*2.2. The oscaf1 Mutant Exhibited Defects in Chloroplast Development*

The *oscaf1* mutant exhibited an albino seedling phenotype and eventually died after the three leaves stage (Figure 2A). Compared with the wild type (WT), photosynthetic pigment concentrations showed that chlorophyll a (Chla), chlorophyll b (Chlb), and carotenoid (Car) contents decreased significantly in *oscaf1* mutants (Figure 2B). According to the results of chlorophyll fluorescence measurements, the Fv/Fm values of *oscaf1* mutants were low (Figure 2C). The results suggested that *oscaf1* mutants had impaired chloroplast development and photosynthesis. Subsequently, we analyzed the expression levels of chloroplast development and photosynthesis-related genes in WT and *oscaf1* mutants using

qRT-PCR. The results showed that compared with the WT, the transcript levels of *HZMA1*, *PORA*, *CAB1*, *psaA*, *rbcL*, *CHLI*, *CHLH*, *ATPa*, *psbA*, *TaX2*, *ATPB*, and *ATPE* in *oscaf1* mutants decreased, while the relative expression levels of *YGLI*, *HSA1*, *HSA2*, *RPOB*, *RPOC1*, and *RPOC2* increased (Figure 2D). Particularly, in *oscaf1* mutants, the expression levels of *HZMA1*, *PORA*, *CAB1*, *psaA*, *rbcL*, *CHLH,* and *psbA* decreased significantly, and the expression levels of *YGLI*, *HSA1*, *RPOB*, *RPOC1,* and *RPOC2* increased markedly in *oscaf1* mutants (Figure 2D). Overall, the results suggested that OsCAF1 influences chloroplast development and photosynthesis in rice.

**Figure 2.** *oscaf1* showed defects in chloroplast development and photosynthesis. (**A**) Phenotype observations of WT (left) and *oscaf1* mutant (right). Scale bar, 1 cm. (**B**) Pigment content of WT and *oscaf1* at seedling stage. (**C**) Photochemical efficiency of PSII in the light (Fv/Fm) of WT and *oscaf1* mutants at seedling stage. (**D**) Relative expression levels of chloroplast development and photosynthesis genes in WT and *oscaf1* mutants. The data represent mean ± SE from three independent biological duplicate, \* and \*\* indicated *p* < 0.05 and *p* < 0.01, respectively.

To investigate chloroplast development in *oscaf1* mutants further, we observed the ultrastructure of chloroplasts at the three leaves stages of WT and *oscaf1* mutants using transmission electron microscopy (TEM). We observed that the chloroplast number in the mesophyll cells of the *oscaf1* mutant was less than in WT (Figure 3B). In addition, the chloroplasts in WT mesophyll cells were well developed, with normal-looking and distinctly stacked grana and thylakoids (Figure 3A,C). Conversely, the *oscaf1* mutant had abnormal chloroplast architecture along with abnormally structured thylakoid and abnormally stacked grana (Figure 3B,D). The results suggested that OsCAF1 plays a key role in early chloroplast development in rice.

**Figure 3.** Chloroplast ultrastructure observation of WT (**A**,**C**) and *oscaf1* mutants (**B**,**D**) mesophyll cell at the three leaves stage. cp, chloroplast, gl, grana lamella. Bars = 2.0 μm.

*2.3. Increased Reactive Oxygen Species (ROS) Contents and Reduced ROS-scavenging Gene Expression Levels in oscaf1 Mutant*

In plants, the overproduction of ROS might cause cell death in leaves [24]. The *oscaf1* mutants died after the three leaves stage; therefore, we tested the ROS levels in leaves of the WTs and *oscaf1* mutants. The *oscaf1* mutant leaves displayed a more intense brown color following 3,3-diaminobenzidine (DAB) staining, indicating that the levels of H2O2 in *oscaf1* mutant leaves were higher than in WT leaves (Figure 4A). In addition, following nitroblue tetrazolium (NBT) staining, *oscaf1* mutant leaves had larger blue leaf areas than WT leaves. The results suggested that oscaf1 mutant leaves generate more O2<sup>−</sup> than WT (Figure 4B). The results of the experiments indicated that oscaf1 leaves accumulated high ROS levels. Moreover, we measured the H2O2 contents in WT and *oscaf1* mutant leaves. The results indicated that the H2O2 content in the mutant was significantly increased compared to that in WT (Figure 4C). This result was consistent with DAB staining. Meanwhile, the ROS-scavenging gene expression levels were investigated in WT and *oscaf1* mutants. According to the qRT-PCR results, the expression levels of *APX1*, *APX2*, *SODA1*, *SODB*, *CatA*, and *CatC* were significantly decreased in *oscaf1* mutants compared to WT (Figure 4B). The ROS-scavenging genes reduced expression in *oscaf1* might have had feedback from lack of OsCAF1. Overall, a decrease in ROS-scavenging gene expression levels would impair the ROS detoxification system, eventually leading to the accumulation of ROS, such as H2O2 in *oscaf1* mutant leaves.

**Figure 4.** DAB, NBT staining, H2O2 contents and gene expression level analysis in WT and *oscaf1* mutants. (**A**) DAB staining of leaves from WT (left) and oscaf1 mutants (right). Scale bar, 1 cm. (**B**) NBT staining of leaves from WT (left) and oscaf1 mutants (right). Scale bar, 1 cm. (**C**) Measurement of H2O2 content in WT and *oscaf1* mutant (**D**) Relative expression levels of genes involved in ROS-scavenging. The data represent mean ± SE from three independent biological duplicate, \* and \*\* indicated *p* < 0.05 and *p* < 0.01, respectively.
