ISSN 10214437, Russian Journal of Plant Physiology, 2014, Vol. 61, No. 4, pp. 543–547. © Pleiades Publishing, Ltd., 2014.
Nitric oxide (NO) is a gaseous signaling molecule
that is involved in a multitude of physiological pro
cesses in plants  and animals . In the late 1980s,
it was discovered that NO is synthesized in animal cells
by the enzyme nitric oxide synthase (NOS) using the
amino acid Larginine as the substrate (arginine path
way). Later, NO was found to also be produced from
nitrite in animals by distinctive mechanisms (nitrite
pathway) . Since the regulation of NO production is
important in physiological responses, the sources of
NO and its production mechanisms are of particular
interest in biology and medicine.
In plants, it has been shown that NO is produced
from nitrite through enzymatic as well as nonenzy
matic mechanisms, whereas the arginine pathway has
yet to be elucidated due to the lack of a NOS homolog
in plants . Nitrate reductase (NR) is the first
enzyme, whose NO producing activity was confirmed
in plants by both in vitro  and in vivo  studies. In
contrast to the arginine pathway that is catalyzed by
NOS enzymes, the nitrite pathway involves multiple
routes and mechanisms. More recently, many plant
enzymes other than NR have been reported to pro
The article is published in original.
duce NO from nitrite: peroxisomal xanthine oxidase
, plasma membrane bound nitrite:NO reductase
, and nonsymbiotic hemoglobin . One electron
reduction of nitrite by electron transport systems also
produces NO in chloroplasts  and mitochondria
. In addition to these enzymatic mechanisms,
nonenzymatic NO production in acidic and reducing
environments, that may occur in the apoplast  and
plastids , has physiological relevance.
Compounds other than Larginine and nitrite have
been shown to induce NO production in plants: the
polyamines spermine and spermidine in
 and hydroxylamine in NRfree plant cells .
These studies imply that plants may have the potential
to utilize a variety of chemicals to produce NO.
Sodium azide (
) has been applied for research
purposes as a vasodilator [15, 16], but it is cytotoxic
because it inhibits a range of metalcontaining enzyme
activities . It is now evident that the vasodilative
activity of azide is due to its function as a precursor of
NO in animals [16, 18]. However, there is no literature
available to confirm the presence of azidedependent
NO production in plants.
In this study we used the floating fern
as a plant model to investigate azidedependent
is a fresh water fern in sym
biotic relationship with the nitrogenfixing cyanobac
. Its small size and
aquatic habitat made
ideally suited for this
AzideDependent Nitric Oxide Emission from the Water Fern
, M. F. Cohen
, and H. Yamasaki
Faculty of Science, University of the Ryukyus, Nishihara 9030213, Japan;
fax: +81988958576; email: firstname.lastname@example.org
Department of Biology, Sonoma State University, Rohnert Park, CA 94928, USA
Received April 4, 2013
—Nitric oxide (NO) is involved in versatile functions in plant growth and development as a signaling
molecule. To date, plants have been reported to produce NO following exposure to nitrite (N the amino
acid Larginine, hydroxylamine, or polyamines. Here we demonstrate azidedependent NO production in
plants. The water fern
emitted NO into air upon exposure to sodium azide (NaN
). The NO
production was dependent on azide concentration and was strongly inhibited by potassium cyanide (KCN).
with the catalase inhibitor 3aminotriazole (3AT) abolished the azidedependent
NO production. Although nitritedependent NO production was inhibited by sodium azide, azidedepen
dent NO production was not affected by nitrite. These results indicate that
NO using azide as a substrate. We suggest that plants are also capable of producing NO from azide by the
action of catalase as previously reported in animals.
Keywords: Azolla pinnata,
nitric oxide, nitrite, azide, catalase, ROS
: 3AT—3aminotriazole; NOS—nitric oxide syn
thase; NR—nitrate reductase.