Background: C2H2 zinc finger proteins (C2H2-ZFPs) are one of the largest transcription factor families in plants and play vital roles in plant organ development and patterning, seed germination, and fruit ripening, as well as responses to biotic and abiotic stresses. Although widely studied in many species, the genome-wide characterization of the C2H2-ZFP family in watermelon (Citrullus lanatus) remains lacking. Methods: In this study, we identified 96 ClZFP genes in the watermelon genome and analyzed their chromosomal positions, gene structures, conserved motifs, and expression profiles. A tissue-specific expression analysis of 12 representative ClZFP genes revealed diverse and organ-preferential expression profiles, indicating functional differentiation during development. Results: Under abiotic stress treatments, four genes were significantly downregulated under drought, while one gene was strongly induced; six genes were inhibited and three genes were activated under low temperature; and most tested genes were upregulated at 72 h under salt stress, with one gene continuously induced throughout the treatment period. Key ClZFP members such as ClZFP36 and ClZFP72 showed specific and strong induction under drought and salt stress, respectively. Conclusions: These results indicate that ClZFPs may be involved in the tolerance of watermelon to various abiotic stresses. This study not only clarifies the evolutionary and expression characteristics of the ClZFP family in watermelon but also provides candidate genes for the genetic improvement of stress tolerance in cucurbit crops.

Tomato flavor quality is governed by a complex metabolic network; however, the effects of organic nutrients on key pathways remain unclear. This study combined metabolomics, relative odor activity value (rOAV) analysis, and sensory evaluation to elucidate the mechanism by which “XINONG” compost tea (T2) regulates tomato flavor metabolism. Compared with conventional inorganic fertilizer (T1), T2 increased fruit soluble solids and suppressed alkaloid biosynthesis, reducing bitter α-tomatine and its intermediates. It also redirected phenylpropanoid flux toward p-coumaric and ferulic acid derivatives, increasing aromatic precursors such as p-coumaroylmalic acid while lowering astringent free cinnamic acids. The rOAVs of key aroma compounds (hexanal, 2-hexenal, and β-ionone) were enhanced, intensifying green, fruity, and floral notes. Sensory evaluation confirmed that T2 improved fruit sweetness and overall acceptability. This study elucidates the metabolic mechanisms by which organic nutrients enhance tomato flavor, providing insights for flavor-directed improvement of agricultural products through molecular-assisted breeding and precision cultivation.

Melatonin plays a crucial role in regulating plant defense against various environmental stresses. However, its role and the underlying mechanism in enhancing plant resistance to aphid infestation remain elusive. In this research, we discovered that aphid infestation notably upregulated the expression of the melatonin biosynthetic gene caffeic acid O-methyltransferase 1 (ClCOMT1) and increased melatonin levels in watermelon (Citrullus lanatus). The overexpression of ClCOMT1 enhanced defensive responses and resistance to aphids in watermelon. This was accompanied by the accumulation of cytosolic free calcium ([Ca2+]cyt), upregulation of the Ca2+-permeable channel gene cyclic nucleotide-gated ion channel 20 (ClCNGC20), and downregulation of calmodulin 7 (ClCaM7). ClCaM7 interacts with ClCNGC20 and suppresses its channel activity. In contrast, the knockout of ClCOMT1 showed opposite effects compared with its overexpression. Pretreatment with LaCl3 (a Ca2+ influx channel inhibitor) or EGTA (a Ca2+ chelator) compromised ClCOMT1 overexpression-induced aphid resistance; whereas CaCl2 or the Ca2+ ionophore A23187 effectively restored aphid resistance in clcomt1 mutants. Moreover, silencing ClCNGC20 impaired ClCOMT1 overexpression-induced [Ca2+]cyt accumulation and aphid resistance; conversely, silencing ClCaM7 restored [Ca2+]cyt accumulation and aphid resistance in the clcomt1 mutant. Overexpression of watermelon ClCNGC20 elevated [Ca2+]cyt levels and improved aphid resistance in transgenic Arabidopsis plants, whereas overexpression of watermelon ClCaM7 had the opposite effect. Collectively, these findings imply the crucial role of ClCNGC20 and ClCaM7 in mediating melatonin-induced [Ca2+]cyt elevation, subsequently activating defensive responses in plants. This study offers insights into the molecular mechanisms underlying melatonin-mediated plant aphid resistance, with potential applications in the breeding or engineering of aphid-resistant cucurbit varieties.

Fusarium wilt, caused by Fusarium oxysporum f. sp. niveum (FON), is a widespread and destructive fungal disease in watermelon. While melatonin helps plants defend against various pathogens, its role and mechanism in combating FON are not well understood. Here, we demonstrated that overexpressing the melatonin biosynthesis gene ClCOMT1 enhances watermelon (Citrullus lanatus) resistance to FON race 1 (FON1), accompanied by increased production of nitric oxide (NO) mediated by nitrate reductase 1 (ClNR1) and H2O2 mediated by respiratory burst oxidase homologue D (ClRBOHD). Conversely, knockout of ClCOMT1 exhibited contrasting effects compared with its overexpression. Deletion or silencing of ClNR1 and ClRBOHD abolished defense responses and resistance induced by exogenous melatonin or ClCOMT1 overexpression. However, exogenous SNP (a NO donor) or H2O2 restored FON1 resistance in ClCOMT1 knockout plants. Upon FON1 infection, exogenous SNP induced H2O2 production, whereas ClNR1 deletion eliminated melatonin-induced H2O2 generation. Furthermore, ClRBOHD deletion inhibited SNP-induced resistance, while H2O2 supplementation rescued resistance in ClNR1 deletion mutants. Overall, these findings indicate that NO-dependent H2O2 signaling is crucial for melatonin-induced watermelon defense responses and resistance to FON1. Given the growing emphasis on reducing pesticide use, this mechanism holds remarkable potential for the sustainable control of Fusarium wilt.

Nitric oxide (NO) is a pivotal gaseous signaling molecule that plays a critical role in regulating plant tolerance to cold stress; however, the underlying mechanisms of signal transduction remain poorly elucidated. In this study, knockout of nitrate reductase 1 (ClNR1), a crucial gene for NO biosynthesis, led to reduced cold tolerance in watermelon (Citrullus lanatus), accompanied by downregulation of cycle nucleotide-gated channel (ClCNGC) 20, a key Ca2+-permeable channel gene, decreased Ca2+ influx, and upregulation of calmodulin (ClCaM) 2/5/7. Conversely, application of the NO donor sodium nitroprusside (SNP) exhibited contrasting effects compared with NR1 knockout. Silencing ClCNGC20 counteracted SNP-induced Ca2+ influx, downregulation of ClCaM 2/5/7, and cold tolerance. Silencing ClCaM2/5/7 alleviated the inhibition on C-REPEAT-BINDING FACTOR (ClCBF) expression and cold tolerance induced by ClNR1 knockout or ClCNGC20 silencing. Multiple experimental approaches revealed the interactions between ClCaM2/5/7 and voltage-dependent anion channel (ClVDAC) 1 proteins. Overexpression of ClVDAC1 hindered the induction of ClCBF expression and cold tolerance triggered by SNP or CaCl2, whereas ClVDAC1 silencing mitigated the inhibition on ClCBF expression and cold tolerance caused by ClNR1 knockout or ClCNGC20 silencing. Additionally, cold stress rapidly triggered Ca2+ influx, which stimulated NO production. These findings suggest that Ca2+ influx promotes NO generation, which leads to further Ca2+ influx via upregulating ClCNGC20, forming a positive feedback loop that enhances cold tolerance. Furthermore, ClCaM2/5/7 interacts with ClVDAC1 to negatively regulate the NO- and Ca2+ signaling-mediated C-repeat-binding factor pathway and subsequent cold tolerance.

Pyrus bretschneideri ‘Xinli No.7’, a progeny of Pyrus sinkiangensis ‘Korla Fragrant Pear’, is an early-maturing, high-quality pear (Pyrus spp.) cultivar. As a dominant variety in China’s pear-producing regions, it holds significant agricultural importance. Investigating its male sterility (MS) mechanisms is critical for hybrid breeding and large-scale cultivation. Integrated cytological, physiological, and transcriptomic analyses were conducted to compare dynamic differences between male sterility (MS, ‘Xinli No.7’) and male-fertile (MF, ‘Korla Fragrant Pear’) plants during anther development. Cytological observations revealed that, compared with ‘Korla Fragrant Pear’, the tapetum of ‘Xinli No.7’ exhibited delayed degradation and abnormal thickening during the uninucleate microspore stage. This pathological alteration compressed the microspores, ultimately leading to their abortion. Physiological assays demonstrated excessive reactive oxygen species (ROS) accumulation, lower proline content, higher malondialdehyde (MDA) levels, and reduced activities of antioxidant enzymes (peroxidase and catalase) in MS plants. Comparative transcriptomics identified 283 co-expressed differentially expressed genes (DEGs). Functional enrichment linked these DEGs to ROS-scavenging pathways: galactose metabolism, ascorbate and aldarate metabolism, arginine and proline metabolism, fatty acid degradation, pyruvate metabolism, and flavonoid biosynthesis. qRT-PCR validated the expression patterns of key DEGs in these pathways. A core transcriptome-mediated MS network was proposed, implicating accelerated ROS generation and dysregulated tapetal programmed cell death. These findings provide theoretical insights into the molecular mechanisms of male sterility in ‘Xinli No.7’, supporting future genetic and breeding applications.

Fusarium wilt, caused by Fusarium oxysporum (Fo), is a destructive fungal disease that reduces crop yield and quality. Hydrogen sulphide (H2S), a critical signalling molecule, modulates plant defence responses; however, its role and mechanism in combating Fo remain elusive. This study reveals that exogenous NaHS (an H2S donor) enhances watermelon resistance to Fusarium oxysporum f. sp. niveum race 2 (FON2), accompanied by elevated hydrogen peroxide (H2O2) and methyl jasmonate (MeJA) levels. Exogenous H2O2 and MeJA also enhance FON2 resistance. Conversely, silencing respiratory burst oxidase homologue F (ClRBOHF) and jasmonic acid carboxyl methyltransferase (ClJMT), key genes for H2O2 and MeJA biosynthesis, respectively, inhibits NaHS-induced resistance to FON2. Deletion of l-cysteine desulfhydrase (ClLCD), a pivotal gene for H2S generation, reduces FON2 resistance, but this reduction is restored by H2O2 or MeJA supplementation. Upon FON2 infection, exogenous H2O2 elevates MeJA levels; however, silencing ClRBOHF suppresses NaHS-induced MeJA accumulation. Furthermore, silencing ClClJMT inhibits H2O2-induced FON2 resistance, while MeJA supplementation rescues the reduced resistance caused by ClRBOHF silencing. Collectively, these findings demonstrate that H2O2-dependent MeJA plays a crucial role in regulating H2S-induced watermelon resistance to FON2. The growing focus on reducing pesticide use highlights the potential of this mechanism for combating Fo sustainably.

Watermelon exhibits deeply lobed leaves, distinct from its close cucurbit relatives such as melon and cucumber. However, the genetic mechanisms underlying lobed-leaf development in watermelon remain elusive. In this study, we successfully delimited the lobed-leaf regulatory gene, ClLL1, into a 29.14-Kb interval on chromosome 4 and verified that its loss-function results in non-lobed leaves. Phylogenetic analysis revealed that ClLL1 is a putative orthologue of AtHB22 within the class I of HD-ZIP transcription factor family, distinct from the well-characterized LMI1/RCO lineage. Notably, ClLL1 displays exceptional sequence conservation among watermelon lobed-leaf germplasms. Mechanistically, we demonstrated that ClLL1 forms homodimers and autoregulates its own expression through direct promoter binding, establishing a dose-dependent relationship with leaf lobation severity. Furthermore, ClLL1 directly activates the auxin efflux carrier gene ClPIN1, linking leaf-shape determination to auxin signaling. This regulatory network was corroborated by exogenous application of the auxin transport inhibitor NPA and auxin (IAA), which significantly altered leaf margin phenotypes. Our results establish a ClLL1-mediated auxin pathway in watermelon leaf-shape formation, providing new insights into the functions of the ClLL1 lineage in Cucurbitaceae.

The phytohormone methyl jasmonate (MeJA) enhances plant cold stress tolerance, but the underlying mechanisms remain elusive. Here, we discovered that MeJA induces a transient Ca2+ influx and elevated cytoplasmic free Ca2+ ([Ca2+]cyt) levels during the watermelon (Citrullus lanatus) response to cold stress. Conversely, silencing jasmonic acid carboxyl methyltransferase (ClJMT), encoding an enzyme that methylates JA to MeJA, led to contrasting effects compared with MeJA application. Upon cold exposure, MeJA rapidly and continuously upregulated 2 Ca2+-permeable channel genes, namely cyclic nucleotide-gated ion channel (ClCNGC) 2 and ClCNGC20. Silencing ClCNGC2 or ClCNGC20 attenuated MeJA-induced Ca2+ influx, [Ca2+]cyt accumulation, C-REPEAT BINDING FACTOR (CBF) pathway activation, and watermelon cold tolerance. Accordingly, ClCNGC2 or ClCNGC20 overexpression increased Ca2+ influx, [Ca2+]cyt levels, and expression of the CBF regulon and improved freezing tolerance in transgenic Arabidopsis thaliana plants. Multiple assays showed that ClCNGC2 and ClCNGC20 do not directly interact. Interestingly, silencing ClCNGC2 or ClCNGC20 abolished MeJA-induced upregulation of ClCNGC20 or ClCNGC2, respectively, in watermelon response to cold, demonstrating their reciprocal activation at the transcriptional level. Collectively, these findings suggest a mutual dependence between ClCNGC2 and ClCNGC20 in mediating MeJA-induced Ca2+ influx followed by [Ca2+]cyt elevation, subsequently activating the CBF pathway and enhancing cold tolerance in plants. This study provides insights into the molecular mechanisms underlying MeJA-mediated plant cold tolerance, holding potential for the breeding or engineering of cold-resistant cucurbit varieties.

As a prominent external feature of watermelon, the stripe pattern exhibits remarkable phenotypic diversity, directly impacting commercial value through consumer preference. However, the genetic and molecular mechanisms underlying this important agronomic trait in watermelon remain poorly understood. In this study, we discovered that the total chlorophyll content in dark green stripes (DGS) was significantly higher than that in light green stripes (LGS) or reticular green stripes (RGS). Moreover, the number and size of chloroplasts were significantly increased in the DGS. Genetic analysis identified the KNOX TF ClSP as the most likely candidate for regulating watermelon dark green stripe formation, whose functional disruption substantially impaired chlorophyll biosynthesis and chloroplast development, converting dark green stripes into reticulate stripes. Through transcriptome analysis, we identified approximately 94 differently expressed genes (DEGs) that contain the KNOX TF binding cis-element 'TGAC' in their promoters. Among these genes, the expression pattern of ARABIDOPSIS PSEUDO RESPONSE REGULATOR 2-LIKE (APRR2-like) TF ClAPRR2 closely mirrored that of ClSP, displaying significantly down-regulated transcriptional expression in LGS compared to DGS. Utilizing Y1H, GUS activity, DLR and EMSA assays, we confirmed that ClSP activates the transcriptional activity of ClAPRR2 through promoter binding. Collectively, we propose a potential working model for the ClSP-ClAPRR2 module, which regulates the chlorophyll synthesis and chloroplast development in watermelon fruits, providing new insights into the mechanisms underlying stripe pattern formation.

Fusarium wilt, caused by the soil-borne fungal pathogen Fusarium oxysporum (Fo), is widely recognized as one of the most devastating fungal diseases, inflicting significant damage on a wide range of agricultural and horticultural crops. Despite melatonin has recently emerged as a potential enhancer of plant resistance against Fo, the underlying mechanisms remain elusive. In this study, our results demonstrate that exogenous melatonin and MeJA enhance watermelon resistance against Fusarium oxysporum f. sp. Niveum race 2 (FON2) in a dose-dependent manner. The optimal concentration for melatonin and MeJA was determined to be 10 μM and 1 μM, respectively. Both melatonin and MeJA inhibited FON2 mycelial growth on PDA medium in a dose-dependent manner. Furthermore, exogenous melatonin significantly stimulated upregulation of MeJA synthesis genes and increased MeJA content upon FON2 infection. However, pretreatment with a MeJA synthesis inhibitor (DIECA) suppressed the induction of melatonin-induced resistance against FON2. Furthermore, MeJA also induced the upregulation of melatonin biosynthetic gene caffeic acid O-methyltransferase 1 (ClCOMT1) and increased melatonin accumulation in response to FON2. Notably, the reduction in FON2 resistance caused by ClCOMT1 deletion was completely restored through exogenous application of MeJA. These results suggest that melatonin facilitates MeJA accumulation, which provides feedback to promote melatonin accumulation, forming a reciprocal positive regulatory loop in response to FON2 infection. Additionally, polyphenol oxidase, phenylalanine ammonia lyase, and lignin are involved in the MeJA-induced resistance against FON2. The growing concern over minimizing pesticide usage and transitioning to sustainable and natural control strategies underscores the significant potential of such a mechanism in combating Fo.

Melatonin is a pivotal bioactive molecule that enhances plant cold stress tolerance, but the precise mechanisms remain enigmatic. Here, we have discovered that overexpressing melatonin biosynthetic gene ClCOMT1 or applying exogenous melatonin activates the C-repeat binding factor (CBF)-responsive pathway and enhances watermelon cold tolerance. This enhancement is accompanied by elevated levels of nitric oxide (NO) and hydrogen sulfide (H2S), along with upregulation of nitrate reductase 1 (ClNR1) and L-cysteine desulfhydrase (ClLCD) genes involved in NO and H2S generation respectively. Conversely, knockout of ClCOMT1 exhibits contrasting effects compared to its overexpression. Furthermore, application of sodium nitroprusside (SNP, a NO donor) and NaHS (a H2S donor) promotes the accumulation of H2S and NO, respectively, activating the CBF pathway and enhancing cold tolerance. However, knockout of ClNR1 or ClLCD abolished melatonin-induced H2S or NO production respectively and abrogated melatonin-induced CBF pathway and cold tolerance. Conversely, supplementation with SNP and NaHS restored the diminished cold response caused by ClCOMT1 deletion. Additionally, deletion of either ClNR1 or ClLCD eliminated NaHS- or SNP-induced cold response, respectively. Overall, these findings suggest a reciprocal positive-regulatory loop between ClNR1-mediated NO and ClLCD-mediated H2S, which plays a crucial role in mediating the melatonin-induced enhancement of cold tolerance.

The integration of regression techniques with remote sensing has proved to be a highly advantageous approach for estimating crop yield in various plant species. This study collected canopy hyperspectral data at multiple growth stages under water and nitrogen stress conditions, and combined with machine learning to predict wheat yield, and evaluated the performance degradation of models across stress. Model performance of Random Forest Regression (RFR), Partial Least Squares Regression (PLSR), and the Multi-random Ensemble on PLSR (MRE-PLSR) algorithms were quantified using the pearson correlation coefficient (PCC) and mean absolute error (MAE). For each dataset composed of canopy hyperspectral data and yield, it was paired with a dataset from the same location under different stress conditions during the same stage to form a combination for validating model performance. Among all combinations, PLSR exhibited superior prediction accuracy compared to RFR. And MRE-PLSR further improved PCC by an average of 14.5 % compared to PLSR. In the combinations where the wheat growth environments differed the most between the training set and testing sets, MRE-PLSR showed significant improvement of PCC, reaching up to 37.5 %. Without setting a random seed, the algorithm was run 100 times on different computers, and the performance remained stable across all combinations, thus validating the replicability of this study. Subsequently, this study validated the transferability of MRE-PLSR. One dataset was designated as the target dataset, and a small number of transfer samples were randomly extracted from another dataset from the same region. These samples were used to update the model trained on a mixture of two datasets from another regions. The results indicate that using the updated model has a better fit to the measured yield compared to using a original model from another location, with an average reduction of 37 t/hm2 in MAE. The proposed method provides a promising solution for predicting wheat yield and its losses.

Melatonin plays a crucial role in regulating plant cold tolerance, but the mechanisms underlying signal transduction remain elusive. In this study, we discovered that overexpression of the melatonin biosynthetic gene caffeic acid O-methyltransferase1 (COMT1) enhanced watermelon (Citrullus lanatus) cold tolerance, accompanied by the accumulation of cytosolic free calcium ([Ca2+]cyt), a stimulation of Ca2+ influx, and upregulation of 4 Ca2+-permeable channel genes (CNGC2/10/17/20). Conversely, the knockout of COMT1 exhibited contrasting effects compared with its overexpression. Knocking out the 4 CNGC genes revealed that only cyclic nucleotide-gated ion channel 20 (CNGC20) mediates melatonin-induced Ca2+ influx in response to cold stimuli. CNGC20 deletion impeded watermelon callus redifferentiation, prompting us to employ a virus-induced gene silencing strategy to suppress its expression. Silencing CNGC20 compromised COMT1 overexpression-induced [Ca2+]cyt accumulation, Ca2+ influx, and watermelon cold tolerance. Yeast 2-hybrid, bimolecular fluorescence complementation, firefly luciferase complementation imaging, and pull-down assays revealed an interaction between CNGC20 and calmodulin7 (CaM7). Overexpressing CaM7 inhibited melatonin-induced [Ca2+]cyt accumulation, Ca2+ influx, and watermelon cold tolerance. Conversely, silencing CaM7 increased [Ca2+]cyt accumulation, Ca2+ influx, and cold tolerance, whereas COMT1 overexpression failed to further enhance these responses in CaM7-silenced plants, indicating the negative regulation role of CaM7 in melatonin-mediated cold responses. Overall, these findings provide insights into the molecular mechanisms underlying melatonin-enhanced plant cold tolerance via Ca2+ signaling, holding potential for breeding/engineering cold-tolerant cucurbit varieties. The interaction between cyclic nucleotide-gated ion channel 20 and calmodulin7 mediates Ca2+ signaling in Citrullus lanatus, playing a pivotal role in melatonin-induced plant tolerance to cold stress.

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Grafting is a common technique used to enhance watermelon yield under biotic and abiotic stress conditions; however, it may influence fruit development and quality. This study evaluated the impact of grafting on the fruit quality attributes and volatile profiles of'Ningnongke huadai' watermelon using 11 commercial rootstocks, encompassing wild watermelons, bottle gourds, and pumpkins. Significant variations were observed in fruit weight, length, rind thickness, firmness of rind and flesh, and central soluble solids content. Notably, wild watermelon grafting increased central soluble solids, while pumpkin-grafted fruits exhibited higher fruit weight but lower central soluble solids. The analysis identified 122 volatile compounds in watermelon samples, primarily ketones, aldehydes, and alcohols. Regarding volatile composition, bottle gourd and wild watermelon grafts did not significantly differ from non-grafted watermelons, whereas pumpkin-grafted fruits showed distinctive volatile profiles characterized by higher aldehyde content and lower levels of alcohols and aromatic hydrocarbons. In conclusion, wild watermelon rootstocks increased soluble solids and had minimal impact on the volatile profiles of grafted fruits, making them potentially suitable for commercial production of'Ningnongke huadai' watermelon. The evident variability in volatile profiles among grafting combinations underscores the need for watermelon rootstock breeding programs to account for the influence of rootstocks on fruit aroma.

High salinity can severely inhibit the growth and development of watermelon(Citrullus lanatus L.).WRKY proteins are believed to mediate the adaptation of plants to abiotic stresses.Here,we identified the ClWRKY61 gene,which positively regulates the tolerance of watermelon to salt stress.Knockout of the ClWRKY61 reduced salt tolerance,while overexpression of the ClWRKY61 enhanced salt tolerance in watermelon according to phenotypic and physiological analyses.Yeast two-hybrid assays revealed that ClWRKY61 interacts with the ClLEA55 protein,and this interaction was further confirmed by luciferase complementation imaging,transient bimolecular fluorescence complementation,and GST pull-down assays.Knockout of the ClLEA55 resulted in lower salt tolerance compared to the wild-type plants.RNA-seq analysis indicated 421 up-regulated and 133 down-regulated genes in the ClWRKY61 knockout line under salt stress,containing 293 differentially expressed genes with W-box in their promoters.Thirteen genes encoding phytoene synthase,MYB transcription factor,sucrose synthase,alpha/beta-hydrolases superfamily protein,glutathione reductase,sugar transporter,LEA protein,WRKY transcription factor,ERF transcription factor,alpha-glucan water dikinase,and calcium-dependent protein kinase showed transcriptional changes in ClWRKY61 knockout line,ClWRKY61 overexpression line,and ClLEA55 knockout line under salt stress.These results provide an opportunity to mediate the regulation of salt stress in watermelon with WRKY proteins.

Numerous studies have proven evidence that melatonin plays an important regulatory role in plant growth, development, and defense against various biotic and abiotic stresses. As well as melatonin, plant hormones and many second messengers, such as calcium (Ca2+), reactive oxygen species (ROS), nitric oxide (NO), and hydrogen sulfide (H2S), also play important roles in the regulation of various physiological processes in plants. In recent years, increasing studies have indicated that melatonin interacts with plant hormones and many second messengers to regulate multiple physiological processes. The role of melatonin in regulating seed germination involves abscisic acid (ABA), gibberellins (GA), Ca2+ signal, and H2O2; in regulating stomatal movement involves ABA, Ca2+, ROS, and H2S; in regulating rhizogenesis involves auxin, ROS, and NO; in regulating fruit ripening involves ethylene (ETH), ROS, and NO; in regulating plant senescence involves ABA, cytokinins (CKs), Ca2+, ROS, and NO; in regulating tolerance to abiotic stress involves CK, ABA, ETH, jasmonic acid (JA), and all secondary signals mentioned above; in regulating disease resistance involves ETH, JA, salicylic acid (SA), ROS, and NO. Plant hormones and the second messengers also interact with each other, forming complex regulatory networks, to regulate multiple physiological processes in plants.

Male sterility is a valuable trait for watermelon breeding, as watermelon hybrids exhibit obvious heterosis. However, the underlying regulatory mechanism is still largely unknown, especially regarding the related non-coding genes. In the present study, approximately 1035 differentially expressed genes (DEGs), as well as 80 DE-lncRNAs and 10 DE-miRNAs, were identified, with the overwhelming majority down-regulated in male-sterile floral buds. Enrichment analyses revealed that the general phenylpropanoid pathway as well as its related metabolisms was predicted to be altered in a mutant compared to its fertile progenitor. Meanwhile, the conserved genetic pathway DYT1 - TDF1 - AMS - MS188 - MS1 , as well as the causal gene ClAMT1 for the male-sterile mutant Se18, was substantially disrupted during male reproductive development. In addition, some targets of the key regulators AMS and MS188 in tapetum development were also down-regulated at a transcriptional level, such as ABCG26 ( Cla004479 ), ACOS5 ( Cla022956 ), CYP703A2 ( Cla021151 ), PKSA ( Cla021099 ), and TKPR1 ( Cla002563 ). Considering lncRNAs may act as functional endogenous target mimics of miRNAs, competitive endogenous RNA networks were subsequently constructed, with the most complex one containing three DE-miRNAs, two DE-lncRNAs, and 21 DEGs. Collectively, these findings not only contribute to a better understanding of genetic regulatory networks underlying male sterility in watermelon, but also provide valuable candidates for future research.