Identification And Functional Analyses Of LAR And ANR Encoding Key Enzymes In Proanthocyanidins From Mulberry

Proanthocyanidins are important secondary metabolites that are widely found in plants.They play important role in plant resistance and participate in plant physiological and biochemical processes.They also have various activities such as anti-oxidant,anti-tumor,and anti-thrombosis activity.An appropriate amount of proanthocyanidins in forage can reduce swelling disease in ruminants.Therefore,it is of great significance to study the biosynthetic pathway of proanthocyanidins to improve the quality of mulberry.We studied two important genes in the proanthocyanin synthesis pathway of Morus notabilis were studied: LAR,encoding leucoanthocyanidin reductase,and ANR,encoding anthocyanidin reductase.The main experimental results can be summarized follows:1? Cloning and Bioinformatics analysis of MnANR and MnLAR genesIn bioinformatics analyses,one ANR and one LAR gene were identified in the Mulberry genome database,and the corresponding genes were cloned from various tissues of M.notabilis.Gene structural analyses of ANR and LAR revealed that the number and location of introns were the same as those in their homologs in other plants.A multi-sequence alignment revealed their high similarity to homologous genes in other species.In an evolutionary analysis MnANR and MnLAR had close relationship with homologous genes of dicotyledonous woody plants.2? Expression analysis of proanthocyanidin biosynthesis-related genes during maturation of different colored mulberry fruitsAnalyses of tissue-specific expression in mulberry showed that ANR and LAR transcript levels were the highest in leaves,followed by fruit,and that both genes were expressed in other tissues as well each tissue,the transcript level of LAR was much higher than that of ANR.This difference in expression suggests that there may be different in function and regulation among different members of the same gene family.We compared the the expression patterns of ANR and LAR genes during the mulberry fruit ripening process between Yue Shen Da 10(purple fruit)and Zhen Zhu Bai(white fruit).The results showed that the transcript levels of LAR and ANR decreased during fruit ripening.At various stages during fruit ripening,the contents of catechin,epicatechin,which are the products of reactions catalyzed by ANR and LAR,were determined by UPLC.The results showed that the total accumulation pattern of catechin and epicatechin was first accumulated and then decreased during the full ripening period and lowest in the full maturity period,the accumulation trend of metabolites first accumulated and then decreased with the decrease of gene expression.The higher the expression level of the gene,the higher the amount of metabolite accumulation,indicating that the transcript levels of ANR and LAR were related to proanthocyanidin accumulation.Both ANR and LAR were up-regulated by Botrytis cinerea,ultraviolet radiation,polyethylene glycol,and methyl jasmonate treatments.3? Preliminary study on functions of genes MnANR and MnLAR encoding key enzymes in proanthocyanidin biosynthesis in mulberryOverexpression of MnANR and MnLAR in tobacco resulted in higher accumulation of proanthocyanidins in the leaves of transgenic lines than in wild-type tobacco.In terms of phenotype,the flowers of the transgenic lines were lighter or white.To explore the reasons for the change in flower color,we analyzed the transcript levels of genes in the flavonoid synthesis pathway,and quantified proanthocyanidins in tobacco flowers.The results showed that the expression of most genes in the flavonoid biosynthetic pathway was inhibited in the transgenic plants.This explained the lighter flower color of the transgenic tobacco plants.The contents epicatechin and catechin were increased in both overexpressing lines.The results of previous studies have suggested that MnANR and MnLAR may have epimerase activity,which could convert anthocyanins into catechins and epicatechins.Alternatively,catechins and epicatechins may be epimerized,and multimeric proanthocyanidins underwent non-stereospecific depolymerization.We evaluated the disease resistance of the wild-type and transgenic tobacco lines,and found that the transgenic lines were resistant to B.cinerea.In in vitro experiments,the growth of B.cinerea was inhibited more strongly by proanthocyanidin extracts from leaves of transgenic plants than by those from wild-type tobacco.Together,these results indicated that overexpression of MnANR and MnLAR can improve the disease resistance of tobacco.