Comprehensive Genomic Characterization and Expression Analysis of the Lipoxygenase Gene Family in Watermelon under Hormonal Treatments

: Lipoxygenases (LOXs) are non-haem iron-containing dioxygenases and play vital roles in a variety of plant biological processes. Here, we ﬁrst carried out the genome-wide identiﬁcation of LOX genes in watermelon. A total of 16 LOX genes were identiﬁed, which could be classiﬁed into two categories according to phylogenetic analysis: the 9-LOXs (ClLOX1–4, 12, and 15) and 13-LOXs (ClLOX5–11, 13, 14, and 16). Furthermore, the protein structures, intrachromosomal distributions, and gene structures were thoroughly analyzed. Cis -element analysis of the promoter regions indicated that the expression of ClLOX genes may be inﬂuenced by stress and plant hormones. Bioinformatic and expression analyses revealed that the expression of ClLOX genes is tissue-speciﬁc and hormone-responsive. The detected LOX genes exhibited distinctive expression patterns in various tissues. Di ﬀ erent ClLOX genes showed di ﬀ erent responses to methyl jasmonate (MeJA), salicylic acid (SA), and ethylene (ET) treatments, particularly ClLOX7 , which exhibited the most active response to the above treatments. This study provides valuable information for a better understanding of the functions of LOX genes and further exploration of the LOX gene family in watermelon.

Plant LOXs have a highly conserved lipoxygenase domain at the C-terminus and a PLAT/LH2 (polycystin-1, lipoxygenase, α-toxin domain, or lipoxygenase homology) domain at the N-terminus [8]. According to whether the substrate is oxygenated at carbon atom 9 or 13 of the fatty acid hydrocarbon backbone, plant LOXs are generally divided into two categories of 9-LOXs and 13-LOXs [2]. In addition, based on their primary structure and overall sequence similarity, plant LOXs can also be classified order of the corresponding chromosomes ( Table 1). The gDNA and CDS lengths of the ClLOX family genes varied from 2913 to 9824 bp and from 1686 to 2787 bp, respectively, encoding proteins ranging from 561 to 928 amino acids (aa) in length with an average length of 810.06 aa. In addition, the calculated MW, pI, and GRAVY values varied from 85.95 to 105.04 kDa, from 5.13 to 8.83, and from −0.581 to −0.187, respectively ( Table 1). The subcellular localization analysis showed that the watermelon LOX proteins were located in cytoplasm and chloroplast (Table 1).

Evolutionary Relationship among LOX Family Members in Various Plant Species
To reveal the phylogenetic relationships of LOX family members in watermelon and other plant species, a phylogenetic tree was constructed based on the protein sequences of watermelon, pepper [13], tomato [18], Arabidopsis, and rice [17]. As a result, these LOX proteins could be clearly divided into two categories of 9-LOXs and 13-LOXs. Among the watermelon LOX proteins, six members (ClLOX1-4, 12, and 15) were grouped into the 9-LOX category, while other 10 members fell into the 13-LOX category ( Figure 1). It is worth noting that ClLOX1-4 were clustered in the 9-LOX category, while ClLOX5-11 and ClLOX13 were clustered together in the 13-LOX category (Figure 1), suggesting that the ClLOX proteins may be evolutionarily conserved.

Evolutionary Relationship among LOX Family Members in Various Plant Species
To reveal the phylogenetic relationships of LOX family members in watermelon and other plant species, a phylogenetic tree was constructed based on the protein sequences of watermelon, pepper [13], tomato [18], Arabidopsis, and rice [17]. As a result, these LOX proteins could be clearly divided into two categories of 9-LOXs and 13-LOXs. Among the watermelon LOX proteins, six members (ClLOX1-4, 12, and 15) were grouped into the 9-LOX category, while other 10 members fell into the 13-LOX category ( Figure 1). It is worth noting that ClLOX1-4 were clustered in the 9-LOX category, while ClLOX5-11 and ClLOX13 were clustered together in the 13-LOX category (Figure 1), suggesting that the ClLOX proteins may be evolutionarily conserved.  Table S2.

Characterization and Conserved Domain Analysis of Watermelon LOX Proteins
To determine the evolutionarily conserved domains, the full-length amino acid sequences of ClLOX proteins were submitted to pfam and SMART tools. The results showed that all watermelon 9-LOXs and 13-LOXs harbored both a conserved LH2/PLAT and a LOX domain, while ClLOX9  Table S2.

Characterization and Conserved Domain Analysis of Watermelon LOX Proteins
To determine the evolutionarily conserved domains, the full-length amino acid sequences of ClLOX proteins were submitted to pfam and SMART tools. The results showed that all watermelon 9-LOXs and 13-LOXs harbored both a conserved LH2/PLAT and a LOX domain, while ClLOX9 seemed to have a truncated LOX domain (Figure 2A,B). demonstrated to play a vital role in iron binding and is necessary for enzyme stability and activity [5,32,33]. The 10 motifs were widely present in all ClLOX proteins, and their distributions exhibited certain degrees of specificity ( Figure 2). The majority of ClLOX proteins harbored motif 1, except for ClLOX6 ( Figure 2C). Besides, some other ClLOX proteins were also lack of certain conserved motifs. For example, motif 4, motif 5, motif 6, and motif 9 were absent in ClLOX13, while motif 10 was not found in ClLOX4. In addition, ClLOX1 and ClLOX2 had an additional motif 8 in their N-terminus ( Figure 2C). These differences in motif arrangement may account for the functional differentiation among LOX proteins in watermelon.
To further characterize the structures of watermelon LOX proteins, all full-length ClLOX protein sequences were aligned by MAFFT, and the representative motif of 38 amino acids was shown in Figure S2. The watermelon 13-LOXs had a conserved F residue that is indicative of the 13-LOX activity of LOX enzymes, while the 9-LOXs had V/H/L in the corresponding position (Supplementary Figure S2).

Intrachromosomal Localization and Structural Analysis of Watermelon LOX Genes
The 16 ClLOX genes were mapped on five of the eleven chromosomes in watermelon genome, and the number of genes on each chromosome was highly uneven (Figure 3). Chromosome 2 comprised the largest number of ClLOX genes (11 genes). Chromosome 9 included two ClLOX genes, while each of chromosomes 6, 7, and 11 only had one ClLOX gene ( Figure 3).
Further, we carried out a synteny analysis of LOX genes between watermelon and Arabidopsis. All Arabidopsis LOX genes had syntenic copies in watermelon (Supplementary Figure S3), and these syntenic relationships may originate from whole genome triplication or segmental duplication events [15]. To further trace the evolutionary history of ClLOX genes, we investigated the duplication information of them using MCScanX. The results showed that a total of 10 ClLOX genes (ClLOX1, 2, 3-10) could be determined as tandemly duplicated genes. The structures of the ClLOX proteins were further examined by using the MEME server. A total of 10 motifs (designated as motif 1-10) were identified for the 16 ClLOX proteins ( Figure 2C). Amongst them, motifs 1-9 corresponded to the LOX domain, while motif 10 was part of the LH2/PLAT domain (Supplementary Figure S1). Motif 1 included a representative motif of 38 amino acids [His-(X)4-His-(X)4-His-(X)17-His-(X)8-His] with five conserved His (H) residues, which was demonstrated to play a vital role in iron binding and is necessary for enzyme stability and activity [5,32,33]. The 10 motifs were widely present in all ClLOX proteins, and their distributions exhibited certain degrees of specificity ( Figure 2). The majority of ClLOX proteins harbored motif 1, except for ClLOX6 ( Figure 2C). Besides, some other ClLOX proteins were also lack of certain conserved motifs. For example, motif 4, motif 5, motif 6, and motif 9 were absent in ClLOX13, while motif 10 was not found in ClLOX4. In addition, ClLOX1 and ClLOX2 had an additional motif 8 in their N-terminus ( Figure 2C). These differences in motif arrangement may account for the functional differentiation among LOX proteins in watermelon.
To further characterize the structures of watermelon LOX proteins, all full-length ClLOX protein sequences were aligned by MAFFT, and the representative motif of 38 amino acids was shown in Figure  S2. The watermelon 13-LOXs had a conserved F residue that is indicative of the 13-LOX activity of LOX enzymes, while the 9-LOXs had V/H/L in the corresponding position (Supplementary Figure S2).

Intrachromosomal Localization and Structural Analysis of Watermelon LOX Genes
The 16 ClLOX genes were mapped on five of the eleven chromosomes in watermelon genome, and the number of genes on each chromosome was highly uneven (Figure 3). Chromosome 2 comprised the largest number of ClLOX genes (11 genes). Chromosome 9 included two ClLOX genes, while each of chromosomes 6, 7, and 11 only had one ClLOX gene ( Figure 3).   Further, we carried out a synteny analysis of LOX genes between watermelon and Arabidopsis. All Arabidopsis LOX genes had syntenic copies in watermelon (Supplementary Figure S3), and these syntenic relationships may originate from whole genome triplication or segmental duplication events [15]. To further trace the evolutionary history of ClLOX genes, we investigated the duplication information of them using MCScanX. The results showed that a total of 10 ClLOX genes (ClLOX1, 2, 3-10) could be determined as tandemly duplicated genes.

Cis-Element Analysis in the Promoter Regions of ClLOX Genes
The structural features of ClLOX genes were examined by the GSDS tool. Generally, all ClLOX genes harbored introns in their genomic sequences, and the intron number varied from two to eight ( Figure 4). All 9-LOX genes (6 out of 9) contained eight introns. Most of the 13-LOX genes comprised 7-8 introns, except for ClLOX7, ClLOX10, and ClLOX14, which had 6, 5, and 6 introns, respectively ( Figure 4). The structural features of ClLOX genes were examined by the GSDS tool. Generally, all ClLOX genes harbored introns in their genomic sequences, and the intron number varied from two to eight ( Figure 4). All 9-LOX genes (6 out of 9) contained eight introns. Most of the 13-LOX genes comprised 7-8 introns, except for ClLOX7, ClLOX10, and ClLOX14, which had 6, 5, and 6 introns, respectively ( Figure 4).

Cis-Element Analysis in the Promoter Regions of ClLOX Genes
To reveal the possible transcriptional regulation patterns of the ClLOX genes, the 2.0-kb sequence upstream of the ATG site of ClLOX genes was retrieved and analyzed by using the PlantCARE program. A total of 15 kinds of cis-elements involved in responses to stresses and plant hormones were identified, and all ClLOX genes contained 2-8 kinds of cis-elements in their promoter regions, with the exception of ClLOX7 ( Figure 5). Among the stress-and hormone-related cis-elements, anaerobic induction element (ARE) and ET responsive element (ERE) were the most frequently detected in ClLOX genes. In addition, 1, 3, 3, 4, 5, and 5 ClLOX genes had the dehydration-responsive element (DRE), low temperature responsive element (LTR), defense and stress-responsive elements (TC-rich repeats), MYB binding site involved in drought and stress (MBS), WRKY binding site (W-box), and wound-responsive element (WUN-motif), respectively ( Figure 5), implying the possible roles of ClLOX genes in responses to various stresses. Besides the ERE element, seven other important hormone-related cis-elements were also widely identified, including ABRE, CGTCA-motif, TCA-element, AuxRR-core, TGA-element, P-box, and GARE-motif, suggesting that the ClLOX genes may be regulated by various plant hormones. Notably, over half of ClLOX genes (nine out of 16) contained the TCA-element in their promoters, and ABA responsive element (ABRE), MeJA responsive element (CGTCA-motif), auxin-responsive element (AuxRR-core and TGA-element), and gibberellin responsive element (P-box and GARE-motif) were found in the promoter regions of 7, 5, 4, and 6 ClLOX genes, respectively ( Figure 5).
Agriculture 2020, 10, x FOR PEER REVIEW 9 of 16 To reveal the possible transcriptional regulation patterns of the ClLOX genes, the 2.0-kb sequence upstream of the ATG site of ClLOX genes was retrieved and analyzed by using the PlantCARE program. A total of 15 kinds of cis-elements involved in responses to stresses and plant hormones were identified, and all ClLOX genes contained 2-8 kinds of cis-elements in their promoter regions, with the exception of ClLOX7 ( Figure 5). Among the stress-and hormone-related ciselements, anaerobic induction element (ARE) and ET responsive element (ERE) were the most frequently detected in ClLOX genes. In addition, 1, 3, 3, 4, 5, and 5 ClLOX genes had the dehydrationresponsive element (DRE), low temperature responsive element (LTR), defense and stress-responsive elements (TC-rich repeats), MYB binding site involved in drought and stress (MBS), WRKY binding site (W-box), and wound-responsive element (WUN-motif), respectively ( Figure 5), implying the possible roles of ClLOX genes in responses to various stresses. Besides the ERE element, seven other important hormone-related cis-elements were also widely identified, including ABRE, CGTCA-motif, TCA-element, AuxRR-core, TGA-element, P-box, and GARE-motif, suggesting that the ClLOX genes may be regulated by various plant hormones. Notably, over half of ClLOX genes (nine out of 16) contained the TCA-element in their promoters, and ABA responsive element (ABRE), MeJA responsive element (CGTCA-motif), auxin-responsive element (AuxRR-core and TGA-element), and gibberellin responsive element (P-box and GARE-motif) were found in the promoter regions of 7, 5, 4, and 6 ClLOX genes, respectively ( Figure 5).

Expression Analysis of ClLOX Genes in Different Tissues and during Fruit Development
Four ClLOX genes (one from 9-LOXs and three from 13-LOXs) were selected to examine their expression levels in various tissues, including leaves, roots, stems, flowers, and fruit. The qRT-PCR results indicated that some ClLOX genes may have tissue-specific expression patterns. For example, ClLOX14 was highly expressed in fruit; ClLOX4 and ClLOX7 exhibited the most abundant transcripts in flowers; while ClLOX8 showed evident expression specificity in roots ( Figure 6). We further determined the expression patterns of ClLOX genes during fruit development in watermelon based on RNA-seq data by referring to our previous study [6]. During the development of flesh, ClLOX5 and ClLOX15 showed decreases in transcript levels. During rind development, ClLOX4, ClLOX5, and ClLOX16 displayed increases in transcripts at some time points, particularly at 26 DAP (Supplementary Figure S4).

Expression Analysis of ClLOX Genes in Different Tissues and during Fruit Development
Four ClLOX genes (one from 9-LOXs and three from 13-LOXs) were selected to examine their expression levels in various tissues, including leaves, roots, stems, flowers, and fruit. The qRT-PCR results indicated that some ClLOX genes may have tissue-specific expression patterns. For example, ClLOX14 was highly expressed in fruit; ClLOX4 and ClLOX7 exhibited the most abundant transcripts in flowers; while ClLOX8 showed evident expression specificity in roots ( Figure 6). We further determined the expression patterns of ClLOX genes during fruit development in watermelon based on RNA-seq data by referring to our previous study [6]. During the development of flesh, ClLOX5 and ClLOX15 showed decreases in transcript levels. During rind development, ClLOX4, ClLOX5, and ClLOX16 displayed increases in transcripts at some time points, particularly at 26 DAP (Supplementary Figure S4).

Expression Analysis of Several Watermelon ClLOX Genes in Response to JA, SA and ET Treatment
Considering that the ClLOX genes harbor a large number of hormone-related cis-elements, qRT-PCR was carried out to investigate the expression patterns of selected ClLOX genes under JA, SA, and ET treatments. Upon JA treatment, the expression levels of ClLOX7, ClLOX12, and ClLOX14 showed notable increases in both the leaf and root, with the most significant increases being observed for ClLOX7, suggesting that ClLOX7 plays a primary role in JA signaling in leaves and roots ( Figure  7A,B). However, ClLOX4 and ClLOX16 showed different expression patterns in leaves and roots under JA treatment. The expression of ClLOX4 observably decreased in leaves but gradually increased and reached the highest level at 9 h in roots ( Figure 7A,B). For ClLOX16, the expression increased and peaked at 24 h in leaves, while exhibited a significant decrease in roots at 3 h ( Figure  7A,B). Upon SA treatment, ClLOX7 and ClLOX16 showed similar expression patterns, and their

Expression Analysis of Several Watermelon ClLOX Genes in Response to JA, SA and ET Treatment
Considering that the ClLOX genes harbor a large number of hormone-related cis-elements, qRT-PCR was carried out to investigate the expression patterns of selected ClLOX genes under JA, SA, and ET treatments. Upon JA treatment, the expression levels of ClLOX7, ClLOX12, and ClLOX14 showed notable increases in both the leaf and root, with the most significant increases being observed for ClLOX7, suggesting that ClLOX7 plays a primary role in JA signaling in leaves and roots ( Figure 7A,B). However, ClLOX4 and ClLOX16 showed different expression patterns in leaves and roots under JA treatment. The expression of ClLOX4 observably decreased in leaves but gradually increased and reached the highest level at 9 h in roots ( Figure 7A,B). For ClLOX16, the expression increased and peaked at 24 h in leaves, while exhibited a significant decrease in roots at 3 h ( Figure 7A,B). Upon SA treatment, ClLOX7 and ClLOX16 showed similar expression patterns, and their transcripts dramatically increased and peaked at 3 h, while the expression of ClLOX14 showed remarkable declines at certain time points ( Figure 7C). Upon ET treatment, the expression of ClLOX7 and ClLOX16 showed significant increases, while that of ClLOX4 and ClLOX12 displayed significant decreases at certain time points ( Figure 7D). In addition, the expression of ClLOX14 was observably induced at earlier time points (1 h), but showed sharp decreases thereafter ( Figure 7D). These results indicated that ClLOX genes may be involved in diverse hormonal responses.
Agriculture 2020, 10, x FOR PEER REVIEW 11 of 16 transcripts dramatically increased and peaked at 3 h, while the expression of ClLOX14 showed remarkable declines at certain time points ( Figure 7C). Upon ET treatment, the expression of ClLOX7 and ClLOX16 showed significant increases, while that of ClLOX4 and ClLOX12 displayed significant decreases at certain time points ( Figure 7D). In addition, the expression of ClLOX14 was observably induced at earlier time points (1 h), but showed sharp decreases thereafter ( Figure 7D). These results indicated that ClLOX genes may be involved in diverse hormonal responses.

Discussion
LOX proteins are widely present in plants, and are generally encoded by a multigene family. In this study, a total of 16 LOX proteins were identified in watermelon through HMMER combined with BLASTP search (Table 1), and several proteins possessing either one LOX domain or the PLAT/LH2 domain were excluded based on the criteria in previous reports [11,13]. Watermelon has a comparatively larger number of LOX family members relative to other plant species, such as Arabidopsis (six members) [12], pepper (eight members) [13], tea plant (11 members) [14], tomato (14 members) [18], and melon (18 members) [20], and the number of LOX genes in these plants is not proportional to their genome sizes. Notably, 11 out of the 16 ClLOX genes were located on chromosome 2 and formed two distinct tandem duplicate gene clusters (Figure 3). Moreover, over half of the ClLOX genes (10 in 16) were identified as tandemly duplicated genes, indicating that tandem duplication events are the main driving force for the evolution of ClLOX genes. This feature has also been found in some other plants, such as poplar [11], maize [16], tomato [18], cucumber [34], and radish [15]. In addition, according to the collinear module of watermelon and Arabidopsis, all of the six AtLOX genes have syntenic copies in ClLOX genes (Supplementary Figure S3). The larger number of AtLOX-ClLOX orthologous events indicates that ClLOX genes may have similar structure and function to AtLOX genes.

Discussion
LOX proteins are widely present in plants, and are generally encoded by a multigene family. In this study, a total of 16 LOX proteins were identified in watermelon through HMMER combined with BLASTP search (Table 1), and several proteins possessing either one LOX domain or the PLAT/LH2 domain were excluded based on the criteria in previous reports [11,13]. Watermelon has a comparatively larger number of LOX family members relative to other plant species, such as Arabidopsis (six members) [12], pepper (eight members) [13], tea plant (11 members) [14], tomato (14 members) [18], and melon (18 members) [20], and the number of LOX genes in these plants is not proportional to their genome sizes. Notably, 11 out of the 16 ClLOX genes were located on chromosome 2 and formed two distinct tandem duplicate gene clusters ( Figure 3). Moreover, over half of the ClLOX genes (10 in 16) were identified as tandemly duplicated genes, indicating that tandem duplication events are the main driving force for the evolution of ClLOX genes. This feature has also been found in some other plants, such as poplar [11], maize [16], tomato [18], cucumber [34], and radish [15]. In addition, according to the collinear module of watermelon and Arabidopsis, all of the six AtLOX genes have syntenic copies in ClLOX genes (Supplementary Figure S3). The larger number of AtLOX-ClLOX orthologous events indicates that ClLOX genes may have similar structure and function to AtLOX genes.
Through a phylogenetic analysis of the LOX gene family in watermelon and other plant species, LOX family members can be divided into the categories of 9-LOXs and 13-LOXs (Figure 1), which is in accordance with the classification of LOXs in previous reports [2,7,13]. All the watermelon 13-LOX members contain the F residue at the active site ( Figure S2), which is indicative of the 13-LOX activity of LOX enzymes [35,36]. However, the 9-LOXs had V/H/L in the corresponding position instead ( Figure S2). The conserved V residue associated with 9-LOX activity is a characteristic of LOX enzymes, while some 9-LOXs possess no V residue at the position [37][38][39]. In addition, the analysis of conserved motif distribution and exon-intron arrangement further supported the phylogenetic results. The LOXs clustered together tended to have similar conserved motif distributions and structural features (Figures 2 and 4). However, some phylogenetically related ClLOX genes had similar exon/intron structures but variable numbers of introns, such as ClLOX12 and ClLOX13, ClLOX17 and ClLOX19 (Figure 4), suggesting that gain or loss of introns may occur during the evolution of ClLOX genes, which may be responsible for their indispensable roles in watermelon.
Previous studies have shown that some LOX genes have distinctive expression patterns, which may provide important clues for understanding their physiological functions. For example, over half of the PtLOX genes were found to be preferentially expressed in mature leaves and male catkins [11]. Most of the GhLOX genes were expressed in vegetative tissues, while several GhLOX genes were only expressed in specific tissues, such as GhLOX4, GhLOX5, and GhLOX15 in root, GhLOX7 in stem, and GhLOX17 and GhLOX19 in stigma [32]. In the present study, the identified ClLOX genes also displayed significantly higher expression only in specific tissues, such as ClLOX14 in fruit, ClLOX4 and ClLOX7 in flowers, and ClLOX8 in roots ( Figure 6), suggesting their specific roles in these tissues. In addition, two tandemly duplicated genes, ClLOX7 and ClLOX8, exhibited diverse tissue expression patterns ( Figure 6), implying that they are functionally distinctive and have undergone non-functionalization, sub-functionalization, or neofunctionalization [40,41]. In addition, several ClLOX genes showed decreases in transcript abundance during fruit development (Supplementary Figure S4), and similar results were also found in kiwifruit [42], grape [19], melon [20], and tomato [18,43]. The higher expression levels of these LOX genes indicate that plants may require the LOX activity for cell division and fruit enlargement during early developmental periods. Instead, ClLOX4, ClLOX5, and ClLOX16 showed increases in transcripts during the late stage of rind development, implying their regulatory roles in fruit ripening. Previous reports have revealed that the LOX activity is associated with membrane degradation during fruit ripening, and ethyl and butyl acetates would increase with fruit ripening [20,44]. Therefore, the continuous increase in the expression of LOX genes during fruit development may be associated with cell degradation and senescence during fruit ripening.
The LOX pathway is involved in the early steps of plant responses to pathogen and insect attacks. The analysis of cis-elements uncovered many hormone-related cis-elements in the promoter regions of the ClLOX genes ( Figure 5). Therefore, we determined the expression profiles of some selected ClLOX genes upon different plant hormone treatments. Under JA treatment, the expression of all the genes was altered, particularly ClLOX7, which exhibited more dramatic increases of expression in both leaves and roots relative to ClLOX genes ( Figure 7A,B). ClLOX7 is orthologous to AtLOX2 in the 13-LOX group (Figure 1), both of which were localized in chloroplasts (Table 1). Therefore, compared with other ClLOX genes, ClLOX7 might play a primary role similar to that of AtLOX2, which can provide linolenic acid hydroperoxide substrates for JA biosynthesis in vivo [45]. JA, SA, and ET are three main defense-associated phytohormones that mediate signal transduction to combat attackers such as pathogens and herbivorous insects, and the SA-and ET/JA-mediated defense response pathways were reported to act antagonistically, synergistically, or additively [46][47][48]. In this study, the selected ClLOX genes were also found to be regulated in response to SA and ET treatments ( Figure 7C,D). Our previous reports have shown that other oxylipin pathway genes, such as ClAOCs, ClAOS and ClHPLs, are also regulated in response to JA, SA, and ET, which may play important roles in watermelon defense against root-knot nematode (RKN) infection [6,30]. The roles of LOXs in the defense against pathogens and pests might be associated with the synthesis of a number of compounds from the oxylipin pathway [1,7]. For example, OsLOX1 could lead to the production of more JA, (Z)-3-hexenal, and colneleic acid after brown plant hopper (BPH) feeding, and transgenic rice plants with enhanced OsLOX1 expression were more resistant to BPH attack [49]. Tea plant CsLOX1 was induced in response to feeding by the tea green leafhopper, and the expression profile showed a clear association with the emission pattern of LOX-derived volatiles [50]. ZmLOX10 provides substrate to several LOX branches and produces a series of 13-oxylipin products, and the zmlox10 plants were unable to produce green leaf volatiles (GLVs) and JA, resulting in a dramatic decline in herbivore-induced plant volatiles and attractiveness to parasitoid wasps [51,52]. A recent study has revealed that GLVs and JA contribute to maize susceptibility to Colletotrichum graminicola due to the suppression of SA-related defense [53]. Hence, the ClLOX genes may participate in the protection of plants from biotic stresses by catalyzing the synthesis of some oxylipins through the regulation of the SA-and ET/JA-mediated defense response pathways. In addition, some DRE and ABRE and ERE cis-elements were found in the promoters of LOXs, suggesting that these LOXs may participate in watermelon defense against abiotic stress. A recent report has revealed that tomato LOXs were up-regulated or down-regulated in response to heat, salt, or drought stress [54]. In future work, we will focus on the functions of watermelon LOX genes in defense against abiotic stress.

Conclusions
In summary, a total of 16 LOX genes were identified in watermelon, which could be divided into two categories: 9-LOXs (ClLOX1-4, 12, and 15) and 13-LOXs (ClLOX5-11, 13, 14, and 16). Their phylogenetic relationships, protein structures, intrachromosomal distributions, gene structures, and cis-element compositions in the promoters were thoroughly analyzed in this work. The results improve our understanding of the LOX gene family in watermelon. In addition, the expression analysis of some selected ClLOX genes showed that their expression is tissue-specific as well as hormone-responsive. These findings may expand the understanding of the functions of ClLOX genes and lay a foundation to select candidate genes for watermelon genetic improvement.
Supplementary Materials: The following are available online at http://www.mdpi.com/2077-0472/10/10/429/s1. Figure S1. Logo of 10 motifs of watermelon LOX proteins by MEME. Figure S2. Multiple sequence alignments of the representative 38 amino acid motif of watermelon LOX proteins. The representative 38 amino acid motif is boxed with purple. The five essential His residues involved in the binding of the iron atom in the active site are denoted by asterisks. The residues indicative of LOX enzymes with 9-or 13-LOX activity are boxed with red. Figure S3. Collinear relationships of LOX genes in watermelon and Arabidopsis. Figure S4. Expression patterns of ClLOX genes during the development of flesh and rind at different stages. The expression levels are indicated as log2-based FPKM+1 values. DAP, days after pollination.