The phased short-interfering RNA siRD29(-) regulates GIBBERELLIN 3-OXIDASE 3 during stolon-to-tuber transitions in potato.

Phased short-interfering RNAs (phasiRNAs) fine tune various stages of growth, development, and stress responses in plants. Potato (Solanum tuberosum) tuberization is a complex process, wherein a belowground modified stem (stolon) passes through developmental stages like swollen stolon and minituber before it matures to a potato. Previously, we identified several phasiRNA-producing loci (PHAS) from stolon-to-tuber transition stages. However, whether phasiRNAs mediate tuber development remains unknown. Here, we show that a gene encoding NB-ARC DOMAIN-CONTAINING DISEASE RESISTANCE PROTEIN (StRGA4; a PHAS locus) is targeted by Stu-microRNA482c to generate phasiRNAs. Interestingly, we observed that one of the phasiRNAs, referred as short-interfering RNA D29(-), i.e. siRD29(-), targets the gibberellin (GA) biosynthesis gene GIBBERELLIN 3-OXIDASE 3 (StGA3ox3). Since regulation of bioactive GA levels in stolons controls tuber development, we hypothesized that a gene regulatory module, Stu-miR482c-StRGA4-siRD29(-)-StGA3ox3, could govern tuber development. Through transient expression assays and small RNA sequencing, generation of siRD29(-) and its phase was confirmed in planta. Notably, the expression of StGA3ox3 was higher in swollen stolon compared to stolon, whereas siRD29(-) showed a negative association with StGA3ox3 expression. Antisense (AS) lines of StGA3ox3 produced more tubers compared to wild type. As expected, StRGA4 overexpression (OE) lines had high levels of siRD29(-) and mimicked the phenotypes of StGA3ox3-AS lines, indicating the functionality of this module in potato. In vitro tuberization assays (with or without a GA inhibitor) using StGA3ox3 antisense lines and overexpression lines of StGA3ox3 or StRGA4 revealed that StGA3ox3 controls the tuber stalk development. Taken together, our findings suggest that a phasiRNA, siRD29(-), mediates the regulation of StGA3ox3 during stolon-to-tuber transitions in potato.

Development of aerial and belowground tubers in potato is governed by photoperiod and epigenetic mechanism.

Plants exhibit diverse developmental plasticity and modulate growth responses under various environmental conditions. Potato (Solanum tuberosum), a modified stem and an important food crop, serves as a substantial portion of the world's subsistence food supply. In the past two decades, crucial molecular signals have been identified that govern the tuberization (potato development) mechanism. Interestingly, microRNA156 overexpression in potato provided the first evidence for induction of profuse aerial stolons and tubers from axillary meristems under short-day (SD) photoperiod. A similar phenotype was noticed for overexpression of epigenetic modifiers-MUTICOPY SUPRESSOR OF IRA1 (StMSI1) or ENAHNCER OF ZESTE 2 (StE[z]2), and knockdown of B-CELL-SPECIFIC MOLONEY MURINE LEUKEMIA VIRUS INTEGRATION SITE 1 (StBMI1). This striking phenotype represents a classic example of modulation of plant architecture and developmental plasticity. Differentiation of a stolon to a tuber or a shoot under in vitro or in vivo conditions symbolizes another example of organ-level plasticity and dual fate acquisition in potato. Stolon-to-tuber transition is governed by SD photoperiod, mobile RNAs/proteins, phytohormones, a plethora of small RNAs and their targets. Recent studies show that polycomb group proteins control microRNA156, phytohormone metabolism/transport/signaling and key tuberization genes through histone modifications to govern tuber development. Our comparative analysis of differentially expressed genes between the overexpression lines of StMSI1, StBEL5 (BEL1-LIKE transcription factor [TF]), and POTATO HOMEOBOX 15 TF revealed more than 1,000 common genes, indicative of a mutual gene regulatory network potentially involved in the formation of aerial and belowground tubers. In this review, in addition to key tuberization factors, we highlight the role of photoperiod and epigenetic mechanism that regulates the development of aerial and belowground tubers in potato.

The Polycomb group methyltransferase StE(z)2 and deposition of H3K27me3 and H3K4me3 regulate the expression of tuberization genes in potato.

Polycomb repressive complex (PRC) group proteins regulate various developmental processes in plants by repressing target genes via H3K27 trimethylation, and they function antagonistically with H3K4 trimethylation mediated by Trithorax group proteins. Tuberization in potato has been widely studied, but the role of histone modifications in this process is unknown. Recently, we showed that overexpression of StMSI1, a PRC2 member, alters the expression of tuberization genes in potato. As MSI1 lacks histone-modification activity, we hypothesized that this altered expression could be caused by another PRC2 member, StE(z)2, a potential H3K27 methyltransferase in potato. Here, we demonstrate that a short-day photoperiod influences StE(z)2 expression in the leaves and stolons. StE(z)2 overexpression alters plant architecture and reduces tuber yield, whereas its knockdown enhances yield. ChIP-sequencing using stolons induced by short-days indicated that several genes related to tuberization and phytohormones, such as StBEL5/11/29, StSWEET11B, StGA2OX1, and StPIN1 carry H3K4me3 or H3K27me3 marks and/or are StE(z)2 targets. Interestingly, we observed that another important tuberization gene, StSP6A, is targeted by StE(z)2 in leaves and that it has increased deposition of H3K27me3 under long-day (non-induced) conditions compared to short days. Overall, our results show that StE(z)2 and deposition of H3K27me3 and/or H3K4me3 marks might regulate the expression of key tuberization genes in potato.

Genome-wide transcriptome analysis reveals small RNA profiles involved in early stages of stolon-to-tuber transitions in potato under photoperiodic conditions.

BACKGROUND: Small RNAs (sRNAs), especially miRNAs, act as crucial regulators of plant growth and development. Two other sRNA groups, trans-acting short-interfering RNAs (tasiRNAs) or phased siRNAs (phasiRNAs), are also emerging as potential regulators of plant development. Stolon-to-tuber transition in potato is an important developmental phase governed by many environmental, biochemical and hormonal cues. Among different environmental factors, photoperiod has a major influence on tuberization. Several mobile signals, mRNAs, proteins and transcription factors have been widely studied for their role in tuber formation in potato, however, no information is yet available that describes the molecular signals governing the early stages of stolon transitions or cell-fate changes at the stolon tip before it matures to potato. Stolon could be an interesting model for studying below ground organ development and we hypothesize that small RNAs might be involved in regulation of stolon-to-tuber transition process in potato. Also, there is no literature that describes the phased siRNAs in potato development. RESULTS: We performed sRNA profiling of early stolon stages (4, 7 and 10 d) under long-day (LD; 16 h light, 8 h dark) and short-day (SD; 8 h light, 16 h dark) photoperiodic conditions. Altogether, 7 (out of 324) conserved and 12 (out of 311) novel miRNAs showed differential expression in early stolon stages under SD vs LD photoperiodic conditions. Key target genes (StGRAS, StTCP2/4 and StPTB6) exhibited differential expression in early stolon stages under SD vs LD photoperiodic conditions, indicative of their potential role in tuberization. Out of 830 TAS-like loci identified, 24 were cleaved by miRNAs to generate 190 phased siRNAs. Some of them targeted crucial tuberization genes such as StPTB1, POTH1 and StCDPKs. Two conserved TAS loci, referred as StTAS3 and StTAS5, which share close conservation with members of the Solanaceae family, were identified in our analysis. One TAS-like locus (StTm2) was validated for phased siRNA generation and one of its siRNA was predicted to cleave an important tuber marker gene StGA2ox1. CONCLUSION: Our study suggests that sRNAs and their selective target genes could be associated with the regulation of early stages of stolon-to-tuber transitions in a photoperiod-dependent manner in potato.

MiRNA160 is associated with local defense and systemic acquired resistance against Phytophthora infestans infection in potato.

To combat pathogen infection, plants employ local defenses in infected sites and elicit systemic acquired resistance (SAR) in distant tissues. MicroRNAs have been shown to play a significant role in local defense, but their association with SAR is unknown. In addition, no such studies of the interaction between potato and Phytophthora infestans have been reported. We investigated the role of miR160 in local and SAR responses to P. infestans infection in potato. Expression analysis revealed induced levels of miR160 in both local and systemic leaves of infected wild-type plants. miR160 overexpression and knockdown plants exhibited increased susceptibility to infection, suggesting that miR160 levels equivalent to those of wild-type plants may be necessary for mounting local defense responses. Additionally, miR160 knockdown lines failed to elicit SAR, and grafting assays indicated that miR160 is required in both local and systemic leaves to trigger SAR. Consistently, SAR-associated signals and genes were dysregulated in miR160 knockdown lines. Furthermore, analysis of the expression of defense and auxin pathway genes and direct regulation of StGH3.6, a mediator of salicylic acid-auxin cross-talk, by the miR160 target StARF10 revealed the involvement of miR160 in antagonistic cross-talk between salicylic acid-mediated defense and auxin-mediated growth pathways. Overall, our study demonstrates that miR160 plays a crucial role in local defense and SAR responses during the interaction between potato and P. infestans.