Rapeseed (Brassica napus) is an important oil seed crop in the Brassicaceae family. Chemical induced male sterility (CIMS) is one of the widely used method to produce the hybrids in B. napus. Identification of the key genes and pathways that involved in CIMS were important to understand the underlying molecular mechanism. In the present report, a multi-omics integrative analysis, including of the proteomic, transcriptomic and miRNAs, combined with morphological and physiological analysis were conducted. Earlier degeneration of the tapetosomes and elaioplasts, aberrantly stacking in tapetal cells and incompletely deposition in tryphine of pollen wall were observed in chemical hybridization agent (CHA) of SX-1 treated B. napus through SEM and TEM analysis. It was revealed that the deficiencies in protein processing in endoplasmic reticulum (ER) and flavonoids biosynthesis were occurred at early stage in the SX-1 treated materials. Subsequently, plant hormone signal transduction, biosynthesis of amino acids, fatty acids and steroid in anther at later stages were identified down-regulated after SX-1 treatment. 144 transcript factors (TFs) were also indentified to down-regulated at early stage, which suggested the early regulation in anther and pollen wall development were disordered in CHA treated B. napus. In addition, 7 important miRNAs were identified and 2 of the predicted target genes of miRNAs were Rf-like genes. Taken together, an interaction network of candidate genes and the putative metabolism pathways were constructed based on the multi-omics integrative analysis, it provided a new insight into the male sterility induced by CHA of SX-1 in B. napus.

Polygonatum hunanense H.H. Liu & B.Z. Wang (syn. P. kingianum var. grandifolium), is a perennial Liliaceae species with ethnopharmaceutical and nutraceutical value. It exhibits traditional medicinal properties such as qi-tonifying, yin-nourishing, kidney-benefiting, and antioxidant activities. P. hunanense maintains evergreen foliage year-round and shows significantly higher biomass accumulation than congener taxa. These traits are linked to phytohormone regulation, as indicated by previous studies. The absence of validated reference genes in P. hunanense has hindered molecular-level investigations in this species to date. In this study, 11 candidate reference genes (GAPDH, H2A, EF-1α, ACT, TUA, UBC, RPL, PP2A, 18S, TUB, and UBQ) were identified from the P. hunanense transcriptome dataset. Expression profiles of these genes were analyzed by qRT-PCR across three experimental conditions: different tissues, hormone treatments, and abiotic stresses. Gene expression stability was analyzed using GeNorm, NormFinder, Delta Cᴛ, BestKeeper, and RefFinder software. The stability of candidate reference genes was validated through expression analysis of target genes (KO, KAM, GA13-ox, GA44-dio, ALDH7A1, IPM) involved in phytohormone metabolic pathways. Overall, the stability of the 11 candidate reference genes varied among different P. hunanense samples. EF-1α, 18S and ACT were identified as the most stable in tissue samples. EF-1α, H2A and PP2A showed the highest stability under hormone treatments, and 18S, UBC, and EF-1α exhibited consistent stability in abiotic stresses. This study establishes EF-1α as the optimal pan-experimental reference gene for qRT-PCR normalization in P. hunanense, demonstrating consistent expression stability across diverse tissues, hormone treatments, and abiotic stresses, thereby providing a significant reference for future gene expression studies.