The AtACL protein sequences of Arabidopsis thaliana was retrieved from NCBI (https://www.ncbi.nlm.nih.gov/) and the CitACL protein sequences of Citrus reticulata were from the Citrus Pan-genome to Breeding Database of Huazhong Agricultural University (citrus.hzau.edu.cn/orange/) (Hu, 2015; Hu et al., 2015). These sequences were aligned using Blast against the Melon Genomics Database (http://cucurbitgenomics.org/) to search for candidate melon proteins (CmACL proteins). The CmACL proteins were also identified using the conserved domains of ACL proteins. The proteins identified using these two methods were deduplicated and verified by NCBI CDD (http://www.ncbi.nlm.nih.gov/cdd) with an e-value of 10⁻⁴. The CmACL protein sequences were obtained.

The physicochemical properties of the CmACL proteins were determined. Amino acid number, molecular weight, isoelectric point, and total average hydrophilicity were calculated using Expasy (https://www.expasy.org/). The subcellular localization of the CmACL proteins was predicted online using the Cell-PLoc 2.0 package (http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/).

Phylogenetic analysis was performed using the identified CmACL protein sequences and the ACL protein sequences of A. thaliana and C. reticulata. Based on multiple sequence alignment, a phylogenetic tree was constructed using the Neighbor-Joining method in MEGA v11.0.13.

The data on the chromosomal locations of the CmACL genes were downloaded from the Melon Genomics Database and visualized using an online tool (http://mg2c.iask.in/mg2c_v2.1/).

The DNA and CDS sequences of the CmACL genes were downloaded from the Melon Genomics Database. The structure of the CmACL genes was analyzed using the online tool Gene Structure Display Server 2.0 (http://gsds.gao-lab.org/). The conserved motifs of the CmACL proteins were predicted using MEME 5.5.1 (https://meme-suite.org/meme/tools/meme). The length of the conserved motifs was set to 6–50 amino acids and the number of conserved motifs was set to 20. The 2-kb sequences upstream of the four genes were used as the promoter sequences, and the cis-acting elements were identified using PlantCare (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). Conserved motifs and cis-acting elements were visualized with TBtools.

The interactions of the CmACL proteins were predicted online using STRING (https://cn.string-db.org). The target genes with the highest default Blast scores were selected to construct the protein interaction network. Optimising protein-protein interation networks with Cytoscape (Doncheva et al., 2019; Yu et al., 2022).

The melon selfing line Bailangua P7 was provided by Vegetable Research Institute, Gansu Academy of Agricultural Sciences. The plant material were planted in the greenhouse with the day/night temperature of 23–28°C/18–22°C at the melon breeding base in Gaolan county (36°16′49″, 103°37′57″), China, on March 18, 2022. The plant were grown with a 50 cm distance between seedlings and a 60 cm distance between rows and were under the same local management practices (soil management, irrigation, fertilization, and disease control).

Fruit samples were collected at 10, 20, 30, 35, and 40 d after pollination. At each time point, three fruits were harvested from three different plants and used as a replicate, and three replicates were used for the following analyses. The flesh was obtained from the center-equatorial portion of each fruit after removing the pericarp. Then the samples were immediately transferred to liquid nitrogen and stored at –80°C.

The content of soluble sugars (glucose, fructose, sucrose, raffinose, stachyose, trehalose, maltose, galactose, lactose, trehalose, rhamnose, and arabinose) at different fruit developmental stages was determined using ion chromatography (Thermo iCS5000 ion chromatography system and electrochemical detector).

The content of organic acids (citric acid, malic acid, shikimic acid, oxalic acid, tartaric acid, quinic acid, lactic acid, maleic acid, and succinic acid) at different fruit developmental stages was determined using high-performance liquid chromatography (Waters e2695 separation module and Waters 2998 detector).

Quantitative real-time (qRT)-PCR was used to determine the relative expression of the CmACL genes at different developmental stages in the melon material. Primers were designed using the PrimerQuest Tool with the 18S gene as an internal reference. The primer sequences are shown in Table 1 . The RNA reverse transcription was performed using the TUREscript 1st Stand cDNA Synthesis Kit (Aidlab), and fluorescence quantification was performed using the abm^® EvaGreen qPCR Master Mix-ROX Kit. Each treatment had three technical replicates, and the 2^−ΔΔCt method was used to calculate relative gene expression.

IBM SPSS Statistics 26 software was used for T-test significance analysis and Pearson correlation analysis. Heat map and column chart were generated using GraphPad Prism 9 software.

A total of four CmACL proteins were identified in the melon genome using Blast with the ACL protein sequences of A. thaliana and C. reticulata ( Table 1 ). To elucidate the phylogenetic relationships of the ACL proteins, a phylogenetic tree was constructed using the Neighbor-Joining method for 12 ACL protein sequences from A. thaliana, C. reticulata, and C. melo ( Figure 1A ). The ACL proteins were divided into α and β subfamilies. MELO3C015245.2.1 clustered with CitACLα1, and MELO3C010675.2.1 clustered with CitACLα2. Therefore, MELO3C015245.2.1 and MELO3C010675.2.1 were renamed as CmACLα1 and CmACLα2, respectively. MELO3C011482.2.1 and MELO3C021268.2.1 clustered with the ACL proteins in the β subfamily and were renamed as CmACLβ1 and CmACLβ2.

Chromosomal localization ( Figure 1B ) showed one CmACL gene on Chr2, one on Chr11, and two on Chr3. The number of amino acids was 423 and 608 in the α and β subfamilies, respectively ( Table 2 ). The relative molecular weight was 46653.68–66017.74 Da, and the isoelectric point was 5.34–8.23. The hydrophilicity ranged between −0.124 and −0.036, and the four CmACL proteins were hydrophilic. The instability index ranged between 30.52 and 42.15, indicating that the CmACL proteins were stable except for CmACLα1. The four CmACL proteins were localized in the nucleus.

The function of a protein is determined by its number and type of conserved motifs. The conserved motifs of the CmACL proteins were predicted by MEME and visualized using TBtools. As shown in Figure 2A , both CmACLα1 and CmACLα2 of the α subfamily contained 12 conserved motifs with similar positions, including motif1, motif3, motif4, motif6, motif7, motif12, motif13, motif14, motif15, motif16, motif17, and motif18. Both CmACLβ1 and CmACLβ2 of the β subfamily contained 15 conserved motifs with similar positions, including motif1, motif2, motif3, motif4, motif5, motif7, motif8, motif9, motif10, motif11, motif12, motif15, motif16, motif19, and motif20. Analysis of the conserved motifs in the CmACL proteins showed that the members in the same subfamily shared identical conserved motifs indicating that the same subfamily might have similar biological functions.

The structures of four CmACL genes were analyzed to further reveal their phylogenetic diversity ( Figure 2B ). The results showed that the genes in the same subfamily shared similar structures. CmACLα1 and CmACLα2 contained 11 introns and 12 exons, while CmACLβ1 and CmACLβ2 contained 15 introns and 16 exons. The variances in intron and exon distribution may be attributed to evolution, as distinct gene structures have led to different gene functions.

The cis-acting elements of the CmACL genes are shown in Figure 3 . These elements were related to light, abiotic and biotic stress, plant growth and development, and phytohormone. Light responsiveness elements were most abundant and present in the four CmACL genes. Plant abiotic and biotic stress elements included anaerobic induction (CmACLα1 and CmACLβ1), drought-inducibility (CmACLα1/2 and CmACLβ1), defense and stress responsiveness (CmACLα2 and CmACLβ2), and low-temperature responsiveness (CmACLβ1). Plant growth and development elements included circadian control (CmACLα2), palisade mesophyll cells differentiation (CmACLα2), endosperm expression (CmACLα2), seed-specific regulation (CmACLβ2), and zein metabolism regulation (CmACLβ2). Phytohormone elements included MeJA-responsiveness (CmACLα1 and CmACLβ2), abscisic acid responsiveness (CmACLα1/2 and CmACLβ2), gibberellin-responsiveness (CmACLα2 and CmACLβ1/2), salicylic acid responsiveness (CmACLα2 and CmACLβ2), and auxin-responsive (CmACLβ1). These results suggest that CmACL genes may have important roles in plant growth, development, and the stress response.

The protein interaction network was constructed using the online tool STRING ( Figure 4 ). Results showed that all four CmACL proteins interacted with one another. Notably, several proteins such as Aco, XP_008467310.1, XP_008455442.1, XP_008456386.1, XP_008461234.1, XP_008456567.1, XP_008440462.1, XP_008460481.1 and XP_008446580.1, belonging to tricarboxylic acid cycle, glyoxylate cycle, and glycolytic pathway, were showing tight relationships with CmACL, suggesting that CmACL may play an important role in sugar and acid metabolism.

The levels of soluble sugars and organic acids in Bailangua P7 were determined at 10, 20, 30, 35, and 40 d after pollination. Table 3 shows that the melon contained high levels of sucrose, fructose, and glucose; low levels of raffinose, stachyose, trehalose, and maltose; and no detectable levels of galactose, lactose, trehalose, rhamnose, and arabinose. Fructose and glucose increased as the fruit ripened, maximized at 35 d after pollination, and then decreased slightly at 40 d post-pollination. Sucrose was low from 10 to 30 d and accumulated rapidly from 35 to 40 d after pollination, peaking at 40 d post-pollination. Table 4 shows that the melon fruit contained high levels of citric acid and malic acid; low levels of shikimic acid; and no detectable levels of oxalic acid, tartaric acid, quinic acid, lactic acid, maleic acid, and succinic acid. Citric acid was the highest at 10 d after pollination, decreased rapidly from 10 to 20 d, and then increased and decreased from 20 to 40 d. Malic acid decreased with fruit ripening.

The relative expression of CmACL genes was determined at different fruit developmental stages ( Figure 5 ). The expression of four CmACL genes decreased and then increased with fruit ripening. The expression of CmACLα2 decreased from 10 to 20 d and then gradually increased from 20 to 40 d. The expression of CmACLα1, CmACLβ1, and CmACLβ2 decreased from 10 to 30 d and then increased from 30 to 40 d. The expression of CmACLα1 at 40 d after pollination was 2.36-, 2.65-, 2.95-, and 1.33-fold that at 10, 20, 30, and 35 d. The expression of CmACLβ1 at 40 d after pollination was 5.46-, 11.38-, 13.65-, and 4.40-fold that at 10, 20, 30, and 35 d.

The expression of CmACL genes was correlated with the content of sugars and acids in melon ( Table 5 ). The results showed significant correlations between the expression of four genes. For example, the expression of CmACLα1 was significantly and positively correlated with CmACLα2 and CmACLβ1 (correlation coefficients of 0.824 and 0.841, respectively), and the expression of CmACLα2 was significantly and positively correlated with CmACLβ1 (0.665).

There were significant correlations between the expression of CmACL genes and sugar and acid content. The expression of CmACLα1 was significantly negatively correlated with malic acid (−0.584) and significantly positively correlated with glucose, fructose, sucrose, trehalose, and maltose (0.614, 0.687, 0.867, 0.884, and 0.961). The expression of CmACLα2 was significantly positively correlated with fructose, sucrose, trehalose, and maltose (0.545, 0.695, 0.859, and 0.748). The expression of CmACLβ1 was significantly positively correlated with sucrose, trehalose, and maltose (0.970, 0.866, and 0.907). The expression of CmACLβ2 was significantly positively correlated with citric acid, shikimic acid, raffinose, and stachyose (0.822, 0.843, 0.765, and 0.806).