Melon is a useful plant species for studying mitochondrial genetics because it contains one of the largest and structurally diverse mitochondrial genomes among all plant species and undergoes paternal transmission of mitochondria. We used droplet digital (dd) PCR in combination with flow cytometric determination of nuclear DNA quantities to determine the absolute per-cell copy numbers of four mitochondrial genes (nad9, rps1, matR, and atp6) across four stages of melon leaf development. The copy numbers of these mitochondrial genes not only varied during leaf development but also differed among each other, and there was no correlation between the copy numbers of the mitochondrial genes and their transcript levels. The gene copy numbers varied from approximately 36.8 ± 4.5 (atp6 copies in the 15th leaf) to approximately 82.9 ± 5.7 (nad9 copies in the 9th leaf), while the mean number of mitochondria was approximately 416.6 ± 182.7 in the 15th leaf and 459.1 ± 228.2 in the 9th leaf. These observations indicate that the leaf cells of melon do not contain sufficient copies of mitochondrial genes to ensure that every mitochondrion possesses the entire mitochondrial genome. Given this cytological evidence, our results indicate that mtDNA in melon exists as a sub-genomic molecule rather than as a single-master circle and that the copy numbers of individual mitochondrial genes may vary greatly. An improved understanding of the molecular mechanism(s) controlling the relative prevalence and transmission of sub-genomic mtDNA molecules should provide insights into the continuity of the mitochondrial genome across generations.

The rind appearance of melon is one of the most vital commercial quality traits which determines the preferences and behavior of consumers toward the consumption of melon. In this study, we constructed an F₂ population derived from SC (mottled rind) and MG (non-mottled rind) lines for mapping the mottled rind gene(s) in melon. Genetic analysis showed that there were two dominant genes (CmMt1 and CmMt2) with evidence of epistasis controlling the mottled rind. Meanwhile, the phenotypic segregation ratio implied that the immature rind color had an epistatic effect on the mottled rind, which was regulated by CmAPRR2. A Kompetitive Allele-Specific PCR (KASP) DNA marker (CmAPRR2^SNP(G/T)) was developed and shown to co-segregate with rind color, confirming that CmAPRR2 was CmMt1. Using bulked segregant analysis sequencing and KASP assays, CmMt2 was fine-mapped to an interval of 40.6 kb with six predicted genes. Functional annotation, expression analysis, and sequence variation analyses confirmed that AtCPSFL1 homolog, MELO3C026282, was the most likely candidate gene for CmMt2. Moreover, pigment content measurement and transmission electron microscopy analysis demonstrated that CmMt2 might participate in the development of chloroplast, which, in turn, decreases the accumulation of chlorophyll. These results provide insight into the molecular mechanism underlying rind appearance and reveal valuable information for marker-assisted selection breeding in melon.

Increased consumption of vegetables has been recommended worldwide as a part of a healthy diet; therefore, determining gene function among breeding materials is crucial for vegetable improvement to meet the sustainable development of new vegetable varieties. However, genetic transformation is time-consuming and laborious, which limits the exploration of gene function for various vegetable crops. Virus-Induced Gene Silencing (VIGS) can perform large-scale and rapid gene silencing in plants due to a reduction in the experimental period and its independence from the stable genetic transformation, providing an excellent opportunity for functional research. VIGS can accelerate model plant research and make it easier to analyze gene function and validation in vegetable crops. Moreover, with the advent of technologies such as virus-mediated heterologous protein expression and the development of CRISPR/Cas9 technology, virus-mediated genetic tools have ushered in a new era in genetics and crop improvement. This study summarizes recent achievements in VIGS and Virus-Induced Gene Editing (VIGE) in vegetables. We also identify several challenges in the current state of VIGS technology in vegetables, serving as a guide for future research.

Non-Mendelian transmission of mitochondria has been well established across most eukaryotes, however the genetic mechanism that governs this uniparental inheritance remains unclear. Plants in the genus Cucumis, specifically melon and cucumber, exhibit paternal transmission of the mitochondrial (mt) DNA, making them excellent models for exploring the molecular mechanisms underlying mitochondrial transmission. Here, we develop a toolkit to screen for mutants in mitochondrial inheritance (mti), and use fine mapping to successfully identify a mitochondrially targeted endonuclease gene (MTI1) controlling mitochondrial transmission. Knockout of MTI1 results in a shift from paternal to bi-parental inheritance of the mtDNA, confirming the crucial role of MTI1 in uniparental inheritance of mitochondria. Moreover, we demonstrate that MTI1 exhibits robust endonuclease activity both in vitro and in vivo, specifically expresses in mitochondria of the fertilized ovule within 24 h of pollination. Collectively, this study reveals that a nuclear-encoded but mitochondria-targeted gene plays a causative role in governing the non-Mendelian mitochondrial inheritance, revolutionizing our knowledge about mitochondrial DNA transmission.

SMALL AUXIN UP-REGULATED RNA (SAURs) genes are acknowledged as auxin-responsive genes that play crucial roles in modulating adaptive growth under abiotic stress conditions. Low temperatures constitute a primary limiting factor that significantly impairs the development, growth, and fruit quality of watermelon plants during the winter and spring seasons. Despite their potential importance, SAURs have not yet been thoroughly investigated or characterized in watermelon. In this study, we identified a positive regulator of the chilling stress response among watermelon SAURs, designated as ClSAUR1. Subcellular localization analysis demonstrated that the protein is directed to both the nucleus and cytoplasm. Quantitative real-time PCR (qRT-PCR) analysis indicated that ClSAUR1 is ubiquitously expressed across various watermelon tissues, with pronounced expression in the roots and leaves. Moreover, qRT-PCR and promoter::β-glucuronidase (GUS) staining assays revealed that the expression of ClSAUR1 is significantly upregulated in response to exogenous abscisic acid (ABA) and chilling stress. The overexpression of ClSAUR1 in tobacco lines was contrasted and analyzed, revealing an increased tolerance to chilling stress. This was evidenced by a reduced degree of wilting and chlorosis compared to wild-type (WT) plants. Furthermore, the overexpressed lines showed reduced reactive oxygen species (ROS) accumulation and increased antioxidant enzyme activity. The qRT-PCR results further indicated that the expression levels of genes associated with abscisic acid (ABA), antioxidant enzymes, and CBF–COR cold-responsive pathways were upregulated in the transgenic tobacco lines. This study provides new insights into the role of ClSAURs in enhancing the cold resistance of watermelon.

Valine-glutamine (VQ) motif-containing proteins play important roles in diverse plant developmental processes and signal transduction in response to biotic and abiotic stresses. However, no systematic investigation has been conducted on VQ genes in watermelon. In this study, we identified 31 watermelon VQ genes, which were classified into six subfamilies (I–VI). All of the deduced proteins contained a conserved FxxxVQxL/F/VTG motif. Eleven ClVQs were involved in segment duplication, which was the main factor in the expansion of the VQ family in watermelon. Numerous stress- and hormone-responsive cis-elements were detected in the putative promoter region of the ClVQ genes. Green fluorescent protein fusion proteins for ten selected ClVQs were localized in the nucleus, but three ClVQs also showed signals in cell membranes and the cell wall, thus confirming their predicted divergent functionality. Quantitative real-time PCR (qRT-PCR) analysis indicated that the majority of ClVQ genes were specifically or preferentially expressed in certain tissues or organs, especially in the male flower. Analyses of RNA-sequencing data under osmotic, cold, and drought stresses and Cucumber green mottle mosaic virus (CGMMV) infection revealed that the majority of ClVQ genes, especially those from subfamily IV, were responsive to these stresses. The results provide useful information for the functional characterization of watermelon ClVQ genes to unravel their biological roles.