*2.6. E*ff*ect of Chilling Stress on Plastid Proteins during Greening*

To determine whether chilling stress affected plastid protein biosynthesis, immunoblotting analysis was performed (Figure 6). SDS-PAGE showed that the proteins with molecular weight from 20 kDa to 35 kDa under low temperatures were much lower than those at 28 ◦C after 12 h and 48 h of light irradiation (Figure 6C). In etiolated seedlings, PSI (Lhca1, Lhca2, Lhca3, Lhca4, and PsaD) and PSII (D1, D2, CP43, Lchb1, Lchb2, Lchb3, Lchb4, Lchb5, and Lchb6) proteins were undetected, and large amounts of PSI and PSII proteins were rapidly synthesized at 28 ◦C during greening (Figure 6A,B). Chilling stress inhibited the accumulation of PSI and PSII proteins during greening (Figure 6A,B, Supplementary Figures S1 and S2), especially at 12 ◦C, where we hardly detected PSI and PSII proteins.

**Figure 6.** Immunoblot analysis of thylakoid proteins in control and chill-stressed rice seedlings. Six day old etiolated seedlings were treated with 18 ◦C or 12 ◦C chilling stress. Thylakoid proteins were isolated from control and chill-stressed seedlings after 0 h, 0.5 h, 12 h, and 48 h of greening. Immunoblot analyses were performed with antibodies specific for representative photosystem I (PSI) (**A**) and photosystem II (PSII) (**B**). The SDS–PAGE of 20 ug plastid protein stained by Coomassie blue (**C**).

### *2.7. E*ff*ect of Chilling Stress on Chloroplast Biogenesis during Greening*

Chloroplast biogenesis normally depends on a stable supply and correct stoichiometry of chlorophyll and photosynthetic proteins during greening. To further investigate the effect of low temperature on plastid development, plastid morphology was analyzed via transmission electron microscopy (TEM). The results showed that the proplastid developed into the etioplast that contains the prolamellar bodies (PLBs) in etiolated seedlings (Figure 7A). When seedlings were illuminated (120 μmol m−<sup>2</sup> s<sup>−</sup>1) for 48 h at 28 ◦C, PLBs disappeared and thylakoids formed and grana thylakoids stacked regularly (Figure 7B). Grana stacking was inhibited and thylakoids were much looser at 18 ◦C (Figure 7C), and no functional thylakoid structure was formed after 48 h of greening at 12 ◦C (Figure 7D). Taken together, these results suggest that chilling stress inhibited the biogenesis of chloroplast, which might have been due to the lack of chlorophylls and photosynthetic proteins.

Chlorophyll fluorescence is an important indicator of the work status of chloroplasts. Compared with the seedlings grown under low temperature, the seedlings at 28 ◦C showed higher quantitative values of maximum PSII yield (Fv/Fm) and lower non-photochemical quenching (NPQ) (Figure 8). Minimal fluorescence yield (F0) showed no big fluctuations between 28 ◦C, 18 ◦C, and 12 ◦C treatments, but maximal fluorescence yield (Fm) was significantly lower in the chill-stressed seedlings.

**Figure 7.** Effect of chilling stress (18 ◦C and 12 ◦C) on chloroplast biogenesis of rice seedlings. Plastid ultrastructure of etiolated seedlings (**A**); chloroplast ultrastructure after 48 h of greening under normal temperature (28 ◦C) condition (**B**); chloroplast ultrastructure after 48 h of greening at 18 ◦C (**C**); chloroplast ultrastructure after 48 h of greening at 12 ◦C (**D**). G: granum; SL: stroma lamellae; GL: grana lamellae; SG: starch grain. Bar = 1 μm.

**Figure 8.** Effect of chilling stress on chlorophyll fluorescence parameters of rice seedlings. The chlorophyll fluorescence images (**A**) and chlorophyll fluorescence parameters (Fv/Fm, NPQ, F0, Fm) (**B**) after 48 h of greening. Six day old etiolated seedlings were treated with 18 ◦C or 12 ◦C chilling stress. Values are means ± SD from three independent biological replicates. Different letters indicate significant differences according to Duncan's multiple range tests (*p* < *0.05*).

### *2.8. E*ff*ect of Chilling Stress on ROS Accumulation, Oxidation, and Electrolyte Leakage during Greening*

As crucial indexes of oxidative damages under chilling stress, the contents of hydrogen peroxide (H2O2), superoxide anion radical (O2 .−), and malondialdehyde (MDA) were determined. Histochemical detection and quantification analysis showed that H2O2 increased slightly, but the superoxide anion radical (O2 .−) levels remained almost stable at 28 ◦C during greening. An ROS burst occurred in chill-treated leaves, especially at 12 ◦C (Figure 9A–D), indicating that chilling stress could induce ROS accumulation. MDA content and electrolyte leakage (EL) were quantified to examine the lipid peroxidation and the damage to cellular membranes. MDA had a slight increase and EL had no remarkable change at 28 ◦C during greening, but both MDA and EL significantly increased under low temperatures during greening, especially at 12 ◦C (Figure 9E,F). These results indicate that chilling stress induced ROS accumulation and caused lipid peroxidation and finally destroyed the integrity of membranes during greening.

**Figure 9.** Effects of chill-stressed (18 ◦C and 12 ◦C) on H2O2 (**A**,**C**), O2 .<sup>−</sup> (**B**,**D**), MDA content (**E**), and EL (**F**) of rice seedlings. Histochemical detection (**A**,**B**), content of H2O2 (**C**), O2 .<sup>−</sup> (**D**), MDA (**E**), and EL (**F**) 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 ROS levels were measured. Values are means ± SD from three independent biological replicates. Different letters indicate significant differences according to Duncan's multiple range tests (*p* < *0.05*).
