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Ubiquitination-mediated protein degradation plays important roles in ABA signal transduction and delivering responses to chloroplast stress signals in plants, but additional E3 ligases of protein ubiquitination remain to be identified to understand the complex signaling network. Here we reported that ZEITLUPE (ZTL), an F-box protein, negatively regulates abscisic acid (ABA) signaling during ABA-inhibited early seedling growth and ABA-induced stomatal closure in Arabidopsis thaliana. Using molecular biology and biochemistry approaches, we demonstrated that ZTL interacts with and ubiquitinates its substrate, CHLH/ABAR (Mg-chelatase H subunit/putative ABA receptor), to modulate CHLH stability via the 26S proteasome pathway. CHLH acts genetically downstream of ZTL in ABA and drought stress signaling. Interestingly, ABA conversely induces ZTL phosphorylation, and high levels of ABA also induce CHLH proteasomal degradation, implying that phosphorylated ZTL protein may enhance the affinity to CHLH, leading to the increased degradation of CHLH after ABA treatment. Taken together, our results revealed a possible mechanism of reciprocal regulation between ABA signaling and the circadian clock, which is thought to be essential for plant fitness and survival.

Watermelon fruit undergoes distinct development stages with dramatic changes during fruit ripening. To date, the molecular mechanics of watermelon ripening remain unclear. Genetic and transcriptome evidences suggested that the ethylene response factor (ERF) gene ClERF069 may be an important candidate factor affecting watermelon fruit ripening. To dissect the roles of ClERF069 in fruit ripening, structure and phylogenetic analysis were performed using the amplified full-length sequence. Normal-ripening watermelon 97103, non-ripening watermelon PI296341-FR and the RIL population were used to analyze ClERF069 expression dynamics and the correlation with fruit ripening indexs. The results indicated that ClERF069 belongs to ERF family group VI and show high homology (83% identity) to melon ERF069-like protein. ClERF069 expression in watermelon flesh was negatively correlated with fruit lycopene content and sugar content during fruit ripening progress. Further transgenic evidences indicated that overexpression of 35S:ClERF069 in tomato noticeably delayed the ripening process up to 5.2 days. Lycopene, β-carotenoid accumulation patterns were altered and ethylene production patterns in transgenic fruits was significantly delayed during fruit ripening. Taken together, watermelon ethylene response factor ClERF069 was concluded to be a negative regulator of fruit ripening.

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.

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.

There has been no systematic research on the effect of plant growth regulators and silver nitrate treatments on the control of sex expression in watermelon. In this study, we tested sex expression responses of four watermelon forms (monoecism, gynoecism, andromonoecism, and hermaphrodite) to gibberellin, ethephon and silver nitrate treatments. Results have shown that, in monoecious plants, gibberellins (GA3) and ethephon treatments reduced the percentage of female flowers and delayed the occurrence of the first female flower, while silver nitrate induced the formation of bisexual flowers. In gynoecious plants, both ethephon and silver nitrate treatments transformed some female flowers into bisexual flowers, and treatment with ethephon resulted in a mass of abnormal flowers, while no obvious effect of treatment with GA3 was observed. In andromonoecious plants, ethephon and GA3 treatments delayed the occurrence of the first bisexual flower, and GA3 reduced the percentage of bisexual flowers, while no distinct effect for silver nitrate treatment was observed. In hermaphroditic plants, ethephon treatment induced the appearance of numerous abnormal flowers, while no obvious effects for GA3 and silver nitrate treatments were observed. We analyzed the transcription levels of all the expressed aminocyclopropane-1-carboxylic acid synthase (ACS) homologues in two gynoecious mutants and their wild types. We also tested the gene expression of CitACS4 which had been recognized as the andromonoecious gene in all treatments. All these results suggested that the best masculinizing treatment for breeding of the gynoecious line is silver nitrate, which repressed the expression of CitACS4 and induced many bisexual flowers for use in self-fertilization subsequently.

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.

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.

MicroRNAs (miRNAs) are a class of endogenous small non-coding RNAs involved in the post-transcriptional gene regulation and play a critical role in plant growth, development and stresses response. However less is known about miRNAs involvement in grafting behaviors, especially with the watermelon (Citrullus lanatus L.) crop, which is one of the most important agricultural crops worldwide. Grafting method is commonly used in watermelon production in attempts to improve its adaptation to abiotic and biotic stresses, in particular to the soil-borne fusarium wilt disease. In this study, Solexa sequencing has been used to discover small RNA populations and compare miRNAs on genome-wide scale in watermelon grafting system. A total of 11,458,476, 11,614,094 and 9,339,089 raw reads representing 2,957,751, 2,880,328 and 2,964,990 unique sequences were obtained from the scions of self-grafted watermelon and watermelon grafted on-to bottle gourd and squash at two true-leaf stage, respectively. 39 known miRNAs belonging to 30 miRNA families and 80 novel miRNAs were identified in our small RNA dataset. Compared with self-grafted watermelon, 20 (5 known miRNA families and 15 novel miRNAs) and 47 (17 known miRNA families and 30 novel miRNAs) miRNAs were expressed significantly different in watermelon grafted on to bottle gourd and squash, respectively. MiRNAs expressed differentially when watermelon was grafted onto different rootstocks, suggesting that miRNAs might play an important role in diverse biological and metabolic processes in watermelon and grafting may possibly by changing miRNAs expressions to regulate plant growth and development as well as adaptation to stresses. The small RNA transcriptomes obtained in this study provided insights into molecular aspects of miRNA-mediated regulation in grafted watermelon. Obviously, this result would provide a basis for further unravelling the mechanism on how miRNAs information is exchanged between scion and rootstock in grafted watermelon, and its relevance to diverse biological processes and environmental adaptation.

As part of our ongoing efforts to sequence and map the watermelon (Citrullus spp.) genome, we have constructed a high density genetic linkage map. The map positioned 234 watermelon genome sequence scaffolds (an average size of 1.41 Mb) that cover about 330 Mb and account for 93.5% of the 353 Mb of the assembled genomic sequences of the elite Chinese watermelon line 97103 (Citrullus lanatus var. lanatus). The genetic map was constructed using an F(8) population of 103 recombinant inbred lines (RILs). The RILs are derived from a cross between the line 97103 and the United States Plant Introduction (PI) 296341-FR (C. lanatus var. citroides) that contains resistance to fusarium wilt (races 0, 1, and 2). The genetic map consists of eleven linkage groups that include 698 simple sequence repeat (SSR), 219 insertion-deletion (InDel) and 36 structure variation (SV) markers and spans ∼800 cM with a mean marker interval of 0.8 cM. Using fluorescent in situ hybridization (FISH) with 11 BACs that produced chromosome-specifc signals, we have depicted watermelon chromosomes that correspond to the eleven linkage groups constructed in this study. The high resolution genetic map developed here should be a useful platform for the assembly of the watermelon genome, for the development of sequence-based markers used in breeding programs, and for the identification of genes associated with important agricultural traits.

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. [ABSTRACT FROM AUTHOR]

Background: Pumpkin (Cucurbita maxima Duch.) is an economically important crop belonging to the Cucurbitaceae family. However, very few genomic and genetic resources are available for this species. As part of our ongoing efforts to sequence the pumpkin genome, high-density genetic map is essential for anchoring and orienting the assembled scaffolds. In addition, a saturated genetic map can facilitate quantitative trait locus (QTL) mapping. Results: A set of 186 F2 plants derived from the cross of pumpkin inbred lines Rimu and SQ026 were genotyped using the genotyping-by-sequencing approach. Using the SNPs we identified, a high-density genetic map containing 458 bin-markers was constructed, spanning a total genetic distance of 2,566.8 cM across the 20 linkage groups of C. maxima with a mean marker density of 5.60 cM. Using this map we were able to anchor 58 assembled scaffolds that covered about 194.5 Mb (71.7 %) of the 271.4 Mb assembled pumpkin genome, of which 44 (183.0 Mb; 67.4 %) were oriented. Furthermore, the high-density genetic map was used to identify genomic regions highly associated with an important agronomic trait, dwarf vine. Three QTLs on linkage groups (LGs) 1, 3 and 4, respectively, were recovered. One QTL, qCmB2, which was located in an interval of 0.42 Mb on LG 3, explained 21.4 % phenotypic variations. Within qCmB2, one gene, Cma_004516, encoding the gibberellin (GA) 20-oxidase in the GA biosynthesis pathway, had a 1249-bp deletion in its promoter in bush type lines, and its expression level was significantly increased during the vine growth and higher in vine type lines than bush type lines, supporting Cma_004516 as a possible candidate gene controlling vine growth in pumpkin. Conclusions: A high-density pumpkin genetic map was constructed, which was used to successfully anchor and orient the assembled genome scaffolds, and to identify QTLs highly associated with pumpkin vine length. The map provided a valuable resource for gene cloning and marker assisted breeding in pumpkin and other related species. The identified vine length QTLs would help to dissect the underlying molecular basis regulating pumpkin vine growth. [ABSTRACT FROM AUTHOR]

Background Modern watermelon (Citrullus lanatus L.) cultivars share a narrow genetic base due to many years of selection for desirable horticultural qualities. Wild subspecies within C. lanatus are important potential sources of novel alleles for watermelon breeding, but successful trait introgression into elite cultivars has had limited success. The application of marker assisted selection (MAS) in watermelon is yet to be realized, mainly due to the past lack of high quality genetic maps. Recently, a number of useful maps have become available, however these maps have few common markers, and were constructed using different marker sets, thus, making integration and comparative analysis among maps difficult. The objective of this research was to use single-nucleotide polymorphism (SNP) anchor markers to construct an integrated genetic map for C. lanatus. Results Under the framework of the high density genetic map, an integrated genetic map was constructed by merging data from four independent mapping experiments using a genetically diverse array of parental lines, which included three subspecies of watermelon. The 698 simple sequence repeat (SSR), 219 insertion-deletion (InDel), 36 structure variation (SV) and 386 SNP markers from the four maps were used to construct an integrated map. This integrated map contained 1339 markers, spanning 798 cM with an average marker interval of 0.6 cM. Fifty-eight previously reported quantitative trait loci (QTL) for 12 traits in these populations were also integrated into the map. In addition, new QTL identified for brix, fructose, glucose and sucrose were added. Some QTL associated with economically important traits detected in different genetic backgrounds mapped to similar genomic regions of the integrated map, suggesting that such QTL are responsible for the phenotypic variability observed in a broad array of watermelon germplasm. Conclusions The integrated map described herein enhances the utility of genomic tools over previous watermelon genetic maps. A large proportion of the markers in the integrated map are SSRs, InDels and SNPs, which are easily transferable across laboratories. Moreover, the populations used to construct the integrated map include all three watermelon subspecies, making this integrated map useful for the selection of breeding traits, identification of QTL, MAS, analysis of germplasm and commercial hybrid seed detection. [ABSTRACT FROM AUTHOR]

Powdery mildew of cucurbits, incited by Podosphaera xanthii (Castagne) Braun & Shishkoff (syn. Sphaerotheca fuliginea auct. p.p.). is an economically important foliar disease, which is now common in watermelon [Citrullus lanatus (Thunb.) Mastum. & Nakai]. This disease occurs in all watermelon-growing areas and can reduce yields by up to 30%. Finding and breeding for resistance to this disease is important to reduce dependence on fungicides and to use in combination with fungicides to limit the spread of fungicide-resistant P. xanthii. This is the first English report that race 2WF of P. xanthii can infect watermelon. It is the prevalent race of watermelon powdery mildew in Beijing. [ABSTRACT FROM AUTHOR]

Background: Cucumber, Cucumis sativus L., is an economically and nutritionally important crop of the Cucurbitaceae family and has long served as a primary model system for sex determination studies. Recently, the sequencing of its whole genome has been completed. However, transcriptome information of this species is still scarce, with a total of around 8,000 Expressed Sequence Tag (EST) and mRNA sequences currently available in GenBank. In order to gain more insights into molecular mechanisms of plant sex determination and provide the community a functional genomics resource that will facilitate cucurbit research and breeding, we performed transcriptome sequencing of cucumber flower buds of two near-isogenic lines, WI1983G, a gynoecious plant which bears only pistillate flowers, and WI1983H, a hermaphroditic plant which bears only bisexual flowers. Result: Using Roche-454 massive parallel pyrosequencing technology, we generated a total of 353,941 high quality EST sequences with an average length of 175bp, among which 188,255 were from gynoecious flowers and 165,686 from hermaphroditic flowers. These EST sequences, together with ∼5,600 high quality cucumber EST and mRNA sequences available in GenBank, were clustered and assembled into 81,401 unigenes, of which 28,452 were contigs and 52,949 were singletons. The unigenes and ESTs were further mapped to the cucumber genome and more than 500 alternative splicing events were identified in 443 cucumber genes. The unigenes were further functionally annotated by comparing their sequences to different protein and functional domain databases and assigned with Gene Ontology (GO) terms. A biochemical pathway database containing 343 predicted pathways was also created based on the annotations of the unigenes. Digital expression analysis identified ∼200 differentially expressed genes between flowers of WI1983G and WI1983H and provided novel insights into molecular mechanisms of plant sex determination process. Furthermore, a set of SSR motifs and high confidence SNPs between WI1983G and WI1983H were identified from the ESTs, which provided the material basis for future genetic linkage and QTL analysis. Conclusion: A large set of EST sequences were generated from cucumber flower buds of two different sex types. Differentially expressed genes between these two different sex-type flowers, as well as putative SSR and SNP markers, were identified. These EST sequences provide valuable information to further understand molecular mechanisms of plant sex determination process and forms a rich resource for future functional genomics analysis, marker development and cucumber breeding. [ABSTRACT FROM AUTHOR]

Watermelon belongs to the genus Citrullus. There have been continuing interests in breeding of watermelon for economic benefits, but information on the scope and utilization of genetic variations in Citrullus is still limited. The present study was conducted in 2012-13, to evaluate the genetic diversity and population structure of the 1197 line watermelon collection maintained by the Beijing Vegetable Research Center (BVRC), which belongs to seven Citrullus species including Citrullus naudinianus, Citrullus colocynthis, Citrullus rehmii, Citrullus ecirrhosus, Citrullus amarus, Citrullus mucosospermus, and Cirullus lanatus subsp. vulgaris. Twenty-three highly informative microsatellite markers evenly distributed in the watermelon genome were used to assess genetic diversity in this collection. The markers detected on an average of 6.05 alleles per locus with the average value of polymorphism information content (PIC) at 0.49. A high level of gene diversity [Nei's gene diversity index (Nei) = 0.56] and a low observed heterozygosity (Ho = 0.10) were revealed within the collection. Structure analysis grouped the 1197 accessions into two main populations (Pop I and Pop II) and an admixture group. Pop I contained 450 accessions from C. lanatus subsp. vulgaris (446) and C. mucosospermus (4). Pop II comprised 465 accessions, 379 of which belonged to C. lanatus subsp. vulgaris and 86 to C. naudinianus (3), C. ecirrhosus (2), C. rehmii (2), C. colocynthis (11), C. amarus (58), and C. mucosospermus (10). The remaining 282 accessions were classified as an admixture group. The two main populations were further subdivided into four subgroups. The groupings were consistent with the estimation of F statistics (Fst) and Nei's genetic distances in collections. We confirmed the distinct genetic backgrounds between American and East Asian ecotypes. Subsequently, we defined a core set consisting of 130 accessions including 47 from Pop I, 68 from Pop II, and 15 from the Admixture group. This core set was able to capture all 133 alleles detected by 23 simple sequence repeats (SSRs) in 1197 accessions. These results will facilitate efficient use of genetic variations in Citrullus in watermelon breeding and help optimization of accessions in genomewide association studies. [ABSTRACT FROM AUTHOR]

Because of the growing threat of global warming, drought stress could severely affect the normal growth and development of crop plants. To alleviate such an adverse effect, there is a need to screen watermelon germplasm collections to identify genetic sources for potential drought tolerance, in the present study, 820 accessions of USDA's Citrullus PIs and 246 watermelon breeding lines were evaluated for their drought tolerance at the seedling stage under extreme water stress conditions in a greenhouse. Significant variations in drought tolerance were observed in the Citrullus germplasm collections. Using fast clustering analysis, the tested watermelon materials could be assigned into four groups, including tolerant, intermediate tolerant, moderately sensitive, and sensitive, respectively. The most drought-tolerant Citrullus germplasm, including 13 Citrullus lanatus var. lanatus and 12 C. lanatus var. citroides accessions, were originated from Africa. These genetic materials could be used for rootstock breeding or for developing drought-tolerant watermelon cultivars. [ABSTRACT FROM AUTHOR]