A molecular framework of ethylene-mediated fruit growth and ripening processes in tomato

Abstract

Although the role of ethylene in tomato (Solanum lycopersicum) fruit ripening has been intensively studied, its role in tomato fruit growth remains poorly understood. In addition, the relationship between ethylene and the developmental factors NON-RIPENING (NOR) and RIPENING INHIBITOR (RIN) during ripening is under debate. Here, we carried out comprehensive genetic analyses of genome-edited mutants of tomato ETHYLENE INSENSITIVE 2 (SlEIN2), four EIN3-like genes (SlEIL1-4), and three EIN3 BINDING F-box protein genes (SlEBF1-3). Both slein2-1 and the high-order sleil mutant (sleil1 sleil2 sleil3/SlEIL3 sleil4) showed reduced fruit size, mainly due to decreased auxin biosynthesis. During fruit maturation, slein2 mutants displayed the complete cessation of ripening, which was partially rescued by slebf1 but not slebf2 or slebf3. We also discovered that ethylene directly activates the expression of the developmental genes NOR, RIN, and FRUITFULL1 (FUL1) via SlEIL proteins. Indeed, overexpressing these genes partially rescued the ripening defects of slein2-1. Finally, the signal intensity of the ethylene burst during fruit maturation was intimately connected with the progression of full ripeness. Collectively, our work uncovers a critical role of ethylene in fruit growth and supports a molecular framework of ripening control in which the developmental factors NOR, RIN, and FUL1 act downstream of ethylene signaling.

Figure 1

CRISPR/Cas9-mediated mutagenesis of ethylene signaling genes. A, Schematic diagram of the indicated SlEIN2 gene (drawn to scale). Black bars represent exons and lines represent introns. The sequences of the regions targeted by the sgRNAs are shown in the expanded regions. In these sequences, the sgRNAs-target sequences are underlined, and the PAM sequences are colored in purple. Sequences of the isolated mutant alleles are aligned to the WT. The in/del nucleotides are colored. Arrowheads indicate the sites of frameshift in the translated mutant proteins. B, Immunoblotting assay showing the levels of endogenous SlEIN2 and SlEILs proteins in WT and slein2-1 fruits at the MG stage. Actin was used as an internal protein control. C, Relative mRNA levels of four SlEIL (SlEIL1-SlEIL4) genes and three SlEBF (SlEBF1-SlEBF3) genes in WT and slein2-1 fruits at the MG stage. Error bars indicate sd (n = 3), **P < 0.01, Student’s t test. D and E, Triple response assay and statistical analysis. D, WT and slein2-1 seedlings were grown on 1/2 MS medium with or without 5-μM ACC for 3 days in the dark (bar = 1 cm). E, Hypocotyl and root length of 3-day-old seedlings. Error bars indicate sd (n = 24), **P < 0.01, Student’s t test. F, Epinasty assay of WT and slein2-1 plants. One-month-old plants were incubated in 50 ppm ethylene for 24 h. No symptom of epinasty was observed in slein2-1 plants. G and H, Lateral root number of WT and slein2-1. G, 15-day-old seedlings grown on 1/2 MS medium. H, Statistics of lateral root number of WT and slein2-1 seedlings. Error bars indicate sd (n = 24), **P < 0.01, Student’s t test. I, Phenotypes of 80-day-old and 110-day-old WT and slein2-1 plants under natural pollination conditions. J, Representative WT and slein2-1 30-dpa fruits and their respective cross sections (Bar = 1 cm). K, Pollination-independent fruit development of slein2-1. WT and slein2-1 flowers were emasculated at −1 dpa. All 11 emasculated WT ovaries aborted, while 9 of 13 emasculated slein2-1 flowers produced parthenocarpic fruit (Bar = 1 cm). L, Phenotypes of 80-day-old and 110-day-old WT and slein2-1 plants under manual pollination conditions. To eliminate the impact of facultative parthenocarpic fruit, manual pollination was performed for every single WT and slein2-1 flower at the anthesis stage. M, Representative 38 dpa and 52 dpa fruits of WT, sleil1, sleil2, sleil3, sleil4, slebf1, slebf2, slebf3, SlEILs CS, and SlEBF3 OE (Bar = 1 cm).

Figure 2

Ethylene positively regulates fruit growth. A, Cross-fertilization assay. Emasculated WT flowers were fertilized with WT or slein2-1 pollen. Conversely, WT or slein2-1 pollen was used to fertilize emasculated slein2-1 flowers. Each plant bears the same number of fruits. The 45-dpa fruits and statistics of fruit diameters were shown (n = 12, sd). B, Statistics of viable seed number of fruits harvested in (A). The seed number was determined at 55 dpa. For each genotype, n = 12. **P < 0.01, Student’s t test. C, Statistics of WT, slein2-1, sleil1 sleil2 sleil3/SlEIL3 sleil4 fruit diameter at different growth stages (10, 20, 30, and 40 dpa) with manual pollination. Each plant bears the same number of fruits. For each genotype, n = 12. **P < 0.01, Student’s t test. D, Expression data of SlEIN2, SlEIL1, SlEIL2, SlEIL3, and SlEIL4 in pericarps at different growth stages (0, 5, 10, 20, and 30 dpa, MG). Data were extracted from the Tomato Expression Atlas platform (https://tea.solgenomics.net/). RPM, reads per million mapped reads. E, Histological analysis of transverse sections of 5-dpa WT and slein2-1 fruit pericarps, and statistics of their respective number of cell layers (n = 10, SD). Student’s t test. Bar = 20 μm. F, GUS staining analysis of the auxin-responsive promoter DR5 fused to the GUS reporter gene (DR5:GUS) in WT and slein2-1 young fruits (1 and 3 dpa). Black arrowheads indicate pericarps. White arrowheads indicate seeds. G, Relative mRNA levels of three auxin-related genes (SlARF5, SlARF9, and SlGH3.2) and two cell cycle genes (SlCycB1.2 and SlCDKB2.1) in WT and slein2-1 young fruits (3 dpa). Error bars indicate sd (n = 3), **P < .01. Student’s t test. H, IAA content in WT and slein2-1 young fruits (3 dpa). Error bars indicate sd (n = 3), **P < 0.01, Student’s t test. I, Statistics of WT and slein2-1 fruit diameter at 30 dpa and the diameter of 30-dpa slein2-1 fruits treated with 20-μM 2,4-D once a week after fertilization. Error bars indicate sd (n = 12), **P < 0.01, Student’s t test.

Figure 3

rin and slein2-1 fruits show comparable ripening features. A, Different stages of fruit ripening of WT, Nr, rin, and slein2-1. Fruits of rin and slein2-1 show complete ripening cessation. The first signs of carotenoid accumulation on the external surface of the fruit were taken to define the breaker stage. B, Ethylene production of WT, Nr, rin, and slein2-1 fruits at different ripening stages. Error bars indicate sd (n = 12). **P < 0.01, Student’s t test; n.s., not significantly different. C, Carotenoid levels in WT, Nr, rin, and slein2-1 fruits at the Br+10 stage. Error bars indicate sd (n = 3), **P < 0.01, Student’s t test. n.d., not detectable. D–G, Relative mRNA levels of tomato SlACS2 (D), SlACS4 (E), PSY (F), and Pl (G) in WT, Nr, rin, and slein2-1 fruits at different ripening stages. Error bars indicate sd (n = 3), **P < 0.01, Student’s t test. Br + 0, color breaker stage; Br + 3, 3-day postcolor breaker stage; Br + 7, 7-day postcolor breaker stage; Br + 10, 10-day postcolor breaker stage.

Figure 4

rin but not slein2 responds to exogenous ethylene treatment. A, Effect of exogenous ethylene treatment on WT, Nr, rin, and slein2-1 fruits. MG fruits were incubated in a sealed chamber with air or 50 ppm ethylene. Daily observation was performed to identify the days to color breaker, and photographs were taken at the indicated times. B, Statistics of days to color breaker of fruits in (A). Error bars indicate sd (n = 12), **P < 0.01, Student’s t test. C, Relative mRNA levels of SlACS2 and SlACS4 in WT, rin, and slein2-1 fruits responding to ethylene at different stages (RF, Br + 3 fruits). Error bars indicate sd (n = 3), **P < 0.01, Student’s t test. D, Relative mRNA levels of NR, SlETR4, SlEBF2, SlEBF3, E4, and E8 in WT, rin, and slein2-1 fruits responding to ethylene at the MG stage. Error bars indicate sd (n = 3), **P < 0.01, Student’s t test. E, Relative mRNA levels of NR, SlETR4, SlEBF2, SlEBF3, E4, and E8 in WT, rin, and slein2-1 fruits responding to ethylene at the Br + 3 ripening stage. Error bars indicate sd (n = 3), **P < 0.01, Student’s t test.

Figure 5

Ripening-related TF genes act downstream of ethylene signaling during fruit ripening. A, Relative mRNA levels of RIN, FUL1, FUL2, NOR, TAGL1, HB-1, and NOR-like1, in WT, rin, and slein2-1 fruits at the Br + 3 ripening stage. Error bars indicate sd (n = 3), **P < 0.01, Student’s t test. B, EMSA showing the binding of tomato EIL protein SlEIL1 to the promoters of RIN, NOR, and FUL1. Biotin-labeled probes (10 fmol) were used in each reaction. Cold probe indicates unlabeled probe, and 5 pmol of cold probe was used for competition with biotin-labeled probe. C and D, Transient dual-LUC reporter assay illustrating the activation of RIN, NOR, and FUL1 transcription by SlEIL1. Error bars indicate sd (n = 3), **P < 0.01, Student’s t test. E, Representative fruits of the indicated genotypes at the ripening stage. F–H, Relative mRNA levels of ripening-related TF genes (RIN, FUL1, or TAGL1) in WT, slein2, and slein2 35S:NOR (F), slein2 35S:RIN (G), and slein2 35S:FUL1 (H) fruits at the Br + 3 ripening stage. Error bars indicate sd. Different letters above the bars indicate statistically significant differences between the samples (n = 3, P < 0.01, Student’s t test).

Figure 6

The signal intensity of system-2 ethylene mediates the efficiency of the tomato ripening machinery. A, Statistics of days from anthesis to the color breaker stage in fruits of the WT and corresponding mutant alleles. Error bars indicate sd. Different letters above the bars indicate statistically significant differences between the samples (n = 20, P < 0.01, Student’s t test). B, Relative mRNA levels of ripening-associated ethylene signaling marker genes E4 and E8 in WT, sleil1, sleil1 sleil4, sleil1 sleil3 sleil4, and slein2-1 fruits at the Br + 10 stage. Error bars indicate sd. Different letters above the bars indicate statistically significant differences between the samples (n = 3, P < 0.01). C, Statistics of days from the color breaker to red ripening stage in WT, sleil1, sleil1 sleil4, sleil1 sleil3 sleil4, and slein2-1 fruits. Error bars indicate sd. Different letters above the bars indicate statistically significant differences between the samples (n = 20, P < 0.01, Student’s t test). n.d., no data available. D, Ethylene production of WT, sleil1, sleil1 sleil4, and sleil1 sleil3 sleil4 fruits at different ripening stages. Error bars indicate sd (n = 12). E and F, Relative mRNA levels of SlACS2 (E) and RIN (F) of WT, sleil1, sleil1 sleil4, and sleil1 sleil3 sleil4 fruits at different ripening stages. Error bars indicate sd. Different letters above the bars indicate statistically significant differences between the samples (n = 3, P < 0.01, Student’s t test). G, Br + 10 fruits that were harvested from WT, sleil1, sleil1 sleil4, sleil1 sleil3 sleil4, and slein2-1 plants and stored at room temperature for two months. H, Physiological loss of water (weight loss %) in WT, sleil1, sleil1 sleil4, sleil1 sleil3 sleil4, and slein2-1 fruits during different stages of storage. The weight loss per fruit was calculated at 10, 20, 30, and 60 days after storage. Error bars indicate sd. Different letters above the bars indicate statistically significant differences between the samples (n = 20, P < 0.01, Student’s t test).