Integrated Analysis of Small RNA, Transcriptome, and Degradome Sequencing Reveals the MiR156, MiR5488 and MiR399 Are Involved in the Regulation of Male Sterility in PTGMS Rice

A photoperiod- and thermo-sensitive genic male sterile (PTGMS) line is the basic material for two-hybrid rice and is an important genetic breeding resource. Peiai64S (PA64S) is an important germplasm resource of PTGMS rice, and it has been applied to two-line hybrid rice systems in China. Pollen fertility in PA64S is regulated by the temperature and photoperiod, but the mechanism of the fertility transition is unclear. In this study, we obtained the male fertile plant PA64S(F) and the male sterile plant PA64S(S) by controlling different temperatures under long light conditions and used the male fertile and sterile plants to investigate the role of microRNAs (miRNAs) in regulating male fertility in rice. We performed the small RNA library sequencing of anthers from PA64S(S) and PA64S(F). A total of 196 miRNAs were identified—166 known miRNAs among 27 miRNA families and 30 novel miRNAs. In the transcriptome analysis, the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of differentially expressed genes revealed significant enrichment in the synthesis and metabolism of fatty acids and some secondary metabolism pathways such as fatty acid metabolism and phenylalanine metabolism. With a comprehensive analysis of miRNA, transcriptome, and degradome sequencing, we identified that 13 pairs of miRNA/target genes regulated male fertility in rice by responding to temperature change, among which the miR156, miR5488, and miR399 affect the male fertility of PA64S by influencing SPLs, the lignin synthesis of anther walls, and the flavonoid metabolism pathway. The results provide a new understanding of PTGMS rice, which will help us better understand the potential regulatory mechanisms of male sterility in the future.

Genome-Wide Identification of lncRNAs Involved in Fertility Transition in the Photo-Thermosensitive Genic Male Sterile Rice Line Wuxiang S

Long non-coding RNAs (lncRNAs) act as universal regulators of various biological processes, but no genome-wide screening of lncRNAs involved in the fertility transition of the photo-thermosensitive genic male sterile (PTGMS) rice line has been reported. Here, we performed strand-specific RNA sequencing at three developmental stages of a novel PTGMS line Wuxiang S (WXS). A total of 3,948 lncRNAs were identified; 622 of these were detected as differentially expressed lncRNAs (DE-lncRNAs) between male-sterile WXS (WXS-S) and male-fertile WXS (WXS-F). A large proportion of lncRNAs differentially expressed at the stage of pollen mother cells meiosis, suggested that it may be the most critical stage for fertility transition of WXS. Furthermore, functional annotation of the cis- and trans- targets of DE-lncRNAs showed that 150 targets corresponding to 141 DE-lncRNAs were identified as involved in anther and pollen development. Moreover, computational analysis predicted 97 lncRNAs as precursors for 72 miRNAs, and 94 DE-lncRNAs as potential endogenous target mimics (eTMs) for 150 miRNAs. Finally, using the dual luciferase reporter assays, we demonstrated that two lncRNAs act as eTMs to regulate the expression of the SPL and GRF genes by competing for the shared osa-miR156 and osa-miR396, respectively. These genomic characteristics, differential expression, and interaction of lncRNAs with miRNAs and mRNAs contribute to our understanding of the roles of lncRNAs during the fertility transition in PTGMS rice lines.

Comparative Analysis of Proteomics and Transcriptomics during Fertility Transition in a Two-Line Hybrid Rice Line Wuxiang S

The two-line hybrid rice is an important factor of a global crop, but its fertility transition mechanism is unclear. Here, a comparative proteomics and transcriptomics analysis was completed on the two-line hybrid rice line Wuxiang S (WXS) to explore its molecular mechanism and protein regulation during fertility transition. A total of 340 differentially abundant proteins (DAPs) were identified using iTRAQ between the pollen mother cell formation stage (P2) and the meiosis stage (P3). There were 3541 and 4247 differentially expressed genes (DEGs) in P2 and P3 between WXS (Sterile, S)-WXS(S) and WXS (Fertile, F)-WXS(F), respectively, of which 92 and 71 DEGs had corresponding DAPs. Among the DAPs and DEGs, 65 (SP2 vs. FP2) and 55 (SP3 vs. FP3) corresponding DEGs and DAPs (cor-DEGs-DAPs) showed the same expression trend, indicating the cor-DEGs-DAPs genes might play vital roles in WXS fertility transition. Further analysis indicated that cor-DEGs-DAPs proteins were related to energy metabolism-related proteins in anther development and were accompanied by the activation of the stress response pathway and modifications to the cell wall, which ultimately affected the fertility transition of the PTGMS rice line WXS.