Anther development and pollen fertility of cytoplasmic male sterility (CMS) conditioned by Gossypium harknessii cytoplasm (CMS-D2) restorer lines are susceptible to continuous high-temperature (HT) stress in summer, which seriously hinders the large-scale application of "three-line" hybrids in production. Here, integrated small RNA, transcriptome, degradome, and hormone profiling was performed to explore the roles of microRNAs (miRNAs) in regulating fertility stability in mature pollens of isonuclear alloplasmic near-isogenic restorer lines NH and SH under HT stress at two environments. A total of 211 known and 248 novel miRNAs were identified, of which 159 were differentially expressed miRNAs (DEMs). Additionally, 45 DEMs in 39 miRNA clusters (PmCs) were also identified, and most highly expressed miRNAs were significantly induced in SH under extreme HT, especially four MIR482 and six MIR6300 family miRNAs. PmC28 was located in the fine-mapped interval of the Rf1 gene and contained two DEMs, gra-miR482_L-2R + 2 and gma-miR2118a-3p_R + 1_1ss18TG. Transcriptome sequencing identified 6281 differentially expressed genes, of which heat shock protein (HSP)-related genes, such as HSP70, HSP22, HSP18.5-C, HSP18.2 and HSP17.3-B, presented significantly reduced expression levels in SH under HT stress. Through integrating multi-omics data, we constructed a comprehensive molecular network of miRNA–mRNA–gene-KEGG containing 35 pairs of miRNA/target genes involved in regulating the pollen development in response to HT, among which the mtr-miR167a_R + 1, tcc-miR167c and ghr-miR390a, tcc-miR396c_L-1 and ghr-MIR169b-p3_1ss6AG regulated the pollen fertility by influencing ARF8 responsible for the auxin signal transduction, ascorbate and aldarate metabolism, and the sugar and lipid metabolism and transport pathways, respectively. Further combination with hormone analysis revealed that HT-induced jasmonic acid signaling could activate the expression of downstream auxin synthesis-related genes and cause excessive auxin accumulation, followed by a cascade of auxin signal transduction, ultimately resulting in pollen abortion. The results provide a new understanding of how heat-responsive miRNAs regulate the stability of fertility restoration for CMS-D2 cotton under heat stress.

中文翻译：

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热响应性microRNA参与调节高温胁迫下CMS-D2恢复系花粉育性稳定性

哈氏棉细胞质雄性不育(CMS-D2)恢复系的花药发育和花粉育性易受到夏季持续高温胁迫，严重阻碍了“三不育”的大规模应用。 line”混合动力车正在生产中。在这里，进行了整合的小RNA、转录组、降解组和激素分析，以探讨在两种环境下的HT胁迫下，微小RNA（miRNA）在调节同核异质近等基因恢复系NH和SH成熟花粉育性稳定性中的作用。总共鉴定了 211 个已知 miRNA 和 248 个新 miRNA，其中 159 个是差异表达 miRNA (DEM)。此外，还鉴定了 39 个 miRNA 簇 (PmC) 中的 45 个 DEM，并且大多数高表达的 miRNA 在极端高温下的 SH 中显着诱导，特别是 4 个 MIR482 和 6 个 MIR6300 家族 miRNA。PmC28 位于 Rf1 基因的精细映射区间，包含两个 DEM：gra-miR482_L-2R + 2 和 gma-miR2118a-3p_R + 1_1ss18TG。转录组测序鉴定出6281个差异表达基因，其中热休克蛋白（HSP）相关基因，如HSP70、HSP22、HSP18.5-C、HSP18.2和HSP17.3-B，在SH条件下表达量显着降低。高温应力。通过整合多组学数据，我们构建了一个全面的miRNA-mRNA-gene-KEGG分子网络，其中包含35对参与调节花粉发育响应HT的miRNA/靶基因，其中mtr-miR167a_R + 1，tcc -miR167c和ghr-miR390a、tcc-miR396c_L-1和ghr-MIR169b-p3_1ss6AG分别通过影响负责生长素信号转导、抗坏血酸和醛二酸代谢以及糖和脂质代谢和运输途径的ARF8来调节花粉育性。进一步结合激素分析发现，HT诱导的茉莉酸信号可以激活下游生长素合成相关基因的表达，导致生长素过度积累，随后发生生长素信号转导级联反应，最终导致花粉败育。该结果为热响应miRNA如何调节CMS-D2棉花在热胁迫下育性恢复的稳定性提供了新的认识。