BjuB.CYP79F1 Regulates Synthesis of Propyl Fraction of Aliphatic Glucosinolates in Oilseed Mustard Brassica juncea: Functional Validation through Genetic and Transgenic Approaches

Abstract

Among the different types of methionine-derived aliphatic glucosinolates (GS), sinigrin (2-propenyl), the final product in 3C GS biosynthetic pathway is considered very important as it has many pharmacological and therapeutic properties. In Brassica species, the candidate gene regulating synthesis of 3C GS remains ambiguous. Earlier reports of GSL-PRO, an ortholog of Arabidopsis thaliana gene At1g18500 as a probable candidate gene responsible for 3C GS biosynthesis in B. napus and B. oleracea could not be validated in B. juncea through genetic analysis. In this communication, we report the isolation and characterization of the gene CYP79F1, an ortholog of A. thaliana gene At1g16410 that is involved in the first step of core GS biosynthesis. The gene CYP79F1 in B. juncea showed presence-absence polymorphism between lines Varuna that synthesizes sinigrin and Heera virtually free from sinigrin. Using this presence-absence polymorphism, CYP79F1 was mapped to the previously mapped 3C GS QTL region (J16Gsl4) in the LG B4 of B. juncea. In Heera, the gene was observed to be truncated due to an insertion of a ~4.7 kb TE like element leading to the loss of function of the gene. Functional validation of the gene was carried out through both genetic and transgenic approaches. An F2 population segregating only for the gene CYP79F1 and the sinigrin phenotype showed perfect co-segregation. Finally, genetic transformation of a B. juncea line (QTL-NIL J16Gsl4) having high seed GS but lacking sinigrin with the wild type CYP79F1 showed the synthesis of sinigrin validating the role of CYP79F1 in regulating the synthesis of 3C GS in B. juncea.

Genome-wide association mapping within a single Arabidopsis thaliana population reveals a richer genetic architecture for defensive metabolite diversity

“…This is consistent with the finding that among all biosynthetic enzymes, CYP79F2 exerts the strongest effect on pathway flux, with an outsized effect on propyl GSLs (i.e., GSLs with 3C side-chain lengths) [12]. Functional polymorphism at a CYP79F gene also underlies a QTL affecting the propyl fraction of GSLs in Brassica juncea [51], and separately underlies adaptive variation in the proportion of GSLs derived from branched-chain amino acids relative to methionine in Boechera stricta [52]. The association at CYP79F paralogs was recovered in our re-analysis of one European Arabidopsis dataset (Fig.…”

Exogenous Methyl Jasmonate and Salicylic Acid Induce Subspecies-Specific Patterns of Glucosinolate Accumulation and Gene Expression in Brassica oleracea L.

“…In Arabidopsis , MeJA was reported to induce some CYP genes that enhanced the indolic glucosinolate production by 3- to 4-fold [24]. However, in some recent studies the BjuCYP83A1 and CYP79F1 genes were found to regulate aliphatic glucosinolate biosynthesis in B. juncea [51,52].…”

Comparative analysis of glucosinolates and metabolite profiling of green and red mustard (brassica juncea) hairy roots

“…The genes involved in the GSL biosynthesis pathways have been identified in many plant species. However, only a few genes, such as cytochrome P450 83A1 (CYP83A1) (Meenu et al 2015), dihomomethionine N-hydroxylase (CYP79F1) (Sharma et al 2016), and transcription factor MYB28 (Augustine et al 2013), have been reported in mustard.…”