Acyl substrate preferences of an IAA-amido synthetase account for variations in grape (Vitis vinifera L.) berry ripening caused by different auxinic compounds indicating the importance of auxin conjugation in plant development

Abstract

Nine Gretchen Hagen (GH3) genes were identified in grapevine (Vitis vinifera L.) and six of these were predicted on the basis of protein sequence similarity to act as indole-3-acetic acid (IAA)-amido synthetases. The activity of these enzymes is thought to be important in controlling free IAA levels and one auxin-inducible grapevine GH3 protein, GH3-1, has previously been implicated in the berry ripening process. Ex planta assays showed that the expression of only one other GH3 gene, GH3-2, increased following the treatment of grape berries with auxinic compounds. One of these was the naturally occurring IAA and the other two were synthetic, α-naphthalene acetic acid (NAA) and benzothiazole-2-oxyacetic acid (BTOA). The determination of steady-state kinetic parameters for the recombinant GH3-1 and GH3-2 proteins revealed that both enzymes efficiently conjugated aspartic acid (Asp) to IAA and less well to NAA, while BTOA was a poor substrate. GH3-2 gene expression was induced by IAA treatment of pre-ripening berries with an associated increase in levels of IAA-Asp and a decrease in free IAA levels. This indicates that GH3-2 responded to excess auxin to maintain low levels of free IAA. Grape berry ripening was not affected by IAA application prior to veraison (ripening onset) but was considerably delayed by NAA and even more so by BTOA. The differential effects of the three auxinic compounds on berry ripening can therefore be explained by the induction and acyl substrate specificity of GH3-2. These results further indicate an important role for GH3 proteins in controlling auxin-related plant developmental processes.

Fig. 1.

Delayed ripening of Shiraz berries after treatment with different auxins. (A) Changes in TSS, measured as degrees Brix, in field-grown Shiraz berries treated twice (24 d and 9 d preveraison) with 80 mg l⁻¹ IAA, 50 mg l⁻¹ NAA, 20 mg l⁻¹ BTOA in 0.1% (v/v) Chemwet 1000 or 0.1% (v/v) Chemwet 1000 (Control), sampled at 1, 2, 7, 17, 27, 36, 49, 62, 65, 73, 78, and 84 dpis. (B) The same berry samples were used to measure anthocyanin levels (absorbance at 520 nm) and (C) changes in berry weight. All data represent means ±STERR (n=3).

Fig. 2.

Phylogenetic relationship of GH3 protein sequences from grapevine and other plants. The unrooted tree was generated with the PHYLIP program (Felsenstein, 1989) using the Neighbor–Joining method and a bootstrap test with 1000 iterations (bootstrap values are indicated at each node). The predicted grapevine proteins are highlighted with a shaded background. Asterisks indicate functionally characterized proteins and the scale bar indicates genetic distance based on branch length. I–III, Functional groups of GH3 proteins described for Arabidopsis (Staswick et al., 2002, 2005); At, Arabidopsis thaliana; Cc, Capsicum chinense; Gm, Glycine max; Mt, Medicago truncatula; Nt, Nicotiana tabacum; Os, Oryza sativa; Pi, Pinus pinaster; Pp, Physcomitrella patens; Pt, Populus trichocarpa; Vv, Vitis vinifera; Zm, Zea mays. Accession numbers of the protein sequences used in this analysis are provided in Supplementary Table S1 at JXB online.

Fig. 3.

Changes in mRNA levels of five group II GH3 genes during grape berry development and in response to different auxins. (A) The expression of GH3-2–GH3-6 in flower and berry tissue of field grown Cabernet Sauvignon plants was analysed by qRT-PCR at the indicated time points (0=anthesis). The asterisks indicate veraison and all data represent means ±STERR of n=3. (B) Expression patterns of GH3-2–GH3-6 upon treatment with 0.5 μM of three auxins or auxin-like compounds in ex planta berry tissue analysed by qRT-PCR. For each treatment 20 berries (22 d and 12 d preveraison) were placed on 0.8% agar plates containing the indicated auxin concentrations and the plates were kept in the dark at room temperature for 24 h. Bars represent means ±STERR (n=3) and are denoted by a different letter if the means differ significantly (P <0.05) using one-way ANOVA followed by Duncan's post hoc test (a–d, 22 d preveraison; a'–d', 12 d preveraison).

Fig. 4.

In vitro activity of recombinant GH3-2. (A) The expression of recombinant GH3-2 protein was tested by separating 10 μl of His GraviTrap column elution fractions (E1-E3) on a 4–12% polyacrylamide gel followed by Coomassie Brilliant Blue staining (upper panel) or immunodetection using a monoclonal antibody raised against poly-histidine (lower panel). The band with the size of approximately 70 kDa corresponds to the His-tagged GH3-2 protein. (B) TLC analysis of GH3-2 enzyme reactions with 20 amino acids (single letter code). The spot near the origin for the reactions with Trp represents the unbound amino acid. Plates were stained with Ehmann's reagent to detect indole compounds. (C) TLC analysis of GH3-2-catalysed IAA–Asp and IAA–Trp formation with indicated variations of the reaction mixture.

Fig. 5.

Example of Michaelis–Menten kinetics obtained for the formation of IAA–Asp by GH3-1 and GH3-2. Enzyme assays were performed using standard conditions (0.5 μg recombinant GH3-1 or GH3-2 per sample) with IAA as the varying substrate. Reactions were stopped and extracted after 0, 2, 5, and 10 min and IAA–Asp formation was quantified by LC-MS. Initial velocities were plotted versus the various concentrations of IAA and the data was fitted to the Michaelis–Menten equation using non-linear regression (SigmaPlot 11.0). All data represent means ±STERR of n=3.

Fig. 6.

Changes in levels of IAA and IAA–Asp and expression of GH3-1 and GH3-2 in preveraison berries in response to IAA application. (A) IAA and (B) the IAA-amino acid conjugate IAA–Asp were quantified by LC-MS/MS in tissue of preveraison Shiraz berries 1, 2, and 7 d after treatment with a Control solution (0.1% (v/v) Chemwet 1000) or 80 mg l⁻¹ IAA in 0.1% (v/v) Chemwet 1000. FW, fresh weight. (C) The expression of GH3-1 and (D) GH3-2 in the same berry samples was analysed by qRT-PCR. All data represent means ±STERR (n=3). Asterisks indicate significant differences of the mean values of IAA-treated samples from the mean values of Control samples as determined with Student's t test (*P <0.05, **P <0.01, ***P <0.001).