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Constitutively and highly expressed Oryza sativa polyamine oxidases localize in peroxisomes and catalyze polyamine back conversion

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Abstract

Polyamine oxidases (PAOs) are FAD-dependent enzymes involved in polyamine (PA) catabolism. Recent studies have revealed that plant PAOs are not only active in the terminal catabolism of PAs as demonstrated for maize apoplastic PAO but also in a polyamine back-conversion pathway as shown for most Arabidopsis PAOs. We have characterized Oryza sativa PAOs at molecular and biochemical levels. The rice genome contains 7 PAO isoforms that are termed OsPAO1 to OsPAO7. Of the seven PAOs, OsPAO3, OsPAO4, and OsPAO5 transcripts were most abundant in 2-week-old seedlings and mature plants, while OsPAO1, OsPAO2, OsPAO6, and OsPAO7 were expressed at very low levels with different tissue specificities. The more abundantly expressed PAOs—OsPAO3, OsPAO4, and OsPAO5—were cloned, and their gene products were produced in Escherichia coli. The enzymatic activities of the purified OsPAO3 to OsPAO5 proteins were examined. OsPAO3 favored spermidine (Spd) as substrate followed by thermospermine (T-Spm) and spermine (Spm) and showed a full PA back-conversion activity. OsPAO4 substrate specificity was similar to that of OsPAO5 preferring Spm and T-Spm but not Spd. Those enzymes also converted Spm and T-Spm to Spd, again indicative of PA back-conversion activities. Lastly, we show that OsPAO3, OsPAO4, and OsPAO5 are localized in peroxisomes. Together, these data revealed that constitutively and highly expressed O. sativa PAOs are localized in peroxisomes and catalyze PA back-conversion processes.

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Abbreviations

AO:

Amine oxidase

Arg:

Arginine

AtPAO:

Arabidopsis thaliana polyamine oxidase

DAS:

Day(s) after sowing

FAD:

Flavin adenine dinucleotide

GFP:

Green fluorescent protein

HvPAO:

Barley polyamine oxidase

NorSpm:

Norspermine

N1-AcSpm:

N1-acetylspermine

Orn:

Ornithine

PA:

Polyamine

PAO:

Polyamine oxidase

PCR:

Polymerase chain reaction

OsPAO:

Oryza sativa polyamine oxidase

PTS:

Peroxisomal targeting signal

Put:

Putrescine

Spd:

Spermidine

Spm:

Spermine

T-Spm:

Thermospermine

ZmPAO:

Maize polyamine oxidase

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Acknowledgments

We thank to Drs. K. A. Roubelakis-Angelakis and N. -H. Chua for kindly providing mCherry-AtPAO3 and pGFP2 plasmids, respectively. We also thank to Dr. E. Agostinelli and the anonymous reviewer(s) who contributed to improving this manuscript. This work was supported in part by Grant-in-Aids from the Japan Society for the Promotion of Science (JSPS) to TK (21380063) and YT (21780087, 23780345), and by the research funding program “LOEWE—Landes-Offensive zur Entwicklung Wissenschaftlich-ökonomischer Exzellenz” of Hesse’s Ministry of Higher Education, Research, and the Arts to TB.

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Correspondence to Tomonobu Kusano or Yoshihiro Takahashi.

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Yusuke Ono and Dong Wook Kim were contributed equally to this work.

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Ono, Y., Kim, D.W., Watanabe, K. et al. Constitutively and highly expressed Oryza sativa polyamine oxidases localize in peroxisomes and catalyze polyamine back conversion. Amino Acids 42, 867–876 (2012). https://doi.org/10.1007/s00726-011-1002-3

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