Abstract
BackgroundFruit ripening and softening are key traits that have an effect on food supply, fruit nutritional value and consequently, human health. Since ethylene induces ripening of climacteric fruit, it is one of the main targets to control fruit over ripening that leads to fruit softening and deterioration. The characterization of the ethylene pathway in Arabidopsis and tomato identified key genes that control fruit ripening.Methodology/Principal FindingsTo engineer melon fruit with improved shelf-life, we conducted a translational research experiment. We set up a TILLING platform in a monoecious and climacteric melon line, cloned genes that control ethylene production and screened for induced mutations that lead to fruits with enhanced shelf life. Two missense mutations, L124F and G194D, of the ethylene biosynthetic enzyme, ACC oxidase 1, were identified and the mutant plants were characterized with respect to fruit maturation. The L124F mutation is a conservative mutation occurring away from the enzyme active site and thus was predicted to not affect ethylene production and thus fruit ripening. In contrast, G194D modification occurs in a highly conserved amino acid position predicted, by crystallographic analysis, to affect the enzymatic activity. Phenotypic analysis of the G194D mutant fruit showed complete delayed ripening and yellowing with improved shelf life and, as predicted, the L124F mutation did not have an effect.Conclusions/SignificanceWe constructed a mutant collection of 4023 melon M2 families. Based on the TILLING of 11 genes, we calculated the overall mutation rate of one mutation every 573 kb and identified 8 alleles per tilled kilobase. We also identified a TILLING mutant with enhanced fruit shelf life. This work demonstrates the effectiveness of TILLING as a reverse genetics tool to improve crop species. As cucurbits are model species in different areas of plant biology, we anticipate that the developed tool will be widely exploited by the scientific community.
Highlights
Melon (Cucumis melo) belongs to the Cucurbitaceae family that contains about 800 species mainly distributed in tropical and subtropical regions [1]
Low seed production was observed at 3% ethyl methanesulfonate (EMS) with 37% of the plants producing less than 40 seeds per fruit
Female flowers were hand pollinated with male flowers from the same plants, bagged and the fruits left to develop to maturity
Summary
Melon (Cucumis melo) belongs to the Cucurbitaceae family that contains about 800 species mainly distributed in tropical and subtropical regions [1]. The most common techniques to produce altered or loss of function mutations are T-DNA or transposon insertional mutagenesis [18] and RNA interference [19]. Because these methods are mainly based on the ability of a given plant to be transformed, their usefulness as general reverse genetics methods is limited to very few plant species. TILLING (Targeting Induced Local Lesions IN Genomes) combines advantages of random chemical mutagenesis and high throughput mutation. The characterization of the ethylene pathway in Arabidopsis and tomato identified key genes that control fruit ripening
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