*2.5. E*ff*ect of Chilling Stress on Transcriptional Expression of Chlorophyll Biosynthetic Genes*

To decipher the effects of chilling stress on transcription levels of chlorophyll biosynthetic genes, we analyzed the relative expression of four key chlorophyll biosynthetic genes by RT-qPCR. At 28 ◦C, the expression of *HEMA* was increased initially, and then decreased after 12 h of light exposure (Figure 5A). This phenomenon might have been due to avoidance of the oxidative stress caused by excessive tetrapyrrole intermediate accumulation. However, the chilling stressed rice seedlings accumulated more transcript of *HEMA* after 12 h of light exposure. At 28 ◦C, the RNA of *CHLH* after 0.5 h of light exposure was slightly higher than that in etiolated seedlings, but the expression of *CHLH* was much higher at 18 ◦C (Figure 5B). The expression of most chlorophyll biosynthetic genes was inhibited in the dark, but *PORA* transcripts accumulated in the etiolated seedlings but were degraded rapidly upon illumination (Figure 5C). In contrast, *PORB* mRNA level did not fluctuate at 28 ◦C after illumination. Interestingly, the mRNA level of *PORB* was very high at 18 ◦C and significantly lowered

at 12 ◦C (Figure 5D). The expression of *DVR* increased at 28 ◦C and 18 ◦C after 12 h of light irradiation, but at 12 ◦C it had no significant difference from that in the dark (Figure 5E). When the temperature was below 18 ◦C, the expressions of *CHLH*, *PORB*, and *DVR* were severely repressed. Taken together, the repressed expression of chlorophyll biosynthetic genes might be responsible for the inhibition of chlorophyll biosynthesis under low temperatures.

**Figure 5.** Effect of chilling stress on relative expression of chlorophyll biosynthetic genes *HEMA* (**A**), *CHLH* (**B**), *PORA* (**C**), *PORB* (**D**), and *DVR* (**E**). The expression levels of genes at 28 ◦C after 48 h of light exposure were set to 1. *OsACTIN1* was used as an internal standard. Values are means ± SD from three independent biological replicates. Different letters indicate significant differences according to Duncan's multiple range tests (*p* < *0.05*).
