Diversity and Characterization of Curcumin Biosynthetic Genes and Transcription Factors from Curcuma Spp.

Abstract

Diversity and characterization of curcumin biosynthetic genes and transcription factors from Curcuma Spp.

Abstract Turmeric, one of the widely used spices and herbal medicines, is rich in biologically active curcuminoids, a phenylpropanoid derivative. Identification of genes and transcription factors involved in curcumin biosynthesis is necessary for genetic manipulation. Transcriptome sequencing could be judiciously used as a cost-effective method for developing genomic information in non-model species. RNA-seq data generated in our study enable identification of DEGs involved in specific pathway of curcumin, paralogs of genes involved in the pathway as well as identified molecular biomarkers for curcumin. This, study has focused on only two important genes phenylalanine ammonia lyase and O-methyltransferase due to limitations in time and resources. The candidate isoform involved in the pathway were identified by co-expression analysis that correlated gene expression to curcumin content under different field experiments. Invariably all the identified candidate genes correlated with curcumin under all conditions. These genes were cloned fully, structure of gene and protein analysed by in silico, and docking studies, to further support the co-expression analyses. The study enabled optimization of several techniques like SMARTER PCR, RACE PCR, co expression models etc. which may be extended to other candidate genes and crops. Curcuminoids are phenylpropanoid derivatives and the biosynthesis pathway is controlled by several transcription factors (TFs). bHLH, WD40 and MYB TFs could be the most important TFs regulating curcumin biosynthesis in turmeric as evident but the presence of 20 TFs that showed differential expression in comparative transcriptomes under contrasting curcumin. Among these, two bHLH and one WD40 TFs showed maximum comparative fold change and negative correlation vis-à-vis curcumin in qRT-PCR based co expression analysis. The results of comparative transcriptome and qRT-PCR analysis were in congruence, indicating their putative role as negative regulators. The characteristics of the TFs by full length gene mining, phylogenetic tree analysis and molecular modelling confirmed their regulatory capacities and protein structures. MicroRNA are a class of small endogenous non coding RNAs (sRNA) that could play a major role in gene regulation. miRNA mediated regulation of biosynthesis of curcumin is an unexplored area. We have identified through transcriptome analysis, about 29 miRNAs that showed differential expression with respect to curcumin in turmeric accessions with contrasting curcumin content. Expression of one of the conserved miRNAs viz., miR319 showed a negative corelation to curcumin when plants were grown under different light regimes favouring differential curcumin accumulation in rhizomes. This miRNA is a potential candidate for further studies on regulation of biosynthesis of curcumin. The iTRAQ mediated analysis of whole protein profiles helped in identifying proteins such as phenylalanine ammonia lyase, caffeoyl CoA O-methyltransferase, diketide CoA synthase and curcumin synthase 1 differentially expressed from rhizomes of turmeric. The study indicated an obvious difference in profiles of the differentially expressed proteins among the high and low curcuminlines. These differentially expressed proteins, correlated well with the corresponding gene counterparts identified from a comparative transcriptome based on contrasting curcumin.These proteins and genes can be considered as biomarkers to indicate high curcumin levels.Being dedicated candidates of curcumin biosynthesis they may be used individually or combined to indicate the curcumin status of the crop. Further works aimed at deciphering the functions of these proteins may add greater insights into their roles in biosynthesis. We developed a qRT-PCR based simple screening strategy for high and low curcumin lines and Ct values of ClPKS11, ClPAL2, ClOMT2 and ClOMT3 used for screening high/low curcumin accessions. It can be concluded that all high curcumin lines may give a Ct value below 22, while low curcumin lines show Ct values above 25. The method is non-destructive, requires very low quantities of plant tissue and can be completed within a day. Methodology is simple and may be used by the pharmaceutical industries, farmers and breeders for screening high/low curcumin lines at early stages of development. The curcumin-based biomarkers may be used singly or together for more accuracy. The technique developed could be employed as a useful tool in different scientific fields for characterising and estimating the curcumin status in plants during development.