Gibberellin regulation of fruit set and growth in tomato

Abstract

The role of gibberellins (GAs) in tomato (Solanum lycopersicum) fruit development was investigated. Two different inhibitors of GA biosynthesis (LAB 198999 and paclobutrazol) decreased fruit growth and fruit set, an effect reversed by GA(3) application. LAB 198999 reduced GA(1) and GA(8) content, but increased that of their precursors GA(53), GA(44), GA(19), and GA(20) in pollinated fruits. This supports the hypothesis that GA(1) is the active GA for tomato fruit growth. Unpollinated ovaries developed parthenocarpically in response to GA(3) > GA(1) = GA(4) > GA(20), but not to GA(19), suggesting that GA 20-oxidase activity was limiting in unpollinated ovaries. This was confirmed by analyzing the effect of pollination on transcript levels of SlCPS, SlGA20ox1, -2, and -3, and SlGA3ox1 and -2, encoding enzymes of GA biosynthesis. Pollination increased transcript content of SlGA20ox1, -2, and -3, and SlCPS, but not of SlGA3ox1 and -2. To investigate whether pollination also altered GA inactivation, full-length cDNA clones of genes encoding enzymes catalyzing GA 2-oxidases (SlGA2ox1, -2, -3, -4, and -5) were isolated and characterized. Transcript levels of these genes did not decrease early after pollination (5-d-old fruits), but transcript content reduction of all of them, mainly of SlGA2ox2, was found later (from 10 d after anthesis). We conclude that pollination mediates fruit set by activating GA biosynthesis mainly through up-regulation of GA20ox. Finally, the phylogenetic reconstruction of the GA2ox family clearly showed the existence of three gene subfamilies, and the phylogenetic position of SlGA2ox1, -2, -3, -4, and -5 was established.

Figure 1.

Fruit set and growth inhibition of pollinated ovaries with inhibitors of GA biosynthesis and its reversal by GA₃ application. A, Effect of time of emasculation and removal of petals, anthers, and style on number of seeds and fruit growth of pollinated ovaries (at day 0). B, Effect of different doses of LAB 198999. C, Effect of different doses of paclobutrazol (PCB). Pollination was carried out at day 0. LAB 198999 was applied directly to the ovary in 10 μL solution, 2 d after anthesis, after emasculation and petal removal. Paclobutrazol was applied to the roots in the nutrient solution, every 2 d, from 7 d before anthesis to 15 d after anthesis. GA₃ (2,000 ng) was applied to the ovary in 10 μL solution at anthesis. Fruits were collected 20 d after treatment. Values are data from eight fruits ±se. One-hundred percent of fruits developed in all treatments, except those marked with figures in parentheses (number of fruits developed over eight treated).

Figure 2.

Response of unpollinated tomato ovaries to GA₁, GA₃, GA₄, GA₁₉, and GA₂₀ (2,000 ng per ovary) application. Fruits were collected 20 d after treatment and values are means of eight fruits ±se. Values of pollinated ovaries are also included as control. Poll., Pollinated.

Figure 3.

Distribution of transcript levels of SlCPS, SlGA20ox1, -2, and -3, and SlGA3ox1 and -2 in different organs of tomato. Semiquantitative transcript analysis was carried out by RT-PCR, as described in “Materials and Methods,” using total RNA from roots (R), young leaves before flowering (YLp), young and old leaves from plants at flowering (YL, OL), young and old internodes (YI, OI), flowers (Fl), ovary at anthesis (O), stamens (St), sepals (Se), petals (Pe), and 20-d-old fruit (Fr). For each gene, figures below the blots mean normalized values of gene expression versus that of Actin (used as an internal control; flower expression set at 1.0). Data come from a representative experiment out of two biological replicates with similar results.

Figure 4.

Effect of pollination on transcript levels of SlCPS, SlGA20ox1, -2, and -3, and SlGA3ox1 and -2 genes. Semiquantitative transcript analysis was carried out by RT-PCR, as described in “Materials and Methods,” using total RNA from unpollinated (days 0, 5, 10, and 20) and pollinated (day 5, 10, and 20) ovaries. E, Entire ovary; P, pericarp; S, seeds. For each gene, figures below the blots mean normalized values of gene expression versus that of Actin (used as an internal control; expression of entire 20-d-old pollinated fruits set at 1.0 for all the genes but for SlGA3ox2, where expression of day 0 unpollinated ovaries was used as reference). Data come from a representative experiment out of two biological replicates with similar results.

Figure 5.

Maximum-likelihood phylogenetic tree based on comparison of GA2ox protein sequences from different species. The tree was rooted using AtGA20ox1 as outgroup and branch lengths are proportional to the estimated sequence divergence. Bootstrap values above 50% are shown, whereas asterisks indicate statistical significance according to the weighted least-squares likelihood ration test (**, P < 0.01; *, P < 0.05). The three GA2ox subfamilies I, II, and II are indicated, and genes that have been shown to codify for multicatalytic enzymes are underlined. The five genes characterized in this study are shown in bold type. Accession numbers corresponding to the sequences in the tree are the following: AtGA20ox1, X83379; AtGA2ox1, AJ132435; AtGA2ox2, AJ132436 (Thomas et al., 1999); AtGA2ox3, AJ322437 (Thomas et al., 1999); AtGA2ox4, NM103695; AtGA2ox6, NM100121; AtGA2ox7, AC079284; AtGA2ox8, AL021960; CmGA2ox1, AJ315663; HvGA2ox4, AY551432; HvGA2ox5, AY551433; Ls2ox1, AB031206; Ls2ox2, AB031207; LtGA2ox1, DQ324114; NoGA2ox1, AY594291; NoGA2ox2, AY594292; NoGA2ox3, AY588978 (Ubeda-Tomás et al., 2006); NsGA2ox1, Ay242858; NtGA2ox1, AB125232; NtGA2ox2, AB125233; OsGA2ox1, AB059416; OsGA2ox2, AB092484; OsGA2ox3, AB092485 (Sakai et al., 2003); OsGA2ox4, AC132485; OsGA2ox5, BAC10398; OsGA2ox6, AL662958; PcGA2ox1, AJ132438 (Thomas et al., 1999); PsGA2ox1, AF056935 (Martin et al., 1999); PsGA2ox2, AF100954; PttGA2ox1, AY392094; RpGA2ox1, DQ641499; SlGA2ox1, EF441351; SlGA2ox2, EF441352; SlGA2ox3, EF441353; SlGA2ox4, EF441354; SlGA2ox5, EF441355; SoGA2ox1, AF506281 (Lee and Zeevaart, 2002); SoGA2ox2, AF506282; SoGA2ox3, AY935713; VaGA2oxA1, AB181372; VaGA2oxA2, AB181373; VaGA2oxB1, AB181374; VaGA2oxB2, AB181375; VaGA2oxB3, AB181376; VaGA2oxC1, AB181377.

Figure 6.

Distribution of transcript levels of SlGA2ox1, -2, -3, -4, and -5 in different organs of tomato. Semiquantitative transcript analysis was carried out by RT-PCR, as described in “Materials and Methods,” using total RNA from roots (R), young leaves before flowering (YLp), young and old leaves from flowering plants (YL, OL), young and old internodes (YI, OI), flowers (Fl), ovary at anthesis (O), stamens (St), sepals (Se), petals (Pe), and 20-d-old fruit (Fr). For each gene, figures below the blots mean normalized values of gene expression versus that of Actin (used as an internal control; flower expression set at 1.0 for all the genes, except for SlGA2ox5, where expression of YI was used as reference). Data come from a representative experiment out of two biological replicates with similar results.

Figure 7.

Effect of pollination on transcript levels of SlGA2ox1, -2, -3, -4, and -5 genes. Semiquantitative transcript analysis was carried out by RT-PCR, as described in “Materials and Methods,” using total RNA from unpollinated (days 0, 5, 10, and 20) and pollinated (days 5, 10, and 20) ovaries. E, Entire ovary; P, pericarp; S, seeds. For each gene, figures below the blots mean normalized values of gene expression versus that of Actin (used as an internal control; expression of unpollinated day 5 and 10 ovaries set at 1.0 for SlGA2ox1, -3, and -4, of pollinated day 5 ovaries for SlGA2ox2, and seeds from day 10 pollinated ovaries for SlGA2ox5). Data come from a representative experiment out of two biological replicates with similar results.