*4.3. Promoter Analysis of EgrVQ Genes*

The 2,000-bp upstream sequences of the transcriptional start sites of *EgrVQ*genes were submitted to PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) [43] to identify the putative *cis*-elements.

## *4.4. Phylogenetic Analysis and Multiple Sequence Alignment of EgrVQ Proteins*

Multiple sequence alignment of 27 VQ full-length protein sequences from *Eucalyptus* was performed using ClustalX2.11 [44]. A phylogenetic tree was constructed with the neighbor-joining (NJ) method in MEGA 7 (https://www.megasoftware.net/home) [45], with 1,000 bootstrap replicates. All VQ protein sequences from *A. thaliana*, poplarand rice were conducted using ClustalX2.11 and MEGA7 software.

## *4.5. Gene Structure and Conserved Motifs Analysis of EgrVQ Genes*

The exon-intron structures of the *EgrVQ* genes were investigated using Gene Structure Display Server (GSDS: http://gsds.cbi.pku.edu.ch) [46] by aligning cDNA to their corresponding genomic region. In addition, the Multiple Expectation Maximization for Motif Elicitation (MEME) program (http://meme-suite.org/tools/meme) [47] was used to identify the conserved motifs. The parameters were set, as follows: the optimum motif width ranged from 6 to 200, the maximum number of motifs was 20, and other parameters were set at default.

## *4.6. Plant Material and Treatments*

We used approximately 20 cm tall, six-week-old *E. grandis* GL1 clones in hydroponics conditions. The trees were grown in the greenhouse of the Research Institute of Tropical Forestry, Chinese Academy of Forestry, Guangzhou, China.

Hormone treatments were carried out by spraying the leaves of individual plants with 100 nM Epi-Brassinosteroid (Epi-BR) and 100 µM ABA, SA, and MeJA, respectively. For the salt stress treatment, the *E. grandis* plants were transferred to 200 mM NaCl solution and cultured for a total of 168 h; leaves were collected after 0, 1, 6, 24, and 168 h. For cold and heat stress treatments, the plants were transferred into 4 ◦C and 42 ◦C growth chamber. Then leaves were collected after 0, 1, 6, 24, 48, and 168 h of treatment. All of the samples were immediately frozen in liquid nitrogen and stored at −80 ◦C for subsequent total RNA extraction. The leaves from at least three plants were collected, and all treatments were performed in triplicate.
