**1. Introduction**

The VQ protein family either positively or negatively regulates diverse developmental processes, including plant immunity, abiotic stresses, and growth and development [1]. The expression of genes, which are related to biological processes and abiotic stresses in plants, is altered in response to either internal or external signals [1,2]. The *VQ* genes are a type of plant-specific proteins with a dramatically conserved VQ motif, which possesses five conserved amino acids in its main protein sequences FxxVQxhTG [3,4]. Recently, *VQ* genes have been identified by genome-wide analysis in numerous plants. There are 34, 39, 51, 29, and 74 members in Arabidopsis [5], rice [6], poplar [7], moso bamboo [8], and tea [9], respectively. Previous studies have shown that *VQ* genes played important roles in resistance to abiotic and biotic stressors [10]. Meanwhile, accumulating researches showed that many VQ proteins interacted with WRKY transcription factors [1,11]. The WRKY transcription factors, which harbor a highly conserved WRKYGQK amino acid sequence that is followed by a zinc-finger motif at the N-terminal domain, are ubiquitous among higher plants [11,12]. The VQ proteins that contain 50–60 conserved amino acids interact with WRKY transcription factors by the residues V and Q [13,14]. For example, AtVQ15 plays a negative role in response to osmotic stress [15]. AtVQ23 and AtVQ16 transcript levels were strongly induced by *Botrytis cinerea*. A study has shown

that AtVQ22 negatively mediates AtWRKY28 and AtWRKY51 and it is involved with JA (jasmonic acid) defense [16]. In addition, the transcript levels of many *VQ* genes in rice differed when exposed to drought [17]. Moreover, *VQ* genes can regulate multiple biological processes including plant growth and development, senescence, and hormone signaling. The GmVQ1, GmVQ6, and GmVQ53 transcripts are highly expressed during seed development in soybean [18]. AtVQ14 and MINI3 interacted and regulated the abundance of mRNA-encoding proteins that participate in the process of seed development [16]. Specifically, *VQ* genes function by interacting with the group I or IIc WRKY transcription factors [4,19,20]. AtVQ23 and AtVQ16 activated AtWRKY33 by binding its C-terminal WRKY domain and further induced plant defenses [21]. Under salt stress, AtVQ9 expression was increased and it then interacted with WRKY8 for inhibiting the expression of its target genes to modulate salinity stress tolerance [22].

The *Eucalyptus* species are tropical/subtropical woody plants that belong to the Myrtaceae family of angiosperms. It is also the world's leading source of woody biomass [23]. Specifically, *Eucalyptus* not only provides fuel biomass and directly reduces atmospheric carbon dioxide levels [23,24], but it also performs a variety of indirect services through its essential oils that are used as a pesticidal agent and pest repellent [25]. However, *Eucalyptus* can be affected by a variety of biotic and abiotic stressors during growth and development, including heat, cold, salt stress, and disease. *VQ* genes played a vital role in resistance to abiotic and biotic stressors. Thus, it is necessary to understand the characteristics and functions of the *VQ* genes. However, there have been no reports on *VQ* genes in *E. grandis* until now. Fortunately, the availability of the complete *E. grandis* genome provides an opportunity to conduct a comprehensive analysis of *VQ* genes in *E. grandis* [26].

In this study, *VQ* genes in *E. grandis* were identified, and then a bioinformatics analysis was conducted, including phylogenetic relationships, conserved motifs, homologous pairs, gene structures, and promoter analysis. Furthermore, the expression of *EgrVQ* genes was investigated under biotic stress conditions, such as brassinosteroids (BRs), methyl jasmonate (MeJA), salicylic acid (SA), abscisic acid (ABA) treatments, and abiotic stresses, such as cold, heat, and NaCl treatments. These results will provide a solid foundation for elucidating the function of *EgrVQ* genes in response to biotic and abiotic stress and further molecular breeding in *E. grandis*.

### **2. Results**

#### *2.1. Identification of the VQ Gene Family in the E. grandis*

In our study, 27 *VQ* genes were identified, which contained VQ domains using *AtVQ* genes performing BLASTP search in the *E. grandis* genome and VQ conserved domain (PF05678) identification. Table 1 lists detailed information regarding the *VQ* genes in *E. grandis*. These 27 *EgrVQ* genes were named from *EgrVQ1* to *EgrVQ27*. Their translated proteins ranged from 101 to 348 amino acids (AA), with an average of 217 AAs. The predicted molecular weight of the proteins varied from 11.3 to 37.6 kDa, and the pI values ranged from 5.38 to 10.66 (Table 1). By predicting their subcellular localization, it was also found that 12 *EgrVQ* genes were located in the chloroplasts, and 14 *EgrVQ* genes were located in other compartments.

## *2.2. Mapping EgrVQ Genes on Chromosomes, Gene Duplication, and Analysis of Paralogs and Orthologs*

The chromosomal location illustrated that 27 *EgrVQ* genes were randomly and unequally distributed on nine chromosomes (Figure 1). Specifically, chromosome 6 contained eight *VQ* genes and chromosome 8 contained five genes, respectively. Meanwhile, the *EgrVQ13*/*EgrVQ14*, *EgrVQ19*/*EgrVQ20*, and *EgrVQ19*/*EgrVQ21*showed tandem duplication. Consistent with the gene duplication analysis, we also found that *EgrVQ13/EgrVQ14*, *EgrVQ19/EgrVQ20*, *EgrVQ19*/*EgrVQ21*, and *EgrVQ20*/*EgrVQ21* were paralogous pairs. Details from the analysis of paralogs and orthologs with *A. thaliana*, poplar, and rice are presented in Table 2.


**Table 1.** The summary of 27 identified *EgrVQ* genes.

**Figure 1.** Chromosomal location of *E. grandis VQ* genes. Chromosome numbers were indicated above each chromosome. The size of a chromosome was indicated by its relative length. Gene positions and chromosome sizes were given in megabases (Mb) to the left of the figure. Tandem duplicated genes **Figure 1.** Chromosomal location of *E. grandis VQ* genes. Chromosome numbers were indicated above each chromosome. The size of a chromosome was indicated by its relative length. Gene positions and chromosome sizes were given in megabases (Mb) to the left of the figure. Tandem duplicated genes were underlined in red.

were underlined in red. **Table 2.** The summary of paralogous (*Egr–Egr*) and orthologous (*Egr–At*, *Egr–Os* and *Egr-Pt*) gene pairs.


 *EgrVQ15/ AtVQ21 EgrVQ16/ AtVQ11 2.3. Identification of Cis-Elements in the Promoter Regions of EgrVQ Genes*

most of the *EgrVQ* genes might respond to plant biotic and abiotic stresses.

 *EgrVQ24AtVQ31 2.3. Identification of Cis-Elements in the Promoter Regions of EgrVQ Genes*  In this study, the identification of *cis*-regulatory elements was performed in the promoter regions of *EgrVQ* genes (Table 4). The results showed that many stress type *cis*-elements were widespread in the promoter region of *EgrVQ* genes. Table 3 shows the details information. ABA-stress, MeJA-stress, SA-stress, drought-stress, low temperature-stress, dehydration, and salt stress response elements were found in the *EgrVQs* promoter. Interestingly, all of the *EgrVQs* contained a CGTCA-motif and a TGACG-motif, which were involved in the response to MeJA. Moreover, all but *EgrVQ21* contained an ABRE element. In addition, the MBS, TCA-element, and In this study, the identification of *cis*-regulatory elements was performed in the promoter regions of *EgrVQ* genes (Table 4). The results showed that many stress type *cis*-elements were widespread in the promoter region of *EgrVQ* genes. Table 3 shows the details information. ABA-stress, MeJA-stress, SA-stress, drought-stress, low temperature-stress, dehydration, and salt stress response elements were found in the *EgrVQs* promoter. Interestingly, all of the *EgrVQs* contained a CGTCA-motif and a TGACG-motif, which were involved in the response to MeJA. Moreover, all but *EgrVQ21* contained an ABRE element. In addition, the MBS, TCA-element, and LTR *cis*-elements, which were involved in the response to SA, drought, and low-temperature, respectively, were presented in most of the *EgrVQs* promoter regions. These results indicated that most of the *EgrVQ* genes might respond to plant biotic and abiotic stresses.

LTR *cis*-elements, which were involved in the response to SA, drought, and low-temperature, respectively, were presented in most of the *EgrVQs* promoter regions. These results indicated that


**Table 3.** The number of structural analysis of conserved motif FxxVQxxxG in *E. grandis* and other plants. **Table 3.**The number of structural analysis of conserved motif FxxVQxxxG in *E. grandis* and other plants

*Int. J. Mol. Sci.* **2019**, *20*, x 5 of 17

#### *2.4. Multiple Sequence Alignment and Phylogenetic of EgrVQ Proteins 2.4. Multiple Sequence Alignment and Phylogenetic of EgrVQ Proteins*

To obtain the phylogenetic relationship of *E. grandis* VQ proteins, an unrooted tree was constructed, including 27 EgrVQ, 51 PtVQ, 34 AtVQ, and 40 OsVQ proteins (Figure 2). The results showed that EgrVQ proteins were divided into seven sub-families (I–VII). The multiple alignment analysis showed that the EgrVQ proteins presented four conserved motif variations: FxxVQxLTG(20/27), FxxVQxFTG(4/27), FxxVQxVTG(2/27), and FxxVQxLSG(1/27) (Figure 3). Among these motifs, LTG, FTG, and VTG were extensively presented in *A. thaliana* [20], rice [17], and poplar [7]. However, it was found that EgrVQ7 protein presented an LSG motif, which was not reported in previous studies. The details of the conserved motifs in *E. grandis* are presented in Table 3 and Figure 3. To obtain the phylogenetic relationship of *E. grandis* VQ proteins, an unrooted tree was constructed, including 27 EgrVQ, 51 PtVQ, 34 AtVQ, and 40 OsVQ proteins (Figure 2). The results showed that EgrVQ proteins were divided into seven sub-families (I–VII). The multiple alignment analysis showed that the EgrVQ proteins presented four conserved motif variations: FxxVQxLTG(20/27), FxxVQxFTG(4/27), FxxVQxVTG(2/27), and FxxVQxLSG(1/27) (Figure 3). Among these motifs, LTG, FTG, and VTG were extensively presented in *A. thaliana* [20], rice [17], and poplar [7]. However, it was found that EgrVQ7 protein presented an LSG motif, which was not reported in previous studies. The details of the conserved motifs in *E. grandis* are presented in Table 3 and Figure 3.

**Figure 2.** Phylogenetic tree of VQ proteins from *E. grandis*, Arabidopsis, rice, and poplar. ClustalW softwarealigned the complete amino acid sequences of 27 *E. grandis* (prefixed with 'Egr'), 34 Arabidopsis (prefixed with 'At'), 40 rice (prefixed with 'Os'), and 51 (prefixed with 'Pt') VQ proteins, and MEGA 7, with 1000 bootstrap replicates, constructed the neighbor-joining tree. **Figure 2.** Phylogenetic tree of VQ proteins from *E. grandis*, Arabidopsis, rice, and poplar. ClustalW softwarealigned the complete amino acid sequences of 27 *E. grandis* (prefixed with 'Egr'), 34 Arabidopsis (prefixed with 'At'), 40 rice (prefixed with 'Os'), and 51 (prefixed with 'Pt') VQ proteins, and MEGA 7, with 1000 bootstrap replicates, constructed the neighbor-joining tree.
