Plant specialized metabolites are being used worldwide as therapeutic agents against several diseases. Since the precursors for specialized metabolites come through primary metabolism, extensive investigations have been carried out to understand the detailed connection between primary and specialized metabolism at various levels. Stress regulates the expression of primary and specialized metabolism genes at the transcriptional level via transcription factors binding to specific cis-elements. The presence of varied cis-element signatures upstream to different stress-responsive genes and their transcription factor binding patterns provide a prospective molecular link among diverse metabolic pathways. The pattern of occurrence of these cis-elements (overrepresentation/common) decipher the mechanism of stress-responsive upregulation of downstream genes, simultaneously forming a molecular bridge between primary and specialized metabolisms. Though many studies have been conducted on the transcriptional regulation of stress-mediated primary or specialized metabolism genes, but not much data is available with regard to cis-element signatures and transcription factors that simultaneously modulate both pathway genes. Hence, our major focus would be to present a comprehensive analysis of the stress-mediated interconnection between primary and specialized metabolism genes via the interaction between different transcription factors and their corresponding cis-elements. In future, this study could be further utilized for the overexpression of the specific transcription factors that upregulate both primary and specialized metabolism, thereby simultaneously improving the yield and therapeutic content of plants.

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Catharanthus roseus is a commercial source for anti-cancer terpenoid indole alkaloids (TIAs: vincristine and vinblastine). Inherent levels of these TIAs are very low, hence research studies need to focus on enhancing their levels in planta. Since primary metabolism provides precursors for specialized-metabolism, elevating the former can achieve higher amounts of the latter. Cell Wall Invertase (CWIN), a key enzyme in sucrose-metabolism catalyses the breakdown of sucrose into glucose and fructose, which serve as carbon-skeleton for specialized-metabolites. Understanding CWIN regulation could unravel metabolic-engineering approaches towards enhancing the levels of TIAs in planta. Our study is the first to characterize CWIN at gene-expression level in the medicinal plant, C. roseus. The CWINs and their inter-relationship with sucrose and TIA metabolism was studied at gene and metabolite levels. It was found that sucrose-supplementation to C. roseus leaves significantly elevated the monomeric TIAs (vindoline, catharanthine) and their corresponding genes. This was further confirmed in cross-species, wherein Nicotiana benthamiana leaves transiently-overexpressing CrCWIN2 showed significant upregulation of specialized-metabolism genes: NbPAL2, Nb4CL, NbCHS, NbF3H, NbANS, NbHCT and NbG10H. The specialized metabolites- cinnamic acid, coumarin, and fisetin were significantly upregulated. Thus, the present study provides a valuable insight into metabolic-engineering approaches towards augmenting the levels of therapeutic TIAs.

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The role of Melatonin in influencing diverse genes in plants has gained momentum in recent years and many reports have employed qRT-PCR for their quantification. Relative quantification of gene expression relies on accurate normalization of qRT-PCR data against a stably-expressing internal reference-gene. Although researchers have been using commonly available reference-genes to assess Melatonin-induced gene expression, but to-date, there have been no attempts to validate the reference-gene stability under Melatonin-supplementation in planta. In this study, we performed stability assessment of common reference-genes under Melatonin-supplementation and abiotic stress in leaves and seedlings of Catharanthus roseus using geNorm, NormFinder, BestKeeper, ΔCt and RefFinder algorithms. Nine candidate reference-genes were tested for stability in C. roseus (FBOX, CACS, TIP, RSP9, EXP, EXPR, SAND, F17M5, ACT) and our study inferred that while EXP and EXPR were the most-stable, F17M5 was the lowest-stable gene in the leaf-fed samples. Among seedlings of C. roseus, F17M5 and TIP were the most, while ACT was the least-stable gene. The suitability of selected stable reference-gene pairs was demonstrated by assessing the transcript levels of the Melatonin-biosynthesis gene SNAT under same conditions. Our study is the first to comprehensively analyze the stability of commonly-used reference-genes under Melatonin-induced conditions in C. roseus.