Pollination services to crops of watermelon (Citrullus lanatus) and green tomato (Physalis ixocarpa) in the coastal region of Jalisco, Mexico.

Bees play a pivotal role as pollinators in crops essential for human consumption. However, the global decline in bee populations poses a significant threat to pollination services and food security worldwide. The loss and degradation of habitats due to land use change are primary factors contributing to bee declines, particularly in tropical forests facing high deforestation rates. Here, we evaluate the pollination services provided to crops of watermelon (Citrullus lanatus) and green tomato (Physalis ixocarpa) in three municipalities in the state of Jalisco, Mexico, a place with Tropical Dry Forest, during years 2008, and 2014 to 2017. Both crops are cultivated in the dry season, approximately during the months of November to March. We describe the composition of the pollinator community and their visitation frequency (measured through the number of visits per flower per hour), and we assess the impact of pollinators on plant reproductive success and the level of pollinator dependence for each crop species (measured through the number of flowers that developed into fruits). We also evaluate how the landscape configuration (through the percentage of forest cover and distance to the forest) influences richness and abundance of pollinators (measured as number of species and individuals of pollinators per line of 50 m), and we use the model Integrated Valuation of Ecosystem Services and Tradeoffs (InVEST) to map and value the pollination service in both crops. InVEST Crop pollination model is a simulation focuses on wild pollinators providing the pollinator ecosystem service. Our findings indicate that Apis mellifera was the primary pollinator of both crops, one of the few abundant pollinators in the study region during the dry season. In experiments where pollinators were excluded from flowers, watermelon yielded no fruits, while green tomato experienced a 65% reduction in production. In the case of green tomato, fruit set showed a positive correlation with pollinator abundance. A positive association between forest cover and total pollinator abundance was observed in green tomato in 2008, but not in watermelon. Additionally, a positive relationship was observed between the abundance of bees predicted by the InVEST model and the abundance of bees observed in green tomato flowers in 2008. In the study region, green tomato and watermelon rely on pollinators for fruit production, with honeybees (from feral and managed colonies) acting as the primary provider of pollination services for these crops. Consequently, the conservation of natural areas is crucial to provide food and nesting resources for pollinators. By doing so, we can ensure the diversity and abundance of pollinators, which in turn will help secure food security. The findings of this study underscore the critical need for the conservation of natural areas to support pollinator populations. Policymakers should prioritize the protection and restoration of habitats, particularly tropical forests, which are essential for maintaining the diversity and abundance of pollinators.

Mapping the genetic architecture of low-temperature stress tolerance in citron watermelon.

Sweet-fleshed watermelon (Citrullus lanatus) is an important vegetable crop of the tropical origin. It is widely grown and consumed around the world for its hydration and nutritional quality values. Low-temperature stress can affect early planting, seedling establishment, and expansion of crop production to new areas. A collection of 122 citron watermelon (Citrullus amarus) accessions were obtained from the USDA's National Plant Germplasm Repository System gene bank in Griffin, GA. The accessions were genotyped using whole genome resequencing to generate single nucleotide polymorphisms (SNPs) molecular markers and screened under cold-stressed and non-stressed control conditions. Four low-temperature stress tolerance related traits including shoot biomass, vine length, maximum quantum efficiency of photosystem II, and chlorophyll content were measured under cold-stressed and non-stressed control treatment conditions. Correlation analysis revealed the presence of positive relationships among traits. Broad-sense heritability for all traits ranged from 0.35 to 0.73, implying the presence of genetic contributions to the observed phenotypic variation. Genomic regions underlying these traits across several citron watermelon chromosomes were identified. Four low-temperature stress tolerance related putative candidate genes co-located with the peak SNPs from genome-wide association study. These genomic regions and marker information could potentially be used in molecular breeding to accelerate genetic improvements for low-temperature stress tolerance in watermelon.

Transcriptome Profiling Reveals the Effects of Nitric Oxide on the Growth and Physiological Characteristics of Watermelon under Aluminum Stress.

Excessive aluminum ions (Al3+) in acidic soil can have a toxic effect on watermelons, restricting plant growth and reducing yield and quality. In this study, we found that exogenous application of nitric oxide (NO) could increase the photochemical efficiency of watermelon leaves under aluminum stress by promoting closure of leaf stomata, reducing malondialdehyde and superoxide anion in leaves, and increasing POD and CAT activity. These findings showed that the exogenous application of NO improved the ability of watermelon to withstand aluminum stress. To further reveal the mitigation mechanism of NO on watermelons under aluminum stress, the differences following different types of treatments-normal growth, Al, and Al + NO-were shown using de novo sequencing of transcriptomes. In total, 511 differentially expressed genes (DEGs) were identified between the Al + NO and Al treatment groups. Significantly enriched biological processes included nitrogen metabolism, phenylpropane metabolism, and photosynthesis. We selected 23 genes related to antioxidant enzymes and phenylpropane metabolism for qRT-PCR validation. The results showed that after exogenous application of NO, the expression of genes encoding POD and CAT increased, consistent with the results of the physiological indicators. The expression patterns of genes involved in phenylpropanoid metabolism were consistent with the transcriptome expression abundance. These results indicate that aluminum stress was involved in the inhibition of the photosynthetic pathway, and NO could activate the antioxidant enzyme defense system and phenylpropane metabolism to protect cells and scavenge reactive oxygen species. This study improves our current understanding by comprehensively analyzing the molecular mechanisms underlying NO-induced aluminum stress alleviation in watermelons.

Identification and characterization of a natural SNP variant in ALTERNATIVE OXIDASE gene associated with cold stress tolerance in watermelon.

Alternative oxidase (AOX) is a mitochondrial enzyme encoded by a small nuclear gene family, which contains the two subfamilies, AOX1 and AOX2. In the present study on watermelon (Citrullus lanatus), only one ClAOX gene, belonging to AOX2 subfamily but having a similar gene structure to AtAOX1a, was found in the watermelon genome. The expression analysis suggested that ClAOX had the constitutive expression feature of AOX2 subfamily, but was cold inducible, which is normally considered an AOX1 subfamily feature. Moreover, one single nucleotide polymorphism (SNP) in ClAOX sequence, which led to the change from Lys (N) to Asn (K) in the 96th amino acids, was found among watermelon subspecies. Ectopic expression of two ClAOX alleles in the Arabidopsis aox1a knock-out mutant indicated that ClAOXK-expressing plants had stronger cold tolerance than aox1a mutant and ClAOXN-expressing plants. Our findings suggested watermelon genome contained a single ClAOX that possessed the expression features of both AOX1 and AOX2 subfamilies. A naturally existing SNP in ClAOX differentiated the cold tolerance of transgenic Arabidopsis plants, impling a possibility this gene might be a functional marker for stress-tolerance breeding.

Hormonal and metabolites responses in Fusarium wilt-susceptible and -resistant watermelon plants during plant-pathogen interactions.

BACKGROUND: Fusarium oxysporum f. sp. niveum (FON) causes Fusarium wilt in watermelon. Several disease-resistant watermelon varieties have been developed to combat Fusarium wilt. However, the key metabolites that mount defense responses in these watermelon varieties are unknown. Herein, we analyzed hormones, melatonin, phenolic acids, and amino acid profiles in the leaf tissue of FON zero (0)-resistant (PI-296341, Calhoun Grey, and Charleston Grey) and -susceptible (Sugar Baby) watermelon varieties before and after infection. RESULTS: We found that jasmonic acid-isoleucine (JA-Ile) and methyl jasmonate (MeJA) were selectively accumulated in one or more studied resistant varieties upon infection. However, indole-3-acetic acid (IAA) was only observed in the FON 0 inoculated plants of all varieties on the 16th day of post-inoculation. The melatonin content of PI-296341 decreased upon infection. Conversely, melatonin was only detected in the FON 0 inoculated plants of Sugar Baby and Charleston Grey varieties. On the 16th day of post-inoculation, the lysine content in resistant varieties was significantly reduced, whereas it was found to be elevated in the susceptible variety. CONCLUSIONS: Taken together, Me-JA, JA-Ile, melatonin, and lysine may have crucial roles in developing defense responses against the FON 0 pathogen, and IAA can be a biomarker of FON 0 infection in watermelon plants.

Nanoparticle-Mediated Seed Priming Improves Germination, Growth, Yield, and Quality of Watermelons (Citrullus lanatus) at multi-locations in Texas.

Seed priming uses treatments to improve seed germination and thus potentially increase growth and yield. Low-cost, environmentally friendly, effective seed treatment remain to be optimized and tested for high-value specialty crop like watermelon (Citrullus lanatus) in multi-locations. This remains a particularly acute problem for triploids, which produce desirable seedless watermelons, but show low germination rates. In the present study, turmeric oil nanoemulsions (TNE) and silver nanoparticles (AgNPs) synthesized from agro-industrial byproducts were used as nanopriming agents for diploid (Riverside) and triploid (Maxima) watermelon seeds. Internalization of nanomaterials was confirmed by neutron activation analysis, transmission electron microscopy, and gas chromatography-mass spectrometry. The seedling emergence rate at 14 days after sowing was significantly higher in AgNP-treated triploid seeds compared to other treatments. Soluble sugar (glucose and fructose) contents were enhanced during germination in the AgNP-treated seeds at 96 h. Seedlings grown in the greenhouse were transplanted at four locations in Texas: Edinburg, Pecos, Grapeland, and Snook in 2017. At Snook, higher yield 31.6% and 35.6% compared to control were observed in AgNP-treated Riverside and Maxima watermelons, respectively. To validate the first-year results, treated and untreated seeds of both cultivars were sown in Weslaco, Texas in 2018. While seed emegence and stand establishments were enhanced by seed priming, total phenolics radical-scavenging activities, and macro- and microelements in the watermelon fruits were not significantly different from the control. The results of the present study demonstracted that seed priming with AgNPs can enhance seed germination, growth, and yield while maintaining fruit quality through an eco-friendly and sustainable nanotechnological approach.

QTL mapping of resistance to Fusarium oxysporum f. sp. niveum race 2 and Papaya ringspot virus in Citrullus amarus.

KEY MESSAGE: A Citrullus amarus mapping population segregating for resistance to Fusarium oxysporum f. sp. niveum race 2 and Papaya ringspot virus was used to identify novel QTL, important for the improvement in watermelon disease resistance. Multiple disease screens of the USDA Citrullus spp. germplasm collection have highlighted the value of Citrullus amarus (citron melon or wild watermelon) as a resource for enhancing modern watermelon cultivars (Citrullus lanatus) with resistance to a broad range of fungal, bacterial and viral diseases of watermelon. We have generated a genetic population of C. amarus segregating for resistance to two important watermelon diseases: Fusarium wilt (caused by the fungus Fusarium oxysporum f. sp. niveum; Fon race 2) and Papaya ringspot virus-watermelon strain (PRSV-W). QTL mapping of Fon race 2 resistance identified seven significant QTLs, with the major QTL representing a novel genetic source of resistance and an opportunity for gene pyramiding. A single QTL was associated with resistance to PRSV-W, which adhered to expectations of a prior study indicating a single-gene recessive inheritance in watermelon. The resistance loci identified here provide valuable genetic resources for introgression into cultivated watermelon for the improvement in disease resistance.

Genome-wide identification and expression analysis of aquaporin gene family related to abiotic stress in watermelon.

The plant aquaporins (AQPs) are highly conserved integral membrane proteins that participate in multiple developmental processes and responses to various stresses. In this study, a total of 35 AQP genes were identified in the watermelon genome. The phylogenetic analysis showed that these AQPs can be divided into five types, including 16 plasma membrane intrinsic proteins (PIPs), eight tonoplast intrinsic proteins (TIPs), eight nodulin 26-like intrinsic proteins (NIPs), two small basic intrinsic proteins (SIPs), and one uncategorized X intrinsic protein (XIP). A number of cis-elements related to plant responses to hormones and stresses were detected in the promoter sequences of ClAQP genes. Chromosome distribution analysis revealed that the genes are unevenly distributed on eight chromosomes, with chromosomes 1 and 4 possessing the most genes. Expression analysis at different developmental stages in flesh and rind indicated that most of ClAQPs have tissue-specific expression. Meanwhile, some other AQP genes showed differential expression in response to cold, salt, and ABA treatments, which is consistent with the organization of the stress-responsive cis-elements detected in the promoter regions. Our results lay a foundation for understanding the specific functions of ClAQP genes to help the genetic improvement of watermelon.

Manipulation of the rhizosphere microbial community through application of a new bio-organic fertilizer improves watermelon quality and health.

Bio-organic fertilizers (BOFs) combine functional microbes with a suitable substrate and have been shown to effectively suppress soil-borne diseases and promote plant growth. Here, we developed a novel bio-organic fertilizer (BOF) by fermentation of a cow plus chicken manure (M) compost using Fen-liquor Daqu (FLD) as a fermentation starter and compared the compositions of bacterial and fungal communities in the rhizosphere soil of watermelon plants after treatment with different fertilizers. Further, we aimed to explore the mechanisms underlying plant-promoting and disease (Fusarium wilt)-suppressing activities of each rhizosphere microbial community. The microbial communities of soil amended with cow plus chicken manure compost (S+M), soil amended with the BOF (S+BOF), and untreated control soil (S) without plants were analyzed through sequence analysis using the Illumina MiSeq platform. The results showed that a new microbial community was formed in the manure compost after fermentation by the Daqu. Application of the BOF to the soil induced remarkable changes in the rhizosphere microbial communities, with increased bacterial diversity and decreased fungal diversity. Most importantly, S+BOF showed the lowest abundance of Fusarium. Moreover, watermelon quality was higher (P < 0.05) in the S+BOF than in the S+M treatment. Thus, application of the BOF favorably altered the composition of the rhizosphere microbial community, suppressing Fusarium wilt disease and promoting plant quality.

Comparative effects of ethylene inhibitors on Agrobacterium-mediated transformation of drought-tolerant wild watermelon.

Ethylene (C2H4), a phytohormone that is produced in response to both abiotic and biotic stresses, is an important factor influencing the efficiency of Agrobacterium-mediated transformation. In this study, effects of various ethylene inhibitors on the efficiency of Agrobacterium-mediated genetic transformation in drought-tolerant wild watermelon was comparatively examined. Consequently, in comparison to the application of chemical inhibitors such as AgNO3 and aminoethoxyvinylglycine (AVG), lower ethylene level was observed when the infecting Agrobacterium contained a gene for 1-aminocyclopropane-carboxylic acid (ACC) deaminase (acdS), which cleaves ethylene precursor ACC into α-ketobutyrate and ammonia. GUS histochemical and spectrophotometric enzyme assays showed that acdS was more effective in enhancing gene transfer than the chemical ethylene inhibitors. Efficiency of transgenic shoots formation was higher in acdS- and AVG-treated explants. These observations demonstrated that controlling the ethylene level during co-cultivation and shoot formation, particularly using the acdS-harboring Agrobacterium, is advantageous for enhancing the transformation efficiency in this plant.

Identification and expression analyses of WRKY genes reveal their involvement in growth and abiotic stress response in watermelon (Citrullus lanatus).

Despite identification of WRKY family genes in numerous plant species, a little is known about WRKY genes in watermelon, one of the most economically important fruit crops around the world. Here, we identified a total of 63 putative WRKY genes in watermelon and classified them into three major groups (I-III) and five subgroups (IIa-IIe) in group II. The structure analysis indicated that ClWRKYs with different WRKY domains or motifs may play different roles by regulating respective target genes. The expressions of ClWRKYs in different tissues indicate that they are involved in various tissue growth and development. Furthermore, the diverse responses of ClWRKYs to drought, salt, or cold stress suggest that they positively or negatively affect plant tolerance to various abiotic stresses. In addition, the altered expression patterns of ClWRKYs in response to phytohormones such as, ABA, SA, MeJA, and ETH, imply the occurrence of complex cross-talks between ClWRKYs and plant hormone signals in regulating plant physiological and biological processes. Taken together, our findings provide valuable clues to further explore the function and regulatory mechanisms of ClWRKY genes in watermelon growth, development, and adaption to environmental stresses.

Abscisic acid pathway involved in the regulation of watermelon fruit ripening and quality trait evolution.

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.

Feasible sampling plan for Bemisia tabaci control decision-making in watermelon fields.

BACKGROUND: The silverleaf whitefly Bemisia tabaci is one of the most important pests of watermelon fields worldwide. Conventional sampling plans are the starting point for the generation of decision-making systems of integrated pest management programs. The aim of this study was to determine a conventional sampling plan for B. tabaci in watermelon fields. RESULTS: The optimal leaf for B. tabaci adult sampling was the 6th most apical leaf. Direct counting was the best pest sampling technique. Crop pest densities fitted the negative binomial distribution and had a common aggregation parameter (Kcommon ). The sampling plan consisted of evaluating 103 samples per plot. This sampling plan was conducted for 56 min, costing US$ 2.22 per sampling and with a 10% maximum evaluation error. CONCLUSIONS: The sampling plan determined in this study can be adopted by farmers because it enables the adequate evaluation of B. tabaci populations in watermelon fields (10% maximum evaluation error) and is a low-cost (US$ 2.22 per sampling), fast (56 min per sampling) and feasible (because it may be used in a standardized way throughout the crop cycle) technique. © 2017 Society of Chemical Industry.

Genome-wide identification and expression analysis of ClLAX, ClPIN and ClABCB genes families in Citrullus lanatus under various abiotic stresses and grafting.

BACKGROUND: Auxin plays an important role in regulating plant growth and development as well as in the response of plants to abiotic stresses. Auxin is transported by three kinds of major protein families, including the AUXIN RESISTANT 1/LIKE AUX1 (AUX/LAX) influx carriers, the PIN-FORMED (PIN) efflux carriers and the ATP binding cassette B/P-glycoprotein/Multidrug-resistance (ABCB/MDR/PGP) efflux/condition carriers. The biological function of several auxin transporter genes has been well characterized in Arabidopsis thaliana. However, their function in response to exogenous auxin and abiotic stresses in watermelon (Citrullus lanatus. L) remained unknown. RESULTS: Here, the latest updated watermelon genome was used to characterise the ClLAX, ClPIN and ClABCB family genes from watermelon. The genome-wide analysis of the ClLAX, ClPIN and ClABCB family genes, including chromosome localisation, gene structure, and phylogenic relationships, was carried out. Seven ClLAXs, 11 ClPINs and 15 ClABCBs were mapped on 10 watermelon chromosomes. The expression profiles of the ClLAX, ClPIN and ClABCB genes under exogenous indole-3-acetic acid and various abiotic stresses (salt, drought, and cold stresses) treatments were performed by quantitative real-time PCR (qRT-PCR). The transcriptional level of majority ClLAX, ClPIN and ClABCB genes were changed by abiotic stresses in both shoots and roots. We also analysed the expression levels of ClLAX, ClPIN and ClABCB genes in graft response. CONCLUSION: Analysis of the expression patterns of ClLAX, ClPIN and ClABCB genes under salt, drought, cold treatment and grafting response helps us to understand the possible roles of auxin transporter genes in watermelon adaptation to environmental stresses.

Comparative transcriptome profiling of chilling stress responsiveness in grafted watermelon seedlings.

Rootstock grafting may improve the resistance of watermelon plants to low temperatures. However, information regarding the molecular responses of rootstock grafted plants to chilling stress is limited. To elucidate the molecular mechanisms of chilling tolerance in grafted plants, the transcriptomic responses of grafted watermelon under chilling stress were analyzed using RNA-seq analysis. Sequencing data were used for digital gene expression (DGE) analysis to characterize the transcriptomic responses in grafted watermelon seedlings. A total of 702 differentially-expressed genes (DEGs) were found in rootstock grafted (RG) watermelon relative to self-grafted (SG) watermelon; among these genes, 522 genes were up-regulated and 180 were down-regulated. Additionally, 164 and 953 genes were found to specifically expressed in RG and SG seedlings under chilling stress, respectively. Functional annotations revealed that up-regulated DEGs are involved in protein processing, plant-pathogen interaction and the spliceosome, whereas down-regulated DEGs are associated with photosynthesis. Moreover, 13 DEGs were randomly selected for quantitative real time PCR (qRT-PCR) analysis. The expression profiles of these 13 DEGs were consistent with those detected by the DGE analysis, supporting the reliability of the DGE data. This work provides additional insight into the molecular basis of grafted watermelon responses to chilling stress.

In vitro assessment of physiological changes of watermelon (Citrullus lanatus) upon iron oxide nanoparticles exposure.

With the rapid development of nanotechnology, developing nano iron fertilizer is an important strategy to alleviate Fe deficiency and elevate Fe fertilization effect in agricultural applications. In this study, watermelon seedlings were grown in soil amended with iron oxide nanoparticles (γ-Fe2O3 NPs) at different concentrations (0, 20, 50, 100 mg/L). The content of soluble sugar and protein, content of chlorophyll and malondialdehyde (MDA), and activity of antioxidant enzymes of watermelon leaves were determined in five successive weeks to evaluate the physiological changes of watermelon plants after γ-Fe2O3 NPs exposure. Transmission electron microscope (TEM) observations indicated that γ-Fe2O3 NPs could enter root cell of watermelon. Results showed that 20 mg/L γ-Fe2O3 NPs didn't cause any oxidative stress on watermelon and 50 mg/L γ-Fe2O3 NPs could increase soluble sugar, soluble protein and chlorophyll content in the growth of plants. In addition, 50 and 100 mg/L γ-Fe2O3 NPs caused oxidative stress on watermelon leaves, but this NP-induced stress was removed with the growth of watermelon. It is noteworthy that we found γ-Fe2O3 NPs might possess an intrinsic peroxidase-like activity. The variation trend of physiological parameters was correlated with the nutritional requirements of plants. It can be concluded that γ-Fe2O3 NPs at proper concentrations have the ability to improve iron deficiency chlorosis and promote the growth of watermelon plants. To the best of the author's knowledge, this is the first holistic study focusing on the impact of γ-Fe2O3 NPs in long-term experiment of watermelon plants.

Potential involvement of drought-induced Ran GTPase CLRan1 in root growth enhancement in a xerophyte wild watermelon.

Enhanced root growth is known as the survival strategy of plants under drought. Previous proteome analysis in drought-resistant wild watermelon has shown that Ran GTPase, an essential regulator of cell division and proliferation, was induced in the roots under drought. In this study, two cDNAs were isolated from wild watermelon, CLRan1 and CLRan2, which showed a high degree of structural similarity with those of other plant Ran GTPases. Quantitative RT-PCR and promoter-GUS assays suggested that CLRan1 was expressed mainly in the root apex and lateral root primordia, whereas CLRan2 was more broadly expressed in other part of the roots. Immunoblotting analysis confirmed that the abundance of CLRan proteins was elevated in the root apex region under drought stress. Transgenic Arabidopsis overexpressing CLRan1 showed enhanced primary root growth, and the growth was maintained under osmotic stress, indicating that CLRan1 functions as a positive factor for maintaining root growth under stress conditions.

The Ethylene Biosynthesis Gene CitACS4 Regulates Monoecy/Andromonoecy in Watermelon (Citrullus lanatus).

Monoecious and andromonoecious cultivars of watermelon are characterised by the production of male and female flower or male and hermaphrodite flowers, respectively. The segregation analysis in the offspring of crosses between monoecious and andromonoecious lines has demonstrated that this trait is controlled by a single gene pair, being the monoecious allele M semi-dominant to the andromonoecious allele A. The two studied F1 hybrids (MA) had a predominantly monoecious phenotype since both produced not only female flowers, but also bisexual flowers with incomplete stamens, and hermaphrodite flowers with pollen. Given that in other cucurbit species andromonoecy is conferred by mutations in the ethylene biosynthesis genes CmACS7, CsACS2 and CpACS27A we have cloned and characterised CitACS4, the watermelon gene showing the highest similarity with the formers. CitACS4 encoded for a type ACS type III enzyme that is predominantly expressed in pistillate flowers of watermelon. In the andromonoecious line we have detected a missense mutation in a very conserved residue of CitACS4 (C364W) that cosegregates with the andromonoecious phenotype in two independent F2 populations, concomitantly with a reduction in ethylene production in the floral buds that will develop as hermaphrodite flowers. The gene does not however co-segregates with other sex expression traits regulated by ethylene in this species, including pistillate flowering transition and the number of pistillate flowers per plant. These data indicate that CitAC4 is likely to be involved in the biosynthesis of the ethylene required for stamen arrest during the development of female flowers. The C364W mutation would reduce the production of ethylene in pistillate floral buds, promoting the conversion of female into hermaphrodite flowers, and therefore of monoecy into andromonoecy.

The Andromonoecious Sex Determination Gene Predates the Separation of Cucumis and Citrullus Genera.

Understanding the evolution of sex determination in plants requires the cloning and the characterization of sex determination genes. Monoecy is characterized by the presence of both male and female flowers on the same plant. Andromonoecy is characterized by plants carrying both male and bisexual flowers. In watermelon, the transition between these two sexual forms is controlled by the identity of the alleles at the A locus. We previously showed, in two Cucumis species, melon and cucumber, that the transition from monoecy to andromonoecy results from mutations in 1-aminocyclopropane-1-carboxylic acid synthase (ACS) gene, ACS-7/ACS2. To test whether the ACS-7/ACS2 function is conserved in cucurbits, we cloned and characterized ClACS7 in watermelon. We demonstrated co-segregation of ClACS7, the homolog of CmACS-7/CsACS2, with the A locus. Sequence analysis of ClACS7 in watermelon accessions identified three ClACS7 isoforms, two in andromonoecious and one in monoecious lines. To determine whether the andromonoecious phenotype is due to a loss of ACS enzymatic activity, we expressed and assayed the activity of the three protein isoforms. Like in melon and cucumber, the isoforms from the andromonoecious lines showed reduced to no enzymatic activity and the isoform from the monoecious line was active. Consistent with this, the mutations leading andromonoecy were clustered in the active site of the enzyme. Based on this, we concluded that active ClACS7 enzyme leads to the development of female flowers in monoecious lines, whereas a reduction of enzymatic activity yields hermaphrodite flowers. ClACS7, like CmACS-7/CsACS2 in melon and cucumber, is highly expressed in carpel primordia of buds determined to develop carpels and not in male flowers. Based on this finding and previous investigations, we concluded that the monoecy gene, ACS7, likely predated the separation of the Cucumis and Citrullus genera.