Temporal regulation of cell-wall pectin methylesterase and peroxidase isoforms in cadmium-treated flax hypocotyl

Abstract

Background and aims: In hypocotyls of flax (Linum usitatissimum) cadmium-induced reorientation of growth (i.e. an increase in expansion and a decrease in elongation) coincides with marked changes in the methylesterification and cross-linking of homogalacturonans within various cell-wall (CW) domains. The aim of the present study was to examine the involvement of pectin methylesterase (PME) and peroxidase (PER) in this cadmium-induced CW remodelling.

Methods: CW proteins were extracted from hypocotyls of 10- and 18-d-old flax that had been treated or not treated with 0.5 mm Cd(NO(3))(2). PME and PER expression within these extracts was detected by LC/MS, by isoelectric focusing and enzyme activity assays. Transcript expression by RT-PCR of known flax PME and PER genes was also measured in corresponding samples.

Key results: In cadmium-treated seedlings, PME activity increased as compared with controls, particularly at day 10. The increased activity of PME was accompanied by increased abundance of both a basic protein isoform (B2) and a particular transcript (Lupme5). In contrast, induction of PER activity by cadmium was highest at day 18. Among the four reported PER genes, Flxper1 and 3 increased in abundance in the presence of cadmium at day 18.

Conclusions: The temporal regulation of Lupme and Flxper genes and of their respective enzyme activities fits the previously reported cadmium-induced structural changes of homogalacturonans within the CWs. After PME-catalysed de-esterification of homogalacturonans, their cross-linking would depend on the activity of PERs interacting with calcium-dimerized blocks and reinforce the cell cohesion during the cadmium-induced swelling.

Fig. 1.

SDS–PAGE of the cell-wall ionically bound proteins of the hypocotyl. Seedlings were grown in the presence (+) or absence (−) of cadmium and harvested at 10 and 18 d old, and 1 µg of proteins from − Cd and + Cd were loaded per well. This figure is representative of three SDS–PAGE repeats. Note the high variability in the distribution of the silver-stained polypeptides especially in the ranges of 60 kDa, 43–50 kDa and 30–37 kDa.

Fig. 2.

Pectin methylesterase isoenzymes ionically bound to the cell wall of the hypocotyl. Seedlings were grown in the presence (+) or absence (−) of cadmium and harvested at 10 and 18 d old. (A) IEF and (B) transfer of the gel onto a pectin–agarose gel. PME activity was revealed by ruthenium-red staining de-esterified homogalacturonans. A similar amount of PME (0·5 nkatal) activity was loaded per well. The pH values measured with a surface electrode are given on the left. Note the decrease of B1b (arrow) which paralleled the increased of B2 at day 10. (C) Expression of Lupme genes compared with the elongation factor gene LuEF1 α (EF1) as seen by PCR. All gel figures were representative of three to five repeats. N and B are neutral and basic isoforms, respectively.

Fig. 3.

Peroxidase isoenzymes ionically bound to the cell wall of the hypocotyl. Seedlings were grown in the presence (+) or absence (−) of cadmium and harvested at 10 and 18 d old. (A, B) After IEF, the peroxidase activity was revealed in the presence of gaïacol (A) or chloronaphthol (B). A similar amount of PER (0·1 nkatal) activity was loaded per well, each sample being repeated in two successive wells. The pH values measured with a surface electrode are given on the left. Note the increase with age and Cd of the isoforms N and Ba. (C) Expression of Flxper genes compared with elongation factor gene LuEF1 α (EF1) as seen by PCR. All gel figures were representative of three to five repeats. A, N and B are acidic, neutral and basic isoforms, respectively.