Body of Abstract: Understanding the molecular basis of tuberization is crucial to devise strategies for maximizing potato yield. During tuberization, a belowground modified stem (stolon) passes through various developmental transitions, such as swollen stolon and mini-tuber before it matures to a potato. Although a number of mobile RNAs and proteins, transcription factors, and phytohormones are shown to govern tuber development, our knowledge regarding early stages of potato development is limited. Phased secondary siRNAs (phasiRNAs) are shown to fine-tune various growth and development processes in plants. We previously identified 830 PHAS loci from stolon-to-tuber transition stages, which could target key tuberization genes in potato. Thus, we hypothesize that phasiRNAs may regulate early tuber developmental stages. Reduction of gibberellin (GA) levels in stolon is an important step for re-orientation of cell division planes to facilitate tuberization. In this investigation, we demonstrate a microRNA482c that cleaves StRGA4 PHAS locus resulting in the generation of a novel phasiRNA [siRD29(-)] targeting a GA biosynthesis gene, StGA3ox3 in potato. Both siRD29(-) and StGA3ox3 exhibited inverse expression patterns during stolon-to-swollen stages, suggesting that a novel gene regulatory module (miR482c-StRGA4-siRD29(-)-StGA3ox3) could govern early tuberization stages. StGA3ox3 antisense (AS) and StRGA4 overexpression (OE) lines exhibited higher number of tubers compared to wild-type. Further, StRGA4-OE lines had higher expression of siRD29(-) accompanied by a reduced level of StGA3ox3. In vitro tuberization assays using StGA3ox3-OE and -AS lines treated with a GA inhibitor showed the role of StGA3ox3 in the tuber-stalk development. Interestingly, we observed that StBEL5 (a mobile mRNA in potato) induces the expression of StGA3ox3 by binding to its promoter through characteristic TGAC core motifs. In silico analysis suggested that this module is also conserved in tomato. Taken together, our results suggest that phasiRNA siRD29(-) mediates a novel gene regulatory module (miR482c-StRGA4-siRD29(-)-StGA3ox3) in stolon-to-tuber transitions by balancing GA levels in developing stolon.
.jpg "Nilam N. Malankar, PhD student (she/her/hers) photo")