Photoperiod- and thermosensitive genic male sterility (PTGMS) rice is a vital germplasm resource consisting of two-line hybrid rice in which light and temperature strictly control their fertility changes. Variable environmental conditions present huge risks to the two-lines hybrid seed production. Explaining the regulatory mechanism of male fertility in rice PTGMS lines is an essential prerequisite to ensuring food security production. A group of near-isogenic lines (NILs) of a rice PTGMS line unique to this research group was used for this study. These lines have the same genetic background and regulate male fertility by responding to different temperature changes. Transcriptomic analysis revealed that 315 upregulated genes and 391 regulated genes regulated male fertility in response to temperature changes, and differentially expressed genes (DEGs) were mainly characterized in enrichment analysis as having roles in the metabolic pathways of sugar, lipid and phenylpropanoid. Electron microscopy analysis revealed that a lack of starch accumulation in sterile pollen grains induced by high temperature, with an abnormal exine development and a lack of inner pollen grains. Defective processes for sporopollenin synthesis, sporopollenin transport and pollen wall formation in sterile anthers were verified using qPCR. Targeted metabolomics analysis revealed that most lipids (phospholipids, sphingolipids and fatty acids) and flavonoids (flavones and flavanones) were upregulated in fertile anthers and involved in pollen wall development and male fertility formation, while lignin G units and C-type lignin were the major contributors to pollen wall development. The coding genes for trehalose 6-phosphate phosphatase, beta-1,3-glucanase, phospholipase D and 4-coumarate-CoA ligase are considered essential regulators in the process of male fertility formation. In conclusion, our results indicated that the expression of critical genes and accumulation of metabolites in the metabolism of sugar, lipid, and phenylpropanoid are essential for male fertility formation. The results provide new insights for addressing the negative effects of environmental variation on two-line hybrid rice production.

Two-line hybrid rice systems represent a new technical approach to utilizing the advantages of rice hybrids. However, the mechanism underlying the male sterile-line fertility transition in rice remains unclear. Peiai 64S (PA64S) is a photoperiod- and thermo-sensitive genic male sterile (PTGMS) line in which male sterility manifests at an average temperature above 23.5 °C under long-day (LD) conditions. Nongken 58S (NK58S) is a LD-sensitive genic male sterile (PGMS) rice that is sterile under LD conditions (above 13.75 h-day). In contrast, D52S is a short-day (SD)-PGMS line that manifests male sterility under SD conditions (below 13.5 h-day). In this study, we obtained fertile and sterile plants from all three lines and performed transcriptome analyses on the anthers of the plants. Gene ontology (GO) analysis suggested that the differentially expressed genes identified were significantly enriched in common terms involved in the response to jasmonic acid (JA) and in JA biosynthesis. On the basis of the biochemical and molecular validation of dynamic, tissue-specific changes in JA, indole-3-acetic acid (IAA) levels, gibberellin (GA) levels, and JA biosynthetic enzyme activities and expression, we proposed that JA could play a pivotal role in viable pollen production through its initial upregulation, constant fluctuation and leaf-spikelet signaling under certain fertility-inducing conditions. Furthermore, we also sprayed methyl jasmonate (MEJA) and salicylhydroxamic acid (SHAM) on the plants, thereby achieving fertility reversal in the PGMS lines NK58S and D52S, with 12.91–63.53% pollen fertility changes. Through qPCR and enzyme activity analyses, we identified two key enzymes—allene oxide synthase (AOS) and allene oxide cyclase (AOC)—that were produced and upregulated by 20–500-fold in PGMS in response to spraying; the activities of these enzymes reversed pollen fertility by influencing the JA biosynthetic pathway. These results provide a new understanding of hormone interactions and networks in male-sterile rice based on the role of JA that will help us to better understand the potential regulatory mechanisms of fertility development in rice in the future.

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.