*4.6. Expression Analysis of Trihelix Transcription Factor Family*

Total RNA was extracted from rice tissues by the TRIzol method (Thermo Fisher Scientific, Waltham, MA, USA) and treated with DNase to eliminate any DNA contamination. RNA quality was assessed by electrophoresis and stored at −80 ◦C until use. First-strand cDNA (10 µL) was synthesized according to the instructions for the PrimeScript™ RT Master Mix (Takara Biomedical Technology (Beijing) Co., Ltd., Beijing, China). Primers were designed with Primer Premier v. 5.0

(PREMIER Biosoft International, Palo Alto, CA, USA) and were based on the trihelix gene family transcript sequences. Gene specific primers for quantitative real-time PCR are listed in Table S6. Primer amplification specificity was verified in the rice genome database using Blast from NCBI (https://www.ncbi.nlm.nih.gov/) [51]. Rice β-actin was the internal reference gene. Quantitative real-time PCR was performed in the ABI 7300 Real Time PCR System (Applied Biosystems, Foster City, CA, USA) using SYBR Green chemistry and reaction mix consists of 10 µL SYBR qPCR Master Mix (Vazyme Biotech Co.,Ltd., Nanjing, China), 0.4 µL upstream and downstream primers respectively, 0.4 µL ROX, 2 µL cDNA (10 times dilution) and 6.8 µL ddH2O to 20 µL. The PCR reaction protocol was 95 ◦C for 5 min; 95 ◦C for 10 s; 60 ◦C for 20 s; 72 ◦C for 20 s; 45 cycles. Gene expression levels were calculated by the 2-∆∆CT method: ∆∆CT = (CTtarget <sup>−</sup> CTactin) at time x <sup>−</sup> (CTtarget <sup>−</sup> CTactin) at time 0 [52]. The test was repeated three times. Expression data for the rice trihelix family genes were retrieved from the Expression Atlas database (https://www.ebi.ac.uk/gxa/home) [53]. Heatmaps were created in HemI v.1.0 (The CUCKOO Workgroup, Hubei, China) and based on the expression data [54].
