Identification and expression profiling of flowering-related microRNAs and their targets reveals complex regulatory networks associated with bolting time in spinach

Abstract

Bolting is one of the most important limiting traits in vegetable crops, like spinach, when spurt to flowering before harvesting. The evidence indicates bolting is controlled by various environmental factors including temperature, photoperiod, stresses, and genetic factors such as lncRNAs and miRNAs, but there is no information about miRNA-mediated gene expressions and their roles in flowering-time regulatory networks in spinach. Therefore, to gain more insights into the molecular mechanisms of flowering and bolting, we undertook a comprehensive analysis of miRNAs and their expression profiles in two distinct spinach accessions, namely Kashan (early bolting) and Viroflay (late bolting) by employing cutting-edge high-throughput sequencing technology. Our investigation identified 720 and 750 miRNAs in Kashan and Viroflay, respectively. Remarkably, amid the conserved miRNAs, a convergence of 15 families, exemplified by miR156, miR160, miR164, miR166, miR169, and miR399, renowned for their pivotal roles in flowering/bolting temporal control, was discerned across both accessions. Concerning differentially expressed miRNAs (DEMs), 64 target genes were identified, of which 40 genes were classified into seven flowering-related pathways including age, autonomous, circadian clock, hormone, integrator, photoperiodism, and vernalization. Moreover, novel crosstalks between miRNAs and various flowering-associated genes operating in flowering-related pathways, such as miR156 and squamosa promoter-binding proteins, and miR159 and GAMYB genes in the GA flowering pathway, were identified. Additionally, miR172 influenced MADS-box and AP2-like, while miR319 family, miR164, miR160, and miR169 played distinct roles in modulating key factors associated with flowering processes, including Flowering locus T, FLOWERING PROMOTING FACTOR 1, NAC domain, auxin response factor genes, zinc finger genes, and inorganic phosphate transporter genes, showcasing their intricate regulatory involvement in flowering and bolting pathways. The findings of this study will extend our knowledge of miRNA-mediated flowering modulation and can be used in breeding programs aimed at manipulating the spinach floral regulation system.