Watermelon (Citrullus lanatus) is an important horticultural crop worldwide, but peel cracking caused by peel hardness severely decreases its quality. Lignification is one of the important functions of class III peroxidase (PRX), and its accumulation in the plant cell wall leads to cell thickening and wood hardening. For in-depth physiological and genetical understanding, we studied the relationship between peel hardness and lignin accumulation and the role of PRXs affecting peel lignin biosynthesis using genome-wide bioinformatics analysis. The obtained results showed that lignin accumulation gradually increased to form the peel stone cell structure, and tissue lignification led to peel hardness. A total of 79 ClPRXs (class III) were identified using bioinformatics analysis, which were widely distributed on 11 chromosomes. The constructed phylogenetics indicated that ClPRXs were divided into seven groups and eleven subclasses, and gene members of each group had highly conserved intron structures. Repeated pattern analysis showed that deletion and replication events occurred during the process of ClPRX amplification. However, in the whole-protein sequence alignment analysis, high homology was not observed, although all contained four conserved functional sites. Repeated pattern analysis showed that deletion and replication events occurred during ClPRXs’ amplification process. The prediction of the promoter cis-acting element and qRT-PCR analysis in four tissues (leaf, petiole, stem, and peel) showed different expression patterns for tissue specificity, abiotic stress, and hormone response by providing a genetic basis of the ClPRX gene family involved in a variety of physiological processes in plants. To our knowledge, we for the first time report the key roles of two ClPRXs in watermelon peel lignin synthesis. In conclusion, the extensive data collected in this study can be used for additional functional analysis of ClPRXs in watermelon growth and development and hormone and abiotic stress response.

Fusarium wilt is one of the most destructive and less controllable diseases in melon, which is usually caused byfusarium oxysporum. In this study, transcriptome sequencing and Yeast Two-Hybrid (Y2H) methods were used for quantification of differentially expressed genes (DEGs) involved infusarium oxysporum(f. sp.melonisrace 1) stress-induced mechanisms in contrasted melon varieties (M4-45 “susceptible” and MR-1 “resistant”). The interaction factors ofFom-2resistance genes were also explored in response to the plant-pathogen infection mechanism. Transcriptomic analysis exhibited total 1,904 new genes; however, candidate DEGs analysis revealed a total of 144 specific genes (50 upregulated and 94 downregulated) for M4-45 variety and 104 specific genes (71 upregulated and 33 downregulated) for MR-1 variety, respectively. The analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway depicted some candidate DEGs, including Phenylalanine metabolism, phenylpropane biosynthesis, plants-pathogen interaction, and signal transduction of plant hormones, which were mainly involved in disease resistance metabolic pathways. The weighted gene co-expression network analysis (WGCNA) analysis revealed a strong correlation module and exhibited the disease resistance-related genes encoding course proteins, transcription factors, protein kinase, benzene propane biosynthesis path, plants-pathogen interaction pathway, and glutathione S-transferase. Meanwhile, the resistance-related specific genes expression was relatively abundant in MR-1 compared to the M4-45, and cell wall-associated receptor kinases (MELO3C008452andMELO3C008453), heat shock protein (Cucumis_melo_newGene_172), defensin-like protein (Cucumis_melo_newGene_5490), and disease resistance response protein (MELO3C016325), activator response protein (MELO3C021623), leucine-rich repeat receptor protein kinase (MELO3C024412), lactyl glutathione ligase (Cucumis_melo_newGene_36), and unknown protein (MELO3C007588) were persisted by exhibiting the upregulated expressions. At the transcription level, the interaction factors between the candidate genes in response to thefusarium oxysporuminduced stress, and Y2H screening signified the main contribution of MYB transcription factors (MELO3C009678andMELO3C014597), BZIP (MELO3C011839andMELO3C019349), unknown proteins, and key enzymes in the ubiquitination process (4XM334FK014). The candidate genes were further verified in exogenously treated melon plants withf. oxysporum(Fom-2, Race 1), Abscisic acid (ABA), Methyl Jasmonite (MeJA), and Salicylic acid (SA), using the fluorescence quantitative polymerase chain reaction (qRT-PCR) analysis. The overall expression results indicated that the SA signal pathway is involved in effective regulation of theFom-2gene activity.

The KT/HAK/KUP is the largest K+ transporter family in plants, playing crucial roles in K+ absorption, transport, and defense against environmental stress. Sweet watermelon is an economically significant horticultural crop belonging to the genus Citrullus, with a high demand for K+ during its growth process. However, a comprehensive analysis of the KT/HAK/KUP gene family in watermelon has not been reported. 14 KT/HAK/KUP genes were identified in the genomes of each of seven Citrullus species. These KT/HAK/KUPs in watermelon were unevenly distributed across seven chromosomes. Segmental duplication is the primary driving force behind the expansion of the KT/HAK/KUP family, subjected to purifying selection during domestication (Ka/Ks < 1), and all KT/HAK/KUPs exhibit conserved motifs and could be phylogenetically classified into four groups. The promoters of KT/HAK/KUPs contain numerous cis-regulatory elements related to plant growth and development, phytohormone response, and stress response. Under K+ deficiency, the growth of watermelon seedlings was significantly inhibited, with cultivated watermelon experiencing greater impacts (canopy width, redox enzyme activity) compared to the wild type. All KT/HAK/KUPs in C. lanatus and C. amarus exhibit specific expression responses to K+-deficiency and drought stress by qRT-PCR. Notably, ClG42_07g0120700/CaPI482276_07g014010 were predominantly expressed in roots and were further induced by K+-deficiency and drought stress. Additionally, the K+ transport capacity of ClG42_07g0120700 under low K+ stress was confirmed by yeast functional complementation assay. KT/HAK/KUP genes in watermelon were systematically identified and analyzed at the pangenome level and provide a foundation for understanding the classification and functions of the KT/HAK/KUPs in watermelon plants.

The genetic regulatory basis of qualitative and quantitative phenotypes of watermelon is being investigated in different types of molecular and genetic breeding studies around the world. In this study, biparental F₂ mapping populations were developed over two experimental years, and the collected datasets of fruit and seed traits exhibited highly significant correlations. Whole-genome resequencing of comparative parental lines was performed and detected single nucleotide polymorphism (SNP) loci were converted into cleaved amplified polymorphic sequence (CAPS) markers. The screened polymorphic markers were genotyped in segregating populations and two genetic linkage maps were constructed, which covered a total of 2834.28 and 2721.45 centimorgan (cM) genetic lengths, respectively. A total of 22 quantitative trait loci (QTLs) for seven phenotypic traits were mapped; among them, five stable and major-effect QTLs (PC-8-1, SL-9-1, SWi-9-1, SSi-9-1, and SW-6-1) and four minor-effect QTLs (PC-2-1 and PC-2-2; PT-2-1 and PT-2-2; SL-6-1 and SSi-6-2; and SWi-6-1 and SWi-6-2) were observed with 3.77–38.98% PVE. The adjacent QTL markers showed a good fit marker-trait association, and a significant allele-specific contribution was also noticed for genetic inheritance of traits. Further, a total of four candidate genes (Cla97C09G179150, Cla97C09G179350, Cla97C09G180040, and Cla97C09G180100) were spotted in the stable colocalized QTLs of seed size linked traits (SL-9-1 and SWi-9-1) that showed non-synonymous type mutations. The gene expression trends indicated that the seed morphology had been formed in the early developmental stage and showed the genetic regulation of seed shape formation. Hence, we think that our identified QTLs and genes would provide powerful genetic insights for marker-assisted breeding aimed at improving the quality traits of watermelon.

Ascorbic acid (AsA), also known as vitamin C, is a well-known antioxidant found in living entities that plays an essential role in growth and development, as well as in defensive mechanisms. GDP-L-galactose phosphorylase (GGP) is a candidate gene regulating AsA biosynthesis at the translational and transcriptional levels in plants. In the current study, we conducted genome-wide bioinformatic analysis and pinpointed a single AsA synthesis rate-limiting enzyme gene in melon (CmGGP1). The protein prediction analysis depicted that the CmGGP1 protein does not have a signaling peptide or transmembrane structure and mainly functions in the chloroplast or nucleus. The constructed phylogenetic tree analysis in multispecies showed that the CmGGP1 protein has a highly conserved motif in cucurbit crops. The structural variation analysis of the CmGGP1 gene in different domesticated melon germplasms showed a single non-synonymous type-base mutation and indicated that this gene was selected by domestication during evolution. Wild-type (WT) and landrace (LDR) germplasms of melon depicted close relationships to each other, and improved-type (IMP) varieties showed modern domestication selection. The endogenous quantification of AsA content in both the young and old leaves of nine melon varieties exhibited the major differentiations for AsA synthesis and metabolism. The real-time quantitative polymerase chain reaction (qRT-PCR) analysis of gene co-expression showed that AsA biosynthesis in leaves was greater than AsA metabolic consumption, and four putative interactive genes (MELO3C025552.2, MELO3C007440.2, MELO3C023324.2, and MELO3C018576.2) associated with the CmGGP1 gene were revealed. Meanwhile, the CmGGP1 gene expression pattern was noticed to be up-regulated to varying degrees in different acclimated melons. We believe that the obtained results would provide useful insights for an in-depth genetic understanding of the AsA biosynthesis mechanism, aimed at the development of improving crop plants for melon.

Carotenoids are the main pigments in watermelon (Citrullus lanatus L.) fruit and contribute to its aesthetic and nutritional value. Phytoene synthase (PSY) is reported to be the first rate-limiting enzyme in carotenogenesis and controls the carotenoid flux. This study aimed to identify PSY genes responsible for carotenoid biosynthesis in the red-fleshed watermelon cultivar LSW-177. The PSY gene members ClPSY1, ClPSY2 and ClPSY3 were characterized and their catalytic activities were displayed in the heterologous complementation assay. The transcript levels of ClPSY genes at the different developmental stages of LSW-177 fruit and the promoter sequence of ClPSY1 were also analyzed. Transcription factors involved in regulating the ClPSY1 expression were scanned with previous RNA-seq data of the different stages during fruit ripening. Results showed that the PSY proteins from watermelon LSW-177 contained the conserved PSY domains and exhibited the ability to condense GGPP into phytoene in E. coli. ClPSY1 is the dominant carotenogenic gene during fruit ripening; and can be induced by light and hormones. Furthermore, Cla013914 and Cla007950 that, respectively encode the transcription factors WD40-like protein and bZIP, likely upregulate ClPSY1 during fruit ripening. In conclusion, ClPSY1 play a dominant role in carotenoid biosynthesis during watermelon fruit ripening and is regulated by complex light and hormone-responsive networks.