SELF-PRUNING Acts Synergistically with DIAGEOTROPICA to Guide Auxin Responses and Proper Growth Form

Abstract

The SELF PRUNING (SP) gene is a key regulator of growth habit in tomato (Solanum lycopersicum). It is an ortholog of TERMINAL FLOWER1, a phosphatidylethanolamine-binding protein with antiflorigenic activity in Arabidopsis (Arabidopsis thaliana). A spontaneous loss-of-function mutation (sp) has been bred into several industrial tomato cultivars, as it produces a suite of pleiotropic effects that are favorable for mechanical harvesting, including determinate growth habit, short plant stature, and simultaneous fruit ripening. However, the physiological basis for these phenotypic differences has not been thoroughly explained. Here, we show that the sp mutation alters polar auxin transport as well as auxin responses, such as gravitropic curvature and elongation of excised hypocotyl segments. We also demonstrate that free auxin levels and auxin-regulated gene expression patterns are altered in sp mutants. Furthermore, diageotropica, a mutation in a gene encoding a cyclophilin A protein, appears to confer epistatic effects with sp Our results indicate that SP affects the tomato growth habit at least in part by influencing auxin transport and responsiveness. These findings suggest potential novel targets that could be manipulated for controlling plant growth habit and improving productivity.

Figure 1.

Additive phenotype of the sp and dgt mutations in tomato cv MT. A, Representative plants of SP DGT, SP dgt, sp DGT (cv MT), and sp dgt at 90 d after germination (dag). Note the simultaneous fruit ripening in sp compared with SP, a well-known effect of the sp mutation. The dgt mutation delays fruit ripening (at least in part due to its late flowering, as indicated in B) in either genetic background. Bar = 5 cm. B, Chronological time to flowering in sp and dgt mutants. The percentage of plants (n = 15) with at least one open flower is shown. The cv MT (sp DGT) plants flower earlier than the wild-type plants (SP DGT), whereas dgt mutants are late flowering. C, Developmental time to flowering in sp and dgt mutants. The number of leaves produced before the first inflorescence was reduced in sp DGT (cv MT) and increased in genotypes carrying the functional allele of SP. Letters indicate statistically significant differences (Dunn’s multiple comparisons test, P < 0.05). D, sp and dgt alter the expression of the flowering inducer SFT. The dgt mutation leads to lower SFT expression and, thus, delays flowering. A minor influence from SP reducing SFT levels also is noticeable. Asterisks indicate statistically significant differences from the wild-type SP DGT (Student’s t test, P < 0.05). E, Effects of sp and dgt on side branching. Pie charts depict the distribution of side branches in each genotype at 60 dag (n = 15 plants). Gray denotes absence of an axillary bud, yellow denotes a visible bud (greater than 1 cm), and dark green denotes a full branch (with one or multiple leaves). Letters indicate statistically significant differences (Dunn’s multiple comparisons test, P < 0.05).

Figure 2.

Auxin levels in tomato seedlings are affected synergistically by the sp and dgt mutations. A, Representative 7-d-old seedling showing the dissection points for auxin quantitation (bar = 1 cm). B to D, Free IAA levels in leaves + cotyledons (B), hypocotyls (C), and roots (D). Data are means ± se (n = 10). Different letters indicate statistically significant differences (Tukey’s test, P < 0.05) among genotypes. FW, Fresh weight.

Figure 3.

A, The sp mutation exacerbates defective PAT in hypocotyls caused by dgt. Basipetal [³H]IAA transport is shown in 10-mm hypocotyl sections of the wild type (SP DGT), SP dgt, sp DGT (cv MT; also the negative control treated with 1-N-naphthylphthalamic acid [NPA]), and double mutant sp dgt roots. Data are means ± se (n = 10). Letters indicate statistically significant differences between treatments (Tukey's test, P < 0.05). B to E, Vascular patterning in sp and dgt stems. Cross sections of the fifth internode taken at 45 dag are shown. Bars = 100 µm. F and G, Vessel density (F) and mean vessel size (G) in sp and dgt stems. Letters indicate significant differences (P < 0.05, ANOVA and Tukey’s test). H, Vessel size distribution in the xylem of sp and dgt mutants. The x axis shows the upper values of cross-sectional area for each vessel size category. The bars within each category represent a single individual plant (n = 4 per genotype).

Figure 4.

Impact of the sp mutation on auxin responses in planta. A, Kinetics of the gravitropic response in the shoot. Shoot angle is shown after placing plants horizontally at time point 0 (n = 5). B, Elongation of excised hypocotyls in response to naphthaleneacetic acid (NAA). Six-millimeter hypocotyl sections were incubated in the indicated NAA concentration for 24 h before measurement (n = 15). C and D, Time course of in vitro root elongation of seedlings in control and 10 µm NAA-containing Murashige and Skoog (MS) medium (n = 25). In all graphs, error bars indicate se and asterisks indicate statistically significant differences between SP and sp plants harboring the same DGT allele (*, P ≤ 0.05 and **, P ≤ 0.01, Student’s t test).

Figure 5.

Effects of SP on the auxin signaling and transport machinery in planta. A, Expression of the GUS reporter driven by the auxin-inducible DR5 promoter. Representative wild-type (SP) and mutant (sp) seedlings (bars = 2 cm) and their root tips (bars = 250 µm) are shown in the absence or presence of exogenous auxin (20 µm IAA, 3 h) at 15 dag. B to D, Fluorimetric quantification of GUS precipitate. Seedlings were sampled at 15 dag, after treatment with exogenous auxin (20 µm IAA, 3 h) or mock solution. Values are means ± se (n = 4). Letters indicate significant differences between genotypes within the same treatment (P < 0.05, ANOVA and Tukey’s test). E to G, Relative gene expression of PIN transporters in roots. Letters indicate significant differences between genotypes within the same treatment (P < 0.05, ANOVA and Tukey’s test).

Figure 6.

SP and auxin signaling gene expression is altered by the dgt mutation. A, Genomic structures of the SP gene in solanaceous species: tomato, its wild relatives Solanum pimpinellifolium and Solanum pennellii, and potato (Solanum tuberosum). The coding sequence is indicated in yellow (exons, thick bars; introns, thin bars). Red blocks indicate the presence of a conserved or degenerate auxin-response element (AuxRE), TGTCNC. B and C, Relative transcript accumulation of SP (B) and auxin signaling genes (C) in sympodial meristems. Tissues were sampled from 10-d-old plants 24 h after 10 µm IAA or mock spray. Asterisks indicate significant differences with respect to the wild-type SP DGT (P < 0.05, Student’s t test).