2.1.2. Minor Warming-Responsive Genes

We identified 6596 minor warming-responsive genes; more of these genes were upregulated in Kenshin and downregulated in Chiifu (Figure 3A; Table S4). Upregulated genes in both lines were enriched in similar categories to those of warming-responsive genes, such as "response to heat", "heat acclimation", "response to light intensity", "response to oxidative stress", and "response to temperature stimulus" (group (c) in Figure 3B,C). Genes upregulated specifically in Kenshin were enriched in the categories "response to stimulus", "response to stress", "transcription factor activity", and "response to water deprivation" (group (b) in Figure 3B,C). Genes in three categories, "response to stimulus", "response to light stimulus", and "response to auxin stimulus", were upregulated in Kenshin but downregulated in Chiifu in response to minor-warming conditions (group (h) in Figure 3B,C). The

category "response to auxin stimulus" was enriched among genes upregulated in Kenshin under both warming and minor-warming conditions, suggesting that the auxin response might contribute to heat tolerance in Kenshin. *Int. J. Mol. Sci.* **2018**, *19*, x FOR PEER REVIEW 4 of 22

**Figure 2.** Analysis of warming-responsive genes from two contrasting inbred lines, Chiifu and Kenshin. (**A**) Venn diagram of DEGs with over 2-fold differences in expression; a–h indicate the groups of genes in each category; (**B**) Heatmap of DEGs in each group based on fold change; (**C**) Gene Ontology (GO) enrichment analysis of DEGs in each group, with *p* values obtained using the agriGO tool (http://bioinfo.cau.edu.cn/agriGO/index.php). C and K of Figure 2B indicate Chiifu and Kenshin, respectively. **Figure 2.** Analysis of warming-responsive genes from two contrasting inbred lines, Chiifu and Kenshin. (**A**) Venn diagram of DEGs with over 2-fold differences in expression; a–h indicate the groups of genes in each category; (**B**) Heatmap of DEGs in each group based on fold change; (**C**) Gene Ontology (GO) enrichment analysis of DEGs in each group, with *p* values obtained using the agriGO tool (http://bioinfo.cau.edu.cn/agriGO/index.php). C and K of Figure 2B indicate Chiifu and Kenshin, respectively. *Int. J. Mol. Sci.* **2018**, *19*, x FOR PEER REVIEW 5 of 22

**Figure 3.** Analysis of minor warming-responsive genes from two contrasting inbred lines, Chiifu and Kenshin. (**A**) Venn diagram of DEGs with over 2-fold differences in expression; a–h indicate the groups of genes in each category; (**B**) Heatmap of DEGs in each group based on fold change; (**C**) GO enrichment analysis of DEGs in each group, with *p* values obtained using the agriGO tool (http://bioinfo.cau.edu.cn/agriGO/index.php). C and K of Figure 3B indicate Chiifu and Kenshin, respectively. **Figure 3.** Analysis of minor warming-responsive genes from two contrasting inbred lines, Chiifu and Kenshin. (**A**) Venn diagram of DEGs with over 2-fold differences in expression; a–h indicate the groups of genes in each category; (**B**) Heatmap of DEGs in each group based on fold change; (**C**) GO enrichment analysis of DEGs in each group, with *p* values obtained using the agriGO tool (http://bioinfo.cau.edu. cn/agriGO/index.php). C and K of Figure 3B indicate Chiifu and Kenshin, respectively.

to be involved in heat tolerance [21]. In addition, we wanted to examine the possible association between LT treatment and the warming response. We identified 5205 LT-responsive genes, for which more genes were upregulated in Kenshin and downregulated in Chiifu (Figure 4; Table S5), implying that Kenshin is more sensitive to LT exposure than Chiifu. Many GO categories were identified as enriched among upregulated genes in both lines, including "response to cold", "response to stress", and "lipid transport and metabolic process". Although similar GO categories were enriched among LT-responsive genes and genes in the two categories mentioned above (warming and minor warming), the expression of warming- and minor warming-responsive genes

The identification of LT-responsive genes was not the main objective of this study, but

2.1.3. LT-Responsive Genes

might not be influenced by LT treatment (Table S5).

respectively.

**Inbred Line** 

Chiifu (22 °C ⟶ 12 °C ⟶ 28 °C)

Kenshin (22 °C ⟶ 12 °C ⟶ 28 °C)

conditions.
