## 3 Results
### Transcription Factor Analysis of DEGs  
Transcription factors (TFs) are widely involved in plant abiotic stress responses [37]. In the present study, 122 TFs of DEGs were identified, as shown in Table 3. These TFs were divided into 22 families, including AP2-EREBP, bHLH, bZIP, C2C2-Dof, C2C2-GATA, C2H2, C3H, CCAAT, GRAS, HB, HSF, LIM, LOB, LUG, MADS, MYB, NAC, Orphans, PLATZ, S1 Fa-like, SBP, SET, SNF2, TCP, TRAF, Trihelix, WRKY, and zf-HD. Among these families, the percentage of MYB (16, 13.11%), AP2-EREBP (12, 9.84%), NAC (12, 9.84%), bHLH (10, 8.2%), HB (9, 7.38%), and SBP (7, 5.74%) was more than 5% (Table 3). Notably, Orphans (4, 3.28%), SNF2 (2, 1.64%), TRAF (2, 1.64%), LUG (1, 0.82%), and SET (1, 0.82%) are transcriptional regulators. The FPKM values of 122 TFs of DEGs between NT and MHT exposed tobacco leaves were shown in Supplementary Table S3. \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Gene Family** & **Number of TFs** & **Percentage (\%)** & **TF Type** \\ \hline MYB & 16 & 13.11 & Transcription factor \\ AP2-EREBP & 12 & 9.84 & Transcription factor \\ NAC & 12 & 9.84 & Transcription factor \\ bHLH & 10 & 8.20 & Transcription factor \\ HB & 9 & 7.38 & Transcription factor \\ SBP & 7 & 5.74 & Transcription factor \\ C2C2-Dof & 5 & 4.10 & Transcription factor \\ LOB & 5 & 4.10 & Transcription factor \\ C3H & 4 & 3.28 & Transcription factor \\ CCAAT & 4 & 3.28 & Transcription factor \\ MADS & 4 & 3.28 & Transcription factor \\ Orphans & 4 & 3.28 & Transcriptional regulator \\ WRKY & 4 & 3.28 & Transcription factor \\ C2C2-GATA & 3 & 2.46 & Transcription factor \\ GRAS & 3 & 2.46 & Transcription factor \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Transcription factor (TF) analysis results of DEGs between normal temperature (NT) and moderately high temperature (MHT) exposed tobacco leaves  
Figure 4: Statistics of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis of DEGs between NT and MHT exposed tobacco leaves. KEGG pathways are plotted on the ordinate, and the enrichment factor (rich factor) is plotted on the abscissa. The color of points represents the q-value, and the size of points represents the number of DEGs mapped to the reference pathway. Legends for the color scale of q-values and size-scaling of the number of DEGs are shown to the right of the plot. Transcription of Solanesol Biosynthesis Genes in Leaves of Tobacco Plants Grown at Normal Temperature and Moderately High Temperature  
The key enzymes in solanesol biosynthesis include DXS, DXR, farnesyl diphosphate synthase (FPS), geranylgeranyl diphosphate synthase (GGPS), and SPS [4,6]. 3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the conversion of HMG-CoA to mevalonate (MVA), which is the first step in the cytosolic pathway for isoprenoid biosynthesis in plants [38]. To understand how MHT affected solanesol biosynthesis, we monitored the relative transcript abundance of six key enzyme genes involved in solanesol biosynthesis, including two MVA (_NUHMGR_ and _NIFPS_) and four MEP (_NtDXS_, _NtDXR_, _NtGGPS_, and _NtSPS_) genes, after MHT treatment. As expected, these genes were not markedly differentially expressed under NT and those under MHT treatment at 0 DAT (\(p>0.05\)). However, at 3, 6, 9, and 12 DAT, the relative transcript abundance of _NUHMGR_, _NtDXR_, _NtGGPS_, and _NtSPS_ was significantly higher, whereas that of _NtDXS_ and _NtFPS_ was significantly lower in MHT- than in NT-exposed tobacco leaves (\(p<0.05\)) (Figure 5). Figure 5: Relative transcript abundance of selected solanesol biosynthesis genes as measured by quantitative real-time PCR (qRT-PCR) using tobacco leaves grown at normal temperature (NT) and  
moderately high temperature (MHT). (**A**) _N. tabacum_ 3-hydroxy-3-methylglutaryl-CoA reductase (_NUHMGR_), (**B**) _N. tabacum_ 1-deoxy-D-xylulose 5-phosphate synthase (_NtDXS_), (**C**) _N. tabacum_ 1-deoxy-D-xylulose 5-phosphate reductoisomerase (_NtDXR_), (**D**) _N. tabacum_ farnesyl diphosphate synthase (_NIFPS_), (**E**) _N.