Engineering Melon Plants with Improved Fruit Shelf Life Using the TILLING Approach

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.

Regulation of ethylene biosynthesis at the level of 1-aminocyclopropane-1-carboxylate oxidase (ACO) gene

“…A marker-and vector-free antisense ACO construct was successfully transformed into melon via the pollen-tube pathway generating transgenic fruits with superior characteristics of greatly reduced respiration rates and endogenous ethylene production thus promising a significantly prolonged shelf-life compared to wild-type ones. A different strategy, totally avoiding plant transformation, was proposed by Dahmani-Mardas et al (2010). They were also able to engineer melon plants with improved fruit shelf-life by means of targeting induced local lesions IN genome (TILLING) which combines advantages of random chemical mutagenesis and high throughput mutation discovery methods.…”

Role of ethylene receptors during senescence and ripening in horticultural crops

“…The approach has been put to use in different crops like tomato, tobacco, potato, carnation, begonia, torenia, cantaloupe, broccoli, canola, melon, etc. Reverse genetics approach using TILLING platform was exploited in melon to identify novel alleles involved in ripening by Mardas et al, 35 Blocking ethylene production by targeting enzymes has been an approach to combat senescence to good extent but with contraindicated repercussions. Another approach that has gathered attention in recent years is reducing the sensitivity of plant for ethylene without altering the biosynthetic pathway.…”

The genome of melon ( Cucumis melo L.)

“…It is particularly important in Mediterranean and East Asian countries, where hybrid varieties have a significant and growing economic value. In line with the scientific and economic interest of the species, a number of genetic and molecular tools have been developed over the last years, including genetic maps (7), ESTs (http://www.icugi.org), microarrays (8), a physical map (9), BAC sequences (10), and reverse genetic tools (11,12). To complete the repertoire of genomic tools, de novo sequencing of the melon genome was undertaken with 454 pyrosequencing.…”