Seed ageing is an important issue for the long-time seed storage of tobacco. Seed priming has been popularly applied in tobacco production. In this study, the development of molecular marker genes encoding proteins L-isoaspartyl methyltransferase NtPIMT1 and 8-oxoG DNA glycosylase 1 NtOGG1 to detect the degree of seed ageing and the effect of seed priming is conducted in tobacco. Quantitative real-time PCR (qRT-PCR) analysis reveals that relatively higher mRNAs of NtPIMT1 and NtOGG1 are observed in the dry and early germinating seeds. The expressions of NtPIMT1 and NtOGG1 are negatively correlated with the degree of seed damage in non-pelleted and pelleted seeds after accelerated ageing treatments. The early best effects of gibberellin (GA₃) priming on speed and uniform germination are observed in 33 h primed seeds, and relatively lower expressions of NtPIMT1 and NtOGG1 are observed in priming seeds. NtPIMT1 and NtOGG1 genes have potential for use as molecular markers in detecting the seed ageing degree and priming effect of tobacco.

The homeotic transformation of stamens into pistil-like structures (pistillody) causes cytoplasmic male sterility (CMS). This phenomenon is widely present in plants, and might be induced by intracellular communication (mitochondrial retrograde signaling), but its systemic regulating mechanism is still unclear. In this study, morphological observation showed that the stamens transformed into pistil-like structures, leading to flat and dehiscent pistils, and fruit set decrease in sua-CMS (MS K326, somatic fusion between Nicotiana. tabacum L. K326 and Nicotiana suaveolens). Transcriptome data analysis presented that the expression levels of B-class MADS genes, including pMADS1, GLO1, GLO2, pMADS2.1, pMADS2.2, significantly reduced in the pistil-like structure of sua-CMS. DEGs were enriched in flavonoid and phenylpropanoid biosynthesis pathways. Transcriptome and metabolomics analysis revealed that the expression levels of CHI/CHS (key enzymes regulating flavonoid synthesis), and the contents of flavonoids reduced significantly in the pistil-like structures of sua-CMS. Chemical fluorescence staining assay showed that reactive oxygen species (ROS) levels were higher in the pistil-like structure of sua-CMS. Application of external flavonoids (hesperetin) reduced the frequency of pistillody and ROS levels. These results suggested that the metabolism of flavonoids played important roles in regulating pistillody through ROS in sua-CMS. Our study provides new insights into the regulatory mechanism of pistillody in plants.