The Grapevine VvPMEI1 Gene Encodes a Novel Functional Pectin Methylesterase Inhibitor Associated to Grape Berry Development

Abstract

Pectin is secreted in a highly methylesterified form and partially de-methylesterified in the cell wall by pectin methylesterases (PMEs). PME activity is expressed during plant growth, development and stress responses. PME activity is controlled at the post-transcriptional level by proteins named PME inhibitors (PMEIs). We have identified, expressed and characterized VvPMEI1, a functional PME inhibitor of Vitis vinifera. VvPMEI1 typically affects the activity of plant PMEs and is inactive against microbial PMEs. The kinetics of PMEI-PME interaction, studied by surface plasmon resonance, indicates that the inhibitor strongly interacts with PME at apoplastic pH while the stability of the complex is reduced by increasing the pH. The analysis of VvPMEI1 expression in different grapevine tissues and during grape fruit development suggests that this inhibitor controls PME activity mainly during the earlier phase of berry development. A proteomic analysis performed at this stage indicates a PME isoform as possible target of VvPMEI1.

Fig 1. Homology tree of several Vitis vinifera protein sequences annotated as putative invertase/pectin methylesterase inhibitors.

In the analysis the functionally characterized PMEIs and INHs are also included. Multiple sequence alignment was performed using DNAman software package (Lynnon Biosoft). Numbers at branch points represent % of identity.

Fig 2. Alignment of amino acid sequences of functionally characterized PMEIs from different plant origins.

The VvPMEI1 amino acid sequence was aligned with PMEIs from Arabidopsis (AtPMEI1: At1g48020, AtPMEI2: At3g17220, AtPMEI3: At5g20740, AtPMEI4: At4g25250, AtPMEI5: At2g31430 and AtPMEI6: At2g47670), kiwi (AdPMEI:P83326), pepper (CaPMEI1: ABG47806), tomato (SolyPMEI: SGN-U601352) and broccoli (BoPMEI:Q45TJ7). Tobacco cell wall and vacuolar invertase inhibitors (NtCIF: CAA73333 and NtVIF: CAA73334) were also added for comparison. The alignment performed using ClustalW was manually adjusted according to PsiPred secondary structure predictions. Alignment was drawn by using the ESPript program. The secondary structure elements, as elucidated in AdPMEI crystal structure, are indicated at the top of the alignment. Invariant residues are red shadowed and similar residues are colored in red and boxed. Numbers 1 and 2 at the bottom denote disulfide bridges connecting the four conserved Cys residues.

Fig 3. SDS-PAGE analysis of purified VvPMEI1.

VvPMEI1 was loaded before (-) and after (+) endo N-glycosidase digestion: (Mw) molecular weight marker. The proteins were separated by SDS-PAGE and gel was stained with silver nitrate.

Fig 4. Target specificity of VvPMEI1.

Gel diffusion assay showing PME activity from various origins in the presence (+) or absence (-) of VvPMEI1. The representative image of at least three independent experiments is shown.

Fig 5. SPR analysis of VvPMEI1 and PME1 interaction.

Different VvPMEI1 concentrations were injected over the surface (300.0, 150.0, 75.0, 37.5, 18.7, 9.4, 4.7, 2.3 nM). (A) and (C) show sensorgrams (black) obtained at pH 5.5 and 7.5 respectively. Data were fit to the simple 1:1 interaction model, and the resulting fit is shown in red. (B) and (D), concentration dependent binding curves for the interaction at pH 5.5 and 7.5 respectively.

Fig 6. Homology model of VvPMEI1.

The model of VvPMEI1 (top) was based on the AcPMEI template using the Swiss Model algorithm. Sequence comparison of VvPMEI1 and AdPMEI (bottom). Residue of the kiwi inhibitor involved in H-bonds (red), Van der Waals contacts (green) and water-mediated H bonds (yellow) with tomato PME1 and conserved in VvPMEI1 are shown. The secondary structure elements, as elucidated in AdPMEI crystal structure, are boxed in black on the sequence.

Fig 7. Analysis of VvPMEI1 expression and PME activity in grapevine tissues and during grape berry development.

(A) Expression analysis of VvPMEI1 in various grapevine organs by real-time PCR. The relative level of gene expression was normalized with respect to EF1 mRNA; (B) PME activity. Bars represent the average ± SD (n = 3).