β-Alanine N-Methyltransferase of Limonium latifolium. cDNA Cloning and Functional Expression of a Novel N-Methyltransferase Implicated in the Synthesis of the Osmoprotectant β-Alanine Betaine

Abstract

␤-alanine (Ala) betaine, an osmoprotectant suitable under saline and hypoxic environments, is found in most members of the halophytic plant family Plumbaginaceae. In Limonium latifolium (Plumbaginaceae), it is synthesized via methylation of ␤-Ala by the action of a trifunctional S-adenosyl l-methionine (Ado-Met): ␤-Ala N-methyltransferase (NMTase). Peptide sequences from purified ␤-Ala NMTase were used to design primers for reverse transcriptase-PCR, and several cDNA clones were isolated. The 5Ј end of the cDNA was cloned using a 5Ј-rapid amplification of cDNA ends protocol. A 500-bp cDNA was used as a probe to screen a -gt10 L. latifolium leaf cDNA library. Partial cDNA clones represented two groups, NMTase A and NMTase B, differing only in their 3Ј-untranslated regions. The full-length NMTase A cDNA was 1,414 bp and included a 1128-bp open reading frame and a 119-bp 5Ј-untranslated region. The deduced amino acid sequence of 375 residues had motifs known to be involved in the binding of Ado-Met. The NMTase mRNA was expressed in L. latifolium leaves but was absent in Limonium sinuatum, a member of the genus that lacks the synthetic pathway for ␤-Ala betaine. NMTase mRNA expression was high in young and mature leaves and was enhanced by light. NMTase cDNA was expressed in yeast (Saccharomyces cerevisiae) under the control of a galactose-inducible promoter. Protein extracts of galactose-induced recombinant yeast had Ado-Met-specific NMTase activities that were highly specific to ␤-Ala, N-methyl ␤-Ala, and N,N-dimethyl ␤-Ala as methyl acceptors. NMTase activities were not detectable in comparable protein extracts of yeast, transformed with vector control. The NMTase protein sequence shared homology with plant caffeic acid O-methyltransferases and related enzymes. Phylogenetic analyses suggested that ␤-Ala NMTase represents a novel family of N-methyltransferases that are evolutionarily related to O-methyltransferases.Drought, salinity, flooding, and freezing adversely affect agricultural productivity (Boyer, 1982). However, many plants have evolved metabolic adaptations to these abiotic stress factors. Accumulation of osmoprotectants is a common adaptation found in stress-tolerant plants, bacteria, and marine algae (Yancey et al., 1982; Bohnert and Sheveleva, 1998). Quaternary ammonium compounds (QACs) represent some of the most effective osmoprotectants known in biology (Anthoni et al., 1991;Rhodes and Hanson, 1993).Because only certain stress-tolerant plants accumulate the common QAC Gly betaine and many crops do not, it was suggested that engineering crops for Gly betaine overproduction could be a way to improve their stress tolerance (McCue and Hanson, 1990). Transgenic plants, overexpressing bacterial and plant pathways for Gly betaine synthesis, accumulated relatively small quantities of the QAC (Rontein et al., 2002), but nonetheless exhibited stresstolerant phenotypes Murata, 2000, 2001;Hibino et al., 2002). However, reiterative metabolic engineering experiments indicated that availability of the substrate...

Putrescine N-methyltransferase in Solanum tuberosum L., a calystegine-forming plant

“…This was not surprising as both genes share 61% overall identity and are even more homologous (71%) comparing only the 750 bp initial PCR fragment. Very low identity rates are found, if amino acid sequence comparison is expanded to other plant N-methyltransferases from alkaloid metabolism (Table 1b), such as coclaurine-N-methyltransferase from Coptis japonica (Choi et al 2002) and xanthine-N-methyltransferases from Coffea arabica (Uefuji et al 2003) or to the stress metabolite forming b-alanine-N-methyltransferase from Limonium latifolium (Raman and Rathinasabapathi 2003). SAM-dependent methyltransferases often share little sequence identity Fig.…”

Metabolic Engineering for Stress Tolerance

“…al., 2003), Limonium latifolium (Rathinasabapathi et. al., 2001;Raman and Rathinasabapathi, 2003), Mesembryanthemum crystallinum (Vera-Esterella et. al., 2004), Atriplex hortensis (Shen et.…”

“…al., 2001). A full length cDNA for beta-alanine N-methyltransferase from L. latifolium was cloned and characterized recently (Raman and Rathinasabapathi, 2003). Experiments in our laboratory showed that in a variety of plant species, pantothenate synthesis was limited by d-pantoate and not by beta-alanine (Rathinasabapathi and Raman, 2005).…”

“…Transgenic cells were characterized by the production of low levels of ectoine (<79 nmol g -1 fresh weight) and salinity tolerance. NMT is a trifunctional beta-alanine N-methyltransferase (Raman and Rathinasabapathi, 2003) and PS is pantothenate synthetase (Genschel et. al., 1999), both presumably cytosolic enzymes…”

Glycine betaine biosynthesis in saltbushes (Atriplex spp.) under salinity stress

“…Further, GB has been shown to play an essential role in maintaining the health of vital organs, such as the liver, heart and kidneys in humans (Craig 2004;Likes et al 2007). GB reduces cardiovascular risk factors and betaine and choline decrease the risk of infant neural tube defects (Raman & Rathinasabapathi 2003).…”