Fig. 1. Phenotypic characterization of wild type (WT, Zhonghua 11) and tsa4 mutant plants. A, Phenotypes of seedlings of WT and tsa4 mutant at 5-day-old grown in the field. B, Phenotypes of seedlings of WT and tsa4 mutant at 2-week-old grown in the field. C, Field temperature data from 1st April to 14th April, 2024. D-G, Phenotypes of WT and tsa4 mutants grown at temperatures of 25 ºC (D and F) and 35 ºC (E and G). H and I, Pigment contents of WT and tsa4 seedlings grown at temperatures of 25 ºC (H) and 35 ºC (I). Chl a, Chlorophyll a; Chl b, Chlorophyll b; Car, Carotenoid. Data are mean ± SD (n = 3). ** indicates significant difference at the 0.01 level. J-Q, Chloroplast ultrastructure in cells of the WT and tsa4 plants at 25 ºC (J-M) and 35 ºC (N-Q). Chloroplast (J, L, N and P, Scale bars are 5 μm) and thylakoid (K, M, O and Q, Scale bars are 2 μm) are clearly detected. CP, Chloroplast; Thy, Thylakoid.

Fig. 2. Map‐based cloning of TSA4 gene and construction and characteristics of tsa4 mutant. A, TSA4 was located between InDel markers S2 and S3 on chromosome 4 (Chr. 4). The locus was further narrowed down to a 158-kb region between InDel markers S6 and S7. The mutation site of TSA4 is shown by a black arrow. Five black boxes indicate five exons of TSA4. ATG and TAG is initiation codon and termination codon of TSA4. B, Mutations of two CRISPR-Cas9 knockout lines with a GG deletion (KO#1) and an A insertion (KO#2). C and D, Phenotypes of WT and TSA4 knockout lines (KO#1 and KO#2) at the seedling stage at 25 ºC (C) and 35 ºC (D). E, Free green fluorescent protein (GFP) and TSA4‐GFP fusion protein were transiently expressed in tobacco epidermal cells. Red signal is chloroplast auto-fluorescence. Scale bars, 20 μm. F and G, Relative expression levels of TSA4 in different rice organs of WT at 25 ºC (F) and in WT and tsa4 leaves grown under different temperatures (G). H-I, Relative expression levels of chloroplast development-related genes in WT and tsa4 mutant grown at 25 ºC (H) and 35 ºC (I). Tubulin gene is served as the internal control. In F-I, Data are mean ± SD (n = 3). * and ** indicate significant difference at the 0.05 and 0.01 probability level, respectively.