Effect of Melatonin on the stability and expression of reference genes in Catharanthus roseus

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.