Tuning of Pectin Methylesterification: PECTIN METHYLESTERASE INHIBITOR 7 MODULATES THE PROCESSIVE ACTIVITY OF CO-EXPRESSED PECTIN METHYLESTERASE 3 IN A pH-DEPENDENT MANNER

Abstract

Pectin methylesterases (PMEs) catalyze the demethylesterification of homogalacturonan domains of pectin in plant cell walls and are regulated by endogenous pectin methylesterase inhibitors (PMEIs). In Arabidopsis dark-grown hypocotyls, one PME (AtPME3) and one PMEI (AtPMEI7) were identified as potential interacting proteins. Using RT-quantitative PCR analysis and gene promoter::GUS fusions, we first showed that AtPME3 and AtPMEI7 genes had overlapping patterns of expression in etiolated hypocotyls. The two proteins were identified in hypocotyl cell wall extracts by proteomics. To investigate the potential interaction between AtPME3 and AtPMEI7, both proteins were expressed in a heterologous system and purified by affinity chromatography. The activity of recombinant AtPME3 was characterized on homogalacturonans (HGs) with distinct degrees/patterns of methylesterification. AtPME3 showed the highest activity at pH 7.5 on HG substrates with a degree of methylesterification between 60 and 80% and a random distribution of methyl esters. On the best HG substrate, AtPME3 generates long non-methylesterified stretches and leaves short highly methylesterified zones, indicating that it acts as a processive enzyme. The recombinant AtPMEI7 and AtPME3 interaction reduces the level of demethylesterification of the HG substrate but does not inhibit the processivity of the enzyme. These data suggest that the AtPME3·AtPMEI7 complex is not covalently linked and could, depending on the pH, be alternately formed and dissociated. Docking analysis indicated that the inhibition of AtPME3 could occur via the interaction of AtPMEI7 with a PME ligand-binding cleft structure. All of these data indicate that AtPME3 and AtPMEI7 could be partners involved in the fine tuning of HG methylesterification during plant development.

Keywords: degree of blockiness; gel diffusion assay; homology modeling; microscale thermophoresis; pectin methylesterase (PME); pectin methylesterase inhibitor (PMEI); plant biochemistry; plant cell wall; protein expression; protein-protein interaction.

FIGURE 1.

PME3 and PMEI7 are expressed in dark-grown hypocotyls at transcriptomic and proteomic levels. A, level of transcripts of PME3 (■) and PMEI7 (□) in dark-grown hypocotyls from 24 to 96 h postgermination determined by RT-quantitative PCR analysis. The relative level of accumulation was measured using the reference genes TIP41 and CLA, but only the results obtained with TIP41 are shown. Relative expression is shown in means of log₁₀ ± S.E. (error bars) of four replicates. The different letters indicate data sets significantly different according to Tukey's range test, preceded by a one-way ANOVA having p < 0.001. B, activity of the promoters of PME3 and PMEI7, revealed by GUS staining of transgenic dark-grown hypocotyls up to 96 h postgermination. Black scale bars, 0.2 mm (24 h), 0.5 mm (48 h), 1 mm (72 h), and 2 mm (96 h). C, identification of PME3 protein in cell wall protein extracts of 5-day-old dark-grown hypocotyls. The PMEI domain is represented in green, the PME domain is shown in dark blue, and putative basic processing motifs (RKLK and RRLL) are shown in red. D, identification of PMEI7 protein in cell wall protein extracts of 5-day-old dark-grown hypocotyls. The inhibitor domain is indicated in dark blue. For both proteins, signal peptide is indicated by a sky blue-colored sequence, and peptides identified by MALDI-TOF MS analysis are underlined.

FIGURE 2.

PME3-His₆ is processed properly and is active in transgenic N. tabacum plants. A, transgenic N. tabacum plants overexpressing PME3-His₆ were generated. Expression of PME3 under the control of a double CaMV 35S promoter and a 35S transcriptional terminator/polyadenylation sequence (T35S) was carried out using a binary vector. The PME coding region was fused to the TEV sequence for enhanced transcription and to the His₆ tag for affinity purification. B, DM in control plants (■) and transgenic tobacco plants overexpressing PME3-His₆ (□). DM was calculated as described (51). Data represent mean values ± S.E. (error bars) from three replicates. No significant differences were shown with non-parametric Wilcoxon-Mann-Whitney test. C, PME activity from total protein extract of control plants (■) and the PME3-His₆-overexpressing line (□). Data represent means ± S.E. from three independent samples. According to Student's t test, no significant differences are shown. D, Ni-NTA-purified PME3-His₆ analysis by SDS-PAGE. MM, molecular mass marker. PME3, Purified PME3-His₆ (*). E, PME activity from Ni-NTA retained the fraction of control plants (■) and a representative PME3-His₆-overexpressing line (□). For determination of PME activity, assays were performed according to the method adapted from Ref. . Data represent mean values ± S.E. from three independent samples. Significant differences were determined with Student's t test (***, p < 0.001).

FIGURE 3.

Substrate specificity and pH dependence of PME3-His₆. A, HG isolation and tailoring. Lime pectin was first saponified before HG isolation. The recovered HG0 was then highly methylated (96%, HG96). HG96 was further chemically (B-series) or enzymatically (P-series) demethylesterified to reach a specific DM. In the BP series, HG96 was first given alkaline base treatment before further action of orange PME. B, purified PME3-His₆ activities assayed at three pH values (pH 7.5, 6.0, and 4.0) on various HG substrates with different lengths and degrees of methylation. Activities were determined colorimetrically using N-methylbenzothiazolinone-2-hydrazone and alcohol oxidase for released methanol oxidation. Data represent means ± S.D. (error bars) from three replicates. C, PME3-His₆ enzyme activity assayed at pH 7.5 on HG substrates (B-series) with different lengths and a similar degree of methylation (HG96B69, HG37B72, and HG12B71). Data represent the means ± S.D. of three replicates. The different letters indicate data sets significantly different according to Tukey's range test, preceded by a one-way ANOVA having p < 0.001.

FIGURE 4.

Expression of PMEI7-His₆ in bacteria. Purification and tests of PME activity inhibition. A, expression of PMEI7-His₆ in E. coli and purification. SDS-PAGE analysis of total protein extracts (left) and Ni-NTA-purified protein extracts (right) of isopropylthio-β-galactoside-induced cultures containing empty vector (EV) or recombined vector (PMEI7). MM, molecular mass markers. B, gel diffusion assay of the inhibitory capacity of the purified PMEI7-His₆ on commercial orange PME at pH 6.0. Experiments were carried out using 1 milliunit of orange PME and various quantities of purified PMEI7-His₆. Results are means ± S.D. (error bars) of six replicates. The different letters indicate data sets significantly different according to Tukey's range test, preceded by a one-way ANOVA having p < 0.001. C, quantification of the pH dependence of the inhibitory capacity of PMEI7-His₆ on total PME activity of cell wall-enriched protein extracts from three Arabidopsis organs. Top, 3-week-old light-grown leaves; middle, 10-day-old light-grown roots; bottom, 4-day-old dark-grown hypocotyls. 3 milliunits of total PME activity was used with either PMEI7-His₆ storage solution (■) or a PME activity/PMEI7-His₆ (μg) ratio of 3:0.25 (dotted bars), 3:0.5 (), and 3:1 (▨). Results are means ± S.D. of six replicates. The different letters indicate data sets significantly different according to Tukey's range test, preceded by a one-way ANOVA having p < 0.001.

FIGURE 5.

Inhibition of PME3-His₆ activity by PMEI7-His₆ due to pH-dependent complex formation. A, quantification, using a gel diffusion assay and a substrate of DM 90%, of the pH dependence of the inhibitory capacity of PMEI7-His₆ on PME3-His₆ enzymatic activity. 1 milliunit of PME3-His₆ activity (corresponding to 2 μg of protein) was used with either PMEI7-His₆ conservation solution (■) or a PME3-His₆ activity (milliunits)/PMEI7-His₆ (μg) ratio of 1:0.25 (lightly dotted bars), 1:0.5 (), 1:1 (), 1:2 (heavily dotted bars), 1:4 (□), and 1:8 (gray bars). Results are means ± S.D. (error bars) of six replicates. The different letters indicate data sets significantly different according to Tukey's range test, preceded by a one-way ANOVA having p < 0.001. At the most acidic pH, the maximum inhibition of PME3-His₆ activity is reached for a ratio of ±60 pmol of PME3-His₆/60 pmol of PMEI7-His₆. B, inhibition of PME3-His₆ activity by PMEI7-His₆ on the HG96B82 substrate. PME3-His₆ and PMEI7-His₆ were preincubated for 30 min at 30 °C at pH 6.0. HG96B82 was added to the mixture and incubated for 30 min at 30 °C. The reaction was stopped at 90 °C for 10 min. PME activity was determined using a procedure adapted from Ref. . 1 μg of PME3-His₆ was used with either PMEI7-His₆ conservation solution (■) or a PME3-His₆ activity/PMEI7-His₆ (μg) ratio of 1:0.5 (), 1:1 (▨), 1:2 (heavily dotted box), or 1:4 (□). Results are the means ± S.D. of two replicates.

FIGURE 6.

Molecular interaction between PME3-His₆ and PMEI7-His₆ according to pH. The interaction between PME3-His₆ and PMEI7-His₆ was determined by MST. For the MST binding assay at various pH, PME3-His₆ was labeled by a fluorescence blue dye, covalently attached to the protein. The concentration of the blue dye-labeled PME3-His₆ was kept constant (333 nm), whereas the different concentrations of the non-labeled PMEI7-His₆ between 33,000 and 1 nm, between 4000 and 0.98 nm, and between 1562 and 0.76 nm were used for pH 7.5 (A), pH 6 (B), and pH 5 (C), respectively. Mixtures of blue dye-labeled PME3-His₆ and titrated non-labeled PMEI7-His₆ were incubated for 15 min at room temperature and loaded into standard capillaries for MST experiments with 90% LED power and 40% MST power as thermophoresis conditions. The fit curve and the resulting dissociation constant (K_d) values were calculated by averaging replicates assimilated using NT analysis software. K_d values represent the mean ± S.D. (error bars) from 6–8 replicates, depending on pH. Concentrations on the x axis are plotted in nm. K_d of 38,000 ± 2030 nm, 95.6 ± 7.19 nm, and 39.4 ± 4.15 nm, respectively, were shown for pH 7.5, 6, and 5.

FIGURE 7.

Proposed model for the inactivation of PME3 by PMEI7. A, PME3 model. Putative amino acid residues involved in the catalytic site are shown in red, and putative important residues involved in both the pectin-binding site and the PME-PMEI interaction are shown in green. B, PMEI7 model. Putative amino acid residues interacting with the PME pectin-binding site are shown in green. Arabidopsis PME3 and PMEI7 models were built using carrot PME (PDB code 1GQ8, chain A) and kiwi PMEI (PDB code 1XG2, chain B) coordinates, respectively. The tertiary structure was modeled according to Ref. . C, interaction between PME3 (gray) and PMEI7 (blue) was evaluated as described (55, 56) using LZerD based on shape complementarity. Model number 8 from the 15 best models was selected based on the literature data and the number of contacting residues. D, electrostatic potential of PMEI7 on PME3.