<i>In vitro</i> activity of recombinant GH3-6.

<p>(A) The expression of recombinant GH3-6 protein, which was used for the inhibition assays shown in Figs. 2 and 4, 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 molecular mass standards (Precision Plus Protein all blue, BioRad) are indicated. The band with the size of approximately 70 kDa corresponds to the His-tagged GH3-6 protein. (B) TLC analysis of GH3-6 enzyme reactions with IAA and 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.</p

AIEP inhibits GH3-1 and GH3-6 in a competitive manner regarding both substrates – MgATP and IAA.

<p>Reciprocal initial velocities (Fig. 2) for two substrate conentrations were replotted against inhibitor concentrations. (A) Dixon plot for GH3-1 and (B) GH3-6 with varying concentrations of inhibitor and 100 µM (◊) and 1000 µM (○) MgATP and for (C) GH3-1 and (D) GH3-6 with varying concentrations of inhibitor and 10 µM (□) and 100 µM (Δ) IAA. All data represent mean ± standard error of the mean (n = 3).</p

Design of AIEP.

<p>(A) gives the structure of AIEP and (B) shows the structure of the proposed reaction intermediate.</p

Inhibition of GH3-1 and GH3-6 by AIEP.

<p>All kinetic parameters are expressed as means ± standard error of the mean (n = 3).</p

Kinetic analysis of the effect of AIEP on the binding of MgATP and IAA by GH3-1 and GH3-6.

<p>The activity of GH3-1 (A) and GH3-6 (B) was determined over a concentration range of MgATP (1 mM IAA, 1 mM Asp) by quantifying the formation of IAA-Asp after 10 min reaction time using LC-ESI-MS/MS. The same analysis was performed over a concentration range of IAA (3 mM MgATP, 1 mM Asp) using either GH3-1 (C) or GH3-6 (D). The inhibitor concentration in the reaction mix was 0 µM (•), 0.1 µM (○), 1 µM (▴), 5 µM (Δ), 10 µM (▪), or 50 µM (□). The plotted initial velocities were fitted to the Michaelis-Menten equation using non-linear regression (SigmaPlot 11.0). All data represent mean ± standard error of the mean (n = 3).</p

Effect of AIEP on auxin levels and the formation of auxin-Asp conjugates in Shiraz berries.

<p>(A) IAA, (B) IAA-Asp and (C) NAA-Asp were quantified by LC-ESI-MS/MS in <i>ex planta</i> Shiraz berry tissues 5 weeks prior to the initiation of ripening which had been exposed to a Control solution, 0.5 µM NAA (N), 20 µM inhibitor (I(20)) and 0.5 µM NAA in combination with 20 µM (I(20)+N), 10 µM (I(10)+N) or 5 µM (I(5)+N) inhibitor. For each treatment 20 berries were placed on 0.8% agar plates containing the indicated compounds and the plates were kept in the dark at room temperature for 6 h (dark grey bars) or 24 h (light grey bars). FW, fresh weight; n.d., not detected. All data represent mean ± standard error of the mean (n = 3). In each subfigure, bars denoted by a different letter differ significantly (p<0.05) using one-way ANOVA to compare the means followed by Duncan's post hoc test (a–c, 6 h; a′–b′, 24 h).</p

Shiraz Wines Made from Grape Berries (<i>Vitis vinifera</i>) Delayed in Ripening by Plant Growth Regulator Treatment Have Elevated Rotundone Concentrations and “Pepper” Flavor and Aroma

Preveraison treatment of Shiraz berries with either 1-naphthaleneacetic acid (NAA) or Ethrel delayed the onset of ripening and harvest. NAA was more effective than Ethrel, delaying harvest by 23 days, compared to 6 days for Ethrel. Sensory analysis of wines from NAA-treated fruit showed significant differences in 10 attributes, including higher “pepper” flavor and aroma compared to those of the control wines. A nontargeted analysis of headspace volatiles revealed modest differences between wines made from control and NAA- or Ethrel-treated berries. However, the concentration of rotundone, the metabolite responsible for the pepper character, was below the level of detection by solid phase microextraction–gas chromatography–mass spectrometry in control wines, low in Ethrel wines (2 ng/L), and much higher in NAA wines (29 ng/L). Thus, NAA, and to a lesser extent Ethrel, treatment of grapes during the preveraison period can delay ripening and enhance rotundone concentrations in Shiraz fruit, thereby enhancing wine “peppery” attributes