Investigating the functions of small non-coding RNAs in stolon-to-tuber transition stages of potato development

Abstract

Small non-coding RNAs (sRNAs) play a major role in the regulation of plant growth and development. Tuberization (potato development) is a complex process, wherein a stolon (an underground modified stem) passes through transitional stages like swollen stolon, mini-tuber to develop into a mature tuber. Previous studies have reported the intricate network of transcription factors and phytohormones, however, the role of small RNAs in tuber development is less explored. Using a small RNA sequencing approach, we identified a plethora of microRNAs and phasiRNAs (phased secondary siRNAs) from stolons of early stages of tuber transitions under photoperiodic conditions. TASI prediction analysis revealed 830 TAS producing loci resulting in numerous phasiRNAs. Key tuberization genes were found to be the putative targets of identified small RNAs. Interestingly, one of the phasiRNA [siRD29(-)] targets GA biosynthesis gene – gibberellin 3-oxidase 3 (StGA3ox3). Regulating bio-active gibberellin levels (GA) is crucial for tuber development. We demonstrate that miR482c targets StRGA4 (a TAS-like locus) to generate siRD29(-) implicating the possible role of a novel gene regulatory module (miR482c-StRGA4-siRD29(-)-StGA3ox3) governing tuber development. siRD29(-) showed a negative correlation with StGA3ox3 expression from stolon to swollen stolon stages. StRGA4 overexpression (OE) lines showed high levels of siRD29(-) and mimicked the phenotype (increased tuber number) of StGA3ox3-AS lines, pointing towards the functionality of the gene regulatory module in potato. In vitro tuberization assays using OE (StGA3ox3 and StRGA4) and AS (StGA3ox-AS) lines with or without GA inhibitor as well as study of stolon lengths in vivo revealed that StGA3ox3 controls tuber-stalk development. Presence of 12 TGAC tandem core motifs (BEL-KNOX binding motif) in StGA3ox3 promoter possibly facilitate binding of StBEL5 and induces GUS activity in the promStGA3ox3::GUS lines implying its role as an activator. GA quantification from transgenic lines indicated StGA3ox3, might catalyse GA1 and GA3 production. As expected, StRGA4-OE lines exhibited decreased bioactive GA3 levels. Overall, our findings suggest that siRD29(-) mediates the regulation of StGA3ox3 during early stolon stages, which is crucial for stolon development and the module appears to fine-tune tuber initiation process by balancing GA levels. Additionally, we have investigated the role of a potato specific miRNA (miR72), whose expression decreased from stolon-to-tuber stages and OE lines exhibited an interesting sessile tuber phenotype as compared to the wild type indicating crucial role of miRNA in the early stolon development. Overall, our study has unraveled the role of novel small RNA regulatory networks governing the tuber development process.