*2.8. Expression Profiling of NcSOD Genes in Pollen and Ovule*

The expression pattern of the *NcSOD* gene family was obtained from our own RNA-seq raw data (unpublished). All 9 *NcSOD* genes' expression levels were explored in 1 day mature pollen, and 0, 1, 2, and 3 days mature ovule. The expression heatmap was constructed using TBtools (V 1.068, https://github.com/CJ-Chen/TBtools/ accessed on 9 March 2023), in which the color bar from light yellow to a dark red exhibited less to high levels of expression, and light blue to dark blue shows less or no expression of *NcSOD* genes.

#### *2.9. Plant Materials and Abiotic Stresses*

To examine how *NcSOD* members respond to different abiotic stresses, we grew *N. colorata* mature plants in water tub filled with tap water under an open environment. The plants were then subjected to various stress treatments, including 250 mM NaCl, 200 μM CuSO4, and 2.5 mM CdCl2, as well as being treated with cold stress at 8 ◦C and heat stress at 42 ◦C. Each treatment was performed with three independent biological replicates, and each sample was collected from at least five individual plants. Leaves from the plantlets were collected at 0, 2, 4, and 6 h for the salt, heat, cold, and heavy metal stress experiments. After collection, all samples were instantly frozen in liquid nitrogen and preserved at −80 ◦C until total RNA isolation.

## *2.10. RNA Isolation and Real-Time Quantitative PCR Expression Analysis*

RNA extraction was performed using the RNAprep Pure Plant Kit (TIANGEN, Beijing, China). The concentration of the samples was determined using a NanoDrop 2000 C spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The genomic DNA, was removed by DNase I treatment, followed by cDNA synthesis using the QuantiTect Reverse Transcription Kit (Qiagen, Shanghai, China). The RT-qPCR expression was performed on the Roche LightCycler 96 PCR system, following the recommended guidelines for the ChamQTM SYBR RT-qPCR Master Mix (Vazyme Biotech Co., Ltd., Sanya, China). For each RT-qPCR, the expression level of the actin gene in *N. colorata* was employed to standardize the RNA samples. Three biological replicates for each sample were employed for RT-qPCR, analysis with actin as internal control. Gene-specific primers for *NcSODs* and Nc-actin in the RT-qPCR system were designed using the online NCBI Primer-BLAST Program and their specificity was confirmed using the Oligo Calculator online tool (http: //mcb.berkeley.edu/labs/krantz/tools/oligocalc.html accessed on 5 April 2023). The primers were synthesized by NANSHAN BIOTECH, (Sanya), and listed in (Table S1). The 2−ΔΔCT method was used to analyze the RT-qPCR gene expression data [44].
