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.Winter survival and crop productivity are influenced by many different winter stresses, such as freezing temperature, length of freezing period, ice encasement, flooding, and oxidative stress caused by low temperature (LT)-induced photoinhibition (Fowler et al., 1999). Exposure of plants to LT produces morphological, physiological, and biochemical changes that are often highly correlated with plant freezing tolerance (FT) and winter survival. These changes are regulated by LT at the gene expression level. Cold-regulated genes and their products have been identified and characterized in many species (Thomashow, 1999). The complexity of the LT response has made it difficult to separate genes responsible for LT acclimation and cold hardiness from those associated with metabolic adjustments to LT. To characterize the genetic components associated with LT response, a better understanding of the LTresponsive genes responsible for adaptation to winter stresses and their interaction with the environment is needed (Fowler et al., 1999).

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An abundant 17 kDa protein which was isolated and characterized from 10-day old healthy root tissue of white lupin (Lupinus albus) proved to have a high sequence similarity to pathogenesis-related proteins found in other species. Subsequently, a corresponding clone (LaPR-10) was identified in a cDNA library prepared from the same tissue that exhibited a high amino acid sequence similarity to a number of the PR-10 family proteins. The clone contains an open reading frame encoding a polypeptide of 158 amino acids, with a predicted molecular mass of 16,905 Da and an isoelectric point of 4.66. Southern blot analysis indicates that LaPR-10 is likely a single-copy gene, or a member of a small gene family. The clone was expressed in Escherichia coli, and its protein product was purified to near homogeneity. Both the native and the recombinant proteins were immunorecognized by antibodies raised against pea PR-10 proteins, and exhibited a ribonucleolytic activity against several RNA preparations, including lupin root total RNA. Characterization of its enzymatic properties indicates that the LaPR-10 protein belongs to the class II ribonucleases. We present evidence that the white lupin 17 kDa protein is constitutively expressed during all stages of root development and, to a lesser extent, in other plant parts. In addition, we demonstrate the presence, in the LaPR-10 amino acid sequence, of a number of motifs that are common to most PR-10 proteins, as well as a RGD motif that is shared only with the alfalfa SRG1 sequence.

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O-Methyltransferases catalyze the transfer of the methyl groups of S-adenosyl-L-methionine to specific hydroxyl groups of several classes of flavonoid compounds. Of the several cDNA clones isolated from a Chrysosplenium americanum library, FOMT3′ encodes the 3′/5′-O-methylation of partially methylated flavonols. The recombinant protein of another clone, FOMTx which differs from FOMT3′ by a single amino acid residue (Ser286Arg) exhibits no enzymatic activity towards any of the flavonoid substrates tested. Replacement of Ser 286 in FOMT3′ with either Ala, Leu, Lys or Thr, almost abolished O-methyltransferase activity. In contrast with FOMT3′, no photoaffinity labeling could be achieved using [ 14 CH 3 ]AdoMet with the mutant recombinant proteins indicating that Ser 286 is also required for cosubstrate binding. These results are corroborated by isothermal titration microcalorimetry measurements. Circular dichroism spectra ruled out any significant conformational differences in the secondary structures of both FOMT3′ and Ser286Arg. Modeling FOMT3′ on the structure of chalcone methyltransferase indicates that serine 286 is greater than 10 Å from any of the residues of the active site or the AdoMet binding site of FOMT3′. At the same time, residues 282 to 290 are conserved in most of the Chrysosplenium americanum OMTs. These residues form a large part of the subunit interface, and at least five of these residues are within 4 Å of the opposing subunit. It would appear, therefore, that mutations in Ser286 exert their influence by altering the contacts between the subunits and that these contacts are necessary for maintaining the integrety of the AdoMet binding site and active site of this group of enzymes.Key words: flavonoids, O-methyltransferase, photoaffinity labeling. Résumé : Les O-méthyltransférases (OMTs) catalysent le transfert de groupes méthyles de la S-adénosyl-méthionine sur des groupes hydroxyles spécifiques de plusieurs classes de flavonoïdes. Parmi plusieurs clones d'ADNc isolés d'une banque deChrysosplenium amiricanum, le FOMT3′ code une enzyme catalysant la 3′-5′-O-méthylation de flavonols partiellement méthylés. La protéine recombinante issue d'un autre clone, FOMTx, qui ne diffère de FOMT3′ que par un seul acide aminé (Ser286Arg), ne démontre aucune activité enzymatique envers les substrats flavonoïdes testés. Le remplacement de la Ser286 de FOMT3′ par une Ala, Leu, Lys ou Thr abolit presque totalement l'activité O-méthyltransférase. Contrairement à la FOMT3′, aucun marquage par photoaffinité n'est obtenu avec le [ 14 CH 3 ]AdoMet sur les protéines recombinantes mutantes, indiquant que la Ser286 est aussi requise pour la liaison du co-substrat. Ces résultats sont corroborés par des mesures de titration microcalorimétrique isothermique. Le spectre en dichroïsme circulaire exclut toute différence de conformation significative dans la structure secondaire de FOMT3′ et le mutant Ser286Arg. La modélisation de FOMT3′ sur la structure de la chalcone méthyltransférase indique que la sérine 286 est é...