Genome-wide identification and expression pattern analysis of lipoxygenase gene family in banana

The LOX genes have been identified and characterized in many plant species, but studies on the banana LOX genes are very limited. In this study, we respectively identified 18 MaLOX, 11 MbLOX, and 12 MiLOX genes from the Musa acuminata, M. balbisiana and M. itinerans genome data, investigated their gene structures and characterized the physicochemical properties of their encoded proteins. Banana LOXs showed a preference for using and ending with G/C and their encoded proteins can be classified into 9-LOX, Type I 13-LOX and Type II 13-LOX subfamilies. The expansion of the MaLOXs might result from the combined actions of genome-wide, tandem, and segmental duplications. However, tandem and segmental duplications contribute to the expansion of MbLOXs. Transcriptome data based gene expression analysis showed that MaLOX1, 4, and 7 were highly expressed in fruit and their expression levels were significantly regulated by ethylene. And 11, 12 and 7 MaLOXs were found to be low temperature-, high temperature-, and Fusarium oxysporum f. sp. Cubense tropical race 4 (FocTR4)-responsive, respectively. MaLOX8, 9 and 13 are responsive to all the three stresses, MaLOX4 and MaLOX12 are high temperature- and FocTR4-responsive; MaLOX6 and MaLOX17 are significantly induced by low temperature and FocTR4; and the expression of MaLOX7 and MaLOX16 are only affected by high temperature. Quantitative real-time PCR (qRT-PCR) analysis revealed that the expression levels of several MaLOXs are regulated by MeJA and FocTR4, indicating that they can increase the resistance of banana by regulating the JA pathway. Additionally, the weighted gene co-expression network analysis (WGCNA) of MaLOXs revealed 3 models respectively for 5 (MaLOX7-11), 3 (MaLOX6, 13, and 17), and 1 (MaLOX12) MaLOX genes. Our findings can provide valuable information for the characterization, evolution, diversity and functionality of MaLOX, MbLOX and MiLOX genes and are helpful for understanding the roles of LOXs in banana growth and development and adaptations to different stresses.

www.nature.com/scientificreports/ been proved to be regulated by some phytohormones and pathogens. For instance, the expression of Arabidopsis AtLOX1 was abscisic acid and JA inducible 14 , and the rice OsLOX3 was MeJA and Magnaporthe Grisea inducible 15 . Their diverse functions during plant growth and developmental and stress response processes have also been experimentally confirmed in various plant species. Arabidopsis AtLOX3 and AtLOX4 double mutant plants showed developmental dysfunctions of higher plant height and increased inflorescence shoots and flowers 16 .
AtLOX2 and AtLOX6 are found to be involved in wound induced JA synthesis in leaves 17,18 . Transgenic plants overexpressing rice OsLOX2 showed shortened seed germination time 19 . Kiwifruit AdLOXs were involved in the formation of fruit aroma 20 . Silencing of CaLOX2 in pepper plants resulted in decreased JA accumulation and reduced thrips resistance 21 . Transgenic tomato plants overexpressing the tomato lipoxygenase D (TomLoxD) gene resulted in enhanced wound-induced JA biosynthesis and increased Helicoverpa armigera and Botrytis cinerea resistance 22 . Transgenic Arabidopsis plant overexpressing persimmon DkLOX3 showed increased salt tolerance and disease resistance 23 .
Banana, as one of the most important and popular fruit, is an herbaceous perennial plant belonging to Musa family. Cultivated banana is generally low in stress resistance and is susceptible to external environmental stresses such as low temperature and Fusarium wilt 24 . There are also several reports on the expression patterns of some banana LOXs using omic techniques, and their roles in banana responses to high temperature, low temperature and Fusarium wilt have been described [25][26][27][28] . Given that LOXs are vital for plant growth and stress resistance and different LOX members' functions varied, it is of great importance to analyze the LOX gene family from whole genome level for the clarification of their diverse potentials in banana. In the present study, whole genome wide LOX gene family identification was performed based on the M. acuminata, M. balbisiana and M. itinerans genome data. Totally, we identified 18 MaLOX, 11 MbLOX, and 12 MiLOX family members, which were then subjected to series of bioinformatics analysis to show the chromosome location, gene structure and gene duplication events of LOX genes and to reveal the physiological and biochemical characteristics, subcellular localization, and phylogenetic relationship of their encoded proteins. Moreover, the expression patterns of MaLOXs were investigated using quantitative real time PCR (qRT-PCR) and transcriptome data. Our preliminary results can extend the knowledge of banana LOX gene family and can provide insights into their roles in banana growth and development and stress responses.

Materials and methods
Plant materials. In our previous study, 'Tianbaojiao' banana (Musa spp., Cavendish, AAA group) plantlets were used for transcriptome profiling to show the transcriptome changes caused by 4 ℃ low temperature in leaves of four-leaf stage plantlets, by 45 ℃ high temperature in leaves of five-stage plantlets, and by FocTR4 inoculation in banana roots. Moreover, transcriptome changes of natural ripening and ethylene treated 'Tianbaojiao' banana fruits at 0, 1, 3, and 5 days were also compared. Moreover, to show the influence of MeJA treatment on the expression of banana LOXs, 'Brizil' banana (Musa acuminata cv. Brazil) plantlets at six-leaf stage were exposed to 100 mM MeJA solution (containing 0.02% (v/v) Tween 20) treatment 9 , treated leaves were sampled at 0, 6, 12, 24 h after MeJA treatment. In addition, in order to further explore the expression of MaLOXs in response to FocTR4 treatment, 'Zhongjiao No.3' banana (Musa acuminata cv. Brazil) plantlets at six-leaf stage were inoculated with 1 × 10 7 /mL FocTR4 spore suspension according to the inoculation method described by Wang et al. 29 . Roots were collected 0 day, 4 days, 2 weeks, and 4 weeks after treatment. Banana plantlets showed no visible symptom in corm until 4 weeks after FocTR4 inoculation. All samples were immediately frozen in liquid nitrogen and stored at − 80 °C for further use. For qRT-PCR analysis, three independent replicates were used for each time point of MeJA and FocTR4 treatments. All the banana materials used in this research were harvested from cultivated varieties ('Tianbaojiao' banana is a famous traditional cultivar in Tianbao county, Fujian province, China. 'Brazil' is one of the most popular banana variety in the world and 'Zhongjiao No.3' is a new banana variety selected from 'Brazil' by Institute of fruit science, Guangdong Agricultural Academy), and their collections complied with relevant institutional, national, and international guidelines and legislation.
Identification of banana LOX genes. The genomic DNA, CDS, and protein sequence files of M. acuminata var. DH-Pahang, M. balbisiana var. DH PKW and M. itinerans var. Yunnan were downloaded from the banana genome databases (https:// banana-genome-hub. south green. fr/). HMMER3.0 software was used to search against the banana protein sequences using The Hidden Markov Model file of Lipoxygenase (PF00305) downloaded from the Pfam database (http:// pfam. xfam. org/) with E-value ≤ 1 × 10 -5 to obtain candidate LOX proteins, which were further submitted to conserved domain database (CDD, https:// www. ncbi. nlm. nih. gov/ cdd) for the confirmation of the existence of the lipoxygenase and PLAT/LH2 domains 10 . Sequences without Lipoxygenase domain and/or PLAT/LH2 domain were removed. The remaining banana LOXs are named sequentially according to the chromosomal location of their corresponding genes. ExPASy (https:// web. expasy. org/ protp aram/) was used to analyze the basic physicochemical properties of LOX proteins. Chloroplast transit peptide and subcellular localization were predicted by ChloroP 1.1 Server (http:// www. cbs. dtu. dk/ servi ces/ Chlor oP/) and WoLF PSORT (https:// wolfp sort. hgc. jp/). The global sequence alignment program Needle (https:// www. ebi. ac. uk/ Tools/ psa/ emboss_ needle/) in the EMBOSS tool was used to perform pairwise alignment of protein sequences to determine the similarity and identity between LOX members. Gene structure of banana LOXs was drawn by GSDS (http:// gsds. cbi. pku. edu. cn/). The conserved motifs of LOXs (20 maximum number of motifs) were analyzed using MEME suite (http:// meme-suite. org/ tools/ meme) and visualized using TBtools software 30 . The CodonW software (version 1.4.2, http:// codonw. sourc eforge. net/) was used to calculate the effective number of codons (ENC), codon adaptation index (CAI), relative synonymous codon usage (RSCU), and other codon preference parameters 6 . Analysis of cis-acting elements and transcription factor binding sites in the promoters of banana LOX genes. The 1500 bp upstream of the start codon of each banana LOX gene was extracted from the banana genome database. Due to the presence of large numbers of CTT repeat sequences on MaLOX5 promoter region from the genome data, PCR was used to verify its true sequence. It was found that CTT repeat sequences were absent, thus the corrected sequence was used for subsequent analysis. The cis-acting elements of the promoter were predicted using the PlantCARE (http:// bioin forma tics. psb. ugent. be/ webto ols/ plant care/ html/). PlantTFDB (http:// planttfdb.cbi.pku.edu.cn/) was used to predict the transcription factor binding sites (TFBSs) on promoters with the parameter set of p-value ≤ 1e −6 . The promoter regions were partitioned to proximal promoter region (500 bp upstream), median promoter region (501-1000 bp upstream) and distal promoter region (1001-1500 bp upstream).
Gene expression analysis using transcriptome data and qRT-PCR. The expression patterns of banana LOX genes under low temperature, high temperature and FocTR4 treatments were analyzed using our previous transcriptome data. The expression values of banana LOX family genes were extracted from the transcriptome data, and heatmap was drawn using HemI1.0 software (http:// hemi. biocu ckoo. org/). qRT-PCR was used to show the expression patterns of all the banana LOX genes under JA treatment. Total RNA was extracted using RNAprep Pure Plant Kit (TIANGEN, China) according to the manufacturer's instructions. A total of 1 μg RNA was used for cDNA synthesis using PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time) (Takara, China). CDNA was diluted tenfold for subsequent experiments. The PCR reaction conditions used were 95 °C for 30 s, 95 °C for 5 s, and 60 °C for 34 s (40 cycles). Relative gene expression levels were determined using the 2 -∆∆Ct method by using MaCAC as an internal reference 35 . Primers were designed using Oligo 7.0, and their specificity was checked using information obtained from the NCBI website. All primers used in this study are listed in Supplemen Table S1. Statistical analysis and figure drawing were conducted using SPSS 25.0 and GraphPad Prism 6.0 software, respectively.
Weighted gene co-expression network analysis (WGCNA). Genes with FPKM value greater than 10 in at least one RNA-Seq sample were subjected to WGCNA (version 1.68) analysis to construct and identify co-expressed gene clusters with MaLOXs 36 . The parameters were set as follows: The optimal β (soft thresholding power) value was 12; the minModuleSize was 30 and the mergeCutHeight was 0.25. Finally, we extracted the coexpression network of all MaLOXs and filtered out the edges with weights below 0.4. We visualized the network connections using the Cytoscape (version 3.8.0, https:// cytos cape. org/) program 37 . The functional enrichment analysis of MaLOXs and co-expressed genes was performed using Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases.

Results
Identification and characterization of banana LOX gene family members. Totally,18,11,and 12 LOX genes were identified from M. acuminata, M. balbisiana, and M. itinerans genome, respectively (Table 1,  Supplementary Table S2). According to their chromosomal location information, the 18 MaLOXs were defined as MaLOX1-MaLOX18, respectively. Among these MaLOXs, MaLOX5 had two transcripts, which was named as MaLOX5a and MaLOX5b, respectively. MbLOXs and MiLOXs were named in concordance with their MaLOXs homologous (Supplementary Figure S1).

Chromosome location and gene duplication. As shown in
In order to explore the gene duplication events of the LOX family, we investigated the collinearity relationships between banana LOXs as well as pairwise relationships analysis of LOXs from M. acuminata, M. balbisiana, M. itinerans, Arabidopsis, and rice ( Fig. 5; Supplementary Figures S8, S9, S10; Table 2; Supplementary Tables S3, S4)  To further understand whether the genes of the LOX family have been subjected to natural selection pressures during the evolution process and to trace the duplication time of banana LOXs, we calculated the ratios of nonsynonymous (Ka) versus synonymous (Ks) mutation of orthologous gene pairs. As shown in Table 2   www.nature.com/scientificreports/ the promoter sequences of their family members for cis-acting element prediction analysis. In total, four categories of cis-acting elements were identified, including light responsiveness, phytohormone responsiveness, stress responsiveness, and plant growth and development-related elements (Fig. 6, Supplementary Figures S11, S12). Therefore, it is speculated that the expression of banana LOXs may be regulated by multiple factors.

Transcription factor binding site (TFBS) prediction.
To investigate the regulation of transcription factors (TFs) on the expression of banana LOXs, transcription factor binding sites (TFBSs) on the promoter were predicted using PlantTFDB online tool. A total of 8 TF families (AP2/ERF, BBR-BPC, bZIP, C2H2, Dof, MIKC_MADS, NAC and WRKY) were identified in the MaLOX promoters, which covers 10, 4, 3, 5, 7, 11, 4, and 2 members, respectively (Fig. 7). BBR-BPC family has the largest number of binding sites (51) (Table 3, Supplementary Table S5). The codon adaptation index (CAI) value of MaLOXs, MbLOXs and MiLOXs ranged respectively from 0.18 to 0.26, from 0.18 to 0.26, and from 0.19 to 0.26, with a mean value of 0.23, 0.23, and 0.22, suggesting that the codon bias of banana LOXs was weak. With the exception of MiLOX12, the average content of C3s and G3s was significantly higher than that of A3s and T3s, and the average content of GC and GC3s was greater than 0.5, which indicated that the banana LOX codons generally prefer to use and end with G/C. Relative synonymous codon usage (RSCU) can intuitively reflect the degree to which specified  www.nature.com/scientificreports/ codons deviate from synonymous codons, and RSCU > 1 indicates that the codons are used more frequently than expected. 27 codons showed strong preference for GC-ending codons based on the above criterion in MaLOXs, MbLOXs, and MiLOXs, respectively (Fig. 8, Supplementary Figures S14, S15). Among these, 11 codons end in G and 16 codons end in C. Fig. 9, MaLOXs showed divergent expression patterns across different tissues (Supplementary Table S6). MaLOX1 was found to be a highly expressed gene in banana leaves, roots, and fruits. MaLOX7 was highly expressed in fruits and leaves. The expression of MaLOX4 in fruits is higher than in leaves and roots. MaLOX17 was predominantly expressed in the root. Under low temperature treatment, 6 MaLOX members (33.33%) were upregulated and 5 members (27.78%) were downregulated. The expression of most members of the 9-LOX subfamily was inhibited, however, MaLOX17 was significantly induced (Fig. 9A). Most members of TypeII 13-LOX were upregulated by low temperature, with MaLOX15 being particularly significant. Under high temperature stress, 3 members (16.67%), including MaLOX12, 15, and 16, were upregulated, in which MaLOX12 was significantly induced (Fig. 9B), and 9 members were downregulated. The expression of MaLOX6, 8,9,13, and 17 increased greatly under FocTR4 treatment, while the expression of MaLOX4 and 12 declined (Fig. 9C).

Expression pattern of MaLOX genes under different stresses. As shown in
MaLOXs expression pattern analysis during natural ripening and ethylene induced ripening was also performed. The expression of the MaLOX1 was downregulated and MaLOX8 showed fluctuation change as fruit ripens (Fig. 9D). MaLOX1, 7, 8, and 18 were upregulated, while MaLOX2 and MaLOX4 were downregulated by ethylene at 0 day compared with the control group, but they were downregulated at following timepoints in comparison to the postharvest naturally ripening stage.  Table 7). The expression level of MaLOX17 is too low that its relative expression level was not shown in Fig. 10. The expression of MaLOX2-4 and MaLOX9 significantly increased after MeJA treatment, while 5 MaLOX members (MaLOX5, 13, 15, 16, and  18) declined significantly. Eight MaLOX members (MaLOX1-4 and 7-10) were significantly induced by MeJA, and their relative expression peaked at 6 h, then began to decline sharply. MaLOX6 was significantly upregulated

Weighted gene co-expression network analysis (WGCNA) of MaLOXs.
To explore the potential interaction and functions between co-expressed genes, WGCNA was applied to construct the co-expression network based on 4 different transcriptome datas, including banana fruit ripening stages, leaves response to high and low temperature, and roots inoculated with FocTR4. We only keep edges with strong connections with weight values ≥ 0.4. A total of 7629 genes were co-expressed with nine MaLOXs. Visualization using Cytoscape software, three co-expression networks models, respectively containing 5 (MaLOX7-11), 3 (MaLOX6, 13, and 17), and 1 (MaLOX12) MaLOX, were constructed (Fig. 12). GO enrichment analysis result revealed that, from the aspect of biological process, the MaLOXs co-expressed genes were mainly enriched in RNA splicing, mRNA splicing via spliceosome, electron transport chain, generation of precursor metabolites and energy, regulation of mRNA splicing via spliceosome, and response to heat (Fig. 13A); from the aspect of molecular function, nuclear speck, plastid membrane, nuclear body, and spliceosomal complex related co-expressed genes were enriched. According to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment results, these MaLOX www.nature.com/scientificreports/ co-expressed genes were found to be enriched in photosynthesis, proteasome, spliceosome, porphyrin and chlorophyll metabolism, and photosynthesis-antenna proteins (Fig. 13B).

Discussion
Comprehensive genome-wide identification of LOXs in banana. Lipoxygenase is a crucial restriction enzyme in the LOX pathway, which catalyzes the fatty acid metabolism of plant, actively participates in growth and development, and resists extreme external environmental conditions 38 . In this study, we identified 18 MaLOX, 11 MbLOX, and 12 MiLOX genes from M. acuminata, M. balbisiana, and M. itinerans genome, respectively. MaLOXs have more members than Arabidopsis (6), rice (14), and tomato (14), but are the same as grapes (18) 39 and melon (18)  Phylogenetic analysis showed that the banana LOXs family could be further divided into three subfamilies, including 9-LOX, Type I 13-LOX, and Type II 13-LOX, which was consistent with the results of poplar 9 and tea plant 10 . The sequence similarity among Type I LOX members ranged from 26.90 to 98.90%. Type II 13-LOX members contained chloroplast transit peptides except MiLOX6 and MiLOX18, and their sequence similarity ranged from 44.90 to 92.60%. Our results are not completely consistent with classification method of Shibata et al. 41 , who put forward that Type I LOX genes exhibit high sequence similarity (more than 75%) and lack of chloroplast transit peptide, while Type II LOX genes show moderate overall sequence similarity (up to 35%) and exist chloroplast transit peptide. But our result was consistent with the melon LOXs 40 , which may be related to the diversity of the evolution process of the LOX genes. The prediction of subcellular localization showed that MaLOX18, MbLOX18 and MiLOX18 were localized in the cytoplasm, while other Type II 13-LOX members were all localized in the chloroplast. This may be due to the poor conservation of the amino acid sequence of the chloroplast transit peptide of banana LOX18 42,43 . The members of this subfamily have similar gene structure and conserved motifs, indicating that the gene function of banana LOX members from the same subfamily showed certain degree of conservativeness. Our study found that codon bias of banana LOXs was weak, preferring to use and end with G/C, which is consistent with the codon preference characteristics of monocotyledon plants 44 and banana genome 45 . Thus, it was hypnotized that in order to cope with environmental pressures, different banana species have formed unique codon usage bias during evolution.
LOXs may play special roles in banana evolution. Gene duplication is a major factor responsible for the amplification in family gene numbers, in which whole genome duplication (WGD) is considered to be an important driving force for expansion and an important source of gene function diversification 4 . There are three pairs of segmental duplication genes and three tandem duplication gene clusters in the poplar LOX family genes 9 . Five tandem repeat pairs were observed in tomato LOX family, and no segmental duplicate pairs 8 . In this study, the four pairs of segmental duplication genes and two pairs of tandem repeat genes were found in MaLOX Table 3. Codon preference parameters of MaLOX family genes. T3s, C3s, A3s, G3s, and GC3s indicate that the third base of the codon is the content of T, C, A, G, and G + C. GC: total GC content in of CDS. CAI: codon adaptation index. ENC: effective number of codons. The mechanism of gene and genome evolution can be understood through a comparative analysis of relatively close between-species genome. This study has found that there are a high conservation level and have a close homology relationship among MaLOX, MbLOX, and MiLOX genes. The ancestor of M. acuminata and Functional prediction of MaLOXs. The cis-acting elements of the promoter combine with specific transcription factors to form transcription initiation complex and initiate gene specific expression 49 . Four types of cis-regulatory elements were identified at the banana LOX promoters, including light, phytohormone, stress, growth and development-related, which is consistent with the report about the functional diversity of LOX genes 50 . Besides, a variety of kinds of TFBSs were found in the banana LOX promoters. Recent research demonstrated that TFs play an important role in banana growth and adversity stress [51][52][53] , and it is further speculated that banana LOX expression is regulated by many TFs.
Lipoxygenase is a kind of oxygenase widely distributed in various organs of plants, and its expression levels in different parts and developmental stages of plants differed, which are closely related to physiological processes such as plant growth, development, maturity, and senescence 10,37 . In this study, each member of the MaLOX family was expressed in at least one organ. MaLOX1 was highly expressed in leaf, root, and fruit, which suggests that the function of MaLOX1 may be diverse. The expression of MaLOX4 in fruit is higher than in leaf and root, and MaLOX7 is highly expressed in fruit and leaf, which means that the functions of different MaLOXs members varied in different organs.
Low temperature can inhibit the transcriptional level of LOX in banana fruit, reduce the banana volatiles, and the inhibition effect is more obvious as the temperature decreases 25 . Under high temperature, there is an overall decrease in the amount of LOX proteins in banana peel 26,27 . Li et al. 28 found that the high expression of LOX was related to higher FocTR4 resistance of resistant mutant. LOX1.1-3 and LOX2.3 were significantly induced in resistant variety (Musa yunnanensis) during early infection with FocTR4 54 . In this study, the analysis of transcriptome data under low temperature, high temperature, and FocTR4 treatment revealed that the expression patterns of MaLOXs under different stresses differed. MaLOX8, 9, and 13 responded significantly to the above three stresses. The expressions of MaLOX1, 8,10,11,14, and 15 were regulated by high and low temperature; MaLOX6 and 17 were induced by low temperature and FocTR4; MaLOX4 and MaLOX12 responded to high temperature and FocTR4. MaLOX7 and 16 were differentially expressed at high temperature and MaLOX18 was www.nature.com/scientificreports/ only induced by low temperature. In addition, this study also found that in the early stage of FocTR4 infection, each member of MaLOXs responded to varying degrees. WGCNA is an effective way to identify clusters of highly correlated genes and can better preserve the characteristics of biological networks and reflect the relationship among functions and different biological processes 55,56 . Most of the adjacent genes of MaLOXs in their co-expression network were related to RNA splicing, generation of precursor metabolites and energy, heat stress, photosynthesis, and proteasome. Besides, the promoter regions of these differentially expressed genes contain a large number of stress-related cis-acting elements and TFBSs. These results indicated that MaLOXs are widely involved in banana growth and development and various stress responses.
LOX regulates the processes of plant ripening and senescence by participating in the synthesis of ethylene or catalyzing polyunsaturated fatty acids to generate superoxide radicals and destroying cell membrane structure 40,57 . And the roles of LOX in fruit ripening and flavor formation have been confirmed in tomato 8 , apple 58 , peach 12 and kiwi 59 . Our study found that MaLOX1 was downregulated during fruit ripening and 6 members (MaLOX1, 2,  4, 7, 8, and 18) were found to be ethylene responsive. It was reported that under ethylene and high-temperature treatment, the content of LOXA, LOX4, and LOX5 (corresponding to MaLOX4, MaLOX8, and MaLOX1 in this  www.nature.com/scientificreports/ study, respectively) decreased in banana fruit peel 26 . During banana fruit ripening, the expression of MaLOX (or named as BanLOX) decreased 60 . After ethylene treatment, however, it was upregulated in the pulp while it did not change significantly in the peel 60 , which is similar to the results of this study. Moreover, MaLOX1, 4, and 7 were predominantly and specifically expressed during fruit ripening and were regulated by ethylene. Therefore, we speculated that these LOX genes may be the candidate genes involved in banana fruit ripening and flavor formation. MeJA/JA, as a signal molecule that affects biological and abiotic reactions in plants, plays an important role in dealing with various external stresses. It was found that the application of exogenous MeJA can induce endogenous JA biosynthesis in plants 61 , and JA biosynthesis mainly depends on the substrate and expression of the genes at the critical steps of the synthesis pathway, such as LOX, AOC, AOS, and OPR 62 . In this study, with the exception of MaLOX11, 16, and 17, most 9-LOX subfamily MaLOX genes were upregulated and reached the maximum expression level at 6 h. And the expression trend of MaLOX14, belonging to Type II 13-LOX subfamily, also showed similar expression pattern. The expression of MaLOX16 is suppressed by MeJA, while MaLOX13, 15,18 were significantly upregulated at 24 h. We also found that most MeJA responsive MaLOXs contain MeJA-responsive elements in their promoters. The expression of poplar 9 , Panax ginseng 63 , pepper 64 , and tomato 8 LOX genes were found to be regulated by MeJA to some extent, which is consistent with our results. In addition, external application of MeJA can induce the expression of MaLOX1 and MaLOX2, enhance the content of endogenous JA, and alleviate banana chilling injury partially 62 . The above results indicate that MeJA can cause the up-regulation of LOX genes, which can increase the content of endogenous JA, thus improve the stress resistance of plants.

Conclusions
In this study, 18, 11, and 12 family members were respectively identified from M. acuminata, M. balbisiana, and M. itinerans genome, which encoded proteins with conserved domains and mainly located in the cytoplasm or chloroplast. The condon usage in banana LOX family members prefer to use and end with G/C. Four segmental duplications and 2 tandem duplications as well as 3 segmental duplications and 3 tandem duplications occurred respectively during M. acuminata and M. balbisiana evolution. Banana LOXs can be divided into three subfamilies, including 9-LOX, Type I 13-LOX, and Type II 13-LOX, and the sequence characteristics between each subfamily members are conservative. The expression of MaLOXs showed certain tissue specificity, and showed different response patterns to MeJA, high temperature, low temperature, and FocTR4 treatments. Moreover, the potential function analysis of the protomer region and the co-expression network of MaLOXs was constructed using WGCNA indicated that MaLOXs might participate in the growth and development and various stress responses in banana. Our present study can extend the knowledge of banana LOX gene family and provide basis for future exploration of their functions.