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How sugar transporters regulate sugar accumulation in fruits is poorly understood and particularly so for species storing high-concentration Suc. Accumulation of soluble sugars in watermelon (Citrullus lanatus) fruit, a major quality trait, had been selected during domestication. Still, the molecular mechanisms controlling this quantitative trait are unknown. We resequenced 96 recombinant inbred lines, derived from crossing sweet and unsweet accessions, to narrow down the size of a previously described sugar content quantitative trait locus, which contains a putative Tonoplast Sugar Transporter gene (ClTST2). Molecular and biochemical analyses indicated that ClTST2 encodes a vacuolar membrane protein, whose expression is associated with tonoplast uptake and accumulation of sugars in watermelon fruit flesh cells. We measured fruit sugar content and resequenced the genomic region surrounding ClTST2 in 400 watermelon accessions and associated the most sugar-related significant single-nucleotide polymorphisms (SNPs) to the ClTST2 promoter. Large-scale population analyses strongly suggest increased expression of ClTST2 as a major molecular event in watermelon domestication associated with a selection sweep around the ClTST2 promoter. Further molecular analyses explored the binding of a sugar-induced transcription factor (SUSIWM1) to a sugar-responsive cis-element within the ClTST2 promoter, which contains the quantitative trait locus (QTL) causal SNP. The functional characterization of ClTST2 and its expression regulation by SUSIWM1 provide novel tools to increase sugar sink potency in watermelon and possibly in other vegetable and fruit crops.

β-Carotene adds nutritious value and determines the color of many fruits, including melon (Cucumis melo). In melon mesocarp, β-carotene accumulation is governed by the Orange gene (CmOr) golden single-nucleotide polymorphism (SNP) through a yet to be discovered mechanism. In Arabidopsis (Arabidopsis thaliana), OR increases carotenoid levels by posttranscriptionally regulating phytoene synthase (PSY). Here, we identified a CmOr nonsense mutation (Cmor-lowβ) that lowered fruit β-carotene levels with impaired chromoplast biogenesis. Cmor-lowβ exerted a minimal effect on PSY transcripts but dramatically decreased PSY protein levels and enzymatic activity, leading to reduced carotenoid metabolic flux and accumulation. However, the golden SNP was discovered to not affect PSY protein levels and carotenoid metabolic flux in melon fruit, as shown by carotenoid and immunoblot analyses of selected melon genotypes and by using chemical pathway inhibitors. The high β-carotene accumulation in golden SNP melons was found to be due to a reduced further metabolism of β-carotene. This was revealed by genetic studies with double mutants including carotenoid isomerase (yofi), a carotenoid-isomerase nonsense mutant, which arrests the turnover of prolycopene. The yofi F2 segregants accumulated prolycopene independently of the golden SNP. Moreover, Cmor-lowβ was found to inhibit chromoplast formation and chloroplast disintegration in fruits from 30 d after anthesis until ripening, suggesting that CmOr regulates the chloroplast-to-chromoplast transition. Taken together, our results demonstrate that CmOr is required to achieve PSY protein levels to maintain carotenoid biosynthesis metabolic flux but that the mechanism of the CmOr golden SNP involves an inhibited metabolism downstream of β-carotene to dramatically affect both carotenoid content and plastid fate.

Summary Carotenoids are critically important to plants and humans. The ORANGE (OR) gene is a key regulator for carotenoid accumulation, but its physiological roles in crops remain elusive. In this study, we generated transgenic tomato ectopically overexpressing the Arabidopsis wild‐type OR (AtORWT) and a ‘golden SNP’‐containing OR (AtORHis). We found that AtORHis initiated chromoplast formation in very young fruit and stimulated carotenoid accumulation at all fruit developmental stages, uncoupled from other ripening activities. The elevated levels of carotenoids in the AtOR lines were distributed in the same subplastidial fractions as in wild‐type tomato, indicating an adaptive response of plastids to sequester the increased carotenoids. Microscopic analysis revealed that the plastid sizes were increased in both AtORWT and AtORHis lines at early fruit developmental stages. Moreover, AtOR overexpression promoted early flowering, fruit set and seed production. Ethylene production and the expression of ripening‐associated genes were also significantly increased in the AtOR transgenic fruit at ripening stages. RNA‐Seq transcriptomic profiling highlighted the primary effects of OR overexpression on the genes in the processes related to RNA, protein and signalling in tomato fruit. Taken together, these results expand our understanding of OR in mediating carotenoid accumulation in plants and suggest additional roles of OR in affecting plastid size as well as flower and fruit development, thus making OR a target gene not only for nutritional biofortification of agricultural products but also for alteration of horticultural traits.

Key messageGr5.1 is the major locus for cauliflower green curd color and mapped to an interval of 236 Kbp with four most likely candidate genes.Cauliflower with colored curd enhances not only the visual appeal but also the nutritional value of the crop. Green cauliflower results from ectopic development of chloroplasts in the normal white curd. However, the underlying genetic basis is unknown. In this study, we employed QTL-seq analysis to identify the loci that were associated with green curd phenotype in cauliflower. A F2 population was generated following a cross between a white curd (Stovepipe) and a green curd (ACX800) cauliflower plants. By whole-genome resequencing and SNP analysis of green and white F2 bulks, two QTLs were detected on chromosomes 5 (Gr5.1) and 7 (Gr7.1). Validation by traditional genetic mapping with CAPS markers suggested that Gr5.1 represented a major QTL, whereas Gr7.1 had a minor effect. Subsequent high-resolution mapping of Gr5.1 in the second large F2 population with additional CAPS markers narrowed down the target region to a genetic and physical distance of 0.3 cM and 236 Kbp, respectively. This region contained 35 genes with four of them representing the best candidates for the green curd phenotype in cauliflower. They are LOC106295953, LOC106343833, LOC106345143, and LOC106295954, which encode UMP kinase, DEAD-box RNA helicase 51-like, glutathione S-transferase T3-like, and protein MKS1, respectively. These findings lay a solid foundation for the isolation of the Gr gene and provide a potential for marker-assisted selection of the green curd trait in cauliflower breeding. The eventual isolation of Gr will also facilitate better understanding of chloroplast biogenesis and development in plants.

Summary Years of selection for desirable fruit quality traits in dessert watermelon (Citrullus lanatus) has resulted in a narrow genetic base in modern cultivars. Development of novel genomic and genetic resources offers great potential to expand genetic diversity and improve important traits in watermelon. Here, we report a high‐quality genome sequence of watermelon cultivar ‘Charleston Gray’, a principal American dessert watermelon, to complement the existing reference genome from ‘97103’, an East Asian cultivar. Comparative analyses between genomes of ‘Charleston Gray’ and ‘97103’ revealed genomic variants that may underlie phenotypic differences between the two cultivars. We then genotyped 1365 watermelon plant introduction (PI) lines maintained at the U.S. National Plant Germplasm System using genotyping‐by‐sequencing (GBS). These PI lines were collected throughout the world and belong to three Citrullus species, C. lanatus, C. mucosospermus and C. amarus. Approximately 25 000 high‐quality single nucleotide polymorphisms (SNPs) were derived from the GBS data using the ‘Charleston Gray’ genome as the reference. Population genomic analyses using these SNPs discovered a close relationship between C. lanatus and C. mucosospermus and identified four major groups in these two species correlated to their geographic locations. Citrullus amarus was found to have a distinct genetic makeup compared to C. lanatus and C. mucosospermus. The SNPs also enabled identification of genomic regions associated with important fruit quality and disease resistance traits through genome‐wide association studies. The high‐quality ‘Charleston Gray’ genome and the genotyping data of this large collection of watermelon accessions provide valuable resources for facilitating watermelon research, breeding and improvement.

Cucumis melo is highly diverse for fruit traits providing wide breeding and genetic research opportunities, including genome-wide association (GWA) analysis. We used a collection of 177 accessions representing the two C. melo subspecies and 11 horticultural groups for detailed characterization of fruit traits variation and evaluation of the potential of GWA for trait mapping in melon. Through genotyping-by-sequencing, 23,931 informative SNPs were selected for genome-wide analyses. We found that linkage-disequilibrium decays at ~100 Kb in this collection and that population structure effect on association results varies between traits. We mapped several monogenic traits to narrow intervals overlapping with known causative genes, demonstrating the potential of diverse collections and GWA for mapping Mendelian traits to a candidate-gene level in melon. We further report on mapping of fruit shape quantitative trait loci (QTLs) and comparison with multiple previous QTL studies. Expansion of sample size and a more balanced representation of taxonomic groups might improve efficiency for simple traits dissection. But, as in other plant species, integrated linkage-association multi-allelic approaches are likely to produce better combination of statistical power, diversity capture and mapping resolution in melon. Our data can be utilized for selection of the most appropriate accessions for such approaches.

Melon is an important crop that exhibits broad variation for fruit morphology traits that are the substrate for genetic mapping efforts. In the post-genomic era, the link between genetic maps and physical genome assemblies is key for leveraging QTL mapping results for gene cloning and breeding purposes. Here, using a population of 164 melon recombinant inbred lines (RILs) that were subjected to genotyping-by-sequencing, we constructed and compared high-density sequence- and linkage-based recombination maps that were aligned to the reference melon genome. These analyses reveal the genome-wide variation in recombination frequency and highlight regions of disrupted collinearity between our population and the reference genome. The population was phenotyped over 3 years for fruit size and shape as well as rind netting. Four QTLs were detected for fruit size, and they act in an additive manner, while significant epistatic interaction was found between two neutral loci for this trait. Fruit shape displayed transgressive segregation that was explained by the action of four QTLs, contributed by alleles from both parents. The complexity of rind netting was demonstrated on a collection of 177 diverse accessions. Further dissection of netting in our RILs population, which is derived from a cross of smooth and densely netted parents, confirmed the intricacy of this trait and the involvement of major locus and several other interacting QTLs. A major netting QTL on chromosome 2 co-localized with results from two additional populations, paving the way for future study toward identification of a causative gene for this trait.

Carotenoids are crucial for plant growth and human health. The finding of ORANGE (OR) protein as a pivotal regulator of carotenogenesis offers a unique opportunity to comprehensively understand the regulatory mechanisms of carotenoid accumulation and develop crops with enhanced nutritional quality. Here, we demonstrated that alteration of a single amino acid in a wild-type OR greatly enhanced its ability to promote carotenoid accumulation. Whereas overexpression ofORfrom Arabidopsis (Arabidopsis thaliana; AtOR) or from the agronomically important crop sorghum (Sorghum bicolor; SbOR) increased carotenoid levels up to 2-fold, expression ofAtORHis (R90H) orSbORHis (R104H) variants dramatically enhanced carotenoid accumulation by up to 7-fold in the Arabidopsis calli. Moreover, we found thatAtORAla (R90A) functioned similarly toAtORHis to promote carotenoid overproduction. Neither AtOR nor AtORHisgreatly affected carotenogenic gene expression. AtORHisexhibited similar interactions with phytoene synthase (PSY) as AtOR in posttranscriptionally regulating PSY protein abundance. AtORHistriggered biogenesis of membranous chromoplasts in the Arabidopsis calli, which shared structures similar to chromoplasts found in the curd of the orange cauliflower (Brassica oleracea) mutant. By contrast, AtOR did not cause plastid-type changes in comparison with the controls, but produced plastids containing larger and electron-dense plastoglobuli. The unique ability ofAtORHis in mediating chromoplast biogenesis is responsible for its induced carotenoid overproduction. Our study demonstratesORHis/Ala as powerful tools for carotenoid enrichment in plants, and provides insights into the mechanisms underlyingORHis -regulated carotenoid accumulation.

Carotenoids, such as β-carotene, accumulate in chromoplasts of various fleshy fruits, awarding them with colors, aromas, and nutrients. The Orange (CmOr) gene controls β-carotene accumulation in melon fruit by posttranslationally enhancing carotenogenesis and repressing β-carotene turnover in chromoplasts. Carotenoid isomerase (CRTISO) isomerizes yellow prolycopene into red lycopene, a prerequisite for further metabolism into β-carotene. We comparatively analyzed the developing fruit transcriptomes of orange-colored melon and its two isogenic EMS-induced mutants, low-β (Cmor) and yofi (Cmcrtiso). The Cmor mutation in low-β caused a major transcriptomic change in the mature fruit. In contrast, the Cmcrtiso mutation in yofi significantly changed the transcriptome only in early fruit developmental stages. These findings indicate that melon fruit transcriptome is primarily altered by changes in carotenoid metabolic flux and plastid conversion, but minimally by carotenoid composition in the ripe fruit. Clustering of the differentially expressed genes into functional groups revealed an association between fruit carotenoid metabolic flux with the maintenance of the photosynthetic apparatus in fruit chloroplasts. Moreover, large numbers of thylakoid localized photosynthetic genes were differentially expressed in low-β. CmOR family proteins were found to physically interact with light-harvesting chlorophyll a–b binding proteins, suggesting a new role of CmOR for chloroplast maintenance in melon fruit. This study brings more insights into the cellular and metabolic processes associated with fruit carotenoid accumulation in melon fruit and reveals a new maintenance mechanism of the photosynthetic apparatus for plastid development.

MAIN CONCLUSION : Storage promotes carotenoid accumulation and converts amylochromoplasts into chromoplasts in winter squash. Such carotenoid enhancement is likely due to continuous biosynthesis along with reduced turnover and/or enhanced sequestration. Postharvest storage of fruits and vegetables is often required and frequently results in nutritional quality change. In this study, we investigated carotenoid storage plastids, carotenoid content, and its regulation during 3-month storage of winter squash butternut fruits. We showed that storage improved visual appearance of fruit flesh color from light to dark orange, and promoted continuous accumulation of carotenoids during the first 2-month storage. Such an increased carotenoid accumulation was found to be concomitant with starch breakdown, resulting in the conversion of amylochromoplasts into chromoplasts. The butternut fruits contained predominantly β-carotene, lutein, and violaxanthin. Increased ratios of β-carotene and violaxanthin to total carotenoids were noticed during the storage. Analysis of carotenoid metabolic gene expression and PSY protein level revealed a decreased expression of carotenogenic genes and PSY protein following the storage, indicating that the increased carotenoid level might not be due to increased biosynthesis. Instead, the increase likely resulted from a continuous biosynthesis with a possibly reduced turnover and/or enhanced sequestration, suggesting a complex regulation of carotenoid accumulation during fruit storage. This study provides important information to our understanding of carotenogenesis and its regulation during postharvest storage of fruits.