis showed that betaxanthins were produced by HpDODAα1 in the yeast cells with either of the two genes expressed.Over-expression of HpDODAα1 in Nicotiana benthamiana or Arabidopsis thaliana,betaxanthins were significantly produced with the addition of L-DOPA compared to that no betaxanthins were produced overexpression of HpDODAα2 in N.benthamiana or A.thaliana.Scale color and betalain contents showed similar phenotypic traits when HpDODAα1 or HpDODAα2 was silenced.Compared to control,pitaya scale color was still green and betalain contents were decreased when HpDODAα1 or HpDODAα2 was silenced in pitaya scales.The effect of silencing of HpDODAα1 was better than that of HpDODAα2.Those results suggested that HpDODAα1 plays an important role in betalain biosynthesis in pitaya.5.Subcellular localization assay showed that HpCYP76AD1,HpB5GT1 and HpCDOPA5GT were localized in the nucleus and plasma membrane.Betalain contents decreased and expression level decreased except for HpCDOPA5GT when HpCYP76AD1,HpB5GT1 and HpCDOPA5GT were silenced in tobacco.Tobacco transient expression analysis showed that betacyanins were not produced in the leaves with either of the genes expressed or both genes co-expressed.Betacyanins were only produced when HpCYP76AD1,HpDODAα1 and HpCDOPA5GT genes were co-expressed.Those results suggested that HpCYP76AD1,HpDODAα1 and HpCDOPA5GT can produce betacyanins in pitaya.6.CYP76AD1,DODAα1 and CDOPA5GT were cloned from the pulps of ’Guanhuahong’and ’Guanhuabai’ pitayas.No different gene loci related with formation of pigment was observed in the cDNA and promoter sequences of CYP76AD1,DODAα1 and CDOPA5GT between ’Guanhuahong’ and ’Guanhuabai’ pitayas.