Molecular and biochemical characterization of a cold-regulated phosphoethanolamine N-methyltransferase from wheat

Abstract

A cDNA that encodes a methyltransferase (MT) was cloned from a cold-acclimated wheat (Triticum aestivum) cDNA library. Molecular analysis indicated that the enzyme WPEAMT (wheat phosphoethanolamine [P-EA] MT) is a bipartite protein with two separate sets of S-adenosyl-L-Met-binding domains, one close to the N-terminal end and the second close to the C-terminal end. The recombinant protein was found to catalyze the three sequential methylations of P-EA to form phosphocholine, a key precursor for the synthesis of phosphatidylcholine and glycine betaine in plants. Deletion and mutation analyses of the two S-adenosyl-L-Met-binding domains indicated that the N-terminal domain could perform the three N-methylation steps transforming P-EA to phosphocholine. This is in contrast to the MT from spinach (Spinacia oleracea), suggesting a different functional evolution for the monocot enzyme. The truncated C-terminal and the N-terminal mutated enzyme were only able to methylate phosphomonomethylethanolamine and phosphodimethylethanolamine, but not P-EA. This may suggest that the C-terminal part is involved in regulating the rate and the equilibrium of the three methylation steps. Northern and western analyses demonstrated that both Wpeamt transcript and the corresponding protein are up-regulated during cold acclimation. This accumulation was associated with an increase in enzyme activity, suggesting that the higher activity is due to de novo protein synthesis. The role of this enzyme during cold acclimation and the development of freezing tolerance are discussed.

Figure 1

Nucleotide and deduced AA sequences of Wpeamt and of mutants generated. A, Nucleotide and deduced AA sequence of Wpeamt. The open reading frame is 1,497 nucleotides. The predicted polypeptide is 498 AAs in length, with a calculated molecular mass of 57 kD and a pI of 5.3. The two putative Ado-Met-binding domains are highlighted in gray. The first domain is in the N-terminal portion of the protein from AA 64 to 164. The Ado-Met-binding domain consists of four motifs: motif I, post-I, motif II, and motif III. The second putative Ado-Met-binding domain also composed of four motifs is in the C-terminal portion of the protein from AA 294 to 391. Bold letters within each motif I indicate the site of the mutations (G–E) that gives rise to M1 or M2. The underlined nucleotides are the sequences recognized by primers to generate H1 (single) and H2 (double). GenBank accession number: AY065971. B, Schematic representation of the WT WPEAMT and its mutations and deletions. WT, WT recombinant WPEAMT; M1, recombinant WPEAMT in which motif I of the first Ado-Met-binding domain is mutated; M2, WPEAMT in which motif I of the second Ado-Met-binding domain is mutated; H1, first one-half of WPEAMT; H2, second one-half of WPEAMT. The motifs that characterize the Ado-Met-binding domain are represented by black squares (I–III). Blank squares represent the motifs in which the mutation is located. The dashed line at the beginning of each polypeptide corresponds to the His tag.

Figure 2

Functional analysis of the recombinant WPEAMT. The WT WPEAMT, its mutants (M1 and M2), and deletions (H1 and H2) were expressed as N-terminal His tagged fusions in E. coli. All proteins (WT, M1, M2, and H1) were induced with isopropyl-β-d-thiogalactoside (IPTG) at 37°C for 3h except for H2, which was induced at 20°C for 16h. A, Upper, Immunoblot with the anti-WPEAMT antibodies. Lower, Protein pattern stained with Coomassie brilliant blue R-250. B, WPEAMT-specific activity using P-EA as substrate. The arrow indicates the location of the N-terminal fusion of the WT WPEAMT and mutants (65 kD). Lane 1, Untransformed E. coli; lane 2, transformed with WT WPEAMT before induction with IPTG; lane 3, WT WPEAMT after induction with IPTG; lane 4, purified WT WPEAMT on a His-bind resin column; lane 5, mutant M1 after induction; lane 6, mutant M2 after induction; lane 7, deletion H1 after induction; lane 8, deletion H2 after induction.

Figure 3

Autoradiogram of reaction products of the P-EA methylation by WPEAMT separated by TLC. The enzyme assay as well as sample preparation, chromatography conditions, and development of TLC plates were described in “Materials and Methods,” except that [¹⁴C] Ado-Met was used as substrate and the assay time was for 2 h. Lane 1, Reaction products of WT WPEAMT. Lane 2, Reaction products of M2. Lane 3, Reaction products of H1.

Figure 4

DNA gel-blot analysis of genomic DNA using Wpeamt as a probe in DT series of wheat cv CS. The DT series of wheat cv CS, in which one homologous pair of chromosome arms is missing in each line, was used to determine which chromosome arms carry the gene. DNA gel blot of two wheat genotypes (cv CS and cv Cheyenne) and rye (Secale cereale L. cv Puma) are also shown. A, B, and D, Three genomes present in hexaploid wheat; S and L, Presence of the short or long chromosome arms, respectively. In all lanes, 1.8 μg of genomic DNA digested with XbaI was used.

Figure 5

Up-regulation of WPEAMT during CA and salt stress. A, Accumulation of Wpeamt mRNAs under different stress conditions. The 28S ribosomal band stained with ethidium bromide is included to show RNA loads (7.5 μg). B, Immunoblot showing the WPEAMT accumulation. Coomassie Brilliant Blue-stained gel shows the Rubisco band as load control. C, WPEAMT activity using P-EA as substrate in soluble fractions of wheat during stress treatments. Values represent the mean ± se from four independent experiments. NA7, Nonacclimated plants grown for 7 d; CA6, 6-d cold-acclimated plants; NaCl, plants treated with 300 mm NaCl for 18 h; ABA, plants treated with 0.1 mm ABA for 18 h; WS, plants exposed to water stress for 18 h; HS, plants exposed to 40°C for 3 h (heat shock); W, wounding stress for 3 h.

Figure 6

Accumulation of Wpeamt mRNAs during CA in cereals. A, Time course and tissue specificity in winter wheat cv Norstar. NA7 and NA12, Nonacclimated plants grown for 7 and 12 d; CA1, CA6, and CA36, cold-acclimated plants for 1, 6, and 36 d; D5, cold-acclimated plants (36 d) were de-acclimated for 5 d. Exposure was for 3 h. B, Wheat genotypes and species differential accumulation. In this study, total RNA from two spring wheat genotypes (cv Glenlea [Glen], LT₅₀ [lethal temperature that kills 50% of the seedlings] of −8°C; and cv Concorde [Con], LT₅₀ of −8°C), four winter wheat genotypes (cv Monopole [Mon], LT₅₀ of −15°C; cv Absolvent [Abs], LT₅₀ of −16°C; cv Fredrick [Fred], LT₅₀ of −16°C; and cv Norstar [Nor], LT₅₀ of −19°C), winter rye (cv Musketeer [Mus], LT₅₀ of −21°C), oat (Avena sativa L. cv Laurent [Lau], LT₅₀ of −6°C), barley (cv Winchester [Win], LT₅₀ of −7°C) were used. NA, Control plants (nonacclimated) grown for 12 d; CA, plants cold acclimated for 36 d. Exposure was for 16h. C, Accumulation in rice and corn. NA, Control (nonacclimated) plants grown under a day/night temperature of 29°C/26°C; CA, (cold-acclimated) plants grown for 24 h under the corresponding day/night temperatures. Exposure was for 3 d. In all lanes, 7.5 μg of total RNA was used. The 28S ribosomal band stained with ethidium bromide is included to show RNA loads.