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Fruit ripening is a highly complicated process that is accompanied by the formation of fruit quality. In recent years, a series of studies have demonstrated post-transcriptional control play important roles in fruit ripening and fruit quality formation. Till now, the post-transcriptional mechanisms for watermelon fruit ripening have not been comprehensively studied. In this study, we conducted PacBio single-molecule long-read sequencing to identify genome-wide alternative splicing (AS), alternative polyadenylation (APA) and long non-coding RNAs (lncRNAs) in watermelon fruit. In total, 6,921,295 error-corrected and mapped full-length non-chimeric (FLNC) reads were obtained. Notably, more than 42,285 distinct splicing isoforms were derived from 5,891,183 intron-containing full-length FLNC reads, including a large number of AS events associated with fruit ripening. In addition, we characterized 21,506 polyadenylation sites from 11,611 genes, 8703 of which have APA sites. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that fructose and mannose metabolism, starch and sucrose metabolism and carotenoid biosynthesis were both enriched in genes undergoing AS and APA. These results suggest that post-transcriptional regulation might potentially have a key role in regulation of fruit ripening in watermelon. Taken together, our comprehensive PacBio long-read sequencing results offer a valuable resource for watermelon research, and provide new insights into the molecular mechanisms underlying the complex regulatory networks of watermelon fruit ripening. [ABSTRACT FROM AUTHOR]

Watermelon (Citrullus lanatus) as non‐climacteric fruit is domesticated from the ancestors with inedible fruits. We previously revealed that the abscisic acid (ABA) signaling pathway gene ClSnRK2.3 might influence watermelon fruit ripening. However, the molecular mechanisms are unclear. Here, we found that the selective variation of ClSnRK2.3 resulted in lower promoter activity and gene expression level in cultivated watermelons than ancestors, which indicated ClSnRK2.3 might be a negative regulator in fruit ripening. Overexpression (OE) of ClSnRK2.3 significantly delayed watermelon fruit ripening and suppressed the accumulation of sucrose, ABA and gibberellin GA4. Furthermore, we determined that the pyrophosphate‐dependent phosphofructokinase (ClPFP1) in sugar metabolism pathway and GA biosynthesis enzyme GA20 oxidase (ClGA20ox) could be phosphorylated by ClSnRK2.3 and thereby resulting in accelerated protein degradation in OE lines and finally led to low levels of sucrose and GA4. Besides that, ClSnRK2.3 phosphorylated homeodomain‐leucine zipper protein (ClHAT1) and protected it from degradation to suppress the expression of the ABA biosynthesis gene 9'‐cis‐epoxycarotenoid dioxygenase 3 (ClNCED3). These results indicated that ClSnRK2.3 negatively regulated watermelon fruit ripening by manipulating the biosynthesis of sucrose, ABA and GA4. Altogether, these findings revealed a novel regulatory mechanism in non‐climacteric fruit development and ripening. [ABSTRACT FROM AUTHOR]

Keywords: watermelon; ClDMP4; doubled haploid technology; haploid induction; CRISPR-Cas9 EN watermelon ClDMP4 doubled haploid technology haploid induction CRISPR-Cas9 1308 1310 3 06/22/23 20230701 NES 230701 Watermelon ( I Citrullus lanatus i , 2 I n i = 2 I x i = 22) is an economically important I Cucurbitaceae i species that is widely cultivated worldwide. Haploid plants are typically sterile and their chromosome number needs to double to develop into fertile diploid homozygous plants. To identify haploid plants among the F1 offspring of the YJX1 × I Cldmp4 i cross, we looked for green fluorescence in peeled seeds as an indicator of diploid hybrid seeds (Figure 1c). [Extracted from the article]

Key message: The mutation of ClZISO identified in EMS-induced watermelon leads to photosensitive flesh in watermelon. Watermelon (Citrullus lanatus) has a colorful flesh that attracts consumers and benefits human health. We developed an ethyl-methanesulfonate mutation library in red-fleshed line '302' to create new flesh color lines and found a yellow-fleshed mutant which accumulated ζ-carotene. The initial yellow color of this mutant can be photobleached within 10 min under intense sunlight. A long-term light-emitting diode (LED) light treatment turned flesh color from yellow to pink. We identified this unique variation as photosensitive flesh mutant ('psf'). Using bulked segregant analysis, we fine-mapped an EMS-induced G-A transversion in 'psf' which leads to a premature stop codon in 15-cis-ζ-carotene isomerase (ClZISO) gene. We detected that wild-type ClZISO is expressed in chromoplasts to catalyze the conversion of 9,15,9'-tri-cis-ζ-carotene to 9,9'-di-cis-ζ-carotene. The truncated ClZISOmu protein in psf lost this catalytic function. Light treatment can partially compensate ClZISOmu isomerase activity via photoisomerization in vitro and in vivo. Transcriptome analysis showed that most carotenoid biosynthesis genes in psf were downregulated. The dramatic increase of ABA content in flesh with fruit development was blocked in psf. This study explores the molecular mechanism of carotenoid biosynthesis in watermelon and provides a theoretical and technical basis for breeding different flesh color lines in watermelon. [ABSTRACT FROM AUTHOR]

Fruit ripening is a highly complicated process, which is modulated by phytohormones, signal regulators and environmental factors playing in an intricate network that regulates ripening-related genes expression. Although transcriptomics is an effective tool to predict protein levels, protein abundances are also extensively affected by post-transcriptional and post-translational regulations. Here, we used RNA sequencing (RNA-seq) and tandem mass tag (TMT)-based quantitative proteomics to study the comprehensive mRNA and protein expression changes during fruit development and ripening in watermelon, a non-climacteric fruit. A total of 6,226 proteins were quantified, and the large number of quantitative proteins is comparable to proteomic studies in model organisms such as Oryza sativa L. and Arabidopsis. Base on our proteome methodology, integrative analysis of the transcriptome and proteome showed that the mRNA and protein levels were poorly correlated, and the correlation coefficients decreased during fruit ripening. Proteomic results showed that proteins involved in alternative splicing and the ubiquitin proteasome pathway were dynamically expressed during ripening. Furthermore, the spliceosome and proteasome were significantly enriched by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, suggesting that post-transcriptional and post-translational mechanisms might play important roles in regulation of fruit ripening-associated genes expression, which might account for the poor correlation between mRNAs and proteins during fruit ripening. Our comprehensive transcriptomic and proteomic data offer a valuable resource for watermelon research, and provide new insights into the molecular mechanisms underlying the complex regulatory networks of fruit ripening. [ABSTRACT FROM AUTHOR]

NAC (NAM, ATAF1/2, and CUC2) transcription factors play important roles in fruit ripening and quality. The watermelon genome encodes 80 NAC genes, and 21 of these NAC genes are highly expressed in both the flesh and vascular tissues. Among these genes, ClNAC68 expression was significantly higher in flesh than in rind. However, the intrinsic regulatory mechanism of ClNAC68 in fruit ripening and quality is still unknown. In this study, we found that ClNAC68 is a transcriptional repressor and that the repression domain is located in the C-terminus. Knockout of ClNAC68 by the CRISPR-Cas9 system decreased the soluble solid content and sucrose accumulation in mutant flesh. Development was delayed, germination was inhibited, and the IAA content was significantly decreased in mutant seeds. Transcriptome analysis showed that the invertase gene ClINV was the only gene involved in sucrose metabolism that was upregulated in mutant flesh, and expression of the indole-3-acetic acid-amido synthetase gene ClGH3.6 in the IAA signaling pathway was also induced in mutant seeds. EMSA and dual-luciferase assays showed that ClNAC68 directly bound to the promoters of ClINV and ClGH3.6 to repress their expression. These results indicated that ClNAC68 positively regulated sugar and IAA accumulation by repressing ClINV and ClGH3.6. Our findings provide new insights into the regulatory mechanisms by which NAC transcription factors affect fruit quality and seed development. [ABSTRACT FROM AUTHOR]

How raffinose (Raf) family oligosaccharides, the major translocated sugars in the vascular bundle in cucurbits, are hydrolyzed and subsequently partitioned has not been fully elucidated. By performing reciprocal grafting of watermelon (Citrullus lanatus) fruits to branch stems, we observed that Raf was hydrolyzed in the fruit of cultivar watermelons but was backlogged in the fruit of wild ancestor species. Through a genome-wide association study, the alkaline alpha-galactosidase ClAGA2 was identified as the key factor controlling stachyose and Raf hydrolysis, and it was determined to be specifically expressed in the vascular bundle. Analysis of transgenic plants confirmed that ClAGA2 controls fruit Raf hydrolysis and reduces sugar content in fruits. Two single-nucleotide polymorphisms (SNPs) within the ClAGA2 promoter affect the recruitment of the transcription factor ClNF-YC2 (nuclear transcription factor Y subunit C) to regulate ClAGA2 expression. Moreover, this study demonstrates that C. lanatus Sugars Will Eventually Be Exported Transporter 3 (ClSWEET3) and Tonoplast Sugar Transporter (ClTST2) participate in plasma membrane sugar transport and sugar storage in fruit cell vacuoles, respectively. Knocking out ClAGA2 , ClSWEET3 , and ClTST2 affected fruit sugar accumulation. Genomic signatures indicate that the selection of ClAGA2 , ClSWEET3 , and ClTST2 for carbohydrate partitioning led to the derivation of modern sweet watermelon from non-sweet ancestors during domestication. [ABSTRACT FROM AUTHOR]

Abscisic acid (ABA) is a critical regulator of seed development and germination. β-glucosidases (BGs) have been suggested to be contributors to increased ABA content because they catalyze the hydrolysis of ABA-glucose ester to release free ABA. However, whether BGs are involved in seed development is unclear. In this study, a candidate gene, ClBG1, in watermelon was selected for targeted mutagenesis via the CRISPR/Cas9 system. Seed size and weight were significantly reduced in the Clbg1-mutant watermelon lines, which was mainly attributed to decreased cell number resulting from decreased ABA levels. A transcriptome analysis showed that the expression of 1015 and 1429 unique genes was changed 10 and 18 days after pollination (DAP), respectively. Cytoskeleton- and cell cycle-related genes were enriched in the differentially expressed genes of wild type and Clbg1-mutant lines during seed development. Moreover, the expression of genes in the major signaling pathways of seed size control was also changed. In addition, seed germination was promoted in the Clbg1-mutant lines due to decreased ABA content. These results indicate that ClBG1 may be critical for watermelon seed size regulation and germination mainly through the modulation of ABA content and thereby the transcriptional regulation of cytoskeleton-, cell cycle- and signaling-related genes. Our results lay a foundation for dissecting the molecular mechanisms of controlling watermelon seed size, a key agricultural trait of significant economic importance. [ABSTRACT FROM AUTHOR]

Grafting cultivation is implemented worldwide mainly to resist abiotic and biotic stresses and is an effective method to improve watermelon production. However, grafting may affect fruit development and quality. In our experiment, pumpkin-grafted (PG) watermelon fruits developed slower and the ripening period was extended compared to self-grafted (SG) fruits. We found that the concentrations of abscisic acid (ABA) among endogenous phytohormones were dramatically reduced by pumpkin grafting. In order to understand these changes at the gene expression level, we performed a comprehensive analysis of the fruit flesh transcriptomes between PG and SG during fruit development and ripening. A total of 1,675 and 4,102 differentially expressed genes (DEGs) were identified between PG and SG. Further functional enrichment analysis revealed that these DEGs were associated with carbohydrate biosynthesis, phytohormone signaling transmission, and cell wall metabolism categories. ABA centric phytohormone signaling and fruit quality-related genes including ABA receptor, PP2C proteins, AP2-EREBP transcription factors, sucrose transporter, and carotenoid isomerase were co-expressed with fruit ripening. These results provide the valuable resource for understanding the mechanism of pumpkin grafting effect on watermelon fruit ripening and quality development. [ABSTRACT FROM AUTHOR]

1 Loss of VST1 restricts sugar loading into the intercellular space for protection against aphid infestation in watermelon. a Leaf curl phenotypes of WT, vst1-1 and vst1-2 lines upon aphid infestation. Remarkably, the OE plants showed severe leaf curl phenotype than WT plants after aphid infestation (Figure S1a), suggesting that the sugar transporter VST1 plays a negative role in aphid tolerance. [Extracted from the article]

Summary: Unloading sugar from sink phloem by transporters is complex and much remains to be understood about this phenomenon in the watermelon fruit.Here, we report a novel vacuolar sugar transporter (ClVST1) identified through map‐based cloning and association study, whose expression in fruit phloem is associated with accumulation of sucrose (Suc) in watermelon fruit. ClVST197 knockout lines show decreased sugar content and total biomass, whereas overexpression of ClVST197 increases Suc content.Population genomic and subcellular localization analyses strongly suggest a single‐base change at the coding region of ClVST197 as a major molecular event during watermelon domestication, which results in the truncation of 45 amino acids and shifts the localization of ClVST197 to plasma membranes in sweet watermelons. Molecular, biochemical and phenotypic analyses indicate that ClVST197 is a novel sugar transporter for Suc and glucose efflux and unloading.Functional characterization of ClVST1 provides a novel strategy to increase sugar sink potency during watermelon domestication. [ABSTRACT FROM AUTHOR]

Summary: To understand sex determination in watermelon (Citrullus lanatus), a spontaneous gynoecious watermelon mutant, XHBGM, was selected from the monoecious wild type XHB. Using map‐based cloning, resequencing and fluorescence in situ hybridization analysis, a unique chromosome translocation between chromosome 2 and chromosome 3 was found in XHBGM. Based on the breakpoint location in chromosome 2, a putative C2H2 zinc finger transcription factor gene, ClWIP1 (gene ID Cla008537), an orthologue of the melon gynoecy gene CmWIP1, was disrupted. Using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated system 9 to edit ClWIP1, we obtained gynoecious watermelon lines. Functional studies showed that ClWIP1 is expressed specifically in carpel primordia and is related to the abortion of carpel primordia in early floral development. To identify the cellular and metabolic processes associated with ClWIP1, we compared the shoot apex transcriptomes of two gynoecious mutants and their corresponding wild types. Transcriptome analysis showed that differentially expressed genes related to the ethylene and cytokinin pathways were upregulated in the gynoecious mutants. This study explores the molecular mechanism of sex determination in watermelon and provides a theoretical and technical basis for breeding elite gynoecious watermelon lines. Significance Statement: We found a unique chromosome translocation in the spontaneous gynoecious watermelon mutant XHBGM that disrupted the ClWIP1 gene and created artificial gynoecious watermelon lines using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated system 9 to edit the ClWIP1 gene. Transcriptome analysis suggests that the ethylene and cytokinin pathways mediate development of the pistil primordium. This study explores the molecular mechanism of sex determination in watermelon and provides a technical basis for breeding gynoecious watermelon lines for hybrid seed production. [ABSTRACT FROM AUTHOR]

Watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai) is a non-climacteric fruit. The modern sweet-dessert watermelon is the result of years of cultivation and selection for fruits with desirable qualities. To date, the mechanisms of watermelon fruit ripening, and the role of abscisic acid (ABA) in this process, has not been well understood. We quantified levels of free and conjugated ABA contents in the fruits of cultivated watermelon (97103; C. lanatus subsp. vulgaris), semi-wild germplasm (PI179878; C. lanatus subsp. mucosospermus), and wild germplasm (PI296341-FR; C. lanatus subsp. lanatus). Results showed that ABA content in the fruits of 97103 and PI179878 increased during fruit development and ripening, but maintained a low steady state in the center flesh of PI296341-FR fruits. ABA levels in fruits were highest in 97103 and lowest in PI296341-FR, but no obvious differences in ABA levels were observed in seeds of these lines. Examination of 31 representative watermelon accessions, including different C. lanatus subspecies and ancestral species, showed a correlation between soluble solids content (SSC) and ABA levels in ripening fruits. Furthermore, injection of exogenous ABA or nordihydroguaiaretic acid (NDGA) into 97103 fruits promoted or inhibited ripening, respectively. Transcriptomic analyses showed that the expression levels of several genes involved in ABA metabolism and signaling, including Cla009779 (NCED), Cla005404 (NCED), Cla020673 (CYP707A), Cla006655 (UGT) and Cla020180 (SnRK2), varied significantly in cultivated and wild watermelon center flesh. Three SNPs (-738, C/A; -1681, C/T; -1832, G/T) in the promoter region of Cla020673 (CYP707A) and one single SNP (-701, G/A) in the promoter of Cla020180 (SnRK2) exhibited a high level of correlation with SSC variation in the 100 tested accessions. Our results not only demonstrate for the first time that ABA is involved in the regulation of watermelon fruit ripening, but also provide insights into the evolutionary mechanisms of this phenomenon. [ABSTRACT FROM AUTHOR]

Key message: CRISPR/Cas9 system can precisely edit genomic sequence and effectively create knockout mutations in T0 generation watermelon plants. Abstract: Genome editing offers great advantage to reveal gene function and generate agronomically important mutations to crops. Recently, RNA-guided genome editing system using the type II clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) has been applied to several plant species, achieving successful targeted mutagenesis. Here, we report the genome of watermelon, an important fruit crop, can also be precisely edited by CRISPR/Cas9 system. ClPDS, phytoene desaturase in watermelon, was selected as the target gene because its mutant bears evident albino phenotype. CRISPR/Cas9 system performed genome editing, such as insertions or deletions at the expected position, in transfected watermelon protoplast cells. More importantly, all transgenic watermelon plants harbored ClPDS mutations and showed clear or mosaic albino phenotype, indicating that CRISPR/Cas9 system has technically 100% of genome editing efficiency in transgenic watermelon lines. Furthermore, there were very likely no off-target mutations, indicated by examining regions that were highly homologous to sgRNA sequences. Our results show that CRISPR/Cas9 system is a powerful tool to effectively create knockout mutations in watermelon. [ABSTRACT FROM AUTHOR]

• Chromoplast development plays a crucial role in controlling carotenoid content in watermelon flesh. Modern cultivated watermelons with colorful flesh are believed to originate from pale-colored and no-sweet progenitors. But the molecular basis of flesh color formation and regulation is poorly understood. • More chromoplasts and released carotenoid globules were observed in the red-fleshed fruit of the 97103 cultivar than in the pale-colored fruits of the PI296341-FR line. Transcriptome profiles of these two materials identified Cla017962, predicted as ClPHT4;2, was dramatically up-regulated during flesh color formation. High ClPHT4;2 expression levels were closely correlated with increased flesh carotenoid contents among 198 representative watermelon accessions. Down-regulation of ClPHT4;2 expression in transgenic watermelons reduced the fruit carotenoid accumulation. • ClPHT4;2 as a function of chromoplast-localized phosophate transporter was tested by heterologous expression into a yeast phosphate-uptake-defective mutant, western blotting, subcellular localization, and immunogold electron microscopy analysis. Two transcription factors, ClbZIP1 and ClbZIP2, were identified, which responded to ABA and sugar signaling to regulate ClPHT4;2 transcription only in cultivated watermelon species. • Our findings suggest that elevated ClPHT4;2 gene expression is necessary for carotenoid accumulation, and may help to characterize the co-development of flesh color and sweetness during watermelon development and domestication. [ABSTRACT FROM AUTHOR]

Although it has been reported previously that ethylene plays a critical role in sex determination in cucurbit species, how the andromonoecy that carries both the male and hermaphroditic flowers is determined in watermelon is still unknown. Here we showed that the watermelon gene 1-aminocyclopropane-1-carboxylate synthase 4 ( CitACS4), expressed specifically in carpel primordia, determines the andromonoecy in watermelon. Among four single nucleotide polymorphism (SNPs) and one InDel identified in the coding region of CitACS4, the C364W mutation located in the conserved box 6 was co-segregated with andromonoecy. Enzymatic analyses showed that the C364W mutation caused a reduced activity in CitACS4. We believe that the reduced CitACS4 activity may hamper the programmed cell death in stamen primordia, leading to the formation of hermaphroditic flowers. [ABSTRACT FROM AUTHOR]

Watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai] is an important vegetable crop world-wide. Watermelon fruit quality is a complex trait determined by various factors such as sugar content, flesh color and flesh texture. Fruit quality and developmental process of cultivated and wild watermelon are highly different. To systematically understand the molecular basis of these differences, we compared transcriptome profiles of fruit tissues of cultivated watermelon 97103 and wild watermelon PI296341-FR. We identified 2,452, 826 and 322 differentially expressed genes in cultivated flesh, cultivated mesocarp and wild flesh, respectively, during fruit development. Gene ontology enrichment analysis of these genes indicated that biological processes and metabolic pathways related to fruit quality such as sweetness and flavor were significantly changed only in the flesh of 97103 during fruit development, while those related to abiotic stress response were changed mainly in the flesh of PI296341-FR. Our comparative transcriptome profiling analysis identified critical genes potentially involved in controlling fruit quality traits including α-galactosidase, invertase, UDP-galactose/glucose pyrophosphorylase and sugar transporter genes involved in the determination of fruit sugar content, phytoene synthase, β-carotene hydroxylase, 9-cis-epoxycarotenoid dioxygenase and carotenoid cleavage dioxygenase genes involved in carotenoid metabolism, and 4-coumarate:coenzyme A ligase, cellulose synthase, pectinesterase, pectinesterase inhibitor, polygalacturonase inhibitor and α-mannosidase genes involved in the regulation of flesh texture. In addition, we found that genes in the ethylene biosynthesis and signaling pathway including ACC oxidase, ethylene receptor and ethylene responsive factor showed highly ripening-associated expression patterns, indicating a possible role of ethylene in fruit development and ripening of watermelon, a non-climacteric fruit. Our analysis provides novel insights into watermelon fruit quality and ripening biology. Furthermore, the comparative expression profile data we developed provides a valuable resource to accelerate functional studies in watermelon and facilitate watermelon crop improvement. [ABSTRACT FROM AUTHOR]

These authors contributed equally: Jinfang Wang, Yanping Wang. Reference 1 Wang J. The NAC transcription factor ClNAC68 positively regulates sugar content and seed development in watermelon by repressing ClINV and ClGH3.6. [Extracted from the article]

Inheritance of sex forms in watermelon ( Citrullus lanatus ) is not well described. In this study, we made five pairs of crosses of watermelon plants with different sex forms and grew progeny in two different seasons to investigate the inheritance of sex forms and the seasonal effect on sex expression. We showed that environmental factors have no effect on sex forms, but they affect sex expression on individual flowers as more pistillate flowers were observed in spring than in autumn. This suggests that short photoperiod and low temperatures promote formation of pistillate flowers in watermelon. In the F 2 population of the cross of andromonoecious (SL3H or AKKZW) × monoecious (XHB), the segregation ratio is 9 monoecious: 3 trimonoecious: 4 andromonoecious, and the segregation ratio in BC 1 P 1 (F 1 × andromonoecious parent) is 1 monoecious: 1 trimonoecious: 2 andromonoecious. The segregation ratio in the F 2 population of the gynoecious (XHBGM) × monoecious (XHB) is 3 monoecious: 1 gynoecious whereas the segregation ratio in the BC 1 P 1 (F 1 × gynoecious parent) is 1 monoecious: 1 gynoecious. The segregation ratio in the F 2 population of gynoecious × andromonecious cross is 27 monoecious: 12 andromonoecious: 9 gynoecious: 9 trimonoecious: 4 hermaphroditic: 3 gynomonoecious. The segregation ratio in the BC 1 P 1 population (F 1 × gynoecious) is 1 monoecious: 1 gynoecious whereas the segregation ratio in the BC 1 P 2 (F 1 × andromonoecious) is 1 monoecious: 1 trimonoecious: 2 andromonoecious. Taken together, the results suggested that three recessive alleles, andromonoecious ( a ), gynoecious ( gy ) and trimonoecious ( tm ) control the sex forms in watermelon, and a allele is epistatic to the tm allele. The following phenotype-genotype relationships are proposed for each of the sex forms in watermelon: monoecious, A_Gy_Tm_ ; trimonoecious , A_Gy_tmtm ; andromonoecious, aaGy_Tm_ or aaGy_tmtm; gynoecious, A_gygyTm_ ; gynomonoecious, A_gygytmtm ; and hermaphroditic, aagygyTm_ or aagygytmtm . [ABSTRACT FROM AUTHOR]