title: Isolation and characterization of a novel peroxisomal choline monooxygenase in barley.
abstract: Glycine betaine (GB) is a compatible solute accumulated by many plants under various abiotic stresses. GB is synthesized in two steps, choline → betaine aldehyde → GB, where a functional choline-oxidizing enzyme has only been reported in Amaranthaceae (a chloroplastic ferredoxin-dependent choline monooxygenase) thus far. Here, we have cloned a cDNA encoding a choline monooxygenase (CMO) from barley (Hordeum vulgare) plants, HvCMO. In barley plants under non-stress condition, GB had accumulated in all the determined organs (leaves, internodes, awn and floret proper), mostly in the leaves. The expression of HvCMO protein was abundant in the leaves, whereas the expression of betaine aldehyde dehydrogenase (BADH) protein was abundant in the awn, floret proper and the youngest internode than in the leaves. The accumulation of HvCMO mRNA was increased by high osmotic and low-temperature environments. Also, the expression of HvCMO protein was increased by the presence of high NaCl. Immunofluorescent labeling of HvCMO protein and subcellular fractionation analysis showed that HvCMO protein was localized to peroxisomes. [(14)C]choline was oxidized to betaine aldehyde and GB in spinach (Spinacia oleracea) chloroplasts but not in barley, which indicates that the subcellular localization of choline-oxidizing enzyme is different between two plant species. We investigated the choline-oxidizing reaction using recombinant HvCMO protein expressed in yeast (Saccharomyces cerevisiae). The crude extract of HvCMO-expressing yeast coupled with recombinant BBD2 protein converted [(14)C]choline to GB when NADPH was added as a cofactor. These results suggest that choline oxidation in GB synthesis is mediated by a peroxisomal NADPH-dependent choline monooxygenase in barley plants.
authors.affiliation: Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya, 464-8601, Japan. mitsuya@agr.nagoya-u.ac.jp
authors.forename: Shiro
authors.initials: S
authors.lastname: Mitsuya
authors.forename: Junko
authors.initials: J
authors.lastname: Kuwahara
authors.forename: Keiko
authors.initials: K
authors.lastname: Ozaki
authors.forename: Eiji
authors.initials: E
authors.lastname: Saeki
authors.forename: Takashi
authors.initials: T
authors.lastname: Fujiwara
authors.forename: Tetsuko
authors.initials: T
authors.lastname: Takabe
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001622:Betaine / Q000378:metabolism*; D050728:Betaine-Aldehyde Dehydrogenase / Q000235:genetics / Q000378:metabolism; D002794:Choline / Q000378:metabolism; D003080:Cold Temperature; D018076:DNA, Complementary / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics*; D018506:Gene Expression Regulation, Plant / Q000235:genetics; D001467:Hordeum / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D009997:Osmotic Pressure; D010084:Oxidation-Reduction; D010105:Oxygenases / Q000235:genetics / Q000378:metabolism*; D020675:Peroxisomes / Q000201:enzymology*; D018515:Plant Leaves / Q000201:enzymology / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified; D012333:RNA, Messenger / Q000235:genetics; D018749:RNA, Plant / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D018724:Spinacia oleracea / Q000235:genetics / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D010940:Plant Proteins; D012333:RNA, Messenger; D018749:RNA, Plant; D001622:Betaine; C105448:choline monooxygenase; D010105:Oxygenases; D050728:Betaine-Aldehyde Dehydrogenase; D002794:Choline