Comparative transcriptomic analysis reveals the stage-specific gene expression profiles related to S-type cytoplasmic male sterility (CMS) in wheat (Triticum aestivum L.)

Context: Wheat cytoplasmic male sterility (CMS), particularly S-type (derived from T. spelta cytoplasm), is crucial for hybrid seed production due to its easy fertility transition and broad restorer compatibility. However, unclear nuclear-cytoplasmic interaction mechanisms limit elite hybrid development. Aims: This study investigated transcriptomic and hormonal dynamics in S-CMS line (S1376A) and maintainer (1376B) anthers to identify sterility regulators and validate candidates via CRISPR/Cas9. Methods: RNA-seq profiled anthers at tetrad, uninucleate, and binucleate stages. High-performance liquid chromatography (HPLC) quantified indole-3-acetic acid (IAA), zeatin riboside (ZR), and abscisic acid (ABA). CRISPR/Cas9 generated TaMYB80L mutants, validated using microscopy and starch staining. Key Results: Transcriptome analysis identified stage-specific differentially expressed genes (DEGs) enriched in energy metabolism, hormone signaling, cell wall biosynthesis, including genes encoding 6 PCD regulators and 7 hormone-responsive factors. At uninucleate stage, 1376B had higher IAA (206.74 vs. 60.07 ng·g⁻¹) and ZR (63.56 vs. 38.62 ng·g⁻¹), while S1376A had 2.5-fold higher ABA (74.14 vs. 29.67 ng·g⁻¹). TaMYB80L mutants (achieved a triple homoeolog editing efficiency of 8.57%) displayed complete (100%) pollen abortion, phenocopying the S1376A sterility. Conclusions: TaMYB80L is a key regulator of S-type CMS, coordinating tapetal programmed cell death (PCD) and hormonal crosstalk. Our integrated analysis elucidates critical nuclear-cytoplasmic interactions underlying sterility. Implications: Insights into TaMYB80L advance CMS understanding and provide precise targets for hybrid wheat breeding.