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Isolation and characterization of a novel peroxisomal choline monooxygenase in barley

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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. [14C]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 [14C]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.

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Abbreviations

BADH:

Betaine aldehyde dehydrogenase

CMO:

Choline monooxygenase

ER:

Endoplasmic reticulum

GB:

Glycine betaine

GFP:

Green fluorescent protein

mRFP:

Monomeric red fluorescent protein

PEG:

Polyethylene glycol

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Acknowledgments

We thank Dr. Hitoshi Mori (Nagoya University, Japan) for advice on subcellular fractionation, Miss Nicola S. Skoulding (University of Leeds, UK) for critical reading of the manuscript, Dr. Masayoshi Maeshima (Nagoya University, Japan), Dr. Mikio Nishimura (National Institute for Basic Biology, Japan), Dr. Mitsutaka Taniguchi (Nagoya University, Japan) and Dr. Teruhiro Takabe (Meijo University, Japan) for providing yeast strain BJ5458 and the antibodies anti-pumpkin catalase, anti-Eleusine coracana aspartate aminotransferase and anti-spinach CMO, respectively, and Mr. Yasuki Tahara of Nagoya University Farm for harvesting the barley seeds used in this investigation. This work was supported by a Grant-in-Aid for scientific research (No. 20380177 and No. 18880013) from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to T.T. and S.M., respectively) and The Salt Science Research Foundation (to S.M.).

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Authors and Affiliations

  1. Graduate School of Bioagricultural Sciences, Nagoya University, Chikusa, Nagoya, 464-8601, Japan

    Shiro Mitsuya, Junko Kuwahara, Keiko Ozaki, Eiji Saeki, Takashi Fujiwara & Tetsuko Takabe

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  1. Shiro Mitsuya
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  2. Junko Kuwahara
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  3. Keiko Ozaki
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  4. Eiji Saeki
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  5. Takashi Fujiwara
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  6. Tetsuko Takabe
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Correspondence to Shiro Mitsuya.

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Mitsuya, S., Kuwahara, J., Ozaki, K. et al. Isolation and characterization of a novel peroxisomal choline monooxygenase in barley. Planta 234, 1215–1226 (2011). https://doi.org/10.1007/s00425-011-1478-9

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