*2.4. E*ff*ect of Chilling Stress on Enzyme Activities in Chlorophyll Biosynthesis*

To further investigate the inhibitory mechanism of chlorophyll biosynthesis, we next examined some key enzymes involved in chlorophyll biosynthesis. Enzymatic activity of glutamate-1-semialdehyde transaminase (GSA-AT), which catalyzes glutamate-1-semialdehyde to ALA, was significantly increased at 28 ◦C and 18 ◦C (Figure 4A) during greening. However, low temperature decreased GSA-AT activity, and the activity of GSA-AT at 12 ◦C had no significant difference from that recorded in the dark (Figure 4A). ALA dehydratase (ALAD) activity was slightly increased after light exposure, and chilling stress had no significant effect on ALAD activity during

greening (Figure 4B). Mg-chelatase is a key enzyme which initiates the Mg branch of the chlorophyll biosynthesis pathway. Mg-chelatase activity was increased during greening, and chilling stress had an inhibitory effect on the activity of Mg-chelatase (Figure 4C). POR is a light-dependent enzyme in angiosperms that converts Pchlide to Chlide. POR activity went down gradually during greening (Figure 4D) and chill-treated seedlings showed lower POR activities, especially at 12 ◦C. In short, the lower ALA content might be attributable to the fact that cold stress inhibited GSA-AT activity, and the inhibition of conversion from Pchlide into Chlide might have been due to the low POR activity under chilling stress.

**Figure 4.** Effects of chilling stress (18 ◦C and 12 ◦C) on activities of enzymes involved in chlorophyll biosynthesis. Glutamate-1-semialdehyde transaminase (GSA-AT, **A**), ALA dehydratase (ALAD, **B**), Mg-chelatase (**C**), protochlorophyllide oxidoreductase (POR, **D**) activities of control (28 ◦C) and chill-stressed (18 ◦C and 12 ◦C) rice seedlings after 0 h, 0.5 h, 12 h, and 48 h greening. Six day old etiolated seedlings were treated with 18 ◦C or 12 ◦C chilling stress. Seedlings were harvested at 0 h, 0.5 h, 12 h, and 48 h of greening and their activities of enzymes involved in chlorophyll biosynthesis were measured. The activities of enzymes at 28 ◦C after 48 h of light exposure were defined as 100%. Values are means ± SD from three independent biological replicates. Different letters indicate significant differences according to Duncan's multiple range tests at *p* < 0.05. Each data point is the average of three replicates. The error bars represent SD.
