*2.1. Plant Materials and Treatments*

*L. megaphylla* trees were grown in a field at the Zhengzhou Botanical Garden located in Zhengzhou city, Henan Province, China. Young seedlings were cultivated in plant growth chambers with LED lighting for temperature treatments. In total, 6 experimental sets were cultivated for RNA extraction. The first experimental set consisted of roots, stems, and leaves from 1-year-old seedlings that displayed robust and consistent growth. The second experimental set consisted of leaf buds, young stems, young seeds, young leaves, and mature leaves from adult trees of *L. megaphylla* that had been growing in the natural environment for approximately 10 years. The third experimental set included leaves at different developmental stages. Growing leaves were collected roughly every 3–7 or 3–15 days from the beginning of leaf bud growth in late March until the leaves were fully mature by the end of July. The specific sampling dates were 24 March, 29 March, 1 April, 4 April, 7 April, 15 April, 22 April, 29 April, 14 May, 23 May, 4 June, 16 June, 1 July, 15 July, and 31 July. The fourth set was exposed to cold stress [41]. Furthermore, 1-year-old seedlings were grown at 65% relative humidity and under 16 h/8 h light/dark conditions in an LED plant growth incubator (Shengyuan Instrument Co., Ltd., Zhengzhou, China). The seedlings were first treated at 25 ◦C for 7 days as control, and leaves were collected. Then, the seedlings were transferred to 4 ◦C for 7 days for long-term chilling acclimation (CA). Next, the seedlings were shifted to 0 ◦C for an additional 7 days for longterm freezing acclimation (FA). Then, the seedlings were again moved to control conditions (25 ◦C) for 7 days for long-term de-acclimation (DA). The fifth and sixth experimental groups consisted of leaves collected from 1-year-old seedlings that were treated with cold and heat, respectively. For cold treatments, the seedlings were cultivated in an LED plant growth incubator at 25 ◦C, 4 ◦C, 0 ◦C, −4 ◦C, or −6 ◦C for 24 h. For heat treatments, the seedlings were cultivated in an incubator at 25 ◦C, 30 ◦C, 35 ◦C, 40 ◦C, or 45 ◦C for 24 h. Data regarding all six sample sets described above are summarized in Table 1. All collected samples were immediately frozen in liquid nitrogen and stored at −80 ◦C. Three independent biological replicates were collected for each sample.


**Table 1.** Six experimental sets of *L. megaphylla*.

#### *2.2. Total RNA Extraction and cDNA Synthesis*

Total RNA was extracted from 0.05 g samples using a Quick RNA isolation kit (HUAYUEYANG Biotechnology, Beijing, China) according to the manufacturer's protocol [42]. The RNA integrity, purity and concentration were assessed using 2% (*w*/*v*) agarose gel electrophoresis and a NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Waltham, MA, USA). Total RNA (1 μg) with A260/A280 and A260/A230 ratios greater than 1.8 was used for first-strand cDNA synthesis using the Evo M-MLV RT Kit with gDNA Clean for qPCR according to the manufacturer's instructions (Accurate Biotechnology, Changsha, China). Specifically, a 10 μL reaction system (1 μg total RNA, 2 μL 5 × gDNA Clean Reaction Mix, to a total volume of 10 μL with RNase-free water) was subjected to 42 ◦C for 2 min. Then, a 20 μL reaction system (10 μL of first reaction solution, 4 μL 5 × *Evo M-MLV* RT Reaction Mix and 6 μL RNase-free water) was subjected to 37 ◦C for 15 min and 85 ◦C for 5 s. Five-fold diluted cDNA was used for subsequent RT-qPCR experiments. All cDNA samples were stored at −20 ◦C until use.

#### *2.3. Selection of Candidate Reference Genes and Design of RT-qPCR Primers*

In total, 20 candidate genes (*TCTP*, *ACT7*, *GAPDH*, *UBC36*, *UBC7*, *EF2*, *CYP20-2*, *UBQ*, *TUA*, *UBC28*, *ICln*, *ubiquinone*, *PPR*, *SDE2*, *EIF4A-3*, *helicase-15*, *PAB2*, *CYP9*, *RHA2A*, and *EF1α*) were selected from the transcriptome database of *L. megaphylla* based on reference genes reported in the literature. All primers were designed using the qPCR primer quest tool (https://sg.idtdna.com/pages/tools/primerquest?returnurl=%2Fprimerquest% 2FHome%2FIndex, accessed on 9 June 2022) based on the coding sequences (CDS) in the transcriptome database of *L. megaphylla* (Supplementary Table S1). Details of these candidate reference genes and primers are shown in Table 2.


**Table 2.** Candidate reference genes and designed primers for RT-qPCR normalization in *L. megaphylla*.


#### **Table 2.** *Cont.*
