Angela R. Davis

Gene expression in developing watermelon fruit

BackgroundCultivated watermelon form large fruits that are highly variable in size, shape, color, and content, yet have extremely narrow genetic diversity. Whereas a plethora of genes involved in cell wall metabolism, ethylene biosynthesis, fruit softening, and secondary metabolism during fruit development and ripening have been identified in other plant species, little is known of the genes involved in these processes in watermelon. A microarray and quantitative Real-Time PCR-based study was conducted in watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai var. lanatus] in order to elucidate the flow of events associated with fruit development and ripening in this species. RNA from three different maturation stages of watermelon fruits, as well as leaf, were collected from field grown plants during three consecutive years, and analyzed for gene expression using high-density photolithography microarrays and quantitative PCR.ResultsHigh-density photolithography arrays, composed of probes of 832 EST-unigenes from a subtracted, fruit development, cDNA library of watermelon were utilized to examine gene expression at three distinct time-points in watermelon fruit development. Analysis was performed with field-grown fruits over three consecutive growing seasons. Microarray analysis identified three hundred and thirty-five unique ESTs that are differentially regulated by at least two-fold in watermelon fruits during the early, ripening, or mature stage when compared to leaf. Of the 335 ESTs identified, 211 share significant homology with known gene products and 96 had no significant matches with any database accession. Of the modulated watermelon ESTs related to annotated genes, a significant number were found to be associated with or involved in the vascular system, carotenoid biosynthesis, transcriptional regulation, pathogen and stress response, and ethylene biosynthesis. Ethylene bioassays, performed with a closely related watermelon genotype with a similar phenotype, i.e. seeded, bright red flesh, dark green rind, etc., determined that ethylene levels were highest during the green fruit stage followed by a decrease during the white and pink fruit stages. Additionally, quantitative Real-Time PCR was used to validate modulation of 127 ESTs that were differentially expressed in developing and ripening fruits based on array analysis.ConclusionThis study identified numerous ESTs with putative involvement in the watermelon fruit developmental and ripening process, in particular the involvement of the vascular system and ethylene. The production of ethylene during fruit development in watermelon gives further support to the role of ethylene in fruit development in non-climacteric fruits.

Genes expressed during the development and ripening of watermelon fruit

A normalized cDNA library was constructed using watermelon flesh mRNA from three distinct developmental time-points and was subtracted by hybridization with leaf cDNA. Random cDNA clones of the watermelon flesh subtraction library were sequenced from the 5′ end in order to identify potentially informative genes associated with fruit setting, development, and ripening. One-thousand and forty-six 5′-end sequences (expressed sequence tags; ESTs) were assembled into 832 non-redundant sequences, designated as “EST-unigenes”. Of these 832 “EST-unigenes”, 254 (∼30%) have no significant homology to sequences published so far for other plant species. Additionally, 168 “EST-unigenes” (∼20%) correspond to genes with unknown function, whereas 410 “EST-unigenes” (∼50%) correspond to genes with known function in other plant species. These “EST-unigenes” are mainly associated with metabolism, membrane transport, cytoskeleton synthesis and structure, cell wall formation and cell division, signal transduction, nucleic acid binding and transcription factors, defense and stress response, and secondary metabolism. This study provides the scientific community with novel genetic information for watermelon as well as an expanded pool of genes associated with fruit development in watermelon. These genes will be useful targets in future genetic and functional genomic studies of watermelon and its development.

EST-PCR markers representing watermelon fruit genes are polymorphic among watermelon heirloom cultivars sharing a narrow genetic base

To date, there are only a few sequenced-tagged site (STS) markers associated with genes controlling fruit quality in watermelon. A normalized cDNA library for watermelon fruit ( Citrullus lanatus var . lanatus ) was constructed. Sequence analysis of the cDNA clones resulted in the development of 4700 non-redundant ESTs (EST unigenes) expressed in watermelon fruit ( http://www.ncbi.nlm.nih.gov , http://www.icugi.org ). One hundred of these EST unigenes [including 40 EST unigenes that contain simple sequence repeat (SSR) motives (EST-SSRs) and 60 customary EST unigenes (not containing SSR motives)] were used for designing primer pairs for PCR experiments. The EST primer pairs were tested in PCR experiments with genomic DNA of 25 watermelon heirloom cultivars and 13 United States Plant Introductions (US PIs) of Citrullus sp., including four C. lanatus var. lanatus , five C. lanatus var. citroides PIs and four Citrullus colocynthis PIs. The 40 EST-SSR and 60 EST primer pairs produced 108 and 142 EST-PCR markers, respectively, among the Citrullus PIs and watermelon cultivars. A large number of the EST-PCR markers were polymorphic between the Citrullus PIs and the watermelon cultivars, but significantly less polymorphic among the cultivars. Of the 108 EST-PCR markers associated with EST-SSRs, 103 exist in the Citrullus PIs and 64 in the cultivars. Of these 64 markers, 45 (70.3%) were polymorphic among the cultivars. Of the 142 EST-PCR markers associated with customary ESTs, 134 exist in the Citrullus PIs and 108 in the cultivars. Of these 108 markers, 86 (79.6%) were polymorphic among cultivars. The results in this study indicate that polymorphism exists in coding regions of genes expressed in fruits of watermelon cultivars. In total, 131 polymorphic EST-PCR markers (45 associated with EST-SSRs and 86 associated with customary ESTs) related to watermelon fruit genes were generated using the above data. These markers should be useful for DNA fingerprinting of cultivars and breeding lines, for assessing genetic relationships and for genetic mapping of watermelon.

The Purification, Physical/Chemical Characterization, and cDNA Sequence of Cantaloupe Fruit Polygalacturonase-Inhibiting Protein

ABSTRACT Polygalacturonase-inhibiting proteins (PGIPs) are believed to aid in plant defense against fungal pathogens by inhibiting polygalacturonases (PGs) secreted by the invading organism. In an effort to better understand this type of plant-pathogen interaction in cucurbits, we have isolated a cantaloupe PGIP (CmPGIP) from 5 to 15 day postanthesis cantaloupe fruit. CmPGIP inhibited crude extracts of PG from two of four fungal pathogens of cantaloupe that were tested. Results from assays for PG activity that utilized rate of substrate viscosity reduction or rate of reducing group formation were consistent with CmPGIP inhibition of endo-PG activity. The M(r) of CmPGIP by sedimentation equilibrium or MALDITOF MS was 38,500. The pI of CmPGIP was approximately 8.2, and its absorptivity at 280 nm was 0.93 ml/mg. The circular dichroism spectrum of native CmPGIP exhibited strong negative ellipticity in the near UV and possessed a far UV spectrum indicative of beta-sheet periodic structure. Amino acid sequences of the N terminus and a cyanogen bromide peptide were used to construct oligonucleotide primers for polymerase chain reaction sequencing. The sequenced open reading frame predicts a mature protein of 307 amino acids with up to 68% identity to other PGIP molecules. Northern blot analysis revealed differential expression during fruit development. The isolation and structural information obtained for CmPGIP by this investigation provide a foundation for the development of molecular strategies for pre- and postharvest crop protection.

Watermelon: From dessert to functional food

Watermelon in the US traditionally has been viewed as a sweet but non-nutritional fruit. Over the last ten years, we have delved into the world of watermelon to find the horticultural, genetic, and environmental linkages of compounds with demonstrated bioactivity in animal models and clinical trials. Watermelon contains large amounts of lycopene and citrulline in addition to ascorbic acid, potassium, flavonoids, and beta carotene. Germplasm greatly influences lycopene content (<1 to 120 mg/kg fresh weight) while environmental effects can enhance pigment content by 10 to 20%. In contrast, the influences of germplasm and environment on citrulline content are less clear, with amounts reported from 0.9 to 4.3 mg/kg fresh weight. Watermelon is now recognized as a horticultural crop providing important nutritional and bioactive benefits.

Consumer acceptability of low-sugar watermelon sweetened with non-calorie sweetener by a Native American community

Watermelons are a good source of lycopene, a carotenoid that exhibits antioxidant activity and may protect against some cancers. However, intake of watermelon may be restricted for individuals who have diabetes or those who limit carbohydrate intake. A low-sugar watermelon was developed at Lane, Oklahoma using traditional plant breeding techniques. The objective of this study was to determine whether the artificially sweetened low-sugar watermelon was acceptable with Native Americans, a group with a high incidence of diabetes. The red flesh from a low-sugar watermelon and a commercial variety of watermelon was removed and cut into cubes. Low and high levels of artificial sweetener were added to the low-sugar watermelon. Students at a Native American school (Grades 1–12) and adults at a Native American Feeding Center were asked to rate how much they liked or disliked the watermelon using a seven-point hedonic scale. Sugar composition, pH, lycopene and other carotenoids were analyzed from samples using established methods. The pH, lycopene, β-carotene and total carotenoid levels were similar among fruit. Artificially sweetened fruit were rated slightly more acceptable in taste than the commercial control watermelons by both age groups. The low-sugar watermelons were lower in sugar composition but were comparable with conventional melons in all other quality factors and were found acceptable in taste by a broad age group of Native American consumers.