A bZIP transcription factor from Picrorhiza kurrooa modulates the expression of terpenoid biosynthesis genes in Nicotiana Benthamiana

Basic leucine zipper (bZIP) transcription factors are DNA-binding proteins which recognize the ACGT motif in the promoter region of genes and regulate their expression. bZIP TFs play significant roles in plant growth and development, defense, metabolic regulation and responses to environmental stresses. Till now, there is no report on the cloning and characterization of bZIP transcription factors in Picrorhiza kurrooa, an endangered medicinal herb of north western Himalayas. The medicinal properties of P. kurrooa are attributed to picrosides synthesized by isoprenoid pathway. The promoters of regulatory genes 1-deoxy-D-xylulose-5-phosphate synthase (pkdxs) and 3-hydroxy-3-methylglutaryl coenzyme A reductase (pkhmgr) of the pathway contain ACGT cis-acting elements (bzip motifs) at different locations. In this study, we showed the functionality of ACGT motifs by electrophoretic mobility shift assay (EMSA). Full length bZIP gene was cloned through RACE method. In silico analysis revealed pkbZIP contained a highly conserved bZIP domain profile (N-X₇-R/K- X₉-L-X₆-L-X₆-L). Quantitative real time PCR (RT-qPCR) was used to analyze tissue-specific expression and the expression changes in response to temperature and continuous light and dark conditions. The gene expression corroborated with the picroside (active medicinal component of the plant) content at tissue specific level, under light conditions and at 15 °C (temperature favouring picroside accumulation). Transient overexpression in tobacco modulated the expression of genes involved in terpenoid biosynthesis in tobacco suggesting in planta functionality of pkbZIP. Collectively, data suggested that pkbZIP might prove as potential candidate for metabolic engineering programs aimed at enhancing picrosides biosynthesis in P. kurrooa.