Constitutive Expression of Arabidopsis SMALL AUXIN UP RNA19 (SAUR19) in Tomato Confers Auxin-Independent Hypocotyl Elongation

Abstract

The plant hormone indole-3-acetic acid (IAA or auxin) mediates the elongation growth of shoot tissues by promoting cell expansion. According to the acid growth theory proposed in the 1970s, auxin activates plasma membrane H⁺-ATPases (PM H⁺-ATPases) to facilitate cell expansion by both loosening the cell wall through acidification and promoting solute uptake. Mechanistically, however, this process is poorly understood. Recent findings in Arabidopsis (Arabidopsis thaliana) have demonstrated that auxin-induced SMALL AUXIN UP RNA (SAUR) genes promote elongation growth and play a key role in PM H⁺-ATPase activation by inhibiting PP2C.D family protein phosphatases. Here, we extend these findings by demonstrating that SAUR proteins also inhibit tomato PP2C.D family phosphatases and that AtSAUR19 overexpression in tomato (Solanum lycopersicum) confers the same suite of phenotypes as previously reported for Arabidopsis. Furthermore, we employ a custom image-based method for measuring hypocotyl segment elongation with high resolution and a method for measuring cell wall mechanical properties, to add mechanistic details to the emerging description of auxin-mediated cell expansion. We find that constitutive expression of GFP-AtSAUR19 bypasses the normal requirement of auxin for elongation growth by increasing the mechanical extensibility of excised hypocotyl segments. In contrast, hypocotyl segments overexpressing a PP2C.D phosphatase are specifically impaired in auxin-mediated elongation. The time courses of auxin-induced SAUR expression and auxin-dependent elongation growth were closely correlated. These findings indicate that induction of SAUR expression is sufficient to elicit auxin-mediated expansion growth by activating PM H⁺-ATPases to facilitate apoplast acidification and mechanical wall loosening.

Figure 1.

35S:GFP-AtSAUR19 expression in tomato. A, Five micrograms of soluble, lower phase-enriched other membrane (OM) or upper phase-enriched plasma membrane (PM) fractions prepared from 8-d-old M82 or 35S:GFP-AtSAUR19 (GFP-S19) seedlings were immunoblotted with α-GFP and α-AHA antibodies. A section of the Ponceau S-stained blot is shown as a loading control. GFP-S19.4 and GFP-S19.7 are two independent transgenic lines expressing the 35S:GFP-AtSAUR19 transgene. B, Confocal image of GFP-AtSAUR19 tomato leaf epidermal cells. C and D, Mean (±sd) hypocotyl lengths of 8-d-old seedlings (n ≥ 15; C) and first internode lengths of 27-d-old plants (n ≥ 12; D). Asterisks indicate significant difference from the wild type: **P ≤ 0.01 and *P ≤ 0.05. E, Three-day-old seedlings were transferred to plates containing the pH indicator dye, bromocresol purple, and incubated an additional 24 h. F, Percentage of root growth inhibition ± sd by 12.5 mm LiCl. **P ≤ 0.01. G, Kinetics of water loss in leaf detachment assays. Data depict relative mean weights of ≥24 leaves/genotype ± sd. Both GFP-AtSAUR19 transgenic lines exhibit significant differences from M82 at all time points after t = 0 (P ≤ 0.05). H, Mean stomatal apertures (±se) of epidermal peels obtained from 27-d-old tomato leaves incubated in opening buffer ± 10 μm ABA for 2 h. Different letters above bars indicate significant (P ≤ 0.01) difference when analyzed by one-way ANOVA with Tukey’s HSD test.

Figure 2.

Arabidopsis SAUR proteins interact with tomato PP2C.D phosphatases. A, Yeast cells harboring LexA DNA binding domain-AtSAUR9 or AtSAUR19 and GAL4 activation domain-SlPP2C fusion constructs were spotted onto –leu, trp and –leu, trp, his plates. Vector control (Vec) is the empty LexA DNA binding domain vector. B, 6xHis-SlPP2C.D proteins were purified from Escherichia coli and tested in phosphatase assays employing pNPP. Values indicate the mean relative activities ± sd of three assays. C, In vitro phosphatase assay examining SlPP2C38-mediated dephosphorylation of yeast-expressed AHA2. Where indicated, recombinant AtSAUR19, AtSAUR9, or commercial BSA was added to a 4-fold molar excess relative to SlPP2C38. AHA2 Thr-947 phosphorylation status was assessed by GST-14-3-3 far-western blotting. SlPP2C38 and AtSAUR9/19 abundance was determined by detection of the S-tag.

Figure 3.

Auxin-independent elongation of GFP-AtSAUR19 hypocotyl segments. A, Elongation of 5-mm auxin-depleted Arabidopsis hypocotyl segments on medium ±10 μm IAA. B, Elongation of 8-mm auxin-depleted tomato hypocotyl segments on medium ±10 μm IAA. C, Elongation of auxin-depleted Arabidopsis hypocotyl segments on medium ±10 μm IAA. For Arabidopsis assays, hypocotyl segments were prepared from 3- to 4-d-old etiolated Col and GFP-AtSAUR19 seedlings. For the D1-OX line, however, seedlings were grown for 11 d in order to obtain hypocotyl segments that were long enough to conduct the assay. Tomato hypocotyl segments were excised from 6-d-old etiolated seedlings. All data points represent the mean relative segment length ± se from a minimum of five hypocotyls/genotype/treatment. All assays were repeated at least three times. D, qRT-PCR analysis of auxin-depleted Arabidopsis hypocotyl segments treated with 10 μm IAA. Values indicate the mean relative expression (±sd) from three biological replicates.

Figure 4.

Altered mechanical properties of GFP-AtSAUR19 tomato hypocotyls. A, Mean (±se) extension curves of M82 and GFP-AtSAUR19 tomato hypocotyl segments. Six-day-old etiolated seedlings were collected and frozen at −80°C. After thawing in ice water, cotyledons and roots were removed and the apical 12 mm of the hypocotyl mechanically extended on a TA.XTplus texture analyzer as described in “Materials and Methods.” B and C, Mean extension at a force of 15g (B) and mean tensile strength (C). Error bars depict se. Asterisks indicate significant (P < 0.01) difference from M82 wild-type hypocotyls as determined using the Wilcoxon rank-sum test.

Figure 5.

Auxin increases extensibility of wild-type but not GFP-AtSAUR19 hypocotyls. A, Mean (±se) extension curves of auxin- or mock-treated tomato hypocotyls. Auxin-depleted hypocotyls were treated with 10 μm IAA or solvent control for 45 min and frozen. Hypocotyl segments were thawed in ice water and extensibility assessed on a TA.XTplus texture analyzer as described in “Materials and Methods.” B and C, Mean extension at a force of 15g (B) and mean tensile strength (C). Error bars depict se. Different letters above bars indicate significant (P < 0.05) differences between samples as determined by one-way ANOVA assessed by Dunnett’s test.