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.

Tissue Localization of the Glycine Betaine Biosynthetic Enzymes in Barley Leaves

“…2, 3). The signal of CMO protein was detected in the dot-like organelles, which is in agreement with our previous report that CMO is localized in peroxisomes in barley (Mitsuya et al, 2011). In addition, the signal of from the Ministry of Education, Culture, Sports, Science and Technology of Japan and by JGC-S Scholarship Foundation (to S.M.…”

“…This cultivar has been used to generate a large number of ESTs at Okayama University, Japan, and a barley bacterial artificial chromosome library (Saisho et al, 2007). In addition, this cultivar is moderately salt-tolerant (Nakamura et al, 1996) and has been used for the research on the salt tolerance of barley (Arakawa et al, 1992;Ishitani et al, 1995;Fujiwara et al, 2008;Hattori et al, 2009;Fujiwara et al, 2010;Mitsuya et al, 2011).…”

“…Protein gel-blot analyses were carried out as described previously (Mitsuya et al, 2011). Primary [rabbit antiHvCMO peptides (Mitsuya et al, 2011) and anti-spinach BADH (Arakawa et al, 1992) IgGs] and secondary [peroxidase-conjugated donkey anti-rabbit IgG (GE Healthcare, Little Chalfont, UK)] antibodies were diluted in Can Get Signal solution (Toyobo, Osaka, Japan) at 1:1,000, 1:5,000, 1:5,000, respectively. Protein concentration was determined with the Bio-Rad Bradford Protein Assay (Bio-Rad, Hercules, CA, USA) using BSA as a standard.…”

“…Immunodetection of CMO and BADH proteins was performed as described previously (Mitsuya et al, 2011). Primary [rabbit anti-HvCMO peptides (Mitsuya et al, 2011) …”

“…We have succeeded in isolating choline monooxygenase (CMO), the choline oxidizing enzyme, from gramineous barley (Mitsuya et al, 2011). Barley CMO (HvCMO) requires NADPH for activity and is localized to peroxisomes.…”