Artificial microRNA mediated silencing of cyclase and aldo-keto reductase genes reveal their involvement in the plumbagin biosynthetic pathway.

Plumbagin and other naphthoquinone derivatives from the Plumbago zeylanica L. (Plumbaginaceae) are known for their anticancer and other medicinal properties. Previous reports suggest that 3-methyl-1,8-naphthalene-diol is an intermediate of the plumbagin biosynthetic pathway and is synthesized from hexaketide backbone; a reaction catalyzed by type III polyketide synthase (PKS) along with certain accessory enzymes. Our earlier transcriptomic and metabolomic studies suggest that along with PKS, putative cyclase and aldo-keto reductase might be involved in the formation of 3-methyl-1,8-naphthalene-diol. The present study probed young leaf transcriptome and identified cyclase and aldo-keto reductase like transcripts that might be involved in the intramolecular aldol condensation of hexaketide intermediate and decarboxylation, carbonyl reduction and hydroxyl elimination of keto or enol forms of hexaketide intermediates respectively. Moreover, sequence alignment of identified cyclase1 possesses signature ß-α-ß-ß-α-α-ß topology, which belongs to the dimeric α + ß barrel (DABB) protein family and is involved in the C2-C11 and C4-C9 intramolecular aldol condensation of hexaketide intermediates. Along with cyclase1, we further identified and characterized P. zeylanica specific aldo-keto reductase1 (AKR1) which is a novel member of the aldo-keto reductase (AKR) multi-gene family that possesses the conserved Asp60, Tyr65, Lys91, and His132 residues and is proposed to be involved in the C1 decarboxylation, C3 carbonyl reduction and C7 hydroxyl elimination of keto or enol form of hexaketide intermediate to form 3-methyl-1,8-naphthalene-diol. Further, the functional characterization using the artificial microRNA mediated transient silencing approach confirmed the involvement of cyclase1 and AKR1 in the plumbagin biosynthetic pathway. This is the first study reporting the identification and functional characterization of cyclase1 and AKR1 genes involved in the plumbagin biosynthetic pathway and general plant polyketide biosynthesis.

Functional genomics-enabled characterization of CYP81B140 and CYP81B141 from Plumbago zeylanica L. substantiates their involvement in plumbagin biosynthesis.

MAIN CONCLUSION: Novel cytochrome P450s, CYP81B140 and CYP81B141 from Plumbago zeylanica were functionally characterized to understand their involvement in polyketide plumbagin biosynthesis. Further, we propose 3-methyl-1-8-naphthalenediol and isoshinanolone as intermediates for plumbagin biosynthesis. Plumbago zeylanica L. (P. zeylanica) is a medicinally important plant belonging to the family Plumbaginaceae. It comprises the most abundant naphthoquinone plumbagin having anti-cancer activity. Only the polyketide synthase (PKS) enzyme has been identified from the biosynthetic pathway which catalyzes iterative condensation of acetyl-CoA and malonyl-CoA molecules. The plumbagin biosynthesis involves hydroxylation, oxidation, hydration and dehydration of intermediate compounds which are expected to be catalyzed by cytochrome P450s (CYPs). To identify the CYPs, co-expression analysis was carried out using PKS as a candidate gene. Out of the eight identified CYPs, CYP81B140 and CYP81B141 have similar expression with PKS and belong to the CYP81 family. Phylogenetic analysis suggested that CYP81B140 and CYP81B141 cluster with CYPs from CYP81B, CYP81D, CYP81E and CYP81AA subfamilies which are known to be involved in the hydroxylation and oxidation reactions. Moreover, artificial microRNA-mediated transient individual silencing and co-silencing of CYP81B140 and CYP81B141 significantly reduced plumbagin and increased the 3-methyl-1-8-naphthalenediol and isoshinanolone content. Based on metabolite analysis, we proposed that 3-methyl-1-8-naphthalenediol and isoshinanolone function as intermediates for plumbagin biosynthesis. Transient silencing, over-expression and docking analysis revealed that CYP81B140 is involved in C-1 oxidation, C-4 hydroxylation and [C2-C3] hydration of 3-methyl-1-8-naphthalenediol to form isoshinanolone, whereas CYP81B141 is catalyzing [C2-C3] dehydration and C-4 oxidation of isoshinanolone to form plumbagin. Our results indicated that both CYP81B140 and CYP81B141 are promiscuous and necessary for plumbagin biosynthesis. This is the first report of identification and functional characterization of P. zeylanica-specific CYPs involved in plumbagin biosynthetic pathway and in general hexaketide synthesis in plants.

Differential transcriptome and metabolome analysis of Plumbago zeylanica L. reveal putative genes involved in plumbagin biosynthesis.

Plumbagin is a pharmacologically active naphthoquinone present in the Plumbago zeylanica L. having important medicinal properties. The root of P. zeylanica is rich and primary tissue of the plumbagin biosynthesis and accumulation. The complete biosynthetic pathway of plumbagin in plant is still obscure. The present study attempts to understand the plumbagin biosynthetic pathway with the help of differential transcriptome and metabolome analysis of P. zeylanica leaf and root. The transcriptome data showed co-expression of Aldo-keto reductase (PzAKR), Polyketide cyclase (Pzcyclase) and Cytochrome P450 (PzCYPs) transcripts along with the Polyketide synthase (PzPKS) transcripts. Their higher expression in root as compared to leaf supports their possible involvement in plumbagin biosynthesis. The metabolome data of leaf and root revealed naphthalene derivative isoshinanolone that could be potential precursor of plumbagin. Pathway elucidation and transcriptome data of P. zeylanica, will enable and accelerate research on naphthoquinone biosynthesis in plants.