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

Context Wheat cytoplasmic male sterility (CMS), particularly S-type (derived from T. spelta cytoplasm), is crucial for hybrid seed production because of 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 six PCD regulators and seven 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), whereas 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.