Biochemical characterization of Pectin Methylesterase Inhibitor 3 from Arabidopsis thaliana

Abstract

The de-methylesterification of the pectic polysaccharide homogalacturonan (HG) by pectin methylesterases (PMEs) is a critical step in the control of plant cell expansion and morphogenesis. Plants have large gene families encoding PMEs but also PME inhibitors (PMEIs) with differ in their biochemical properties. The Arabidopsis thaliana PECTIN METHYLESTERASE INHIBITOR 3 (PMEI3) gene is frequently used as a tool to manipulate pectin methylesterase activity in studies assessing its role in the control of morphogenesis. One limitation of these studies is that the exact biochemical activity of this protein has not yet been determined. In this manuscript we produced the protein in Pichia pastoris and characterized its activity in vitro. Like other PMEIs, PMEI3 inhibits PME activity at acidic pH in a variety of cell wall extracts and in purified PME preparations, but does not affect the much stronger PME activity at neutral pH. The protein is remarkable heat stable and shows higher activity against PME3 than against PME2, illustrating how different members of the large PMEI family can differ in their specificities towards PME targets. Finally, growing Arabidopsis thaliana seedlings in the presence of purified PMEI3 caused a dose-dependent inhibition of root growth associated with the overall inhibition of HG de-methylesterification of the root surface. This suggests an essential in vivo role for PME activity at acidic pH in HG de-methylesterification and growth control. These results show that purified recombinant PMEI3 is a powerful tool to study the connection between pectin de-methylesterification and cell expansion.

Keywords: Cell expansion; GalA, galacturonic acid; HG, homogalacturonan; PME inhibitor; PME, pectin methylesterase; PMEI, PME inhibitor; Pectin; Pectin methylesterase (PME); Pichia pastoris; Root.

Fig. 1

Expression in Pichia pastoris and purification of PMEI3. A. Scheme of the expression construct, B. Immunoblot with anti-His antibodies of 2 culture supernatants (#1 and #2) and positive control (PC) of purified PMEI3 . C. Coomassie stained gel (left) anti-His immunoblot (right) of purified protein. D. PMEI3-matching tryptic fragments (in red) from the purified protein identified by MS-MS.

Fig. 2

pH-sensitive inhibition of PME activity by PMEI3. Relative PME activity measured at pH 5 (A,D,G); 6.3 (B,E,H) and 7.5 (CF,I) in the presence of 0; 1.12 µM; 2.25 µM; 11.24 µM and boiled 11.24 µM PMEI3 in PME preparations from orange (A-C), Arabidopsis thaliana roots (D-F) or flowers (G-I). PME activity is expressed as % of the control without PMEI3. Error bars are SD (n=3) and stars refer to the P-value of the t-tests on the comparison with the 0 control or for the boiled samples for the comparison with non-boiled 11.24 µM samples. *:<0.05, **:<0.01, ***:<0.001 , ns : not significant.

Fig. 3

Specific PMEI3 activity against purified PMEs. Relative PME activity measured in purified PME3 (A-C) and PME2 (D-F) preparations. at pH 5 (A-D); 6.3 (B-E) and 7.5 (C-F) in the presence of 0; 1.12 µM; 2.25 µM; 11.24 µM and boiled 11.24 µM PMEI3. PME activity is expressed as % of the control without PMEI3. Error bars are standard deviations (n=3) and stars refer to the P-value of the t-tests on the comparison with the 0 control or for the boiled samples for the comparison with non-boiled 11.24 µM samples. *:<0.05, **:<0.01, ***:<0.001, ns : not significant.

Fig. 4

In vivo activity of purified PMEI3. A,B: Representative photographs (A) and primary root lengths (B) of 3-day-old Arabidopsis thaliana seedlings. Curve is a second degree polynomal fit (y = 3,3692x² - 3,6899x + 1,007; R² = 0,9947); IC50 = 0.16 µM. C,D: Representative photographs (C) and fluorescence quantification (D) of COS⁶⁴⁷-labeled roots. 0.2 µM sat and 0.6 µM sat refer to the same samples as for 0.2 µM and 0.6 µM but with an increased detection sensitivity. Two-day-old seedlings were transferred to 0; 0.2 µM or 0.63 µM PMEI3 and incubated for 24h before analysis. Error bars are SD (n > 30 for B and n=5 for D). *** t-test, P-value<0.001.