Ethylene insensitivity conferred by the Green-ripe and Never-ripe 2 ripening mutants of tomato

Abstract

The ripening of a fleshy fruit represents the summation of an array of biochemical processes that are regulated by interactions between developmental programs and environmental inputs. Analysis of tomato (Solanum lycopersicum) mutants and inhibitor studies indicate that ethylene is necessary for full development of the ripening program of climacteric fruit such as tomato, yet ethylene alone is not sufficient. This suggests that an interaction between ethylene and nonethylene (or developmental) pathways mediates ripening. In this study, we have examined the physiological basis for ripening inhibition of the dominant Green-ripe (Gr) and Never-ripe 2 (Nr-2) mutants of tomato. Our data suggest that this inhibition is due to ethylene insensitivity in mutant fruit. Further investigation of ethylene responses in Gr and Nr-2 plants also revealed weak ethylene insensitivity during floral senescence and abscission and, during inhibition of root elongation, a phenotype associated with the triple response. However, ethylene-induced inhibition of hypocotyl elongation and petiole epinasty are normal in Gr and Nr-2, suggesting that these loci regulate a subset of ethylene responses. We have mapped both dominant mutations to a 2-cM overlapping region of the long arm of chromosome 1 of tomato, a region not previously linked to any known ethylene signaling loci. The phenotypic similarity and overlapping map location of these mutations suggest Gr and Nr-2 may be allelic and may possibly encode a novel component of the ethylene response pathway.

Figure 1.

Inhibition of fruit ripening in Gr and Nr-2 mutants. A, Fruit phenotype of the accessions LA2453 (Gr/Gr) and LA2455 (Nr-2/Nr-2) at the ripe stage of development. Fruit were photographed at 60 DPA. B, Ripening phenotype of AC, Gr, Nr-2, and Nr NILs. Photograph shows the normal pattern of ripening in AC fruits from the onset of ripening until the red ripe stage in comparison to mutant NILs. Fruits were harvested based on the accumulation of carotenoid pigments. Note that ripening inhibition is most severe in the Nr-2 NIL. Fruits are not comparable ages. The red ripe AC fruit is at 42 DPA, whereas mutant fruits with the most developed ripening are at 60 DPA.

Figure 2.

Characterization of ethylene synthesis and response in Gr and Nr-2. A, Ethylene production in control AC, Gr, and Nr-2 NILs during fruit ripening. Fruit of different developmental stages (1–5) as defined in the methods were sealed in airtight jars and a 1-mL air sample was taken from the jars after 2 h and sampled for ethylene. Values represent means of at least eight individual fruit. Vertical bars represent se. B, Ripening-related gene expression in AC, Gr, and Nr-2 NILs during fruit ripening. Total RNA was extracted from fruit of stages 1 to 5 as defined above and 10 μg was subjected to RNA gel-blot analysis and hybridized to DNA probes for the ripening-related genes E4, E8, polygalacturonase, and phytoene synthase (PSY1). C, Ethylene responsiveness of AC, Gr, Nr-2, and Nr mature green fruits. Total RNA was extracted from mature green fruits of each genotype treated with 20 μL L⁻¹ of ethylene (+) or held in air (−) for 16 h. A total of 15 μg was subjected to RNA gel-blot analysis and hybridized to a DNA probe for the ethylene-regulated gene E4.

Figure 3.

Seedling triple response and petiole epinasty in AC, Gr, Nr-2, and Nr. A, Triple response phenotype of AC, Gr, Nr-2, and Nr seedlings. Seeds were surface sterilized and sown on 1% water agar in the absence (−) or presence (+) of 10 μm ACC and incubated at 25°C in the dark for 7 d. Quantification of ethylene-induced inhibition of hypocotyl (B) and root (C) lengths. Wild-type and mutant seedlings were sterilized and grown as in A. Data is presented as the mean of at least 21 individual seedlings. Vertical bars represent se. D, Dose response curve of hypocotyl growth on ACC. Seeds were treated and grown as in A, except that five different ACC concentrations (0, 0.2, 0.5, 1, and 10 μm) were used. Following growth in the dark for 7 d, hypocotyl lengths were determined. Each data point is the result of measurements of at least 24 seedlings. Vertical bars represent se. E, Ethylene responsiveness of AC, Gr, Nr-2, and Nr seedlings. Total RNA was extracted from 7-d-old dark grown seedlings grown in the presence (+) or absence (−) of 10 μm ACC. A total of 20 μg was subjected to RNA gel-blot analysis and hybridized to a DNA probe for the ethylene-regulated gene E4. F, Petiole epinasty in AC, Gr, Nr-2, and Nr plants. Four-week-old plants were sealed in airtight chambers for 16 h in the presence of 20 μL L⁻¹ of ethylene.

Figure 4.

Ethylene responsiveness of the pedicel abscission zone in AC, Gr, Nr-2, and Nr. Flower trusses were removed from plants and the cut ends immersed in water in conical flasks. Flasks were sealed in airtight chambers in 20 μL L⁻¹ of ethylene and the percentage abscission was monitored every 24 h for 3 d. Data shown is derived from the average of three independent experiments. Total number of flowers examined for each genotype ranged from 134 to 163. Vertical bars represent se.

Figure 5.

Genetic mapping of Gr and Nr-2 loci to tomato chromosome 1. Populations were constructed as described in the text. The Gr map is based upon a population of 220 F₂ individuals. The Nr-2 map is based upon a population of 438 F₂ individuals. The position of each locus between the flanking markers TG260 and TG245 is indicated by hatched bars and the distances between these markers for each population is given in centiMorgans. RFLP markers are depicted on the right of each map and the number of recombinant plants between each adjacent marker is shown on the left.