*2.3. E*ff*ect of Chilling Stress on the Accumulation of Chlorophyll Intermediates in Rice Seedlings during Greening*

To explore how chilling stress inhibits chlorophyll biosynthesis in rice seedlings during greening, the accumulation of chlorophyll intermediates was measured.

ALA is the first intermediate of chlorophyll biosynthesis. As shown in Figure 3A, light promoted ALA accumulation. After 48 h of light exposure, ALA contents were 19.4% lower at 18 ◦C and 46.8% lower at 12 ◦C compared with those at 28 ◦C. These results indicate that chilling stress inhibited the synthesis of ALA during greening.

PBG content at 28 ◦C was increased by 14.8%, 104.9%, and 155.2% after 0.5 h, 12 h, and 48 h of light exposure, respectively (Figure 3B), and there was no significant difference of PBG content between chill-treated rice seedlings and the control seedlings after 12 h and 48 h of greening. Urogen III and coprogen III levels were significantly increased at 28 ◦C during greening (Figure 3C,D). Under low temperatures, the contents of urogen III and coprogen III during greening were higher than those at 28 ◦C. Proto IX level was gradually decreased after exposure to light, and the rate of decline was accelerated under low temperatures (Figure 3E).

**Figure 3.** Chlorophyll biosynthesis intermediates during the greening period. δ-Amino levulinic acid (ALA, **A**), porphobilinogen (PBG, **B**), uroporphyrinogen III (urogen III, **C**), coproporphyrinogen III (coprogen III, **D**), protoporphyrin IX (Proto IX, **E**), Mg-protoporphyrin IX (Mg-proto IX, **F**), Mg-protoporphyrin monomethyl ester (Mpe, **G**), protochlorophyllide (Pchlide, **H**), chlorophyllide *a* (Chlide *a*, **I**), chlorophyllide *b* (Chlide *b*, **J**), chlorophyll *a* (Chl *a*, **K**), chlorophyll *b* (Chl *b*, **L**), Mg-protoporphyrin IX diester (Mpde, **M**), and heme (**N**) content of control (28 ◦C) and cold-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 cold stress. Seedlings were harvested at 0 h, 0.5 h, 12 h, and 48 h of greening and their chlorophyll biosynthesis intermediates contents were measured. The relative content of intermediates at 28 ◦C after 48 h of light exposure was defined as 100%, except Pchlide; the relative content of Pchlide at 0 h was defined as 100% due to its massive accumulation in the dark. The error bars represent standard deviations of three independent biological replicates. Different letters indicate significantly different at *p* < 0.05 according to Duncan's multiple range tests.

Mg-proto IX is the first intermediate of the Mg branch in the chlorophyll biosynthesis pathway. As shown in Figure 3F, Mg-proto IX increased by 34.5%, 97.5%, and 155.3% after 0.5 h, 12 h, and 48 h of light exposure at 28 ◦C, respectively. However, the level of Mg-proto IX was significantly decreased during greening under the cold condition. These results demonstrated that chilling stress also inhibited the synthesis of Mg-proto IX during greening. Mg-Proto monomethyl ester (Mpe) content was increased during greening, and was barely influenced by the low temperature (Figure 3G).

In the dark, massive amounts of Pchlide were accumulated, because the protochlorophyllide oxidoreductase (POR) in angiosperms is strictly light-dependent. The etiolated rice seedlings accumulated plenty of Pchlide (Figure 3H). The content of Pchlide was drastically decreased at 28 ◦C after 0.5 h of light exposure, while Chlide a and Chlide b contents were increased rapidly and then decreased during greening (Figure 3I,J). Compared with 28 ◦C, the levels of Pchlide were higher but the Chlide a and Chlide b contents were lower in chill-treated groups after 0.5 h and 12 h of light exposure. Obviously, the cold treatment lowered the conversion efficiency of Pchlide to Chlide, especially at 12 ◦C.

The contents of Chl *a* and Chl *b* were almost undetectable in etiolated seedlings (Figure 3K,L), which were significantly increased at 28 ◦C during greening. Under low temperatures, the synthesis of Chl *a* and Chl *b* was significantly suppressed during greening, especially at 12 ◦C. The Chl *a* and Chl *b* contents at 12 ◦C showed little difference from those in the dark. These results suggested that chilling stress greatly inhibited the synthesis of Chl *a* and Chl *b* during greening.

Mpe can also convert to Mg-protoporphyrin IX diester (Mpde) and then further form Chlide a ester. Mpde contents at both 28 ◦C and low temperatures were increased during greening (Figure 3M). Heme is the product of the Fe branch, which usually acts as a cofactor in respiration and photosynthesis. Heme contents at different time points had no significant difference during greening (Figure 3N). However, the contents of heme after 48 h of light exposure were decreased by 19.3% and 29.2% at 18 ◦C and 12 ◦C, respectively. These results also indicate that chilling stress inhibited the pathway of Fe2<sup>+</sup> branch during greening.

In summary, the inhibition of chlorophyll biosynthesis under chilling stress may be attributed to inhibited synthesis of ALA and hampered conversion from Pchlide into Chls.
