Access the full text.
Sign up today, get DeepDyve free for 14 days.
Loading next page...
/lp/multidisciplinary-digital-publishing-institute/comprehensive-genomic-characterization-and-expression-analysis-of-the-Xas43Jh6Yn
References
References for this paper are not available at this time. We will be adding them shortly, thank you for your patience.
- Publisher
- Multidisciplinary Digital Publishing Institute
- Copyright
- © 1996-2020 MDPI (Basel, Switzerland) unless otherwise stated Disclaimer The statements, opinions and data contained in the journals are solely those of the individual authors and contributors and not of the publisher and the editor(s). Terms and Conditions Privacy Policy
- ISSN
- 2077-0472
- DOI
- 10.3390/agriculture10100429
- Publisher site
- See Article on Publisher Site
Abstract
agriculture Article Comprehensive Genomic Characterization and Expression Analysis of the Lipoxygenase Gene Family in Watermelon under Hormonal Treatments 1 , 2 2 1 3 1 , Jianping Liu , Yong Zhou , Jingwen Li , Feng Wang and Youxin Yang * Jiangxi Key Laboratory for Postharvest Technology and Nondestructive Testing of Fruits & Vegetables, Collaborative Innovation Center of Post-Harvest Key Technology and Quality Safety of Fruits and Vegetables, College of Agronomy, Jiangxi Agricultural University, Nanchang 330045, China; ziqian1984@163.com (J.L.); 18770911287@163.com (J.L.) College of Bioscience and Bioengineering, Jiangxi Agricultural University, Nanchang 330045, China; yongzhou@jxau.edu.cn College of Horticulture, Shenyang Agricultural University, Shenyang 110866, China; fengwang@syau.edu.cn * Correspondence: yangyouxin@jxau.edu.cn Received: 2 August 2020; Accepted: 21 September 2020; Published: 25 September 2020 Abstract: 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 erent ClLOX genes showed di 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. Keywords: watermelon; lipoxygenase (LOX); phylogenetic analysis; hormone; gene expression 1. Introduction Lipoxygenases (LOXs; EC 1.13.11.12) belong to a family of non-haem iron-containing dioxygenases that are widely present in plants, animals, fungi, and even in bacteria [1–4]. They can catalyze the oxidation of polyunsaturated fatty acids (PUFAs), such as linoleic acid (LA) and -linolenic acid ( -LeA), into unsaturated fatty acid hydroperoxides [2,5]. In plants, LOX-mediated peroxidation of PUFAs undergoes a series of secondary reactions involving several multigene enzyme families, such as Allene oxide synthase (AOS), hydroperoxide lyase (HPL), divinyl ether synthase (DES), and allene oxide cyclase (AOC), and finally produces a large number of biologically active compounds such as jasmonic acid (JA) and its related chemical compounds [1,6,7]. Plant LOXs have a highly conserved lipoxygenase domain at the C-terminus and a PLAT/LH2 (polycystin-1, lipoxygenase, -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 Agriculture 2020, 10, 429; doi:10.3390/agriculture10100429 www.mdpi.com/journal/agriculture Agriculture 2020, 10, 429 2 of 15 into two subfamilies: type I and type II. Type I LOXs have relatively higher sequence similarities with each other than type II LOXs and are lack of plastid transit peptide [2,9]. Type II exclusively comprises 13-LOXs, and all the proteins harbor an extra chloroplast transit peptide at the N-terminus [10,11]. By genome-wide analysis, previous studies have identified six LOX family genes in Arabidopsis [12], eight in Tartary buckwheat [10], eight in pepper [13], 11 in either tea plant [14] or radish [15], 13 in maize [16], 14 in either rice [17] or tomato [18], 18 in either grape [19] or melon [20], 20 in poplar [11], and 23 in either cucumber [21] or pear [22]. In addition, many LOX genes have been cloned and functionally characterized to be involved in various growth and developmental processes in plants. For example, lox3 lox4 double mutants were male sterile and developed more inflorescence shoots and flowers, suggesting that they are essential for male fertility and flower development in Arabidopsis [23]. Down-regulation of a rice LOX gene reduced the co-oxidation of -carotene in carotenoid-enriched transgenic rice seeds during storage [24]. Moreover, some LOX members were found to be associated with resistance against various abiotic and biotic stresses. For example, silencing of pepper CaLOX2 gene reduced jasmonate accumulation and caused higher susceptibility to thrips feeding [25]. Overexpression of persimmon (Diospyros kaki) DkLOX3 gene in Arabidopsis contributed to higher resistance against various abiotic stresses, including osmotic stress, high salinity, and drought, as well as biotic stresses including Pseudomonas syringae pv. tomato DC3000 and Botrytis cinerea [26,27]. Considering that LOX family members have important functions in di erent developmental processes and various stress responses as mentioned above, we conducted a genome-wide analysis of LOX genes in watermelon genome and systematically analyzed their phylogenetic relationships, protein structures, intrachromosomal localizations, and exon-intron arrangements. In addition, the cis-element analysis of the promoter regions was performed, and the expression patterns of watermelon LOX genes in di erent tissues and in response to various hormonal treatments were also determined. The results may lay a foundation for further elucidating the functions of the LOX genes and facilitate the molecular breeding of watermelon. 2. Materials and Methods 2.1. Identification of LOX Gene Family Members in Watermelon To identify all the possible LOX genes in watermelon, the HMM (Hidden Markov Model) profile of the LOX domain (PF00305) was downloaded from the Pfam database (http://pfam.xfam.org/) and searched against the watermelon (97103) v1 proteome (http://cucurbitgenomics.org/organism/1) using the HMMER program with default parameters. Subsequently, the full-length LOX protein sequences in Arabidopsis and rice were downloaded according to a previous study [17], and used as queries to search against the watermelon (Citrullus lanatus subsp. vulgaris cv. 97103) v1 proteome with the BLASTP program. The resulting sequences were further verified by SMART (http://smart.embl-heidelberg.de/) and Pfam to confirm the presence of both the LOX and PLAT/LH2 domains. Several redundant sequences were removed for not having the complete domain or shortness. 2.2. Analysis of Protein Properties, Phylogenetic Tree and Conserved Motifs The isoelectric point (pI), molecular weight (MW) and grand average of hydropathicity (GRAVY) of watermelon LOX proteins were calculated by the ProtParam tool in ExPASy (https://web.expasy.org/ protparam/). The subcellular localization of each member of watermelon LOX proteins was predicted using the ProtComp server (Version 9.0, New York, NY, USA) http://linux1.softberry.com/berry.phtml). Multiple sequence alignment was carried out by MAFFT (https://www.ebi.ac.uk/Tools/msa/ma t/) using the full-length sequences of LOX proteins from watermelon and other plant species, including tomato [18], pepper [13], Arabidopsis, and rice [17]. A phylogenetic tree was constructed with the MEGA 7.0 using the neighbor-joining (NJ) method with a bootstrap value of 1000. The conserved motifs of watermelon LOX proteins were identified using the MEME tool (http://meme-suite.org/tools/meme), Agriculture 2020, 10, 429 3 of 15 and the parameter settings were set as follows: the number of motifs was 10, and the width range was 6–50. The MEME results were illustrated with the TBtools program [28]. 2.3. Analysis of the Gene Structure and Putative Cis-Acting Regulatory Elements The GSDS tool (Gene Structure Display Server, http://gsds.cbi.pku.edu.cn/) was employed to examine the gene structures of watermelon LOX genes by comparing their sequences of coding sequence (CDS) and corresponding genomic DNA (gDNA). To identify the potential stress- and hormone-related cis-elements, the 2000-bp DNA sequences of ATG site of watermelon LOX genes were obtained from the watermelon (97103) v1 genome database (http://cucurbitgenomics.org/organism/1) and analyzed by the PlantCARE server (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). 2.4. Chromosome Mapping, Duplication and Synteny Analysis The chromosomal information of watermelon LOX genes was downloaded from the watermelon genome database and the LOX genes were mapped to chromosomes with the MapChart software. Duplication analysis between the watermelon LOX genes and synteny analysis of LOX genes between watermelon and Arabidopsis were conducted with the MCScanX software (Athens, GA, USA) (http: //chibba.pgml.uga.edu/mcscan2/) by referring to a previous report [29]. 2.5. Expression Analysis of Watermelon LOX Genes Based on RNA-Seq Data The RNA-seq data of the flesh and rind at 10, 18, 26, and 34 days after pollination (DAP) were obtained, and the expression annotation was performed as previously described [6,30]. FPKM (fragments per kilobase of exon model per million mapped reads) values of watermelon LOX genes were log2-transformed, and then the TBtools program was employed to visualize the expression values. 2.6. Plant Materials and Growth Conditions Watermelon (Citrullus lanatus L. cv. Xinong 8) seeds were sown in pots containing nutritional soil within a greenhouse under the conditions of 25 C/19 C (12 h/12 h). For analysis of tissue-specific expression patterns, various tissues including leaves, roots, stems, flowers, and fruit were harvested from 2-month-old watermelon plants. For various hormone treatments, four-leaf stage watermelon seedlings were treated with 100 M methyl jasmonate (MeJA), 1 mM salicylic acid (SA), and 500 M ethylene (ET) by spraying according to our previous study [30]. Then, the leaves and roots were harvested from treated seedlings at 0, 1, 3, 9, and 24 h post-treatment with three biological triplicates. All of the samples were rapidly frozen in liquid nitrogen and stored at 80 C until use. 2.7. RNA Extraction and Quantitative Real-Time PCR (qRT-PCR) Analysis Total RNA was extracted with the total RNA Miniprep Kit (Axygen Biosciences, Union City, CA, USA) following the manufacturers’ protocol. The RNA (1 g) was reverse-transcribed using the ReverTra Ace qPCR-RT Kit (TOYOBO, Osaka, Japan) for the synthesis of cDNA. qRT-PCR was performed on the iCycler iQTM Real-time PCR Detection System (Bio-Rad, Hercules, CA, USA) in three independent biological replicates. The primers used are described in Supplementary Table S1. Watermelon -actin gene (Cla007792) was used as an internal control, and the relative expression was DDCt analyzed using by the 2 method [31]. 3. Results 3.1. Identification of LOX Genes in Watermelon By HMMER search and BLASTP, a total of 19 LOX genes were identified in watermelon (C. lanatus) genome, three of which were removed for shortness and not having the complete domain of LOX as tested by SMART and Pfam. Finally, the 16 LOX genes were denoted as ClLOX1–16 in an ascending Agriculture 2020, 10, 429 4 of 15 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). Agriculture 2020, 10, x FOR PEER REVIEW 6 of 16 3.2. Evolutionary Relationship among LOX Family Members in Various Plant Species 3.2. Evolutionary Relationship among LOX Family Members in Various Plant Species To reveal the phylogenetic relationships of LOX family members in watermelon and other plant 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], species, a phylogenetic tree was constructed based on the protein sequences of watermelon, pepper tomato [18], Arabidopsis, and rice [17]. As a result, these LOX proteins could be clearly divided into two [13], tomato [18], Arabidopsis, and rice [17]. As a result, these LOX proteins could be clearly divided categories of 9-LOXs and 13-LOXs. Among the watermelon LOX proteins, six members (ClLOX1–4, 12, into two categories of 9-LOXs and 13-LOXs. Among the watermelon LOX proteins, six members and 15) were grouped into the 9-LOX category, while other 10 members fell into the 13-LOX category (ClLOX1–4, 12, and 15) were grouped into the 9-LOX category, while other 10 members fell into the (Figure 1). It is worth noting that ClLOX1–4 were clustered in the 9-LOX category, while ClLOX5–11 13-LOX category (Figure 1). It is worth noting that ClLOX1–4 were clustered in the 9-LOX category, and ClLOX13 were clustered together in the 13-LOX category (Figure 1), suggesting that the ClLOX while ClLOX5–11 and ClLOX13 were clustered together in the 13-LOX category (Figure 1), suggesting proteins may be evolutionarily conserved. that the ClLOX proteins may be evolutionarily conserved. Figure 1. Phylogenetic analysis of LOX family members in watermelon and other plant species. The NJ Figure 1. Phylogenetic analysis of LOX family members in watermelon and other plant species. The phylogenetic tree was created based on the full-length amino acid sequences by using MEGA 7.0 with NJ phylogenetic tree was created based on the full-length amino acid sequences by using MEGA 7.0 1000 bootstrap replicates. The information of LOX proteins in various plant species is presented in with 1000 bootstrap replicates. The information of LOX proteins in various plant species is presented Table S2. in Table S2. 3.3. 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). Agriculture 2020, 10, 429 5 of 15 Table 1. Identification and characterization of LOX family genes in watermelon. Protein Properties gDNA (bp) CDS (bp) Nomenclature Locus Predicted LOX Class Chromosomal Position Domain Length MW Subcellular pI GRAVY (aa) (KDa) Localization LH2 LOX ClLOX1 Cla019908 9-LOX Chr2: 25963474 .. 25968681 (+) 5208 2628 875 99.03 40–181 192–858 6.14 0.365 Cytoplasm ClLOX2 Cla019907 9-LOX Chr2: 25991122 .. 25996850 (+) 5729 2640 879 99.70 40–181 192–862 5.41 0.364 Cytoplasm ClLOX3 Cla019897 9-LOX Chr2: 26087261 .. 26097084 ( ) 9824 2589 862 97.49 18–162 173–845 5.67 0.432 Cytoplasm ClLOX4 Cla019896 9-LOX Chr2: 26108726 .. 26117164 ( ) 8439 2259 752 85.95 7–97 82–735 5.13 0.361 Cytoplasm ClLOX5 Cla008520 13-LOX Chr2: 33497808 .. 33502790 ( ) 4983 2709 902 102.74 74–207 218–885 5.76 0.413 Chloroplast ClLOX6 Cla008519 13-LOX Chr2: 33513361 .. 33519238 ( ) 5878 1686 561 63.98 66–187 342–561 6.16 0.187 Cytoplasm ClLOX7 Cla008517 13-LOX Chr2: 33543209 .. 33546121 ( ) 2913 2355 784 89.41 1–89 100–767 5.44 0.447 Chloroplast ClLOX8 Cla008516 13-LOX Chr2: 33565766 .. 33569015 ( ) 3250 2511 836 95.13 3–129 142–819 5.72 0.579 Chloroplast ClLOX9 Cla003211 13-LOX Chr2: 33577155 .. 33581164 ( ) 4010 2493 830 94.30 3–129 140–813 7.02 0.483 Chloroplast ClLOX10 Cla003210 13-LOX Chr2: 33590798 .. 33596027 ( ) 5230 1989 662 75.43 1–68 79–645 6.57 0.448 Chloroplast ClLOX11 Cla003209 13-LOX Chr2: 33605180 .. 33608524 ( ) 3345 2574 857 97.86 7–140 151–840 6.02 0.558 Chloroplast ClLOX12 Cla009402 9-LOX Chr6: 6667738 .. 6672962 (+) 5225 2652 883 100.89 36–180 191–861 6.00 0.352 Cytoplasm ClLOX13 Cla005400 13-LOX Chr7: 27624656 .. 27628437 (+) 3782 1755 584 67.73 4–128 158–584 6.55 0.581 Chloroplast ClLOX14 Cla015542 13-LOX Chr9: 581154 .. 585338 ( ) 4185 2787 928 103.02 80–234 245–911 6.65 0.376 Cytoplasm ClLOX15 Cla014845 9-LOX Chr9: 6640629 .. 6649232 ( ) 8604 2517 838 96.18 16–143 154–816 6.61 0.459 Cytoplasm ClLOX16 Cla022987 13-LOX Chr11: 16214820 .. 16219598 (+) 4779 2787 928 105.04 105–229 240–911 8.83 0.475 Cytoplasm Agriculture 2020, 10, x FOR PEER REVIEW 7 of 16 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. Agriculture 2020, 10, 429 6 of 15 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 3.3. Characterization and Conserved Domain Analysis of Watermelon LOX Proteins among LOX proteins in watermelon. To To further determine chthe aracterize the evolutionarily structure conserved s of waterm domains, elon LOX proteins, the full-length all fu amino ll-length ClLOX protein acid sequences of ClLOX sequences we proteinsre aligned by were submitted MAF toFpfam T, and and the repres SMARTenta tools. tive moti The results f of 38showed amino a that cids wa all watermelon s shown in 9-LOXs Figure and S2. The watermelon 13-LOXs 13-LOXs harbored both a conserved had a conser LH2/PLA ved F residue that is T and a LOX domain, indicative of the while ClLOX913-LO seemed X toahave ctivity of LOX enzymes, whi a truncated LOX domain le th (Figur e 9-Le O2 Xs A,B). had V/H/L in the corresponding position (Supplementary Figure S2). Figure 2. Phylogenetic relationships (A), conserved domains (B) and motif compositions (C) of Figure 2. Phylogenetic relationships (A), conserved domains (B) and motif compositions (C) of ClLOX ClLOX proteins. proteins. The structures of the ClLOX proteins were further examined by using the MEME server. A total 3.4. Intrachromosomal Localization and Structural Analysis of Watermelon LOX Genes of 10 motifs (designated as motif 1–10) were identified for the 16 ClLOX proteins (Figure 2C). The 16 ClLOX genes were mapped on five of the eleven chromosomes in watermelon genome, Amongst them, motifs 1–9 corresponded to the LOX domain, while motif 10 was part of the and the number of genes on each chromosome was highly uneven (Figure 3). Chromosome 2 LH2/PLAT domain (Supplementary Figure S1). Motif 1 included a representative motif of 38 comprised the largest number of ClLOX genes (11 genes). Chromosome 9 included two ClLOX genes, amino acids [His-(X)4-His-(X)4-His-(X)17-His-(X)8-His] with five conserved His (H) residues, which while each of chromosomes 6, 7, and 11 only had one ClLOX gene (Figure 3). was demonstrated to play a vital role in iron binding and is necessary for enzyme stability and Further, we carried out a synteny analysis of LOX genes between watermelon and Arabidopsis. activity [5,32,33]. The 10 motifs were widely present in all ClLOX proteins, and their distributions All Arabidopsis LOX genes had syntenic copies in watermelon (Supplementary Figure S3), and these exhibited certain degrees of specificity (Figure 2). The majority of ClLOX proteins harbored motif 1, syntenic relationships may originate from whole genome triplication or segmental duplication events except for ClLOX6 (Figure 2C). Besides, some other ClLOX proteins were also lack of certain conserved [15]. To further trace the evolutionary history of ClLOX genes, we investigated the duplication motifs. For example, motif 4, motif 5, motif 6, and motif 9 were absent in ClLOX13, while motif 10 was information of them using MCScanX. The results showed that a total of 10 ClLOX genes (ClLOX1, 2, not found in ClLOX4. In addition, ClLOX1 and ClLOX2 had an additional motif 8 in their N-terminus 3–10) could be determined as tandemly duplicated genes. (Figure 2C). These di erences in motif arrangement may account for the functional di erentiation 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). 3.4. 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). Agriculture 2020, 10, 429 7 of 15 Agriculture 2020, 10, x FOR PEER REVIEW 8 of 16 Agriculture 2020, 10, x FOR PEER REVIEW 8 of 16 Figure 3. Locations of the LOX genes in watermelon chromosomes. Figure 3. Locations of the LOX genes in watermelon chromosomes. Further The structur , we carried al fe out ature a ssynteny of ClLOXanalysis genes were exam of LOXingenes ed by the GSDS tool. Gene between watermelon rally, all and ClLOX Arabidopsis . genes harbored introns in their genomic sequences, and the intron number varied from two to eight All Arabidopsis LOX genes had syntenic copies in watermelon (Supplementary Figure S3), and (Figure 4). All 9-LOX genes (6 out of 9) contained eight introns. Most of the 13-LOX genes comprised these syntenic relationships may originate from whole genome triplication or segmental duplication 7–8 introns, except for ClLOX7, ClLOX10, and ClLOX14, which had 6, 5, and 6 introns, respectively events [15]. To further trace the evolutionary history of ClLOX genes, we investigated the duplication (Figure 4). Figure 3. Locations of the LOX genes in watermelon chromosomes. 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 structural features of ClLOX genes were examined by the GSDS tool. Generally, all ClLOX The structural features of ClLOX genes were examined by the GSDS tool. Generally, all ClLOX genes genes harbored introns in their genomic sequences, and the intron number varied from two to eight harbored introns in their genomic sequences, and the intron number varied from two to eight (Figure 4). (Figure 4). All 9-LOX genes (6 out of 9) contained eight introns. Most of the 13-LOX genes comprised All 9-LOX genes (6 out of 9) contained eight introns. Most of the 13-LOX genes comprised 7–8 introns, 7–8 introns, except for ClLOX7, ClLOX10, and ClLOX14, which had 6, 5, and 6 introns, respectively except for ClLOX7, ClLOX10, and ClLOX14, which had 6, 5, and 6 introns, respectively (Figure 4). (Figure 4). Figure 4. Exon-intron arrangements of watermelon LOX genes according to phylogenetic analysis. The exons and introns are shown as blue boxes and black lines, respectively. 3.5. Cis-Element Analysis in the Promoter Regions of ClLOX Genes Figure Figure 4 4. .Exon-intr Exon-intron arrangements of water on arrangements of watermelon melon LO LOX X genes genes ac accor cording ding to phylogenetic to phylogenetic analysis. analysis. The The exons and exons and intr introns are sho ons are shown wn as blue as blue box boxes es and and black black li lines, nes, re respectively spectively. . 3.5. Cis-Element Analysis in the Promoter Regions of ClLOX Genes Agriculture 2020, 10, 429 8 of 15 Agriculture 2020, 10, x FOR PEER REVIEW 9 of 16 3.5. 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 To reveal upstream o the possible f the ATG site o transcriptional f r ClLOX egulation gen patterns es was retr of the ieved and ClLOX genes, analyzed by the 2.0-kbus sequence ing the Pla upstr ntCAR eam of E progra the ATG m.site A tota of ClLOX l of 15genes kinds of was cis retrieved -elemenand ts involved analyzed in by response using the s to PlantCARE stresses and prog plant ram. hormones we A total of 15 kinds re ident of cis ified, -elements and all involved ClLOX gen in r e esponses s contained to str 2–8 esse kinds of s and plant cis-eleme hormones nts in their were pr identified, omoter region and alls, w ClLOX ith the exceptio genes contained n of 2–8 ClLOX kinds 7 of (Fcis igure -elements 5). Among the in their pr stress- an omoter regions, d hormone-re with the exception lated cis- elements, an of ClLOX7 (Figur aerobic ind e 5). Among uction elemen the stress- t (ARE and) and hormone-r ET responsive element (ERE elated cis-elements, anaer ) were t obic induction he most frequently d element (ARE) etected in and ETClLOX responsive genes. element In addi(ERE) tion, 1wer , 3, 3 e, the 4, 5,most and 5 fr ClLOX equently genes detected had the dehydration in ClLOX genes. - responsive In addition, element (DRE 1, 3, 3, 4, 5, ), and low te 5 ClLOX mperatgenes ure responsiv had the e element dehydration-r (LTR), de esponsive fense an element d stress-re (DRE), sponsive low elements (TC-rich repeats), MYB binding site involved in drought and stress (MBS), WRKY binding temperature responsive element (LTR), defense and stress-responsive elements (TC-rich repeats), MYB site (W binding-box), site involved and wound in dr- ought responsive e and stress lement (WUN (MBS), WRKY -mot binding if), respe sitect (W ively ( -box), Fig and ure 5) wound-r , imply esponsive ing the possible roles of ClLOX genes in responses to various stresses. Besides the ERE element, seven other element (WUN-motif), respectively (Figure 5), implying the possible roles of ClLOX genes in responses important hormone-related cis-elements were also widely identified, including ABRE, CGTCA-motif, to various stresses. Besides the ERE element, seven other important hormone-related cis-elements TCA-element, AuxRR-core, TGA-element, P-box, and GARE-motif, suggesting that the ClLOX genes were also widely identified, including ABRE, CGTCA-motif, TCA-element, AuxRR-core, TGA-element, may be regulated by various plant hormones. Notably, over half of ClLOX genes (nine out of 16) P-box, and GARE-motif, suggesting that the ClLOX genes may be regulated by various plant hormones. contained the TCA-element in their promoters, and ABA responsive element (ABRE), MeJA Notably, over half of ClLOX genes (nine out of 16) contained the TCA-element in their promoters, and responsive element (CGTCA-motif), auxin-responsive element (AuxRR-core and TGA-element), and ABA responsive element (ABRE), MeJA responsive element (CGTCA-motif), auxin-responsive element gibberellin responsive element (P-box and GARE-motif) were found in the promoter regions of 7, 5, (AuxRR-core and TGA-element), and gibberellin responsive element (P-box and GARE-motif) were 4, and 6 ClLOX genes, respectively (Figure 5). found in the promoter regions of 7, 5, 4, and 6 ClLOX genes, respectively (Figure 5). Figure 5. Analysis of stress- and hormone-related cis-elements in the promoter regions ( 2000 bp) of Figure 5. Analysis of stress- and hormone-related cis-elements in the promoter regions (−2000 bp) of ClLOX genes. The numbers of cis-elements are boxed. ClLOX genes. The numbers of cis-elements are boxed. Agriculture 2020, 10, 429 9 of 15 Agriculture 2020, 10, x FOR PEER REVIEW 10 of 16 3.6. Expression Analysis of ClLOX Genes in Different Tissues and during Fruit Development 3.6. Expression Analysis of ClLOX Genes in Di erent Tissues and during Fruit Development Four ClLOX genes (one from 9-LOXs and three from 13-LOXs) were selected to examine their 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 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, 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 ClLOX14 was highly expressed in fruit; ClLOX4 and ClLOX7 exhibited the most abundant transcripts in in flowers; while ClLOX8 showed evident expression specificity in roots (Figure 6). We further flowers; while ClLOX8 showed evident expression specificity in roots (Figure 6). We further determined determined the expression patterns of ClLOX genes during fruit development in watermelon based the expression patterns of ClLOX genes during fruit development in watermelon based on RNA-seq data on RNA-seq data by referring to our previous study [6]. During the development of flesh, ClLOX5 by referring to our previous study [6]. During the development of flesh, ClLOX5 and ClLOX15 showed and ClLOX15 showed decreases in transcript levels. During rind development, ClLOX4, ClLOX5, and decreases in transcript levels. During rind development, ClLOX4, ClLOX5, and ClLOX16 displayed ClLOX16 displayed increases in transcripts at some time points, particularly at 26 DAP increases in transcripts at some time points, particularly at 26 DAP (Supplementary Figure S4). (Supplementary Figure S4). Figure 6. qRT-PCR analysis of the expression of selected ClLOX genes (A–D) in di erent watermelon Figure 6. qRT-PCR analysis of the expression of selected ClLOX genes (A–D) in different watermelon tissues. L, leaves; R, roots; S, stems; F, flowers; Fr, fruit. Error bars indicate standard deviation (SD) tissues. L, leaves; R, roots; S, stems; F, flowers; Fr, fruit. Error bars indicate standard deviation (SD) based on three biological replicates. based on three biological replicates. 3.7. Expression Analysis of Several Watermelon ClLOX Genes in Response to JA, SA and ET Treatment 3.7. 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, Considering that the ClLOX genes harbor a large number of hormone-related cis-elements, qRT- qRT-PCR was carried out to investigate the expression patterns of selected ClLOX genes under JA, PCR was carried out to investigate the expression patterns of selected ClLOX genes under JA, SA, SA, and ET treatments. Upon JA treatment, the expression levels of ClLOX7, ClLOX12, and ClLOX14 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 showed notable increases in both the leaf and root, with the most significant increases being observed ClLOX7, suggesting that ClLOX7 plays a primary role in JA signaling in leaves and roots (Figure 7A,B). for ClLOX7, suggesting that ClLOX7 plays a primary role in JA signaling in leaves and roots (Figure However, ClLOX4 and ClLOX16 showed di erent expression patterns in leaves and roots under JA 7A,B). However, ClLOX4 and ClLOX16 showed different expression patterns in leaves and roots treatment. The expression of ClLOX4 observably decreased in leaves but gradually increased and under JA treatment. The expression of ClLOX4 observably decreased in leaves but gradually reached the highest level at 9 h in roots (Figure 7A,B). For ClLOX16, the expression increased and increased and reached the highest level at 9 h in roots (Figure 7A,B). For ClLOX16, the expression peaked at 24 h in leaves, while exhibited a significant decrease in roots at 3 h (Figure 7A,B). Upon SA increased and peaked at 24 h in leaves, while exhibited a significant decrease in roots at 3 h (Figure treatment, ClLOX7 and ClLOX16 showed similar expression patterns, and their transcripts dramatically 7A,B). Upon SA treatment, ClLOX7 and ClLOX16 showed similar expression patterns, and their 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 Agriculture 2020, 10, x FOR PEER REVIEW 11 of 16 Agriculture 2020, 10, 429 10 of 15 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 increases, while that of ClLOX4 and ClLOX12 displayed significant decreases at certain time points and ClLOX16 showed significant increases, while that of ClLOX4 and ClLOX12 displayed significant (Figure 7D). In addition, the expression of ClLOX14 was observably induced at earlier time points (1 h), decreases at certain time points (Figure 7D). In addition, the expression of ClLOX14 was observably but showed sharp decreases thereafter (Figure 7D). These results indicated that ClLOX genes may be induced at earlier time points (1 h), but showed sharp decreases thereafter (Figure 7D). These results involved in diverse hormonal responses. indicated that ClLOX genes may be involved in diverse hormonal responses. Figure 7. qRT-PCR analysis of the expression of selected ClLOX genes under various hormone Figure 7. qRT-PCR analysis of the expression of selected ClLOX genes under various hormone treatments, including JA (A,B), SA (C), and ET (D). Error bars indicate standard deviation (SD) based treatments, including JA (A,B), SA (C), and ET (D). Error bars indicate standard deviation (SD) based on three biological replicates. on three biological replicates. 4. Discussion 4. Discussion LOX proteins are widely present in plants, and are generally encoded by a multigene family. LOX proteins are widely present in plants, and are generally encoded by a multigene family. In In this study, a total of 16 LOX proteins were identified in watermelon through HMMER combined this study, a total of 16 LOX proteins were identified in watermelon through HMMER combined with with BLASTP search (Table 1), and several proteins possessing either one LOX domain or the BLASTP search (Table 1), and several proteins possessing either one LOX domain or the PLAT/LH2 PLAT/LH2 domain were excluded based on the criteria in previous reports [11,13]. Watermelon has a 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 comparatively larger number of LOX family members relative to other plant species, such as (six members) [12], pepper (eight members) [13], tea plant (11 members) [14], tomato (14 members) [18], Arabidopsis (six members) [12], pepper (eight members) [13], tea plant (11 members) [14], tomato (14 and melon (18 members) [20], and the number of LOX genes in these plants is not proportional to their members) [18], and melon (18 members) [20], and the number of LOX genes in these plants is not genome sizes. Notably, 11 out of the 16 ClLOX genes were located on chromosome 2 and formed two proportional to their genome sizes. Notably, 11 out of the 16 ClLOX genes were located on distinct tandem duplicate gene clusters (Figure 3). Moreover, over half of the ClLOX genes (10 in 16) chromosome 2 and formed two distinct tandem duplicate gene clusters (Figure 3). Moreover, over were identified as tandemly duplicated genes, indicating that tandem duplication events are the main half of the ClLOX genes (10 in 16) were identified as tandemly duplicated genes, indicating that driving force for the evolution of ClLOX genes. This feature has also been found in some other plants, tandem duplication events are the main driving force for the evolution of ClLOX genes. This feature such as poplar [11], maize [16], tomato [18], cucumber [34], and radish [15]. In addition, according to has also been found in some other plants, such as poplar [11], maize [16], tomato [18], cucumber [34], the collinear module of watermelon and Arabidopsis, all of the six AtLOX genes have syntenic copies in and radish [15]. In addition, according to the collinear module of watermelon and Arabidopsis, all of ClLOX genes (Supplementary Figure S3). The larger number of AtLOX-ClLOX orthologous events the six AtLOX genes have syntenic copies in ClLOX genes (Supplementary Figure S3). The larger indicates that ClLOX genes may have similar structure and function to AtLOX genes. number of AtLOX-ClLOX orthologous events indicates that ClLOX genes may have similar structure Through a phylogenetic analysis of the LOX gene family in watermelon and other plant species, and function to AtLOX genes. LOX family members can be divided into the categories of 9-LOXs and 13-LOXs (Figure 1), which Agriculture 2020, 10, 429 11 of 15 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 [37–39]. 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 di erent 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 [46–48]. 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 Agriculture 2020, 10, 429 12 of 15 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. 5. 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 di erent stages. The expression levels are indicated as log2-based FPKM+1 values. DAP, days after pollination. Table S1. Primers sequences used in qRT-PCR. Table S2. LOX family proteins from di erent plant species used in this study. Author Contributions: Data curation, J.L. (Jianping Liu), Y.Z., J.L. (Jingwen Li), and Y.Y.; formal analysis, J.L. (Jianping Liu); funding acquisition, Y.Y. and F.W.; methodology, J.L. (Jingwen Li) and Y.Z.; resources, J.L. (Jianping Liu), Y.Z., and Y.Y.; software, J.L. (Jingwen Li) and F.W.; writing—original draft, J.L. (Jianping Liu) and Y.Z.; writing—review and editing, Y.Y. and F.W. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the National Natural Science Foundation of China (31560572 and 31801904), and the Foundation of Jiangxi Educational Committee (GJJ190219), the Liaoning Revitalization Talents Program (XLYC1807020), the Doctoral Research Foundation of Liaoning Province (20180540094), the Innovative Talent Support Program for the Universities in Liaoning Province, the Young and Middle-aged Science and Technology Innovation Talents Program in Shenyang (RC200449). Conflicts of Interest: The authors declare that they have no conflict of interest. References 1. Porta, H.; Rocha-Sosa, M. Plant lipoxygenases. Physiological and molecular features. Plant Physiol. 2002, 130, 15–21. [CrossRef] [PubMed] 2. Feussner, I.; Wasternack, C. The lipoxygenase pathway. Annu. Rev. Plant Biol. 2002, 53, 275–297. [CrossRef] [PubMed] 3. Wennman, A.; Oliw, E.H.; Karkehabadi, S.; Chen, Y. Crystal structure of manganese lipoxygenase of the rice blast fungus Magnaporthe oryzae. J. Biol. Chem. 2016, 291, 8130–8139. [CrossRef] [PubMed] Agriculture 2020, 10, 429 13 of 15 4. Banthiya, S.; Kalms, J.; Yoga, E.G.; Ivanov, I.; Carpena, X.; Hamberg, M.; Kuhn, H.; Scheerer, P. Structural and functional basis of phospholipid oxygenase activity of bacterial lipoxygenase from Pseudomonas aeruginosa. Biochim. Biophys. Acta 2016, 1861, 1681–1692. [CrossRef] 5. Song, H.; Wang, P.; Li, C.; Han, S.; Lopez-Baltazar, J.; Zhang, X.; Wang, X. Identification of lipoxygenase (LOX) genes from legumes and their responses in wild type and cultivated peanut upon Aspergillus flavus infection. Sci. Rep. 2016, 6, 35245. [CrossRef] 6. Zhou, Y.; Guang, Y.; Li, J.; Wang, F.; Ahammed, G.J.; Yang, Y. The CYP74 gene family in watermelon: Genome-wide identification and expression profiling under hormonal stress and root-knot nematode infection. Agronomy 2019, 9, 872. [CrossRef] 7. Viswanath, K.K.; Varakumar, P.; Pamuru, R.R.; Basha, S.J.; Mehta, S.; Rao, A.D. Plant lipoxygenases and their role in plant physiology. J. Plant. Biol. 2020, 63, 83–95. [CrossRef] 8. Tolley, J.P.; Nagashima, Y.; Gorman, Z.; Kolomiets, M.V.; Koiwa, H. Isoform-specific subcellular localization of Zea mays lipoxygenases and oxo-phytodienoate reductase 2. Plant Gene 2018, 13, 36–41. [CrossRef] 9. Andreou, A.; Feussner, I. Lipoxygenases—Structure and reaction mechanism. Phytochemistry 2009, 70, 1504–1510. [CrossRef] 10. Ji, Q.; Zhang, T.; Zhang, D.; Lv, S.; Tan, A. Genome-wide identification and expression analysis of lipoxygenase genes in Tartary buckwheat. Biotechnol. Biotechnol. Equip. 2020, 34, 273–286. [CrossRef] 11. Chen, Z.; Chen, X.; Yan, H.; Li, W.; Li, Y.; Cai, R.; Xiang, Y. The lipoxygenase gene family in poplar: Identification, classification, and expression in response to MeJA treatment. PLoS ONE 2015, 10, e0125526. [CrossRef] [PubMed] 12. Bannenberg, G.; Martinez, M.; Hamberg, M.; Castresana, C. Diversity of the enzymatic activity in the lipoxygenase gene family of Arabidopsis thaliana. Lipids 2009, 44, 85–95. [CrossRef] [PubMed] 13. Sarde, S.J.; Kumar, A.; Remme, R.N.; Dicke, M. Genome-wide identification, classification and expression of lipoxygenase gene family in pepper. Plant Mol. Biol. 2018, 98, 375–387. [CrossRef] 14. Zhu, J.; Wang, X.; Guo, L.; Xu, Q.; Zhao, S.; Li, F.; Yan, X.; Liu, S.; Wei, C. Characterization and alternative splicing profiles of the lipoxygenase gene family in tea plant (Camellia sinensis). Plant Cell Physiol. 2018, 59, 1765–1781. [CrossRef] [PubMed] 15. Wang, J.; Hu, T.; Wang, W.; Hu, H.; Wei, Q.; Wei, X.; Bao, C. Bioinformatics analysis of the lipoxygenase gene family in radish (Raphanus sativus) and functional characterization in response to abiotic and biotic stresses. Int. J. Mol. Sci. 2019, 20, 6095. [CrossRef] 16. Ogunola, O.F.; Hawkins, L.K.; Mylroie, E.; Kolomiets, M.V.; Borrego, E.; Tang, J.D.; Williams, W.P.; Warburton, M.L. Characterization of the maize lipoxygenase gene family in relation to aflatoxin accumulation resistance. PLoS ONE 2017, 12, e0181265. [CrossRef] 17. Umate, P. Genome-wide analysis of lipoxygenase gene family in Arabidopsis and rice. Plant Signal. Behav. 2011, 6, 335–338. [CrossRef] 18. Upadhyay, R.K.; Mattoo, A.K. Genome-wide identification of tomato (Solanum lycopersicum L.) lipoxygenases coupled with expression profiles during plant development and in response to methyl-jasmonate and wounding. J. Plant Physiol. 2018, 231, 318–328. [CrossRef] 19. Podolyan, A.; White, J.; Jordan, B.; Winefield, C. Identification of the lipoxygenase gene family from Vitis vinifera and biochemical characterisation of two 13-lipoxygenases expressed in grape berries of Sauvignon Blanc. Funct. Plant Biol. 2010, 37, 767–784. [CrossRef] 20. Zhang, C.; Jin, Y.; Liu, J.; Tang, Y.; Cao, S.; Qi, H. The phylogeny and expression profiles of the lipoxygenase (LOX) family genes in the melon (Cucumis melo L.) genome. Sci. Hortic. 2014, 170, 94–102. [CrossRef] 21. Liu, S.Q.; Liu, X.H.; Jiang, L.W. Genome-wide identification, phylogeny and expression analysis of the lipoxygenase gene family in cucumber. Genet. Mol. Res. 2011, 10, 2613–2636. [CrossRef] [PubMed] 22. Li, M.; Li, L.; Dunwell, J.M.; Qiao, X.; Liu, X.; Zhang, S. Characterization of the lipoxygenase (LOX) gene family in the Chinese white pear (Pyrus bretschneideri) and comparison with other members of the Rosaceae. BMC Genom. 2014, 15, 444. [CrossRef] [PubMed] 23. Caldelari, D.; Wang, G.; Farmer, E.E.; Dong, X. Arabidopsis lox3 lox4 double mutants are male sterile and defective in global proliferative arrest. Plant Mol. Biol. 2011, 75, 25–33. [CrossRef] 24. Gayen, D.; Ali, N.; Sarkar, S.N.; Datta, S.K.; Datta, K. Down-regulation of lipoxygenase gene reduces degradation of carotenoids of golden rice during storage. Planta 2015, 242, 353–363. [CrossRef] Agriculture 2020, 10, 429 14 of 15 25. Sarde, S.J.; Bouwmeester, K.; Venegas-Molina, J.; David, A.; Boland, W.; Dicke, M. Involvement of sweet pepper CaLOX2 in jasmonate-dependent induced defence against Western flower thrips. J. Integr. Plant Biol. 2019, 61, 1085–1098. [CrossRef] [PubMed] 26. Hou, Y.; Ban, Q.; Meng, K.; He, Y.; Han, S.; Jin, M.; Rao, J. Overexpression of persimmon 9-lipoxygenase DkLOX3 confers resistance to Pseudomonas syringae pv. tomato DC3000 and Botrytis cinerea in Arabidopsis. Plant Growth Regul. 2018, 84, 179–189. [CrossRef] 27. Hou, Y.; Meng, K.; Han, Y.; Ban, Q.; Wang, B.; Suo, J.; Lv, J.; Rao, J. The persimmon 9-lipoxygenase gene DkLOX3 plays positive roles in both promoting senescence and enhancing tolerance to abiotic stress. Front Plant Sci. 2015, 6, 1073. [CrossRef] 28. Chen, C.; Chen, H.; Zhang, Y.; Thomas, H.R.; Frank, M.H.; He, Y.; Xia, R. TBtools—An integrative toolkit developed for interactive analyses of big biological data. Mol. Plant 2020. [CrossRef] 29. Zhou, Y.; Cheng, Y.; Wan, C.; Li, J.; Yang, Y.; Chen, J. Genome-wide characterization and expression analysis of the Dof gene family related to abiotic stress in watermelon. PeerJ 2020, 8, e8358. [CrossRef] 30. Li, J.; Guang, Y.; Yang, Y.; Zhou, Y. Identification and expression analysis of two allene oxide cyclase (AOC) genes in watermelon. Agriculture 2019, 9, 225. [CrossRef] 31. Livak, K.J.; Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and DDCT the 2 method. Methods 2001, 25, 402–408. [CrossRef] [PubMed] 32. Shaban, M.; Ahmed, M.M.; Sun, H.; Ullah, A.; Zhu, L. Genome-wide identification of lipoxygenase gene family in cotton and functional characterization in response to abiotic stresses. BMC Genom. 2018, 19, 599. [CrossRef] [PubMed] 33. Steczko, J.; Donoho, G.P.; Clemens, J.C.; Dixon, J.E.; Axelrod, B. Conserved histidine residues in soybean lipoxygenase: Functional consequences of their replacement. Biochemistry 1992, 31, 4053–4057. [CrossRef] [PubMed] 34. Ding, Y.; Yang, W.; Su, C.; Ma, H.; Pan, Y.; Zhang, X.; Li, J. Tandem 13-lipoxygenase genes in a cluster confers yellow-green leaf in cucumber. Int. J. Mol. Sci. 2019, 20, 3102. [CrossRef] [PubMed] 35. Sloane, D.L.; Leung, R.; Craik, C.S.; Sigal, E. A primary determinant for lipoxygenase positional specificity. Nature 1991, 354, 149–152. [CrossRef] 36. Meng, K.; Hou, Y.; Huber, D.J.; Dong, X.; Ban, Q.; Chang, X.; Zhang, T.; Rao, J. Molecular cloning, structural characterization, and ripening-related expression of lipoxygenase genes from three persimmon cultivars di ering in postharvest ripening rate. Plant Mol. Biol. Rep. 2016, 34, 854–868. [CrossRef] 37. Li, S.T.; Zhang, M.; Fu, C.H.; Xie, S.; Zhang, Y.; Yu, L.J. Molecular cloning and characterization of two 9-lipoxygenase genes from Taxus chinensis. Plant Mol. Biol. Rep. 2012, 30, 1283–1290. [CrossRef] 38. Padilla, M.N.; Hernández, M.L.; Sanz, C.; Martínez-Rivas, J.M. Molecular cloning, functional characterization and transcriptional regulation of a 9-lipoxygenase gene from olive. Phytochemistry 2012, 74, 58–68. [CrossRef] 39. Hornung, E.; Walther, M.; Kühn, H.; Feussner, I. Conversion of cucumber linoleate 13-lipoxygenase to a 9-lipoxygenating species by site-directed mutagenesis. Proc. Natl. Acad. Sci. USA 1999, 96, 4192–4197. [CrossRef] 40. Preston, J.; Hileman, L. Functional evolution in the plant SQUAMOSA-PROMOTER BINDING PROTEIN-LIKE (SPL) gene family. Front. Plant Sci. 2013, 4, 4. [CrossRef] 41. Zhang, J. Evolution by gene duplication: An update. Trends Ecol. Evol. 2003, 18, 292–298. [CrossRef] 42. Zhang, B.; Chen, K.; Bowen, J.; Allan, A.; Espley, R.; Karunairetnam, S.; Ferguson, I. Di erential expression within the LOX gene family in ripening kiwifruit. J. Exp. Bot. 2006, 57, 3825–3836. [CrossRef] 43. Griths, A.; Barry, C.; Alpuche-Solis, A.G.; Grierson, D. Ethylene and developmental signals regulate expression of lipoxygenase genes during tomato fruit ripening. J. Exp. Bot. 1999, 50, 793–798. [CrossRef] 44. Del Ángel-Coronel, O.A.; León-García, E.; Vela-Gutiérrez, G.; Rojas-Reyes, J.O.; Gómez-Lim, M.Á.; García, H.S. Lipoxygenase activity associated to fruit ripening and senescence in chayote (Sechium edule Jacq. Sw. cv. “virens levis”). J. Food Biochem. 2018, 42, e12438. [CrossRef] 45. Bell, E.; Creelman, R.A.; Mullet, J.E. A chloroplast lipoxygenase is required for wound-induced jasmonic acid accumulation in Arabidopsis. Proc. Natl. Acad. Sci. USA 1995, 92, 8675–8679. [CrossRef] 46. Li, N.; Han, X.; Feng, D.; Yuan, D.; Huang, L.J. Signaling crosstalk between salicylic acid and ethylene/jasmonate in plant defense: Do we understand what they are whispering? Int. J. Mol. Sci. 2019, 20, 671. [CrossRef] [PubMed] Agriculture 2020, 10, 429 15 of 15 47. Di, X.; Gomila, J.; Takken, F.L.W. Involvement of salicylic acid, ethylene and jasmonic acid signalling pathways in the susceptibility of tomato to Fusarium oxysporum. Mol. Plant Pathol. 2017, 18, 1024–1035. [CrossRef] [PubMed] 48. Thaler, J.S.; Humphrey, P.T.; Whiteman, N.K. Evolution of jasmonate and salicylate signal crosstalk. Trends Plant Sci. 2012, 17, 260–270. [CrossRef] [PubMed] 49. Wang, R.; Shen, W.; Liu, L.; Jiang, L.; Liu, Y.; Su, N.; Wan, J. A novel lipoxygenase gene from developing rice seeds confers dual position specificity and responds to wounding and insect attack. Plant Mol. Biol. 2008, 66, 401–414. [CrossRef] 50. Liu, S.; Han, B. Di erential expression pattern of an acidic 9/13-lipoxygenase in flower opening and senescence and in leaf response to phloem feeders in the tea plant. BMC Plant Biol. 2010, 10, 228. [CrossRef] [PubMed] 51. Christensen, S.A.; Nemchenko, A.; Borrego, E.; Murray, I.; Sobhy, I.S.; Bosak, L.; DeBlasio, S.; Erb, M.; Robert, C.A.; Vaughn, K.A.; et al. The maize lipoxygenase, ZmLOX10, mediates green leaf volatile, jasmonate and herbivore-induced plant volatile production for defense against insect attack. Plant J. 2013, 74, 59–73. [CrossRef] [PubMed] 52. He, Y.; Borrego, E.J.; Gorman, Z.; Huang, P.C.; Kolomiets, M.V. Relative contribution of LOX10, green leaf volatiles and JA to wound-induced local and systemic oxylipin and hormone signature in Zea mays (maize). Phytochemistry 2020, 174, 112334. [CrossRef] [PubMed] 53. Gorman, Z.; Christensen, S.A.; Yan, Y.; He, Y.; Borrego, E.; Kolomiets, M.V. Green leaf volatiles and jasmonic acid enhance susceptibility to anthracnose diseases caused by Colletotrichum graminicola in maize. Mol. Plant Pathol. 2020, 21, 702–715. [CrossRef] [PubMed] 54. Upadhyay, R.K.; Handa, A.K.; Mattoo, A.K. Solanum lycopersicum transcript abundance patterns of 9- and 13-Lipoxygenase subfamily gene members in response to abiotic stresses (heat, cold, drought or salt) in tomato (Solanum lycopersicum L.) highlights member-specific dynamics relevant to each stress. Genes 2019, 10, 683. © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
Journal
Agriculture – Multidisciplinary Digital Publishing Institute
Published: Sep 25, 2020