Fleshy fruit expansion and ripening are regulated by the Tomato SHATTERPROOF gene TAGL1

Abstract

The maturation and ripening of fleshy fruits is a developmental program that synchronizes seed maturation with metabolism, rendering fruit tissues desirable to seed dispersing organisms. Through RNA interference repression, we show that Tomato AGAMOUS-LIKE1 (TAGL1), the tomato (Solanum lycopersicum) ortholog of the duplicated SHATTERPROOF (SHP) MADS box genes of Arabidopsis thaliana, is necessary for fruit ripening. Tomato plants with reduced TAGL1 mRNA produced yellow-orange fruit with reduced carotenoids and thin pericarps. These fruit are also decreased in ethylene, indicating a comprehensive inhibition of maturation mediated through reduced ACC Synthase 2 expression. Furthermore, ectopic expression of TAGL1 in tomato resulted in expansion of sepals and accumulation of lycopene, supporting the role of TAGL1 in ripening. In Arabidopsis, the duplicate SHP1 and SHP2 MADS box genes regulate the development of separation layers essential for pod shatter. Expression of TAGL1 in Arabidopsis failed to completely rescue the shp1 shp2 mutant phenotypes, indicating that TAGL1 has evolved distinct molecular functions compared with its Arabidopsis counterparts. These analyses demonstrate that TAGL1 plays an important role in regulating both fleshy fruit expansion and the ripening process that together are necessary to promote seed dispersal of fleshy fruit. From this broad perspective, SHP1/2 and TAGL1, while distinct in molecular function, regulate similar activities via their necessity for seed dispersal in Arabidopsis and tomato, respectively.

Figure 1.

Expression of TAGL1 and TAG1 in Tomato Fruit Tissues and in Response to Ethylene. (A) Total RNA gel blot analysis of TAGL1, TAG1, and 18S rRNA (control). r, roots from 9-d-old seedlings, os, primary stem of 6-week-old plants; ys, primary stem of 3-week-old plants; co, cotyledons of 9-d-old seedlings; h, hypocotyls of 9-d-old seedlings; l, leaves of 6-week-old plants; p, anthesis-stage floral pedicel; s, anthesis sepals; pe, anthesis petals; st, anthesis stamens; sty, anthesis style/stigma; c, anthesis carpels. (B) Wild-type, rin, nor, and Nr fruit RNAs: 1, MG (mature green); 2, MGE (MG + 10 ppm ethylene for 12 h); 3, BR (breaker; i.e., early ripening); 4, BR +7d (red ripe in the wild type). (C) RNAs from independently transformed TAGL1 RNAi transgenic lines are designated transgenic lines TAGL1-1, TAGL1-11, and TAGL1-12, respectively. Fruit RNAs: 1, IM (28 DPA); 2, MG; 3, BR; 4, BR +7d (red ripe in the wild type). (D) Lanes 1 to 4 are combined anthesis-stage stamens and style/stigma from TAGL1-1, 11, 12, and the wild type, respectively. Lanes 5 to 8 are anthesis-stage carpels from TAGL1-1, 11, 12, and the wild type, respectively.

Figure 2.

In Situ Expression Analysis of TAGL1 in Wild-Type Floral Buds and Fruit. (A) TAGL1 expression is first detected in stage 2 to 3 floral buds in presumptive stamen and carpel primordia. (B) By stage 5, TAGL1 is expressed in stamen and carpel primordia. (C) At stage 9, expression in stamens is reduced, with TAGL1 expression seen in developing ovules and stigmas of carpels. (D) Stage 12 to 13 carpel with TAGL1 expression detected in the placenta and weakly in ovules. (E) Cross section of a flower at stage 9. TAGL1 is expressed in the styles of the carpel and weakly in stamens. (F) Sense control on stage 9 floral bud. (G) and (H) In 0 DPA fruit, TAGL1 is expressed in the placenta, areas around vascular bundles, and weakly in the pericarp.

Figure 3.

Parsimony Analysis of AG Clade MADS Box Proteins. Consensus tree with bootstrap values greater than or equal to 50% is shown. Tomato proteins are indicated in red and Arabidopsis proteins in green. Gymnosperm sequences were used as outgroups in the analysis. Species names are abbreviated as follows; accession numbers can be found in Supplemental Table 1 online. Asterids: Am, Antirrhinum majus; Ph, Petunia hybrida; Pi, Petunia inflata; Nt, Nicotiana tabacum; Le, Solanum lycopersicum; Dc, Daucus carota; Pg, Panax ginseng; Gh, Gerbera hybrida; Ha, Helianthus annus; Cm, Chrysanthemum × morifolium; In, Ipomoea nil; Eg, Eustoma grandiflorum. Rosids: At, Arabidopsis thaliana; Bn, Brassica napus; Br, Brassica rapa; Cs, Cucumis sativus; Rr, Rosa rugosa; Md, Malus domestica; Jr, Juglans regia; Ca, Corylus pendula; Bp, Betula pendula; Gh, Gossypium hirsutum; Fa, Fragaria × ananassa; Pt, Populus trichocarpa; Mc, Momordica charantia. Vitaceae: Vv, Vitis vinifera. Caryophyllales: Sl, Silene latifolia; Ra, Rumex acetosa; Pha, Phytolacca americana. Saxifragales: Sxc, Saxifraga caryana; La, Liquidamber styraciflua. Sabiaceae: Md, Meliosma dilleniifolia (AG1 and AG2). Ranunculales: Rf, Ranunculales ficaria; Ho, Helleborus orientalis; Cli, Clematis integrifolia; Aqa, Aquilegia alpina; Thd, Thalictrum dioicum; Bg, Berberis gilgiana; Akq, Akebia quinata; Sc, Sanguinaria canadensis. Magnoliales: Mp, Magnolia precossimina. Piperales: Srh, Saruma henryii; Htc, Houttuynia cordata; Ac, Asarum caudigerum. Chloranthaceae: Cs, Chloranthus spicatus (AG1). Liliales: Ll, Lilium longiflorum. Asparagales: Pe, Phalaenopsis equestris; Ho, Hyacinthus orientalis; Ap, Agapanthus praecox; Av, Asparagus virgatus; Cs, Crocus sativus. Poales: Hv, Hordeum vulgare; Ta, Triticum aestivum; Os, Oryza sativa; Zm, Zea mays. Nymphaeales: Nym, Nymphaea sp. Gingkgoales: Gb, Ginkgo biloba. Cycadales: Ce, Cycas edentata. Gnetales: Gg, Gnetum gnemon. Coniferales: Pm, Picea mariana; Pr, Pinus resinosa.

Figure 4.

TAGL1 Repression Phenotypes. Genotypes are TAGL1 RNAi lines (TAGL-1, 11, and 12) and wild-type Ailsa Craig (AC+/+). TAGL1 RNAi repression alters pericarp (fruit outer wall) thickness, ripening-related fruit pigmentation (A), and style trichome density (B).

Figure 5.

Carotenoid Accumulation Profiles in TAGL1 RNAi Fruit Result in Part from Altered Lycopene-β-Cyclase Expression. (A) HPLC analysis of carotenoid accumulation in BR+7 (Breaker plus 7 d) fruit of transgenic TAGL1 RNAi lines presented as percent of control (cv Ailsa Craig [AC]). Standard error is indicated for a minimum of six fruit per sample. (B) Relative quantitative RT-PCR expression analysis of the chloroplast (LYC-B) and chromoplast (CYC-B) lycopene-β-cyclase genes during fruit development show both are upregulated in ripening stage TAGL1 RNAi fruit, accounting for the metabolism of lycopene to β-carotene and lutein.

Figure 6.

Physiological, Morphological, and Metabolic Characterization of TAGL1 RNAi Fruit. (A) Ethylene production (nl/g/h) of transgenic and control fruit at the indicated days after breaker. Standard error is indicated, and a minimum of six fruit per sample were analyzed. (B) Water loss of transgenic and control fruit over 21 d starting from breaker stage expressed as percentage of original weight. (C) Ailsa Craig wild-type and TAGL1 RNAi fruit at 30 d after breaker. (D) Pericarp thickness of breaker (38 DPA) fruit. (E) Average number of cell layers in immature (28 DPA) and breaker (38 DPA) pericarp. (F) Pericarp firmness as measured by compression in immature (28 DPA) and breaker (38 DPA) fruit. (G) Breaker stage fruit fresh weight. (H) HPLC of immature (28 DPA) fruit total chlorophyll (Total Chl) and lutein concentrations. FW, fresh weight. Standard error is indicated for a minimum of six fruit per sample in (A) and (H), 10 fruit per sample in (B) and (G), and with a minimum of three fruit per sample in (D) to (F) (though multiple measurements were made on each sample in [D] to [F]. See Methods for details).

Figure 7.

Ripening and Starch Metabolism Gene Expression in TAGL1 RNAi and Control Fruit. Total fruit RNA was analyzed via gel blot analysis from the indicated genotypes and the following fruit stages: 1, IM (28 DPA); 2, MG; 3, BR; 4, BR + 7d (red ripe in the wild type). Hybridization probes were derived from gene-specific sequences using primers described in Supplemental Table 2 online. Full names and accession numbers for all genes are also listed in Methods. (A) Ripening-associated genes. (B) Starch metabolism genes.

Figure 8.

TAGL1 RNAi Fruit Are Altered in Pericarp Thickness and Starch Accumulation. Representative section types and stains of the indicated fruit stage and genotype are shown. (A) Toluidine blue–stained hand-sections for cell counting of wild-type (Ailsa Craig) and TAGL1-1 (TAGL1 RNAi) fruit. The left pair is 28 DPA immature fruit and those on the right are red ripe stage (breaker + 7 d). (B) Nile blue A stain for starch granules in cryosectioned wild-type (Ailsa Craig) and TAGL1-1 (TAGL1 RNAi) breaker stage fruit. (C) Optical (left and center) and confocal (right) microscopy of cryosectioned wild-type and TAGL1-1 (R) 28 DPA immature fruit. Confocal microscopy employed chloroplast autofluorescence.

Figure 9.

Overexpression of TAGL1 Results in Ripening Sepals and Conversion of Petals to Stamens. (A) to (J) Buds, flowers, and fruit from wild-type ([A] to [E]) and comparably staged 35S:TAGL1 ([F] to [J]) plants. Floral stages are anthesis stage flowers ([A] and [F]), immature inflorescence floral stages 7 to 8 ([B] and [G]), mature inflorescence floral stages 15 to 16 ([C] and [H]), 30 DPA ([D] and [I]), and red ripe ([E] and [J]). Note the following observations. (F) 35S:TAGL1 flowers fail to open at anthesis. (G) 35S:TAGL1 buds at stages 7 to 8 have lighter colored sepals than comparably staged wild-type buds (B). (H) 35S:TAGL1 buds at stages 15 to 16 have sepals that almost fully enclose the inner organs. The sepals show swelling and turn very light green. (I) 35S:TAGL1 fruit at 30 DPA have mutant sepals that resemble green fruit (D). The mutant sepals also have dark green vertical stripes. (J) 35S:TAGL1 mature red fruit have sepals that swell and turn red. (K) Sepal, petal, stamen, and carpel (from left to right) of control flower at anthesis. (L) Sepal, petal, stamen, and carpel (from left to right) of 35S:TAGL1 flower at anthesis. The sepal is already beginning to turn lighter green. The petal is partially transformed into a stamen. The stamen and carpel both appear normal.

Figure 10.

Chlorophyll and Carotenoid Levels in 35S:TAGL1 Overexpression Lines. Chlorophyll (A) and carotenoid (B) levels were analyzed in control (45 DPA) and 35S:TAGL1 green sepals (36 DPA) and red sepals (45 DPA) by HPLC using mean HPLC peak areas (n = 2). Error bars represent se.

Figure 11.

Ectopic Phenotypes Produced by Overexpression of TAGL1, SHP1, and SHP2 in Arabidopsis. (A) to (H) Individual flower ([A] to [D]) and top view of inflorescence ([E] to [H]) of shp1 shp2 double mutant ([A] and [E]), 35S:SHP1-5 shp1 shp2 ([B] and [F]), 35S:SHP2-7 shp1 shp2 ([C] and [G]), and 35S:TAGL1-7 shp1 shp2 ([D] and [H]) plants. (I) Leaf phenotypes produced by (from left to right) wild-type (Landsberg erecta), shp1 shp2 double mutant, 35S:SHP1-5 shp1 shp2, 35S:SHP2-4 shp1 shp2, and 35S:TAGL1-8 shp1 shp2 plants.