Integrated High-Throughput Sequencing, Microarray Hybridization and Degradome Analysis Uncovers MicroRNA-Mediated Resistance Responses of Maize to Pathogen Curvularia lunata.

Curvularia lunata (Wakker) Boed, the causal agent of leaf spot in maize, is prone to mutation, making it difficult to control. RNAi technology has proven to be an important tool of genetic engineering and functional genomics aimed for crop improvement. MicroRNAs (miRNAs), which act as post-transcriptional regulators, often cause translational repression and gene silencing. In this article, four small RNA (sRNA) libraries were generated from two maize genotypes inoculated by C. lunata; among these, ltR1 and ltR2 were from the susceptible variety Huangzao 4 (HZ), ltR3 and ltR4, from the resistant variety Luyuan (LY), and 2286, 2145, 1556 and 2504 reads were annotated as miRNA in these four sRNA libraries, respectively. Through the combined analysis of high-throughput sequencing, microarray hybridization and degradome, 48 miRNAs were identified as being related to maize resistance to C. lunata. Among these, PC-732 and PC-169, two new maize miRNAs discovered, were predicted to cleave mRNAs of metacaspase 1 (AMC1) and thioredoxin family protein (Trx), respectively, possibly playing crucial roles in the resistance of maize to C. lunata. To further confirm the role of PC-732 in the interaction of maize and C. lunata, the miRNA was silenced through STTM (short tandem target mimic) technology, and we found that knocking down PC-732 decreased the susceptibility of maize to C. lunata. Precisely speaking, the target gene of PC-732 might inhibit the expression of disease resistance-related genes during the interaction between maize and C. lunata. Overall, the findings of this study indicated the existence of miRNAs involved in the resistance of maize to C. lunata and will contribute to rapidly clarify the resistant mechanism of maize to C. lunata.

First Report of Enterobacter cloacae Causing Stem, Leaf and Fruit Rot on Tomato in China.

Tomato (Solanum lycopersicum L.), as one of the most economically important and highly nutritious vegetable crops across the world, is widely cultivated in China, one of the largest tomato-concuming countries in the world (Ye et al., 2020; Wang and Liu, 2021). At present, major bacterial diseases in tomato include bacterial speck disease, tomato bacterial wilt and bacterial canker, all of which affect the tomato production around the world (Rosli et al., 2021; Peritore-Galve et al., 2021; Wang et al., 2022). In April 2022, a new bacterial disease was discovered on leaves, stems and fruits of tomato in a farmer's greenhouse located in Longfeng District in DaQing (125°07`-125°15`E, 46°28`-46°32`N), Heilongjiang Province, China. This field had tomato disease incidences approximately 50%. Apparent brown discolorations were found on fruits, leaves and stems in tomato plants. Symptoms were similar to fungal brown spots caused by Phytophthora infestans of tomato (Zhi et al.,2021; Liu et al.,2021) (Supplementary Figure S1). To isolate and identify the pathogen, the tissues of infected fruits, leaves and stems with typical symptoms were excised from diseased plants separately, and were disinfected with 75% ethanol for 10 s followed by 2% NaClO for 3 min and then washed five to eight times with sterile water (Wang et al., 2017). Afterwards, the samples were plated on nutrient agar (NA) solid medium and incubated. After incubation at 30°C for 2-3 days, bacterial colonies were isolated, then purified on nutrient agar (NA) solid medium at least twice by a streak plate method (Dou et al., 2019; Li et al, 2021; Zhao et al., 2022). White colonies grew on the NA medium after incubating for 2 days, showing round, opaque and smooth, which was similar to characteristics described as Enterobacter cloacae (García-González et al., 2018; Li et al, 2021). To further confirm the speculation on the identity of the isolated bacterium, the fragments of 16S rRNA were amplified and sequenced. The sequence of 16S rRNA was uploaded into GeneBank with accession numbers (OP077195.1). BLAST analysis of the sequence showed 97.68% identity with one corresponding sequence of E. cloacae in GeneBank (namely MK937637.1). Furthermore, a phylogenetic tree based on the sequence of 16S rRNA gene revealed that the isolate was grouped in the same clade as E. cloacae (Supplementary Figure S2). Based on Koch postulates to test pathogenicity of isolated bacteria, bacteria were inoculated on 30 day-old healthy tomato plants with three leaves stages, and the re-isolation of bacteria were carried out after 2 days of inoculation. To confirm pathogenicity, the isolates were cultured on LB medium at 30℃ for 2 days to prepare suspensions and adjusted to an optical density (OD) of 0.2 at A600, with a final concentration of 1ⅹ108 CFU/ml. Eight potted tomato plants were sprayed with bacteria suspensions, and eight control potted plants were sprayed with sterile distilled water. These seedlings were incubated in a chamber at 30°C with a 12 h light/dark photoperiod, with 85% relative humidity. After 2 days, inoculated tomato seedlings showed irregular small spots in leaves and brown necrosis at blade tips, and 8 to 10 days later, the leaves of tomato plants browned and died. The symptoms were the same with those of the initial diseased leaves of tomato plants (Supplementary Figure S1). No symptoms were observed on the control leaves (Supplementary Figure S3). Pathogenicity tests were repeated three biological times with same results. Meanwhile, the bacteria strains were re-isolated from symptomatic inoculated seedlings and confirmed as E. cloacae by culture and sequence methods as above. In China, there are no detailed records about the causal agent of this disease on tomato in a published paper in Chinese and English. To our knowledge, this is the first report of Enterobacter leaf brown necrosis caused by E. cloacae on tomato in China. Those results are of great significance for the production and management of tomato in greenhouse and control of the disease.

Drought-induced ABA, H2O2 and JA positively regulate CmCAD genes and lignin synthesis in melon stems.

BACKGROUND: Cinnamyl alcohol dehydrogenase (CAD) is an important enzyme functions at the last step in lignin monomer synthesis pathway. Our previous work found that drought induced the expressions of CmCAD genes and promoted lignin biosynthesis in melon stems. RESULTS: Here we studied the effects of abscisic acid (ABA), hydrogen peroxide (H2O2) and jasmonic acid (JA) to CmCADs under drought stress. Results discovered that drought-induced ABA, H2O2 and MeJA were prevented efficiently from increasing in melon stems pretreated with fluridone (Flu, ABA inhibitor), imidazole (Imi, H2O2 scavenger) and ibuprofen (Ibu, JA inhibitor). ABA and H2O2 are involved in the positive regulations to CmCAD1, 2, 3, and 5, and JA is involved in the positive regulations to CmCAD2, 3, and 5. According to the expression profiles of lignin biosynthesis genes, ABA, H2O2 and MeJA all showed positive regulations to CmPAL2-like, CmPOD1-like, CmPOD2-like and CmLAC4-like. In addition, positive regulations were also observed with ABA to CmPAL1-like, CmC4H and CmCOMT, with H2O2 to CmPAL1-like, CmC4H, CmCCR and CmLAC17-like, and with JA to CmCCR, CmCOMT, CmLAC11-like and CmLAC17-like. As expected, the signal molecules positively regulated CAD activity and lignin biosynthesis under drought stress. Promoter::GUS assays not only further confirmed the regulations of the signal molecules to CmCAD1~3, but also revealed the important role of CmCAD3 in lignin synthesis due to the strongest staining of CmCAD3 promoter::GUS. CONCLUSIONS: CmCADs but CmCAD4 are positively regulated by ABA, H2O2 and JA under drought stress and participate in lignin synthesis.

Lignin synthesized by CmCAD2 and CmCAD3 in oriental melon (Cucumis melo L.) seedlings contributes to drought tolerance.

KEY MESSAGE: CmCAD2 and CmCAD3 function more positively than CmCAD1 in oriental melon for lignin synthesis which is important to ensure internal water status and thus for drought tolerance. Well-lignification may be the guarantee of efficient axial water transport and barrier of lateral water flow in oriental melon tolerating drought stress, however remains to be verified. As an important enzyme in monolignol synthesis pathway, five cinnamyl alcohol dehydrogenase (CAD) genes were generally induced in melon seedlings by drought. Here we further revealed the roles of CmCAD1, 2, and 3 in lignin synthesis and for drought tolerance. Results found that overexpressing CmCAD2 or 3 strongly recovered CAD activities, lignin synthesis and composition in Arabidopsis cadc cadd, whose lignin synthesis is disrupted, while CmCAD1 functioned modestly. In melon seedlings, silenced CmCAD2 and 3 individually or collectively decreased CAD activities and lignin depositions drastically, resulting in dwarfed phenotypes. Reduced lignin, mainly composed by guaiacyl units catalyzed by CmCAD3, is mainly due to the limited lignification in tracheary elements and development of Casparion strip. While CmCAD1 and 2 exhibited catalysis to p-coumaraldehyde and sinapaldehyde, respectively. Compared with CmCAD1, drought treatments revealed higher sensitivity of CmCAD2 and/or 3 silenced melon seedlings, accompanying with lower relative water contents, water potentials and relatively higher total soluble sugar contents. Slightly up-regulated expressions of aquaporin genes together with limited lignification might imply higher lateral water loss in stems of silenced lines. In Arabidopsis, CmCAD2 and 3 transgenic lines enhanced cadc cadd drought tolerance through recovering lignin synthesis and root development, accompanying with decreased electrolyte leakage ratios and increased RWCs, thus improved survival rates. Briefly, lignin synthesized by CmCAD2 and 3 functions importantly for drought tolerance in melon.

Transcriptome analysis of differentially expressed genes during anther development stages on male sterility and fertility in Cucumis melo L. line.

The genic male sterility (MS) plays a major role in melon hybrids production, it could reduce the cost of pollination and increase the yield and quality. However, the molecular mechanism underlying genetic male sterility is yet poorly understood. The morphological differences of flower buds of melon were observed showed that the flower buds were tetrad when they were 1 mm stage and monocyte microspore when they were 2 mm stage. Electron microscopy showed that there was significant difference between MS lines and MF (male fertility) lines. In order to detect the global expression of the genes during the melon anther development and association with MS, 12 DEGs (differentially expressed genes) libraries were constructed from the anther of MS and MF in the bud stage with 1 and 2 mm diameter, respectively. A total of 765 DEGs expressed in anther during different developmental stage (MS 1 mm vs. MS 2 mm), 148 and 309 DEGs were found to be related to MS as compared to MF (MS 1 mm vs. MF 1 mm, and MS 2 mm vs. MF 2 mm) at a false discovery rate FDR <0.01. Among these, 10 DEGs were expressed in all the three comparisons, including transcription factor bHLH genes. Among the DEGs in RNA-seq analysis, 28 were validated by qRT-PCR. Of these, a number of genes were involved in ABC transfactor B family, cytochrome-related genes, hormone-related genes (auxin transporter, gibberellin-regulated protein), MADS-box protein genes, F-box protein genes, peroxidase-related, and Zinc finger protein genes. These genes are involved in many biological pathways, including starch and sucrose metabolism, signal transduction mechanisms and transcription factors, etc. Compared to the same developmental stage of MS and MF, the different developmental stages of MS indicated diverse gene regulation pathways involved in the anther development in MS. These results would provide novel insight into the global network to male sterility in melon.

Transcriptome analysis reveals the effects of grafting on sugar and α-linolenic acid metabolisms in fruits of cucumber with two different rootstocks.

Flavor quality in cucumber is affected by different rootstocks, but the molecular mechanism is largely unclean. To clarify the differences of sugar and aromatic compounds, cucumber (cucumis sativus) fruits from plants of self-grafted (SG) or grafted onto figleaf gourd (Cucurbita ficifolia; G1) or 'Weisheng No.1' rootstock (Cucurbita moschata ⅹCucurbita moschata hybrids; G2) were performed the transcriptome analysis. We obtained 1013 and 920 differentially expressed genes (DEGs) from G1 and G2 compared to SG respectively, in which 453 genes were co-expressed. Functional annotations showed many DEGs were involved in glycolysis/gluconeogenesis metabolism, fructose metabolism and α-Linolenic acid metabolisms, 20 DEGs were selected from the 3 pathways to validate sequencing accuracy by quantitative real-time PCR. The gene relative expression levels were concurrent with RNA-seq results and sugar and aromatic compounds content phenotypes. Moreover, some vital transcript factors and transport proteins were analyzed. These findings indicate that different rootstocks could induce significantly changes in the physiological profiling and transcripts of sugar- and aromatic flavor-related genes. This study provides a novel insight into the molecular mechanisms of fruit quality regulated by candidate genes.

Mapping and Preliminary Analysis of ABORTED MICROSPORES (AMS) as the Candidate Gene Underlying the Male Sterility (MS-5) Mutant in Melon (Cucumis melo L.).

Melon is an important agricultural and economic vegetable crop worldwide. The genetic male sterility mutant (ms-5) has a recessive nuclear gene that controls the male sterility germplasm. Male sterility could reduce the cost of F1 seed production in melon, but heterozygous fertile plants should be removed before pollination. In this study, bulked segregant analysis combined with specific length amplified fragment sequencing was applied to map the single nuclear male sterility recessive gene. A 30-kb candidate region on chromosome 9 located on scaffold 000048 and spanning 2,522,791 to 2,555,104 bp was identified and further confirmed by cleavage amplified polymorphic sequence markers based on parental line resequencing data and classical mapping of 252 F2 individuals. Gene prediction indicated that six annotated genes are present in the 30-kb candidate region. Quantitative RT-PCR revealed significant differences in the expression level of the LOC103498166 ABORTED MICROSPORES (AMS) gene in male-sterile lines (ms-5) and male-fertile (HM1-1) lines during the 2-mm (tetrad) and 5-mm (the first pollen mitosis) periods, and negative regulation of the AMS candidate gene transcription factor was also detected. Sequencing and cluster analysis of the AMS transcription factor revealed five single-nucleotide polymorphisms between the parental lines. The data presented herein suggest that the AMS transcription factor is a possible candidate gene for single nuclear male sterility in melon. The results of this study will help breeders to identify male-sterile and -fertile plants at seeding as marker-assisted selection methods, which would reduce the cost of seed production and improve the use of male-sterile lines in melon.

Melon13-lipoxygenase CmLOX18 may be involved in C6 volatiles biosynthesis in fruit.

To better understand the function role of the melon CmLOX18 gene in the biosynthesis of C6 volatiles during fruit ripening, we biochemically characterized CmLOX18 and identified its subcellular localization in transgenic tomato plants. Heterologous expression in yeast cells showed that the molecular weight of the CmLOX18 protein was identical to that predicted, and that this enzyme possesseed lipoxygenase activity. Linoleic acid was demonstrated to be the preferred substrate for the purified recombinant CmLOX18 protein, which exhibited optimal catalytic activity at pH 4.5 and 30 °C. Chromatogram analysis of the reaction product indicated that the CmLOX18 protein exhibited positional specificity, as evidenced by its release of only a C-13 oxidized product. Subcellular localization analysis by transient expression in Arabidopsis protoplasts showed that CmLOX18 was localized to non-chloroplast organelles. When the CmLOX18 gene was transgenically expressed in tomato via Agrobacterium tumefaciens-mediated transformation, it was shown to enhance expression levels of the tomato hydroperoxide lyase gene LeHPL, whereas the expression levels of six TomLox genes were little changed. Furthermore, transgenic tomato fruits exhibited increases in the content of the C6 volatiles, namely hexanal, (Z)-3-hexanal, and (Z)-3-hexen-1-ol, indicating that CmLOX18 probably plays an important role in the synthesis of C6 compounds in fruits.

The Relationship between CmADHs and the Diversity of Volatile Organic Compounds of Three Aroma Types of Melon (Cucumis melo).

Alcohol dehydrogenase (ADH) plays an important role in aroma volatile compounds synthesis of plants. In this paper, we tried to explore the relationship between CmADHs and the volatile organic compounds (VOCs) in oriental melon. Three different aroma types of melon were used as materials. The principle component analysis of three types of melon fruit was conducted. We also measured the CmADHs expression level and enzymatic activities of ADH and alcohol acyl-transferase (AAT) on different stages of fruit ripening. An incubation experiment was carried out to investigate the effect of substrates and inhibitor (4-MP, 4-methylpyrazole) on CmADHs expression, ADH activity, and the main compounds of oriental melon. The results illustrated that ethyl acetate, hexyl acetate (E,Z)-3,6-nonadien-1-ol and 2-ethyl-2hexen-1-ol were the four principal volatile compounds of these three types of melon. AAT activity was increasing with fruit ripening, and the AAT activity in CH were the highest, whereas ADH activity peaked on 32 DAP, 2 days before maturation, and the ADH activity in CB and CG were higher than that in CH. The expression pattern of 11 CmADH genes from 24 to 36 day after pollination (DAP) was found to vary in three melon varieties. CmADH4 was only expressed in CG and the expression levels of CmADH3 and CmADH12 in CH and CB were much higher than that in CG, and they both peaked 2 days before fruit ripening. Ethanol and 4-MP decreased the reductase activity of ADH, the expression of most CmADHs and ethyl acetate or hexyl acetate contents of CB, except for 0.1 mM 4-MP, while aldehyde improved the two acetate ester contents. In addition, we found a positive correlation between the expression of CmADH3 and CmADH12 and the key volatile compound of CB. The relationship between CmADHs and VOCs synthesis of oriental melon was discussed.

The Alcohol Dehydrogenase Gene Family in Melon (Cucumis melo L.): Bioinformatic Analysis and Expression Patterns.

Alcohol dehydrogenases (ADH), encoded by multigene family in plants, play a critical role in plant growth, development, adaptation, fruit ripening and aroma production. Thirteen ADH genes were identified in melon genome, including 12 ADHs and one formaldehyde dehydrogenease (FDH), designated CmADH1-12 and CmFDH1, in which CmADH1 and CmADH2 have been isolated in Cantaloupe. ADH genes shared a lower identity with each other at the protein level and had different intron-exon structure at nucleotide level. No typical signal peptides were found in all CmADHs, and CmADH proteins might locate in the cytoplasm. The phylogenetic tree revealed that 13 ADH genes were divided into three groups respectively, namely long-, medium-, and short-chain ADH subfamily, and CmADH1,3-11, which belongs to the medium-chain ADH subfamily, fell into six medium-chain ADH subgroups. CmADH12 may belong to the long-chain ADH subfamily, while CmFDH1 may be a Class III ADH and serve as an ancestral ADH in melon. Expression profiling revealed that CmADH1, CmADH2, CmADH10 and CmFDH1 were moderately or strongly expressed in different vegetative tissues and fruit at medium and late developmental stages, while CmADH8 and CmADH12 were highly expressed in fruit after 20 days. CmADH3 showed preferential expression in young tissues. CmADH4 only had slight expression in root. Promoter analysis revealed several motifs of CmADH genes involved in the gene expression modulated by various hormones, and the response pattern of CmADH genes to ABA, IAA and ethylene were different. These CmADHs were divided into ethylene-sensitive and -insensitive groups, and the functions of CmADHs were discussed.

Clg2p interacts with Clf and ClUrase to regulate appressorium formation, pathogenicity and conidial morphology in Curvularia lunata.

Ras is a small GTPase that regulates numerous processes in the cellular development and morphogenesis of many organisms. In this study, we identified and functionally characterized the Clg2p gene of Curvularia lunata, which is homologous with the Ras protein. The Clg2p deletion mutant (ΔClg2p) had altered appressorium formation and conidial morphology and produced fewer, smaller lesions compared with the wild-type strain. When a dominant Clg2p allele was introduced into the mutant, all of these defective phenotypes were completely restored. To further understand the regulation of Clg2p in appressorium formation and conidial morphology, and its role in pathogenicity, seven Clg2p-interacting proteins were screened using a yeast two-hybrid assay. Two of these proteins, Clf, a homologue of Mst11, which corresponds to MAP kinase kinase kinase in Magnaporthe oryzae, and urate oxidase (designated ClUrase) were functionally characterized. Clg2p specifically interacted with Clf through its RA domain to regulate appressorium formation and pathogenicity, whereas the Clg2p-ClUrase interaction regulated conidial morphology without affecting fungal pathogenicity. This report is the first to elucidate the regulatory mechanism of the key Ras protein Clg2p in C. lunata.

Genome-wide identification, classification and expression analysis in fungal-plant interactions of cutinase gene family and functional analysis of a putative ClCUT7 in Curvularia lunata.

Cutinase is described as playing various roles in fungal-plant pathogen interactions, such as eliciting host-derived signals, fungal spore attachment and carbon acquisition during saprophytic growth. However, the characteristics of the cutinase genes, their expression in compatible interactions and their roles in pathogenesis have not been reported in Curvularia lunata, an important leaf spot pathogen of maize in China. Therefore, a cutinase gene family analysis could have profound significance. In this study, we identified 13 cutinase genes (ClCUT1 to ClCUT13) in the C. lunata genome. Multiple sequence alignment showed that most fungal cutinase proteins had one highly conserved GYSQG motif and a similar DxVCxG[ST]-[LIVMF](3)-x(3)H motif. Gene structure analyses of the cutinases revealed a complex intron-exon pattern with differences in the position and number of introns and exons. Based on phylogenetic relationship analysis, C. lunata cutinases and 78 known cutinase proteins from other fungi were classified into four groups with subgroups, but the C. lunata cutinases clustered in only three of the four groups. Motif analyses showed that each group of cutinases from C. lunata had a common motif. Real-time PCR indicated that transcript levels of the cutinase genes in a compatible interaction between pathogen and host had varied expression patterns. Interestingly, the transcript levels of ClCUT7 gradually increased during early pathogenesis with the most significant up-regulation at 3 h post-inoculation. When ClCUT7 was deleted, pathogenicity of the mutant decreased on unwounded maize (Zea mays) leaves. On wounded maize leaves, however, the mutant caused symptoms similar to the wild-type strain. Moreover, the ClCUT7 mutant had an approximately 10 % reduction in growth rate when cutin was the sole carbon source. In conclusion, we identified and characterized the cutinase family genes of C. lunata, analyzed their expression patterns in a compatible host-pathogen interaction, and explored the role of ClCUT7 in pathogenicity. This work will increase our understanding of cutinase genes in other fungal-plant pathogens.

Identification of microRNA-like RNAs from Curvularia lunata associated with maize leaf spot by bioinformation analysis and deep sequencing.

Deep sequencing of small RNAs is a useful tool to identify novel small RNAs that may be involved in fungal growth and pathogenesis. In this study, we used HiSeq deep sequencing to identify 747,487 unique small RNAs from Curvularia lunata. Among these small RNAs were 1012 microRNA-like RNAs (milRNAs), which are similar to other known microRNAs, and 48 potential novel milRNAs without homologs in other organisms have been identified using the miRBase© database. We used quantitative PCR to analyze the expression of four of these milRNAs from C. lunata at different developmental stages. The analysis revealed several changes associated with germinating conidia and mycelial growth, suggesting that these milRNAs may play a role in pathogen infection and mycelial growth. A total of 8334 target mRNAs for the 1012 milRNAs that were identified, and 256 target mRNAs for the 48 novel milRNAs were predicted by computational analysis. These target mRNAs of milRNAs were also performed by gene ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis. To our knowledge, this study is the first report of C. lunata's milRNA profiles. This information will provide a better understanding of pathogen development and infection mechanism.

The cinnamyl alcohol dehydrogenase gene family in melon (Cucumis melo L.): bioinformatic analysis and expression patterns.

Cinnamyl alcohol dehydrogenase (CAD) is a key enzyme in lignin biosynthesis. However, little was known about CADs in melon. Five CAD-like genes were identified in the genome of melons, namely CmCAD1 to CmCAD5. The signal peptides analysis and CAD proteins prediction showed no typical signal peptides were found in all CmCADs and CmCAD proteins may locate in the cytoplasm. Multiple alignments implied that some motifs may be responsible for the high specificity of these CAD proteins, and may be one of the key residues in the catalytic mechanism. The phylogenetic tree revealed seven groups of CAD and melon CAD genes fell into four main groups. CmCAD1 and CmCAD2 belonged to the bona fide CAD group, in which these CAD genes, as representative from angiosperms, were involved in lignin synthesis. Other CmCADs were distributed in group II, V and VII, respectively. Semi-quantitative PCR and real time qPCR revealed differential expression of CmCADs, and CmCAD5 was expressed in different vegetative tissues except mature leaves, with the highest expression in flower, while CmCAD2 and CmCAD5 were strongly expressed in flesh during development. Promoter analysis revealed several motifs of CAD genes involved in the gene expression modulated by various hormones. Treatment of abscisic acid (ABA) elevated the expression of CmCADs in flesh, whereas the transcript levels of CmCAD1 and CmCAD5 were induced by auxin (IAA); Ethylene induced the expression of CmCADs, while 1-MCP repressed the effect, apart from CmCAD4. Taken together, these data suggested that CmCAD4 may be a pseudogene and that all other CmCADs may be involved in the lignin biosynthesis induced by both abiotic and biotic stresses and in tissue-specific developmental lignification through a CAD genes family network, and CmCAD2 may be the main CAD enzymes for lignification of melon flesh and CmCAD5 may also function in flower development.