## 3 Results
### Gene Ontology Functional Enrichment Analysis of Differentially Expressed Genes  
Gene ontology functional annotations of DEGs were compared between the treatment groups to identify biological functions that were significantly affected by MHT treatment [23; 24]. In GO enrichment analysis based on the non-central hyper-geometric distribution [35], the probability of drawing an individual from within a certain group is different from the probability of drawing an individual from outside of that group. The results of GO functional enrichment analysis are shown in Table 2. Under'molecular function' type, 'hydrolase activity, hydrolysis \(O\)-glycosl compounds', 'hydrolase activity, acting on glycosyl bonds', 'copper ion binding', and 'catalytic activity' were significantly enriched in the plants exposed to MHT. Under 'biological process' type, 'carbohydrate metabolic process', 'lipid metabolic process', 'oxidation-reduction process', and'single-organism metabolic process' were significantly enriched in the plants exposed to MHT. Thus, GO functional enrichment analysis indicated that MHT treatment affected genes related to the above processes.  
Kyoto Encyclopedia of Genes and Genomes Functional Enrichment Analysis of Differentially Expressed Genes  
Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis was used to reveal the major metabolic and signal transduction pathways affected by MHT treatment [24; 36]. The 20 most significantly enriched pathways in this study are shown in Figure 4. The q-value for 'glycerophospholipid metabolism' was <0.01, suggesting that genes in the related pathways were significantly enriched in MHT-exposed tobacco leaves. The q-values for 'DNA replication', 'phenylpropanol biosynthesis', 'ether lipid metabolism','starch and sucrose metabolism', and 'amino sugar and nucleotide sugar metabolism' ranged between 0.01 and 0.05, suggesting that genes in these pathways were relatively enriched in MHT samples. The number of DEGs in the categories of 'biosynthesis of secondary metabolites' and'metabolic pathway' were 114 and 199, respectively (Figure 4). Thus, KEGG functional enrichment analysis indicated that MHT treatment affected genes related to the biosynthesis of secondary metabolites and metabolic pathway.  
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline
**GO ID** & **GO Terms** & **Type 1 ** & **Corrected \(p\)-Value** & **Test 2 ** & **Ref. 3 ** \\ \hline GO0004553 & Hydrolase activity, hydrolyzing O-glycosl compounds & P & \(1.43\times 10^{-9}\) & 77 & 1039 \\ GO0016798 & Hydrolase activity, acting on glycosyl bonds & P & \(1.43\times 10^{-9}\) & 80 & 1107 \\ GO0009975 & Carbohydrate metabolic process & F & \(1.13\times 10^{-7}\) & 117 & 2085 \\ GO0006629 & Lipid metabolic process & F & \(6.13\times 10^{-7}\) & 104 & 1831 \\ GO0005507 & Copper ion binding & P & \(6.13\times 10^{-7}\) & 28 & 243 \\ GO0003824 & Catalytic activity & P & \(6.13\times 10^{-7}\) & 810 & 23,582 \\ GO0055114 & Oxidation-reduction process & F & \(2.26\times 10^{-6}\) & 199 & 4547 \\ GO0044710 & Single-organism metabolic process & F & \(4.53\times 10^{-6}\) & 380 & 9926 \\ \hline \hline \end{tabular}  
* GO ontology type: ‘F’ represents ‘biological process’, and ‘P’ represents ‘molecular function’. b Number of DEGs belonging to each GO term. [ENDFOOTNOTE]  
\end{table}
Table 2: Eight statistically enriched gene ontology (GO) terms of DEGs between normal temperature (NT) and moderately high temperature (MHT) exposed tobacco leaves