The tomato FRUITFULL homologs TDR4/FUL1 and MBP7/FUL2 regulate ethylene-independent aspects of fruit ripening

Abstract

Tomato (Solanum lycopersicum) contains two close homologs of the Arabidopsis thaliana MADS domain transcription factor FRUITFULL (FUL), FUL1 (previously called TDR4) and FUL2 (previously MBP7). Both proteins interact with the ripening regulator RIPENING INHIBITOR (RIN) and are expressed during fruit ripening. To elucidate their function in tomato, we characterized single and double FUL1 and FUL2 knockdown lines. Whereas the single lines only showed very mild alterations in fruit pigmentation, the double silenced lines exhibited an orange-ripe fruit phenotype due to highly reduced lycopene levels, suggesting that FUL1 and FUL2 have a redundant function in fruit ripening. More detailed analyses of the phenotype, transcriptome, and metabolome of the fruits silenced for both FUL1 and FUL2 suggest that the genes are involved in cell wall modification, the production of cuticle components and volatiles, and glutamic acid (Glu) accumulation. Glu is responsible for the characteristic umami taste of the present-day cultivated tomato fruit. In contrast with previously identified ripening regulators, FUL1 and FUL2 do not regulate ethylene biosynthesis but influence ripening in an ethylene-independent manner. Our data combined with those of others suggest that FUL1/2 and TOMATO AGAMOUS-LIKE1 regulate different subsets of the known RIN targets, probably in a protein complex with the latter.

Figure 1.

FUL1 and FUL2 Expression in Wild-Type Tomato and FUL1 RNAi Lines, and Interaction of the FUL Proteins with RIN. (A) Relative expression profiles of FUL1 and FUL2 in different MT tissues obtained by qRT-PCR. FL, flower; I, 5-mm fruits; II, 1-cm fruits; L, leaf; FL, flower; I, 5mm fruits; II, 1cm fruits; MG, mature green; Br, breaker; Br+7, 7 days after breaker stage (red ripe). (B) Relative expression profiles of SlFUL1 and SlFUL2 in separated fruit tissues of red ripe fruits. S = seeds; Pu = pulp; C = columella; Pl = Placenta; Sp = septa; M = mesocarp; E+P = exocarp and peel. (C) Confocal scanning laser microscopy pictures of Arabidopsis protoplasts transfected with split-YFP constructs. Top panel, RIN-YFP(C) and FUL1-YFP(N); bottom panel, RIN-YFP(C) and FUL2-YFP(N). (D) Downregulation of FUL1 and FUL2 in the FUL1 RNAi lines R1-10, R1-25, R1-29, and R1-30 compared with the wild type (WT). The relative expression levels were obtained by qRT-PCR. The error bars in (A) to (C) indicate the se based on two biological replicas.

Figure 2.

Phenotypes of the FUL1 and FUL2 Downregulated Fruits. (A) Fruits of FUL1 RNAi lines R1-10 and R1-25 and wild-type (WT) MT at stage Br + 7. (B) Comparison of the carotenoid levels in wild-type and R1-10 fruits at stage MG, B+7 (Br + 7 d), and B+10 (Br + 10 d). AU, absorbance units; FW, fresh weight. The complete analysis is presented in Supplemental Table 1 online. (C) Binocular microscopy pictures of handmade pericarp sections of wild-type and R1-10 fruits at stage Br + 7. (D) Water loss in wild-type and R1-10 and R1-29 fruits after harvesting at stage Br + 7. The y axis depicts the percentage of weight loss since the day of harvesting. (E) Cuticle thickness of wild-type and R1-10 Br + 7 fruits is visible in light microscopy pictures of thin pericarp sections stained with Sudan IV. The side panels show the desiccated tomatoes 40 d after harvesting. (F) Cuticle thickness in wild-type and R1-10 Br + 7 fruits, measured from the top of the epidermal cell to the surface. For both the wild type and R1-10, the cuticles of four different fruits were measured. Error bars indicate the se. (G) Phenotype of line R1-10 in the Ailsa Craig background. FUL1 RNAi line R1-10 was crossed with Ailsa Craig wild type, and the offspring (harboring the MT mutations recessively) were phenotyped. (H) qRT-PCR analysis showing the downregulation of FUL1 and FUL2 in transgenic lines carrying the FUL2 RNAi construct (R2-44 and R2-45) or the specific 3′ UTR constructs for FUL1 (S1-46 and S1-52) or FUL2 (S2-15 and S2-17). The error bars depict the se based on two biological replicas. (I) Jelly of red ripe fruits from lines R2-40, S1-46, S2-17, and Moneyberg wild type. (J) Red ripe fruits of lines R2-44, S1-46, S2-17, and Moneyberg wild type. Note the variation in pigmentation at the blossom end of the transgenic fruits.

Figure 3.

The Link between FUL1/2 and Ethylene, and the Expression of FUL1, FUL2, and TAGL1 in the cnr and rin Mutants. (A) Ethylene concentrations in wild-type (WT) and FUL1 RNAi Br and Br + 3 stage fruits. Error bars indicate the se based on at least four fruits (B) Relative expression of FUL1 and FUL2 in MG fruits after 6 h of treatment with or without ethephon. (C) Relative expression of FUL1, FUL2, and TAGL1 in different stages of Cnr mutant fruits compared with the wild type. (D) Relative expression of FUL1, FUL2, and TAGL1 in different stages of rin mutant fruits compared with the wild type. In (C) and (D), the expression in the MG wild-type stage is set to 1.0 for each individual gene; the expression levels of the different genes cannot be compared with each other. Because the relative fold differences for FUL2 and TAGL1 ([C] and [D]) are hard to read from the large graphs, these data are also presented in separate inserts in the corresponding panels. Error bars in (B) to (D) indicate the se based on two biological replicates.

Figure 4.

Simplified Model of the Fruit Ripening Regulatory Network in Tomato. The connections between the different regulators are based on expression data from this study and previously published work (Eriksson et al., 2004; Giovannoni, 2007; Vrebalov et al., 2009; Itkin et al., 2009; Klee and Giovannoni, 2011; Martel et al., 2011). RIN functions as a dimer together with either TAGL1 or FUL1/2 to regulate complementary sets of downstream ripening genes. The positive interaction between RIN and TAGL1 is not clear (our expression data show a small downregulation of TAGL1 in the rin mutant only at Br stage). However, this interaction may be indirect via CNR and is therefore depicted with a dashed line. Most likely, RIN is also part of a complex when upregulating CNR, TAGL1, and FUL1/2. However, because the nature of this complex is still unclear, only RIN has been depicted in the white box.