## 4 Discussion
### Transcriptome Analysis of Tobacco Leaves Treated with Moderately High Temperature  
Through transcriptome analysis, we identified substantial differences in leaf gene expression between NT and MHT exposed plants. MHT treatment resulted in significant up- and downregulation of 492 and 1440 genes, respectively, as compared to NT (Figure 3). [PERSON] et al. [18] used the GeneChip microarray to investigate the response of developing barley (_Hordeum vulgare_) seeds after 0.5, 3, and 6 h high-temperature stress; 958 induced and 1122 repressed genes exhibited spatial and temporal expression patterns. [PERSON] et al. [17] investigated temperature-associated changes in gene expression in potato leaves and found that differences in functional categories under the elevated temperature regime included genes involved in secondary metabolism, lipid metabolism, hormone metabolism, photosynthesis, and amino acid biosynthesis. In the present study, functional enrichment analysis revealed that most of the DEGs were related to secondary metabolite biosynthesis, metabolic pathway, carbohydrate metabolism, lipid metabolism, hydrolase activity, catalytic activity, and oxidation-reduction process (Figure 4, Table 2). Moreover, 122 TFs of the DEGs were divided into 22 families, including MYB (16, 13.11%), AP2-EREBP (12, 9.84%), NAC (12, 9.84%), bHLH (10, 8.20%), and other TFs (72, 59.02%) (Table 3). 2514 tobacco TFs, including 274 AP2/ERF, 250 MYB, 190 bHLH, and 152 NAC tobacco TFs, have been reported in the study by [PERSON] et al. [43; 44]. Furthermore, TFs also play an important role in regulating terpenoid (e.g., solanesol) biosynthesis, which largely determines the spatio-temporal expression of the enzyme genes and the specificity and efficiency of the induced expression [39]. Therefore, these candidate transcription factors identified in the present study will be helpful to study the molecular mechanisms of temperature regulation of solanesol accumulation. Transcription of Solanesol Biosynthesis Genes in Tobacco Leaves Treated with Moderately High Temperature  
Solanesol biosynthesis mainly involves two main stages: generation of the C5 isopentenyl diphosphate (IPP) precursor and its double-bond isomer dimethylallyl diphosphate (DMAPP), and generation of synthetic precursors (farnesyl diphosphate (FPP), geranylgeranyl diphosphate (GGPP), and solanesyl diphosphate (SPP)) [6]. Isopentenyl diphosphate and its isomer DMAPP are synthesized via two pathways that occur in different subcellular spaces; i.e., the MVA pathway located in the cytoplasm, and the MEP pathway in the plastids. These two pathways are not independent of each other, as IPP is shuttled between the cytoplasm and the plastids [45; 46]. In the MVA pathway, HMGR catalyzes the conversion of HMG-CoA to MVA, which is the precursor of IPP [38; 46]; FPS catalyzes the condensation of two IPP molecules with one DMAPP molecule to form one FPP molecule [6]. [PERSON] et al. [47] overexpressed the _Hevea brasiliensis HMGR_ gene into the tobacco, resulting in an increase in HMGR activity and a six-fold increase in total sterol content. In the present study, MHT treatment led to a significant upregulation of the transcription level of _NUHMGR_ in tobacco leaves, and a significant downregulation of the transcription level of _NUFPS_ at 3, 6, 9, and 12 DAT (Figure 5). The presence of FPS was found in both cytoplasm and chloroplast of plants [48; 49]. Notably, NtFPS has little effect on the rapid biosynthesis of solanesol in tobacco under MHT treatment. By contrast, as the first rate-limiting enzyme in the MVA metabolic pathway, NUHMGR likely plays a vital role in the rapid biosynthesis of solanesol in tobacco under MHT treatment. DXS is the first enzyme of the MEP metabolic pathway that catalyzes the formation of 1-deoxy-d-xylulose 5-phosphate (DXP) from pyruvate and glycerol-3-phosphate, and DXR catalyzes the reduction of DXP to form MEP [5; 6]. Two _N. tabacum DXR_ genes, _NtDXk1_ and _NtDXk2_, were identified by [PERSON] et al. [50]. Overexpression of _Synechosystis_ sp.