*4.3. RNA Extraction and qRT-PCR Analysis*

For RNA extraction and subsequent qRT-PCR analysis, 3 biological replicates were used for each of the three genotypes (i.e., WT, M, and ECE). Each replicate consisted of 3 seedlings pooled together before RNA extraction, and the seedlings were from the same batch used for the metabolomics analysis. Moreover, each qRT-PCR reaction was performed with 3 technical replicates. Total RNA was extracted using Qiagen RNeasy Mini Kit (Cat No. 74904) from 2-week-old seedling shoots without roots according to the manufacturer'sinstructions. The RNA quality was determined by a Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies), and RNA samples with A260/A280 ratios between 1.9 and 2.1 were selected for further analysis. One microgram total RNA was reverse transcribed using Maxima First-Strand cDNA Synthesis Kit (ThermoFisher Scientific, Waltham, MA, USA, Cat No. K1671), as per the manufacturer's instructions. The qRT-PCR analysis was carried out using selected gene-specific primer pairs (Table S3) on *BIO-RAD* CFX384 Real-Time system with denaturation at 95 ◦C for 10 min, followed by 40 cycles of denaturation at 95 ◦C for 15s and annealing/extension at 60 ◦C for 1 min. The amplification of an *ACTIN* gene (*OsACT1*) was used as an internal control to normalize the data. Melting curve analyses were performed to confirm the amplicon specificity [63]. The values were expressed as the average of three independent biological samples, each averaged from its three technical replicates. The relative expression levels were calculated using the 2−ΔΔCt method [64]. Differentially expressed transcripts were derived from two-sided unpaired t-tests, with a *p* value of less than 0.05 considered to be statistically significant. The data analysis and charting (with reference to WT) was performed using Microsoft Excel 2016 (Microsoft, Redmond, WA, USA).
