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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]

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]

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]