*3.4. RNA Extraction and Quantitative Real-time PCR (qRT-PCR) Assay*

Total rice RNA from leaves of *oscaf1* and WT plants was extracted using an RNA extraction Kit (TaKaRa, Japan). First-strand cDNA was synthesized using a ReverTra Ace qPCR RT Kit (TOYOBO, Japan). The qRT-PCR was conducted using a SYBR green real-time PCR master mix (TOYOBO, Japan) on a Bio-Rad CFX96 system according to the manufacturer's instructions. The qRT-PCR procedure was as follows: 5 min at 95 ◦C followed by 40 cycles of 95 ◦C for 10 s and 58 ◦C for 1 min. *OsActin1* were used as internal controls. The fluorescence data were analysis by Lin-RegPCR program [31,32] to calculate primer efficiency and obtain Ct values. The genes relative expression levels were calculated according to previous study [33]. All qRT-PCR primers are listed in Supplementary Table S1. For date statistical significance was analyzed using ANOVA with Tukey post hoc pairwise comparisons. \* and \*\* indicate *p* < 0.05 and *p* < 0.01, respectively.

### *3.5. Chloroplast RNA Splicing Analysis*

The cDNA of WT and *oscaf1* plants seeding leaves were obtained according to the procedures above. The RT-PCR procedure was as follows: 95 ◦C for 5 min, followed by 32 cycles of 95 ◦C for 30 s, 60 ◦C for 30 s, 72 ◦C for 1 min, and a final elongation step at 72 ◦C for 8 min. The corresponding RT-PCR primers were used for chloroplast RNA splicing analysis according to previous studies [18,26].
