Transcriptome analysis and transcription factors response to transplanting and topping time of upper leaf in tobacco (Nicotiana tabacum L.)

Tobacco, as an annual industrial crop, is significantly affected by agronomic practices such as transplanting and topping, which can influence the yield and quality of the upper leaves. However, the processes by which transcription factors integrate both agronomic practices remain unclear. In this study, eight treatments were designed based on varying transplanting and topping periods. RNA-seq was employed to analyze the transcriptional expression and identify key differentially expressed genes (DEGs) and transcription factors (TFs). Subsequently, a rigorous quality inspection and data cleaning process yielded 1,302,152,358 high-quality reads. The comparative analysis of 8 treatments identified a total of 4265 DEGs. Utilizing K-means analysis, these DEGs demonstrated 9 distinct expression patterns. GO and KEGG annotation revealed a significant association of these DEGs with photosynthesis and secondary metabolism. 10 hub genes including AFC2, SIGB, PGSIP8, BIG5, OSP1, RL13, PDC1, NtORF and EBG were identified through WGCNA analysis. 41 differentially expressed TFs were identified through transcription factor analysis. Among them, 10 TFs namely ABR1, ERF26, HAT5, HSF24, LUH, AGL8, TIFY5A, TIFY10A, WRKY4, and WRKY40 are directly associated with plant stress resistance. 5 TFs such as ERF110, IDD7, ATB40, NAC81, and NAC83 have been identified as positive regulators of growth and development, while 3 TFs such as BH130, TCP4, and WRKY53 have been found to negatively impact growth and development. Furthermore, 4 TFs have been implicated in hormone and light signaling pathways, namely CIGR1, BLH1, WRKY42, and EIN4. The identified hub genes and TFs suggest that early transplanting and topping (B1D1C) contribute to enhanced stress resistance in tobacco. This may result in more developed stomata on tobacco leaves, as well as an increase in leaf thickness and a reduction in leaf size.