*2.1. Transcriptome Analysis of Plants under Warming, Minor-Warming, and Low-Temperature (LT) Conditions Using the Br135K Microarray*

To identify putative warming-related (or HT adaptation-related) genes, we carried out Br135K microarray analysis of samples from two inbred lines, Chiifu and Kenshin, under three conditions (22 ◦C, 12 ◦C, and 12 → 28 ◦C) (Figure 1). The experiments were repeated twice; the mean values are summarized in Table S2. The microarray data have been deposited in "NCBI (https://www.ncbi. nlm.nih.gov/)" with [geo] GSE113637. Among the 41,173 genes deposited on the Br135K microarray, 14,222 (35%) showed probe intensity (PI) values <500 in all samples, whereas 26,951 (65%) showed PI values >500 in at least one sample. Of these 26,951 genes, 2104 had no *Arabidopsis* counterpart, i.e., NA (nonannotated genes). We subjected these 26,951 genes to further analysis because genes with a PI value of 500 (cutoff value) can easily be examined using standard RT-PCR. Responsive

genes (i.e., DEGs) were defined as having at least a 2-fold change (cutoff value) in expression between comparative conditions. As shown in Figure 1, three types of comparisons were made: warming (samples treated for 2 days at 12 ◦C vs. samples after 3 h exposure to 28 ◦C), minor-warming (samples grown at 22 ◦C [control] vs. samples after 3 h exposure to 28 ◦C), and low-temperature (LT) treatment (samples grown at 22 ◦C vs. samples treated for 2 days at 12 ◦C). RT-PCR. Responsive genes (i.e., DEGs) were defined as having at least a 2-fold change (cutoff value) in expression between comparative conditions. As shown in Figure 1, three types of comparisons were made: warming (samples treated for 2 days at 12 °C vs. samples after 3 h exposure to 28 °C), minor-warming (samples grown at 22 °C [control] vs. samples after 3 h exposure to 28 °C), and low-temperature (LT) treatment (samples grown at 22 °C vs. samples treated for 2 days at 12 °C).

*Int. J. Mol. Sci.* **2018**, *19*, x FOR PEER REVIEW 3 of 22

**Figure 1.** Temperature treatment and sampling schedule. Collection times are indicated by circles. Red circles (**a**–**c**) represent sampling times for the microarray experiments as well as qRT-PCR analysis. Blue circles indicate that the collected samples were only used for qRT-PCR. Shoots from five individual plants were sampled and frozen in liquid nitrogen. Treatments were as follows: (**a**) (22 °C) to (**b**) (12 °C); low-temperature (LT) conditions; (**a**) (22 °C) to (**c**) (28 °C), minor-warming conditions; and (**b**) (12 °C) to (**c**) (28 °C), warming conditions. **Figure 1.** Temperature treatment and sampling schedule. Collection times are indicated by circles. Red circles (**a**–**c**) represent sampling times for the microarray experiments as well as qRT-PCR analysis. Blue circles indicate that the collected samples were only used for qRT-PCR. Shoots from five individual plants were sampled and frozen in liquid nitrogen. Treatments were as follows: (**a**) (22 ◦C) to (**b**) (12 ◦C); low-temperature (LT) conditions; (**a**) (22 ◦C) to (**c**) (28 ◦C), minor-warming conditions; and (**b**) (12 ◦C) to (**c**) (28 ◦C), warming conditions.

#### 2.1.1. Warming-Responsive Genes 2.1.1. Warming-Responsive Genes

We identified 6862 warming-responsive genes. Similar numbers of these genes were upregulated in Chiifu and Kenshin, but more were downregulated in Chiifu (Figure 2A; Table S3). Genes in the Gene Ontology (GO) biological process categories "response to heat", "heat acclimation", "response to light intensity", "response to oxidative stress", and "response to temperature stimulus" were enriched among upregulated genes in both lines (group (c) in Figure 2B,C). The categories "response to hormone stimulus", "plasma membrane", "defense response", and "lipid biosynthetic process" were enriched among genes that were specifically upregulated in Kenshin (group (b) in Figure 2B,C). Genes in the categories "response to water deprivation", "response to osmotic stress", "response to sucrose stimulus", and "response to cold" were enriched among downregulated genes in both lines (group (f) in Figure 2B,C). Genes in two categories, "transcription regulatory activity" and "response to auxin stimulus", were upregulated in Kenshin but downregulated in Chiifu under warming conditions (group (h) in Figure 2B,C). We identified 6862 warming-responsive genes. Similar numbers of these genes were upregulated in Chiifu and Kenshin, but more were downregulated in Chiifu (Figure 2A; Table S3). Genes in the Gene Ontology (GO) biological process categories "response to heat", "heat acclimation", "response to light intensity", "response to oxidative stress", and "response to temperature stimulus" were enriched among upregulated genes in both lines (group (c) in Figure 2B,C). The categories "response to hormone stimulus", "plasma membrane", "defense response", and "lipid biosynthetic process" were enriched among genes that were specifically upregulated in Kenshin (group (b) in Figure 2B,C). Genes in the categories "response to water deprivation", "response to osmotic stress", "response to sucrose stimulus", and "response to cold" were enriched among downregulated genes in both lines (group (f) in Figure 2B,C). Genes in two categories, "transcription regulatory activity" and "response to auxin stimulus", were upregulated in Kenshin but downregulated in Chiifu under warming conditions (group (h) in Figure 2B,C).
