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Plant and Cell Physiology, 2002, Vol. 43, No. 3 290-297
© 2002 Oxford University Press

Nitric Oxide Production Mediated by Nitrate Reductase in the Green Alga Chlamydomonas reinhardtii: an Alternative NO Production Pathway in Photosynthetic Organisms

Yasuko Sakihama, Soichi Nakamura and Hideo Yamasaki1

Laboratory of Cell and Functional Biology, Faculty of Science, University of the Ryukyus, Nishihara, Okinawa, 903-0213 Japan

Biological activity of nitric oxide (NO) production was investigated in the unicellular green alga Chlamydomonas reinhardtii. An NO specific electrode detected a rapid increase in signal when nitrite (NO2–) was added into a suspension of C. reinhardtii intact cells in the dark. The addition of KCN or the NO quencher bovine hemoglobin completely abolished the signal, verifying that the nitrite-dependent increase in signal is due to enzymatic NO production. L-arginine, the substrate for NO synthase, did not induce detectable NO production and the NOS inhibitor N{omega}-nitro-L-arginine showed no inhibitory effect on the nitrite-dependent production of NO. Illuminating cells showed a significant suppressive effect on NO production. When the photosynthetic electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea was present in the suspension, C. reinhardtii cells produced NO after the addition of nitrite even under illumination. Kinetic and microscopic observations, using the intracellular fluorescent NO probe 4,5-diaminofluorescein-2 diacetate, both demonstrated that NO was produced within the cells in response to the addition of nitrite. The Chlamydomonas mutant cc-2929, which lacks nitrate reductase (NR) activity, did not display any of the responses observed in the wild-type cells. The results presented here provide direct in vivo evidence to confirm that NR is involved in the nitrite-dependent NO production in the green alga.

1 Corresponding author: E-mail, yamasaki@comb.u-ryukyu.ac.jp; Fax, +81-98-895-8576.


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