*2.7. Analysis Related Cis-Elements in the Candidate DlWRKY Genes*

To analyze the potential function of *DlWRKY* genes in response to various responses, the *cis*-elements in the promoter region of the *DlWRKY* genes were further analyzed. Among these 55 genes, 54 genes could perform *cis*-elements analysis except *DlWRKY*45, which only contain 270 promoter bases. All the *DlWRKY* genes shared the light-responsive boxes and stress-responsive boxes in their promoter. Hormone-related *cis*-elements, such as AuxRR-core, TCA-element, CGTCA-motif, GARE-motif, P-box, and ERE (Ethylene-responsive element), existed in the promoter of all *DlWRKY* genes except *DlWRKY*11, *DlWRKY*41, and *DlWRKY*52. Additionally, circadian-related *cis*-elements were found in the promoter of 39 *DlWRKY* genes and Meristem-related *cis*-elements were only presented in the promoter of 20 *DlWRKY* genes (Figure 7, Tables S5 and S6).

*Int. J. Mol. Sci.* **2018**, *19*, x FOR PEER REVIEW 11 of 20

**Figure 6.** The expression patterns of the selected *DlWRKY* genes under various hormonal and abiotic stresses. The x-axis indicates various treatments and the y-axis indicates the relative expression level. Error bars were obtained from three independent biological replicates. Values with the same letter were not significantly different when assessed using Duncan's multiple range test (*p*  < 0.05, *n* = 3). SA represents salicylic acid, JA represents jasmonic acid, HS represents heat stress, CS represents cold stress, DS represents drought stress, and SS represents salinity stress. **Figure 6.** The expression patterns of the selected *DlWRKY* genes under various hormonal and abiotic stresses. The x-axis indicates various treatments and the y-axis indicates the relative expression level. Error bars were obtained from three independent biological replicates. Values with the same letter were not significantly different when assessed using Duncan's multiple range test (*p* < 0.05, *n* = 3). SA represents salicylic acid, JA represents jasmonic acid, HS represents heat stress, CS represents cold stress, DS represents drought stress, and SS represents salinity stress.

*Int. J. Mol. Sci.* **2018**, *19*, x FOR PEER REVIEW 12 of 20

**Figure 7.** The predicted *cis*-elements in the promoter of the *DlWRKY* genes. The 1.5 kb sequences of 55 *DlWRKY* genes were analyzed with the PlantCARE software. **Figure 7.** The predicted *cis*-elements in the promoter of the *DlWRKY* genes. The 1.5 kb sequences of 55 *DlWRKY* genes were analyzed with the PlantCARE software.

#### **3. Discussion 3. Discussion**

The WRKY proteins, an important transcription factor superfamily which is involved in plant development and stress responses, have been widely detected in various organisms from single-celled green algae to monocots and dicots [15]. Recently, the successful genome sequencing of longan makes it possible to analyze WRKY TFs at the whole-genome level [37]. The present study is the first to identify and characterize WRKY proteins from whole-genome sequences of The WRKY proteins, an important transcription factor superfamily which is involved in plant development and stress responses, have been widely detected in various organisms from single-celled green algae to monocots and dicots [15]. Recently, the successful genome sequencing of longan makes it possible to analyze WRKY TFs at the whole-genome level [37]. The present study is the first to identify and characterize WRKY proteins from whole-genome sequences of longan.

longan. In this study, we identified 59 candidate WRKY genes in the longan genome (471.88 Mb) using the HMM and Blastn search methods. These genes included 58 *DlWRKYs*, which were also found by Lin et al. [37], and one gene *Dlo\_*022548.1 (*DlWRKY*36) found in our study. Finally, after the WRKY domain scanning and sequence alignment, 55 *DlWRKY* genes were determined in the longan genome (Table 1). The number of *WRKY* genes in longan was similar to those found in grape (59 *VvWRKYs*), whose genome size is 487 Mb, which is similar to that of the longan genome [29]. However, the size of the WRKY family in longan is smaller than that in *A. thaliana* (72), *Oryza sativa* ssp. *Indica* (102), and the common bean (88), although their genome sizes are similar (*O*. *sativa* ssp. *Indica*, 466 Mb; common bean, 587 Mb) or even smaller (*A. thaliana*, 119 Mb) than the longan genome size (Table S7) [28,38,39]. Therefore, the number of WRKY family members is not necessarily correlated with the genome size. Previous studies showed that the only group I *WRKYs* are present in green algae and all *WRKY* genes originated from the group I C-terminal WRKY domains, whereas group II members were evolved in the common ancestor of land plants, and Group III members emerged in the common ancestor of seed plants [15]. In addition, as a newly defined and the most dynamic group with many duplication events, the differences in the number of *WRKY* genes in Group III are the primary cause of the sizes of *WRKY* gene families [40]. In the present study, the differences in the number of *WRKY* genes between longan and *Arabidopsis* In this study, we identified 59 candidate WRKY genes in the longan genome (471.88 Mb) using the HMM and Blastn search methods. These genes included 58 *DlWRKYs*, which were also found by Lin et al. [37], and one gene *Dlo\_*022548.1 (*DlWRKY*36) found in our study. Finally, after the WRKY domain scanning and sequence alignment, 55 *DlWRKY* genes were determined in the longan genome (Table 1). The number of *WRKY* genes in longan was similar to those found in grape (59 *VvWRKYs*), whose genome size is 487 Mb, which is similar to that of the longan genome [29]. However, the size of the WRKY family in longan is smaller than that in *A. thaliana* (72), *Oryza sativa* ssp. *Indica* (102), and the common bean (88), although their genome sizes are similar (*O*. *sativa* ssp. *Indica*, 466 Mb; common bean, 587 Mb) or even smaller (*A. thaliana*, 119 Mb) than the longan genome size (Table S7) [28,38,39]. Therefore, the number of WRKY family members is not necessarily correlated with the genome size. Previous studies showed that the only group I *WRKYs* are present in green algae and all *WRKY* genes originated from the group I C-terminal WRKY domains, whereas group II members were evolved in the common ancestor of land plants, and Group III members emerged in the common ancestor of seed plants [15]. In addition, as a newly defined and the most dynamic group with many duplication events, the differences in the number of *WRKY* genes in Group III are the primary cause of the sizes of *WRKY* gene families [40]. In the present study, the differences in the number of *WRKY* genes between longan and *Arabidopsis* mainly existed in groups IIc and III, indicating that the group IIc and III *WRKY* genes may play important roles in the functional evolution of *DlWRKYs*.

mainly existed in groups IIc and III, indicating that the group IIc and III *WRKY* genes may play important roles in the functional evolution of *DlWRKYs*. According to the classification scheme for the WRKY family of Eulgem et al. [41], the DlWRKY proteins were divided into three distinct clusters: groups I, II, and III. Group II proteins were further divided into five distinct groups: a–e (Figure 1 and Table 1). In addition, subgroup IIc contained the largest number of WRKY proteins. These results were consistent with the results observed in other species [28,29,42–44]. The WRKY motif was fairly conserved in longan WRKY proteins, and three variants of this motif were observed. All the DlWRKYs, except DlWRKY19 and DlWRKY47, possessed WRKYGQK. DlWRKY19, which belonged to subgroup IIc, possessed WRKYGKK. DlWRKY19, which belonged to subgroup III, possessed WKKYRQK. In the common According to the classification scheme for the WRKY family of Eulgem et al. [41], the DlWRKY proteins were divided into three distinct clusters: groups I, II, and III. Group II proteins were further divided into five distinct groups: a–e (Figure 1 and Table 1). In addition, subgroup IIc contained the largest number of WRKY proteins. These results were consistent with the results observed in other species [28,29,42–44]. The WRKY motif was fairly conserved in longan WRKY proteins, and three variants of this motif were observed. All the DlWRKYs, except DlWRKY19 and DlWRKY47, possessed WRKYGQK. DlWRKY19, which belonged to subgroup IIc, possessed WRKYGKK. DlWRKY19, which belonged to subgroup III, possessed WKKYRQK. In the common bean, the variants WRKYGKK, WRKYGEK, WKKYEDK, and WKKYCEDK are mainly observed in subgroup IIc [28]; in mulberry,

bean, the variants WRKYGKK, WRKYGEK, WKKYEDK, and WKKYCEDK are mainly observed in

WRKYGKK is detected in subgroup IIb [27]. Moreover, in rice, nine variants, most of which belong to groups III and IIc, are observed [45]. Previous studies showed that these variations of the WRKYGQK motif might change the DNA binding specificities of downstream target genes, and WRKY genes with the variations of the WRKYGQK motif may recognize binding sequences other than the W-box element ((C/T)TGAC(C/T)) [15]. Hence, the result suggested that DlWRKY19 and DlWRKY47 may possess different binding specificities and functions from those of other DlWRKY proteins.

WRKY family genes play important roles in diverse plant development and shown a tissue-specific expression in many plant species [15,40]. For example, *AtWRKY*75 exerts a negative effect on root hair development [46]. *SUSIBA*2 [47] and *MINISEED*3 [48] play roles in the regulation of seed development. In grape, nearly half of the 59 *VvWRKY* genes show no significant organ/tissue-related differences in expression, and some clear spatial differences are noted [29]. In mulberry, 13 *WRKY* genes exhibit the highest expression in the *Morus notabilis* root tissue. A maximum of 25 *WRKYs* show the highest expression in the bark tissue, and 10 WRKY genes display the highest expression in other stages [27]. In the present study, the expression profiles of 55 longan *WRKY* genes in nine longan tissues were ascertained by RNA-seq analysis (Figure 4). The results demonstrated variation in the expression pattern of *DlWRKY* genes. In total, 25 *DlWRKY* genes (*DlWRKY*1, 2, 3, 5, 6, 8, 9, 13, 14, 23, 24, 28, 30, 32, 35, 37, 38, 39, 44, 49, 50, 52, 53, and 54) were highly expressed in at least six longan tissues. As highly expressed genes usually play important roles in plant development [44], we concluded that the 25 highly expressed *DlWRKY* genes might be important regulatory factors in longan development. It was found that group I and group IId *WRKY* genes are ancestral to other *WRKY* genes in plants or algae and are more likely to be constitutively expressed in different tissues [15,40]. For instance, most of the highly expressed *SiWRKY* genes belonged to group I and IId [40]. Consistent with these studies, in the present study, most of the members of groups I (9 of 11) and IId (4 of 6) were the highly expressed gene. In contrast, 12 *DlWRKY* genes were expressed at low levels in all tested tissues and these minimally expressed *DlWRKY* genes were distributed in almost all the *WRKY* gene subgroups except for IId. Meanwhile, six *DlWRKY* genes were preferential accumulation in no more than three tissues, implying that these genes might play crucial roles during the development of specific organs. Additionally, these specifically or minimally expressed *DlWRKY* genes could be induced under environment stimuli. For example, *DlWRKY*10, 22, 41, and 47 were not detected in leaves under normal conditions, but they were induced by different abiotic stresses (Figure 6). Similar results were also found in other studies [15,40,49].

Perpetual flowering is a crucial trait for fruit trees as it enlarges the production period [50]. To date, the genetic control of PF has been deciphered in several model plants. For example, In *Arabidopsis*, the PF trait is controlled by *PERPETUAL FLOWERING 1* (*PEP1*), an orthologue of the FLC floral repressor [51]. In the diploid strawberry and rose, the PF trait is due to a mutation in the orthologue of the *TERMINAL FLOWER* 1 (*TFL*1) floral repressor [50,52]. Recent studies showed that the PF trait of some cultivated strawberries is genetically controlled by the major *FaPFRU* locus, which is non-orthologous to *TFL*1 [53,54]. However, the multi-year delay in the onset of flowering and the long juvenile phase hampers the research of PF traits in perennials, such as longan. Although WRKY TFs regulate various plant developments, only a few data are available on whether WRKY TFs are involved in the flowering time regulation. Meanwhile, as a kind of TF, WRKY genes regulated plant flowering by being directly active or inhibiting the downstream target gene. For example, promoter sequences of *FT*, *LFY*, and *AP1* harbor W-boxes (TTTGACT/C); *AtWRKY*71 affects the flowering time of plants by directly regulating these genes [16]. In our study, all the 55 *DlWRKY* genes were constructively expressed in the three test flower induction process of the "SX" longan, while 18 *DlWRKY* genes showed a specific expression in the "SJ" longan (Figure 5a). This result indicated that these 18 *DlWRKY* genes may specifically be involved in the flower induction of "SJ". In summary, we proposed that these 18 *DlWRKY* genes may participate in the forming of the longan PF habit, which further studies are required to verify the function of these genes.

WRKY genes play crucial roles in the response to abiotic and biotic stress-induced defense signaling pathways [15]. Numerous studies have demonstrated that WRKY genes are expressed strongly and rapidly in response to particular abiotic stresses [15,22,29,40,52]. Consistent with these previous studies, our study showed that 44 *DlWRKY* genes (80%) showed up- or down-regulated expression in at least one tested treatment (Figure 6 and Figure S1), thereby highlighting the extensive involvement of *WRKY* genes in environmental adaptation. SA, JA, and Eth play important roles in biotic and abiotic stresses [55]. Many *WRKYs*, such as *AtWRKY*28, *AtWRKY*46, *AtWRKY*70, and *AtWRKY*54, play an important role in SA- and JA-dependent defense signaling pathways [53,56,57]. In the present study, 27 and 18 *DlWRKY* genes were up- or down-regulated by SA and MeJA treatment, respectively. For example, *DlWRKY*25, the orthologue of *AtWRKY*70 and *AtWRKY*54, was regulated by the SA and JA treatments. *AtWRKY*25 and *AtWRKY*33 regulate plant adaptation to salinity stress through an interaction with their upstream or downstream target genes [58]; their orthologue *DlWRKY*8 in longan was regulated by SA, JA, heat, drought, and salinity. In grape [29], *VvWRKY*42 and its orthologue *DlWRKY*11 in our study were up-regulated by salt treatment. Furthermore, we observed same orthologous genes with different expression patterns under stress treatment. *DlWRKY*44 was down-regulated under drought, and its orthologous gene *VvWRKY*35 was up-regulated under this stress treatment. *DlWRKY*19 showed no significant differential expression in response to salinity, and its orthologous gene *VvWRKY*25 was up-regulated [29]. We speculate that these orthologous genes may be involved in the different signaling pathways in different species. Additionally, only one gene (*DlWRKY*52) was significantly highly expressed under all abiotic stresses. These results indicated that the different *DlWRKYs* played different roles in regulating stress response and that further investigation of the functions of these *DlWRKY* genes is necessary. Differential responses of several *WRKYs* are regulated by the presence of *cis*-elements in their promoter region [27,40,49]. For example, *Morus*013217, which contains three LTREs in its promoter regions showed a strong response to cold stress [27]. Similar results were also found in our study. For instance, four HSEs were found in the promoter regions of *DlWRKY*2, which showed a strong response to heat stress. *DlWRKY*36, *DlWRKY*46, and *DlWRKY*48 showed responsiveness to SA treatment and their expressions were all up-regulated, and more than two TCA-elements were found in their promoters. While the *DlWRKY*11 and *DlWRKY*52 hormone-related *cis*-elements existed in their promoter, they showed no response to the SA or MeJA treatments (Figure 6 and Table S6). Thus, these *cis*-elements could provide more evidence of the DlWRKY genes in response to different stresses or hormonal signaling.
