Nitric Oxide: The Versatility of an Extensive Signal Molecule

Abstract

Nitric oxide (NO) is a small highly diffusible gas and a ubiquitous bioactive molecule. Its chemical properties make NO a versatile signal molecule that functions through interactions with cellular targets via either redox or additive chemistry. In plants, NO plays a role in a broad spectrum of pathophysiological and developmental processes. Although nitric oxide synthase (NOS)-dependent NO production has been reported in plants, no gene, cDNA, or protein has been isolated to date. In parallel, precise and regulated NO production can be measured from the activity of the ubiquitous enzyme nitrate reductase (NR). In addition to endogenous NO formation, high NO emissions are observed from fertilized soils, but their effects on the physiology of plants are largely unknown. Many environmental and hormonal stimuli are transmitted either directly or indirectly by NO signaling cascades. The ability of NO to act simultaneously on several unrelated biochemical nodes and its redox homeostatic properties suggest that it might be a synchronizing molecule in plants.

Full text

9 Apr 2003 13:13 AR AR184-PP54-05.tex AR184-PP54-05.sgm LaTeX2e(2002/01/18) P1: FHD
10.1146/annurev.arplant.54.031902.134752
Annu. Rev. Plant Biol. 2003. 54:109–36
doi: 10.1146/annurev.arplant.54.031902.134752
Copyright
c
° 2003 by Annual Reviews. All rights reserved
NITRIC OXIDE: The Versatility of an Extensive
Signal Molecule
Lorenzo Lamattina,Carlos Garc
´
ıa-Mata,
Magdalena Graziano, andGabriela Pagnussat
Instituto de Investigaciones Biol
´
ogicas, Facultad de Ciencias Exactas y Naturales,
Universidad Nacional de Mar del Plata, CC 1245, Argentina; email: lolama@mdp.edu.ar
Key Words chemical messenger, plant hormone, nitrate reductase
■ Abstract Nitric oxide (NO) is a small highly diffusible gas and a ubiquitous
bioactive molecule. Its chemical properties make NO a versatile signal molecule that
functions through interactions with cellular targets via either redox or additive chem-
istry. In plants, NO plays a role in a broad spectrum of pathophysiological and devel-
opmental processes. Although nitric oxide synthase (NOS)-dependent NO production
has been reported in plants, no gene, cDNA, or protein has been isolated to date. In
parallel, precise and regulated NO production can be measured from the activity of the
ubiquitous enzyme nitrate reductase (NR). In addition to endogenous NO formation,
high NO emissions are observed from fertilized soils, but their effects on the physi-
ology of plants are largely unknown. Many environmental and hormonal stimuli are
transmitted either directly or indirectly byNO signaling cascades. The ability of NO to
act simultaneously on several unrelated biochemical nodes and its redox homeostatic
properties suggest that it might be a synchronizing molecule in plants.
CONTENTS
INTRODUCTION .....................................................110
WHEN AND HOW NO WAS IDENTIFIED
AS A CHEMICAL MESSENGER: A BRIEF OVERVIEW ...................111
The History of NO in Animals .........................................111
First Evidence of NO Activity as a Signal Molecule in Plants .................111
CHEMICAL RICHNESS OF A SIMPLE MOLECULE .......................112
Molecular Targets of NO ..............................................112
Antioxidant and Pro-Oxidant Properties of NO ............................114
SOURCE OF NO IN PLANTS ...........................................115
NO Generation and Methods of Measurement .............................115
Structure-Function Comparison Between Animal NO
Synthase (NOS) and Plant Nitrate Reductase (NR) ........................116
Production of NO by Plant NR .........................................118
ACTIONS OF NO IN PLANTS ..........................................119
Nitrogen Fertilization: A New View of an Old Practice ......................119
Pharmacological Effects Versus Physiological Roles ........................120
1040-2519/03/0601-0109$14.00
109
Annu. Rev. Plant Biol. 2003.54:109-136. Downloaded from www.annualreviews.org
by Universidad Nacional de Mar del Plata on 04/08/11. For personal use only.

9 Apr 2003 13:13 AR AR184-PP54-05.tex AR184-PP54-05.sgm LaTeX2e(2002/01/18) P1: FHD
110 LAMATTINA ET AL.
NO Signaling, Processing, and Transduction Pathways ......................120
NO AND CROSS-TALK WITH CLASSICAL
PLANT HORMONES .................................................121
Auxins, NO, and Rooting .............................................121
ABA, NO, and Water Deficit ...........................................122
Cytokinins, Gibberelins, NO, and Phytochrome Pathways ....................124
Ethylene, NO, and Senescence .........................................126
CONCLUDING REMARKS AND FUTURE DIRECTIONS ...................126
Is NO a Synchronizing Chemical Messenger? .............................127
Toward a Regulated Endogenous Production of NO .........................127
INTRODUCTION
Multicellular organisms are highly specialized communities with many different
organs whose functions have to be coordinated very precisely. Communication
between cells and organs must therefore work rapidly and safely, a process that
has been accomplished during evolution through the appearance of a number of
systems, mechanisms, and tools that favor survival.
Plants have a very different lifestyle from animals. Since light is ubiquitous on
the surface of Earth, vascular plants adapted to remain sessile and developed a
differentiated modular structure, whose elements (roots, leaves, shoots, buds, flo-
wers, meristems) ensure that predation and environmental damage can be over-
come through the regeneration of modules or of the individual from modules
(155). Most plant cells have developed a very fine capability to sense environ-
mental stimuli and to process them to produce a whole-plant response. Given the
characteristics of plant lifestyle, one might expect that unique molecules would
have developedfor plant-cell signal transduction. However, with a few remarkable
exceptions, the scenario representing plant cell life can be constructed with actors
chosen from movies about animal cells. That is certainly the case with nitric oxide
(NO)
1
, which has emerged as a new chemical messenger in plant biology after the
characterization of NO functionality in prokaryotes and animals. The biological
activities of NO are numerous and complex and thus are very difficult to integrate
in a simple way. NO is necessary atthe very beginning of life becauseits synthesis
and production are required for the activation of eggs at fertilization (87). NO
governs an impressive number of physiological and pathophysiological reactions
involved in later growth and developmental processes (46, 99). In summary, the
biological activities of NO are diverse and are exerted on phylogenetically distant
species,openingafantasticwindowforanas-yet unexploredfield ofNO’sfunction
in the plant kingdom.
In this review, we present and discuss recent advances in our understanding
of the mechanisms by which NO modulates molecular and physiological plant
responses. The scope of this article is the integration of NO biochemistry in
1
The term NO (without a dot for the unpaired electron) is used for all NO forms, including
NO radical, NO
−
, and NO
+
.
Annu. Rev. Plant Biol. 2003.54:109-136. Downloaded from www.annualreviews.org
by Universidad Nacional de Mar del Plata on 04/08/11. For personal use only.

9 Apr 2003 13:13 AR AR184-PP54-05.tex AR184-PP54-05.sgm LaTeX2e(2002/01/18) P1: FHD
NITRIC OXIDE 111
plant biology processes. The enzymatic mechanism of NO synthesis, the target
molecules involved in NO signaling pathways, the cross-talk with classical plant
hormones, and the intringuing relationships between NO and nitrogen fertilization
are points of intense discussion to open new questions for future research. We do
not focus on NO signaling in the induction of cell death, defense genes, and inter-
play with reactive oxygen species (ROS) and salicylic acid during plant defense
against pathogen attack because excellent reviews are already available on these
subjects (43,83, 157,161).
WHEN AND HOW NO WAS IDENTIFIED
AS A CHEMICAL MESSENGER: A BRIEF OVERVIEW
The History of NO in Animals
It has long been known that the signal molecule acetylcholine dilates blood vessels
in both humans and animals. In most cases, dilation of the vessels is caused by
relaxation of the muscle cells. In 1980, Furchgott discovered that an unknown
substance formed in the endothelium was able to relax the smooth muscle cells
and he named it EDRF (endothelium-derived relaxing factor) (50). Some years
before, Murad found that nitroglycerine activates guanylyl cyclase (GC), which
produces cyclic GMP, and relaxes the muscle fibers. This finding then raised the
question of how the nitroglycerine outside the cell could affect an enzyme inside
the cell. The answer was that nitroglycerine was contaminated with traces of NO.
Murad then bubbled NO gas through smooth muscle cells and GC was activated
(4). Thus, he hypothesized that hormones may influence smooth muscles via NO
even before NO production by eukaryotes was reported (65).
Some years later and working independently, Ignarro showed that NO displays
identical chemical behavior and, in fact, was identical to EDRF (73). Intensive
research on NO biological functions followed worldwide. The fact that NO is an
unstable gas, which in the presence of H
2
O and O
2
is rapidly converted to NO
−
3
and
NO
−
2
, may explain why NO was not discovered earlier in mammals.
NOhassubsequentlybeenidentifiedasacriticalsignalingmoleculein(a)main-
taining blood pressure in the cardiovascular system, (b) stimulating host defenses
in the immune system, (c) regulating neural transmission in the brain, (d) regulat-
ing gene expression, (e) platelet aggregation, ( f ) learning and memory, (g) male
sexual function, (h) cytotoxicity and cytoprotection, and (i) development of arte-
riosclerosis, among others (75,93, 94,103,113).
In 1992, Science named NO “Molecule of the Year” because of its widespread
biological significance, and in 1998, Furchgott, Murad, and Ignarro were awarded
the Nobel Prize in Physiology and Medicine.
First Evidence of NO Activity as a Signal Molecule in Plants
Twenty years ago, NO studies in plant systems were focused on the phytotoxic
properties of the oxides of nitrogen (NO
2
,N
2
O
3
,NO
−
2
,NO
−
3
) and their effect
Annu. Rev. Plant Biol. 2003.54:109-136. Downloaded from www.annualreviews.org
by Universidad Nacional de Mar del Plata on 04/08/11. For personal use only.

9 Apr 2003 13:13 AR AR184-PP54-05.tex AR184-PP54-05.sgm LaTeX2e(2002/01/18) P1: FHD
112 LAMATTINA ET AL.
upon vegetation or on enzymes containing metal groups (97a, 133). Considerable
amounts of NO and N
2
O are produced naturally from nonpolluted terrestrial and
aquaticecosystems,whichrepresent a major contributiontothe natural destruction
of the ozone layer (86). Ozone is produced on the surface of Earth through photo-
chemical processes involving oxides of nitrogen and, as an air pollutant, increases
respiratory illness and general oxidative damages in crop plants (48). However,
in 1990 Welburn raised the question of why atmospheric oxides of nitrogen are
phytotoxic and not alternative fertilizers (160). At the same time, several reports
showed that nitrogen oxides were able to stimulate seed germination (60,61).
More recently, the late 1990s saw an explosion of reports on new and fascinating
effects of NO in plants (8,9,40, 42,56,92), paralleling the discoveries about NO
in animal systems in the late 1980s.
CHEMICAL RICHNESS OF A SIMPLE MOLECULE
Nitric oxide is a free radical lipophilic diatomic gas under atmospheric condi-
tions. Its small Stokes’ radius and neutral charge allows rapid membrane diffusion
(55,145). Molecular mechanisms for precise and controlled NO delivery from
storedcellularcompartmentshaveneverthelessbeenreportedin animals (59,118).
NO rapidly reacts with oxygen to produce a variety of nitrogen oxides. The stabil-
ity and decay of NO depend on its concentration, the redox status of the system,
and the concentration of its target molecules and metals (64). Neutral NO has a
single electron in its 2p-π antibonding orbital (145). The removal of this electron
generates the nitrosonium cation (NO
+
); conversely, the addition of an electron
formsthe nitroxylanion(NO
−
)(Figure 1A).Theadditionof electronsfromNO
+
to
NO
−
decreasesthe bondorder, increasesthebondlength,and decreasesvibrational
energies(145). The different forms of NO share different chemical reactivitiesand
properties. One of the most important chemical properties of NO is, however, the
existence of an unpaired electron, allowing a high reactivity with oxygen (O
2
),
superoxide (O
−
2
), N derivatives, and transition metals.
Molecular Targets of NO
In biological systems, NO reacts with O
2
,O
−
2
, and transition metals (Me
+/2+
), gen-
erating NOx, peroxinitrite (ONOO
−
), and metal-NO adducts, respectively (Figure
1B). The high reactivity of thiols explains the rapid formation of S-nitrosothiol
(RS-NO). Both metal- and thiol-containing proteins are target sites for NO, and
as such are very important cellular vehicles for the orchestration of NO’s bio-
logical function. Among these cellular target proteins are cellular messengers,
ion channels, enzymes, receptors, and transcription factors. For example, solu-
ble GC-mediated NO signaling in smooth muscle cells is based on enzyme ac-
tivation leading to elevation of cytosolic cGMP concentration (95). GC is a het-
erodimer of one α-subunit and one β-subunit, both required for catalytic activity.
Each subunit contains an N-terminal regulatory domain and a C-terminal catalytic
Annu. Rev. Plant Biol. 2003.54:109-136. Downloaded from www.annualreviews.org
by Universidad Nacional de Mar del Plata on 04/08/11. For personal use only.

9 Apr 2003 13:13 AR AR184-PP54-05.tex AR184-PP54-05.sgm LaTeX2e(2002/01/18) P1: FHD
NITRIC OXIDE 113
Figure 1 (A) Chemistry of the interrelated forms of NO with potential biological activity.
Interconversion of NO forms. NO
·
(NO radical) is rapidly oxidized by the removal of one
electron to give nitrosonium cation (NO
+
), or reduced by theaddition of one electron to form
nitroxyl anion (NO
−
), which are important intermediates in the biochemistry of NO. (B)
Chemical reactions of NO produced endogenously or released by NO donors. In the presence
of transition metals (Me
+x
) such as Fe, Cu, or Zn, NO can interact to form metal-nitrosyl
complexes; this chemistry underlies the regulatory effect of NO on transcription factors (TF)
and certain enzymes. NO
+
and NO
·
can also nitrosylate thiol groups of cysteines of proteins
(R-S-NO). In the presence of superoxide (O
−
2
), a rapid peroxynitrite (ONOO
−
) formation
occurs, which can lead to the formation of NO
2
and the potent oxidant hydroxyl radical
(OH
·
). Other chemical reactions engendered by peroxynitrite are the nitration of tyrosines
(Tyr-NO
2
) or oxidation of thiol residues to sulfenic and sulfonic acids. In the presence of
oxygen, NO is oxidized to NO
2
, then NO
2
reacts with NO to give N
2
O
3
and then NO
−
2
/NO
−
3
.
Annu. Rev. Plant Biol. 2003.54:109-136. Downloaded from www.annualreviews.org
by Universidad Nacional de Mar del Plata on 04/08/11. For personal use only.

9 Apr 2003 13:13 AR AR184-PP54-05.tex AR184-PP54-05.sgm LaTeX2e(2002/01/18) P1: FHD
114 LAMATTINA ET AL.
domain. A noncovalently bound heme group is located on the regulatory domain
of each subunit. When NO binds to the heme group, it forms a ferrous-nitrosyl-
heme complex, which is necessary for the enzyme activation. Increase of cGMP
concentration activates a cGMP-dependent protein kinase (cGMP kinase). Two
cGMP kinase isoforms have been described, but only cGMP kinase I is employed
by the NO signaling pathway (68). In plants, no homolog to GC has been found to
date, but there are many examples of enzymes containing metal-bound domains
whose activities could potentially be regulated by NO.
NO promotes both the activation of DNAmethyl transferase (67) and the deam-
ination of 5-methylcytosine with the subsequent formation of thymidine residues
(19). These findings provide an explanation for the known induction of C-T tran-
sitions in genomic DNA upon exposure to NO. Since mitochondria are organelles
highly affected by the presence of and exposure to NO (128,172), the possible
involvementofNOindeaminationeventsleadingtoC-Utransitionsduringthepro-
cess known as “plant mitochondrial mRNA editing” remains to be explored. This
editing involves most mitochondrial transcripts in higher plants and profoundly
alters their expression in the organelle (19a).
Antioxidant and Pro-Oxidant Properties of NO
Since its discovery as an endogenous free radical, NO has been proposed to be
either cytotoxic or cytoprotective (7a, 146). The cytoprotection is based on NO’s
ability to regulate the leveland toxicity of ROS (62). The complexredox chemistry
of NO, which is related to changes in the ambient redox milieu, is hypothesized to
provide a general mechanism for cell redox homeostasis regulation (145). Thus,
NO can exert a protective action against oxidative stress provoked by an increased
concentration of superoxide, hydrogen peroxide, and alkyl peroxides (165). In
addition, the NO molecule itself possesses antioxidant properties (77).
The Fenton-type reaction between H
2
O
2
and redox active metal produces the
hydroxyl radical (OH
•
). This powerful oxidant has the capacity to oxidize a
wide number of biomolecules, a process underlying many diseases (3). The pres-
ence of NO can attenuate the Fenton oxidative damage preventing the forma-
tion of oxidants by scavenging either iron or superoxide and thus limiting
hydroxyl radical formation (10, 11,165). Several studies have also demonstrated
that NO can act as a chain-breaking antioxidant arresting lipid peroxidative re-
actions (69,134). The cytoprotective effects of NO in plants were reported under
strong oxidative conditions during both biotic and abiotic stresses, even under
photo-oxidative situations (8–10). Cytoprotection against the oxidative burst was
clearly observed at different levels of organization such as cell culture, tissue,
organ, and whole plant, and exerted on all tested macromolecules: DNA, RNA,
protein, chlorophyll, and lipids (8–10). Cytoprotection against photo-oxidative
damage in potato leaves (10) and gibberellic acid (GA)-mediated ROS increase
in barley aleurone during germination (7) was accomplished without activation
of either cellular antioxidant enzymes or expression of antioxidant-coding genes
Annu. Rev. Plant Biol. 2003.54:109-136. Downloaded from www.annualreviews.org
by Universidad Nacional de Mar del Plata on 04/08/11. For personal use only.

9 Apr 2003 13:13 AR AR184-PP54-05.tex AR184-PP54-05.sgm LaTeX2e(2002/01/18) P1: FHD
NITRIC OXIDE 115
(7,10). However, reports in other systems showed an NO-mediated activation of
both antioxidant enzymes (41,132) and gene expression of antioxidant enzymes
(111,163).
NO-mediated toxicity is mainly generated by reaction with superoxide anion
(O
−
2
),leading to the formation of thestrongoxidant peroxinitrite (ONOO
−
),which
can oxidize thiol residues to sulfenic and sulfonic acids (72). Peroxinitrite can also
nitrate peptides and proteins at the phenyl side chain of the tyrosine residues (126)
(Figure 1B). The nitration of tyrosine residues can prevent their phosphorylation.
The study of the functional consequences of this peroxinitrite-mediated tyrosine
nitration in vivo and its effects on cellular signaling cascades is an area of future
research.
SOURCE OF NO IN PLANTS
NO Generation and Methods of Measurement
The demonstration of NO formation in vascular endothelial cells opened a new
area of biological research (114). However, NO is an unstable molecule that is
produced at very low concentrations. The need for an accurate and reproducible
analytical method to detect NO in different systems was highlighted.
NO can be measured either directly or indirectly. Direct measurements involve
chemiluminiscence (CM) and electrochemical (EM) methods. The CM is based
on the reaction of NO with H
2
O
2
to form peroxinitrite, and then this oxidizing
species is quantified by reaction with luminol (79, 80). The EM is a protocol that
can be used to monitor concentrations of NO directly and continuously (96). It
is based on the quantification of NO release in the presence of Cu
+/2+
by amper-
ometric measurements. Indirect methods rely on monitoring a molecular species
or a physiological effect, which reflects the presence of NO. For example, the
oxyhemoglobin (HbO
2
) oxidation method is based on the oxidation of HbO
2
to
methemoglobin (MetHb) by NO, and the spectral change of the oxidized form.
However, as HbO
2
is also oxidized by NO
−
2
, it is difficult to differentiate NO
from its product of decomposition, NO
−
2
. Other heme proteins such as horseradish
peroxidase (HRP) have recently been employed for assay of NO. HRP is a heme
protein with ferric iron; it forms a stable complex with the addition of NO that
inducesaspectralchangefrom396.5 to 420 nm. The detection limit is 10 nmol L
−1
(81).
The most commonly used indirect method is the Griess reaction (108), based
on the measurement of NO
−
2
, which is the stable nitrogen oxide formed following
NO decomposition in aqueous solution in vitro. Analysis by the Griess reagent
is based on a two-step diazotization reaction and has a sensitivity limit of about
100 nmol L
−1
. Another indirect method involves the utilization of fluorimetric
probes(102). Oneassayexploitstheability ofNOto generateN-nitrosatingagents.
When the relativelynonfluorescent 2,3-diaminonaphthalene (DAN) reacts with N-
nitrosating agents, it produces the highly fluorescent product, 2,3-naphthotriazole
Annu. Rev. Plant Biol. 2003.54:109-136. Downloaded from www.annualreviews.org
by Universidad Nacional de Mar del Plata on 04/08/11. For personal use only.

17 Apr 2003 13:47 AR AR184-PP54-05.tex AR184-PP54-05.sgm LaTeX2e(2002/01/18) P1: FHD
116 LAMATTINA ET AL.
(NAT). This method allows a detection limit of 10 nmol L
−1
and can be used to
quantify NO generated in vivo. Recently, the development of diamino fluoresceins
(DAFs) has enhanced the specificity and sensitivity for NO quantification under
neutral pH conditions. In addition, DAFs have an excitation wavelength, which
is less damaging to cells and show less interference from the autofluorescence of
biological samples. The detection limit of DAF-2DA is 5 nmol L
−1
(84). Although
in severalsystems fluorescent staining for NO identification seems to be one of the
most sensitive methods, in many other systems the dye is not as reliable because
the negative probe—the NO-insensitive form—highly stains some characteristic
subcellular structures of plant cells such as the cell wall (L. Lamattina & M.
Graziano, unpublished results).
NO can also be detected as a free radical by electron paramagnetic resonance
(EPR) spectroscopy. Samplesare prepared in liquid nitrogen and a spin trap added
such as hemoglobin for in vitro nitrosyl hemoglobin complex formation. The
spectrum of nitrosyl hemoglobin shows a characteristic triplet EPR signal in an
X-band spectrometer (Klystron frequency 9.52205 GHz). A calibration curve with
a known concentration of NO in water and hemoglobin is necessary. The detection
limit of NO by EPR is 500 nmol L
−1
(85).
Structure-Function Comparison Between Animal NO
Synthase (NOS) and Plant Nitrate Reductase (NR)
NO formation in mammals mainly relies on the activity of NOS (EC 1.14.13.39).
NOS is a heme protein that contains four bound prosthetic groups: iron protopor-
phyrin IX, tetrahydrobiopterin (H
4
Bip), flavin adenine dinucleotide (FAD), and
flavin mononucleotide (FMN). NOS also contains binding sites for calmodulin,
L-arginine and NAD(P)H (Figure 2). The FAD-binding region (260 aa long) is
similar to the ferredoxin-NADP
+
reductase (FNR), a member of the flavin oxi-
doreductasefamily.ThisfamilyincludescytochromeP-450reductase,cytochrome
b
5
reductase, and NOS, among others.
The NOS active enzyme is an approximately 260-kD homodimer andcatalyzes
a five-electron oxidation of a terminal guanidine nitrogen of
L-arginine associated
with stoichiometric consumption of dioxygen (O
2
) and NADH to form L-citrulline
and NO (115). H
4
Bip is a redox-active cofactor of aromatic amino acid hydroxy-
lases and an allosteric regulator. The biological function of H
4
Bip for NOS activity
is unclear (148).
Three major NOS isoforms (neuronal NOS–nNOS-, endothelial NOS–eNOS-,
and inducible NOS–iNOS-) have been identified in humans, with different tissue
specificity and Ca
2+
requirements. Whereas constitutive NOS isoforms, nNOS
and eNOS, are Ca
2+
-dependent, iNOS is Ca
2+
-independent (105a). NOS activ-
ity and/or protein have been described in all vertebrate groups, including larval
sea lampreys (138,171). NOS activity is also present in invertebrate phyla (98),
e.g., arthropods such as Drosophila melanogaster (125), and echinoderms, e.g.,
the starfish Asterias rubens (44). In fungi, NOS was described to be involved in
Annu. Rev. Plant Biol. 2003.54:109-136. Downloaded from www.annualreviews.org
by Universidad Nacional de Mar del Plata on 04/08/11. For personal use only.

9 Apr 2003 13:13 AR AR184-PP54-05.tex AR184-PP54-05.sgm LaTeX2e(2002/01/18) P1: FHD
NITRIC OXIDE 117
photoconidiation of Neurospora crassa (109). NOS was found in the plasmodial
slime mold Physarum polycephalum (myxomycetes) (162), in the protozoan Try-
panosoma cruzi (117), and also described in bacterial species such as Nocardia
(29).
In plants, neither the gene or cDNA, nor any protein with high sequence simi-
larity to known NOS, have been found. Data about plant NOS come from its detec-
tion using anti-NOS antibodies and the measurement of NOS activity [reviewed
in (11,161)]. Although the use of antibodies indicated positive immunoreactivity,
NOS protein shares domains with high similarity to cytochrome P-450 reductases
(23) and this increases the potential of an anti-NOS antibody cross-reaction with
that domain present in many oxidoreductases. The determination of NOS activity
in plant tissues by the conversion of
L-arginineto L-citrulline (6,40, 42, 127) or the
sensitivityof NO production to NOS inhibitors (42,49) seems to be weak evidence
unless the protein could be purified and/or the corresponding gene cloned.
In biological systems, NO can be generated by enzymatic and nonenzymatic
reduction of inorganic forms of oxidized nitrogen. In plants, the nonenzymatic re-
action leading to NO formation can be catalyzed by ascorbic acid (AA) below pH
4.0 in the chloroplast and apoplastic space where AA was reported to be present
(70). In fact, reduced ascorbate is synthesized and exported by the aleurone (41a).
The aleurone also has a strong H
+
pump at the plasma membrane allowing the
external pH to fall below pH 3. In the presence of NO
−
2
, NO is produced stoichio-
metrically because of the combination of ascorbate and lowpH (P.C. Bethke,M.R.
Badger, R.L. Jones, manuscript in preparation). In addition, although the redox
conditions needed for the reaction are far from physiological pH, microlocalized
optimal pH conditions within the cell cannot be dismissed conclusively.
A light-mediated NO
−
2
reduction by carotenoids with consequent NO produc-
tion has also been reported (35). Recently, a plasma membrane-bound enzyme was
shown to catalyze the formation of NO from NO
−
2
in tobacco roots (147). This
activity was not NO
−
2
-dependent, as is the cytosolic NR; EC 1.6.6.1, and was not
affected by NR inhibitors.
NR is a cytosolic enzyme involved in primary metabolism that catalyzes a
two-electron transfer reaction from NAD(P)H to NO
−
3
. Because NR is becoming
a central integrating point for the control of carbon and nitrogen metabolism in
higherplants, thebiochemistryandmolecularbiologyofNRhavebeenextensively
revised over recent years (28,37, 71, 76). The enzyme is a redox system with an
internal electron transfer chain. NR is active as a homodimer composed of two
identical∼100-kD subunits,eachcontainingoneequivalent offlavinadeninedinu-
cleotide (FAD), heme-Fe, and Mo-molybdopterin (Mo-MPT) (Figure 2) (27,144).
Recent data on the 3-D structure of NR indicate that it contains five structurally
distinct domains: Mo-MPT, dimer interface, cytochrome b, FAD, and NAD(P)H
(45). During catalytic turnover, the FAD, Fe, and Mo are cyclically reduced and
oxidized, giving two forms of NR, one reduced and the other oxidized. Studies
on NR functionality have led to the characterization of the biochemical properties
of the different domains. For instance, the domains that bind NAD(P)H and FAD
Annu. Rev. Plant Biol. 2003.54:109-136. Downloaded from www.annualreviews.org
by Universidad Nacional de Mar del Plata on 04/08/11. For personal use only.

9 Apr 2003 13:13 AR AR184-PP54-05.tex AR184-PP54-05.sgm LaTeX2e(2002/01/18) P1: FHD
118 LAMATTINA ET AL.
established NR as a member of the FNR structure family of the flavoenzymes (78)
such as NOS and cytochrome P-450 reductase (159). This family of proteins is
interesting because its members have little sequence similarity, whereas they have
a very similar conformation in their FNR-like fragment (36). Given these char-
acteristics, some questions arise: (a) Are similarities between structural domains
of NOS and NR (including FAD, NAD(P)H, Heme-Fe, and Pterin rings) (Figure
2) close enough to lead them to share common regulated pathways and antigenic
epitopes? (b) Could any NOS inhibitors block NR activity under specific condi-
tions? Positive answers would throw doubt on all of immunological and inhibition
studies for defining the presence of NOS in plants.
Production of NO by Plant NR
In 1979, Klepper provided the first evidence of NO production due to NR activity
(82). Later, Harper demonstrated that NOx (NO + NO
2
) evolved from soybean
leaves during in vivo NR assays (63). When using anaerobic gas purging (N
2
or
argon), the production of NOx was greater than that obtained using aerobic gas
purging (air or O
2
). He also found a positive correlation between NOx production
and NO
−
2
level. Because the activitywas lost in denatured leaf tissue, an enzymatic
NOx evolution from NO
−
2
was proposed (63). Later, it was demonstrated that a
soybean mutant lacking constitutive NR activity (named nr1) did not evolve NOx
during in vivoNR assays (107). Furthermore, in 1997 NO emissions were reported
from sugar cane (Saccharum officinarum), sunflower (Helianthus annus), corn
(Zeamays),spinach(Spinaceaoleracea),tobacco(Nicotianatabaccum),andother
species (164), suggesting that in plants, NO-producing capacity could be a general
process not restricted to the legume form of NR. Finally, in in vitro experiments
using a Clarke-type NO sensor, maize NR was shown to catalyze the reduction of
nitrite to NO at a physiological pH range (168). In similar experiments, the authors
reported an NR-dependent and simultaneous production of both NO and reactive
nitrogen species (169).
When does plant NR produce NO? Are there precise physiological conditions
and tissue specificities determining NO production? Does a constitutive and basal
NO production exist? First, NR-dependent NO production apparently relies on
nitrite accumulation (130). Nitrite produced in the cytosol is translocated to the
chloroplastandimmediatelyreducedtoNH
+
4
bynitritereductase(NiR;EC1.6.6.4)
because it is highly toxic (143). Thus, if we assume that in healthy plants, con-
taining an operative photosynthetic electron transfer, the NO
−
2
produced by NR
is immediately reduced, then NO production can no longer be expected. How-
ever, NO
−
2
can be accumulated when photosynthetic activity is inhibited or absent
(82,158) or under anaerobic conditions (20), increasing the production of NR-
dependent NO. When the photosynthetic electron transport system is unable to
supply the reduced form of ferredoxin for the NiR reaction, NO
−
2
is accumulated
in the cytosol together with an overproduction of ROS. At this point, NR would
convert NO
−
2
to NO. Since NO is highly diffusible and membrane permeable,
NO could rapidly attain the stroma of the chloroplast. Thus, the production of
Annu. Rev. Plant Biol. 2003.54:109-136. Downloaded from www.annualreviews.org
by Universidad Nacional de Mar del Plata on 04/08/11. For personal use only.

17 Apr 2003 13:48 AR AR184-PP54-05.tex AR184-PP54-05.sgm LaTeX2e(2002/01/18) P1: FHD
NITRIC OXIDE 119
NR-dependent NO under stress conditions would enhance the ROS-scavenging
chemical capacity of the cell through the ability of NO to react with ROS and
metals. This point is discussed further below.
ACTIONS OF NO IN PLANTS
Nitrogen Fertilization: A New View of an Old Practice
Soils are an important source of NO and contribute to almost 20% ofthe global at-
mospheric NO budget (34). Microbial derived dinitrogen oxide (N
2
O) and
nitrogen oxide (NO) are products of denitrification, nitrification, and reduction
of nitrate to ammonia (33,173) (Figure 3).
Several studies have shown a significant increase in the emission of NO and
N
2
O from soils amended with both biological and inorganic fertilizers compared
with nonfertilized soils (14, 24,116). The annual emission of both N
2
O and NO
from intensively fertilized potato fields (150 Kg N ha
−1
) was 16% and 10.7% of
the total nitrogen (N) applied, respectively (135). For maize and grasslands, which
were used to estimate the global emissions of fertilizer-induced N
2
O, the release
was approximately 1.25% of the N applied (74). Thus, NO emission seems to
be dependent on crop species (131). Other studies have shown that there exists
a correlation between NO release-flux, soil temperature, and water content, in
addition to the relation between total extractable nitrogen (TEN) from soils and
NO emission (38). On the other hand, the soil source of NO is similar in magnitude
to fossil fuel emissions of NO
x
(38).
The Green Revolution, with the increased use of fertilizers and pesticides, has
been mainly responsible for enhanced crop production during the second half
of the past century. For instance, the global application of N fertilizer increased
dramatically from 32 to 80TgNbetween 1970 and 1990 (47), leading to an
increased emission of NOs from soils. To our knowledge, no reliable data are
available to distinguish between the effect of N
2
O/NO emission and the effect of
N per se on the crop yield.
To improve the efficiency of N fertilization, some outstanding questions must
first be resolved. (a) How extensive and sensitive is the perception of gaseous NO
by different plant species? (b) How can we distinguish, among the increased NO
fluxes derived from N fertilization, the NO that is acting as a signal for specific
growth and developmental processes, from that triggering a range of nonspe-
cific responses? (c) Could the concentrations of gaseous NO emitted from the
soil reach the threshold level to induce stomatal closure in natural ecosystems?
(d) What are the adequate temperature and humidity conditions, atmospheric
gaseous composition, light intensity and quality, moisture and biological activ-
ity in soils for beneficial/detrimental NO emission, such that these parameters
can be integrated in forecasting programs? (e) Is the NO derived from N fertil-
ization one of the compounds responsible for the increase of chlorophyll content,
health, and fitness of N-fertilized plants? With regard to this last question, it is well
Annu. Rev. Plant Biol. 2003.54:109-136. Downloaded from www.annualreviews.org
by Universidad Nacional de Mar del Plata on 04/08/11. For personal use only.

17 Apr 2003 13:49 AR AR184-PP54-05.tex AR184-PP54-05.sgm LaTeX2e(2002/01/18) P1: FHD
120 LAMATTINA ET AL.
documented that iron deficiency impairs chlorophyll biosynthesis and chloroplast
development (152). NO promotes a significant increase in chlorophyll content
and chloroplast membrane density in maize plants growing with a very low iron
concentration (58). These results supporta physiological action for NO iniron nu-
trition processes within the plant cell, and point to the need for a careful dissection
and study of the way that N fertilization affects plant physiology.
Pharmacological Effects Versus Physiological Roles
The wide and consistent production of experimental results over the past five years
indicates that NO acts as a signaling molecule in plants. NO mediates a variety of
growthand developmental processes including responses to environmental stimuli
ofbothbioticandabioticorigin(Figure3).However,asignificantquestionremains:
To what extent is NO a real, endogenous regulator and/or a signal mediating the
reported effects?
The determination of NO concentration in vivoremains difficultand unreliable.
Even in animals, there is still uncertainty about the physiological concentrations
of NO (T. Higenbottam, personal communication). Most studies on physiological
NO effects in plants (Figure 4) and animals have been accomplished by exoge-
nous application of NO donors. Concentrations from nanomolar to millimolar of
NO donors have been used. As previously stated, the release of NO into solution
depends on the chemical characteristics and concentrations of the NO donor com-
pound, the pH of the media, and the temperature and concentrations of NO target
molecules (123,151). Thus, the exogenous application of NO makes it very diffi-
cult to discriminate between the physiological relevance and the pharmacological
effects derived from NO biochemical reactivity.
NO Signaling, Processing, and Transduction Pathways
To date, there is no evidence for the existence of DNA elements within analyzed
promoter sequences of eukaryotic genes that bind or directly respond to NO (19).
Consequently, NO activates signaling pathways, which results in changes of gene
expression, through intermediate cellular messengers.
Inbothanimalandplantcells,cGMPisanimportantcomponentofNOsignaling
(4,42, 120,146). The presence of cGMP in plants was shown by mass spectrome-
try and radioimmune assays (119). A cyclic nucleotide phosphodiesterase (PDE)
activityhas been also reported (26,124), although neither GC nor the PDE respon-
sible for cGMP synthesis and degradation, respectively, have been cloned in plants
yet. Various stimuli, such as GA in barley aleurone (119), light stimulation of bean
cells (25), or NO treatment in spruce needles (120), cause a transient increase in
cGMP concentrations. Treatments with NO donors were also reported to induce a
transient increase of endogenous cGMP levels in tobacco (42). Moreover, specific
GC inhibitors were able to suppress the expression of PAL genes stimulated by
NO (42).
Annu. Rev. Plant Biol. 2003.54:109-136. Downloaded from www.annualreviews.org
by Universidad Nacional de Mar del Plata on 04/08/11. For personal use only.

9 Apr 2003 13:13 AR AR184-PP54-05.tex AR184-PP54-05.sgm LaTeX2e(2002/01/18) P1: FHD
NITRIC OXIDE 121
cGMP-dependent protein kinase (cGMP-PK), the potential target for cGMP,
has not been isolated nor cloned in plants. However, cGMP can also act via cyclic
ADP ribose (cADPR) in plants, as occurs in animal cells (57). cADPR appears to
be involved in the NO-induced activation of defense genes in tobacco (42) and to
regulate calcium concentrations in stomatal guard cells in response to ABA (90).
On the other hand, cGMP-independent effects of NO have also been reported.
Calcium release from internal stores induced the expression of plant defense genes
via a cGMP-independent pathway (42). This effect might be explained by the
nitrosylation/activation of ryanodine-sensitive channels, as occurs in skeletal and
cardiac muscle cells, where calcium release is regulated by S-nitrosylation of the
ryanodinereceptor(167).OthertargetsofNOaremitogenactivatedproteinkinases
(MAPKs). As in mammals, where NO modulates MAPKs activities in tumor cells
and neurons (105), their activation was also shown in transgenic tobacco with
recombinant NOS or NO donor treatments (83).
Taken together, the reported data suggest that the components of animal NO
signaling are also functional in plants and reveal the unusual complexity of the cel-
lular response to NO, which may be mediated by phosphorylation-, nitrosylation-,
or calcium-controlled mechanisms.
NO AND CROSS-TALK WITH CLASSICAL
PLANT HORMONES
A long-standing objective of the study of plant hormone action has been to elu-
cidate the molecular basis of signaling systems that are operating to transduce
hormone messages into the physiological responses that they regulate. The steps
along a signal transduction pathway are generally mediated by changes in protein-
protein interactions, activation of gated channels, regulation of catalytic activities,
and combinations of these. The same domains and motifs of proteins are often
involved in different transduction pathways. Since NO can regulate processes re-
lated to plant growthand development,it has been postulated to be a nontraditional
regulatorof plantgrowth(12). Here, wesummarize the cross-talk between NOand
plant hormones and the common cellular messengers involved in their signaling
pathways.
Auxins, NO, and Rooting
Gouvˆea et al. demonstrated the capability of root cells to respond to NO, which
induced the elongation of maize root segments in a dose-dependent manner (56).
The investigators proposed that the auxin indole acetic acid (IAA) and NO might
share common steps in the signal transduction pathways, because both elicit the
same plant response. The dependence on NO in auxin-induced adventitious root
development was recently demonstrated in cucumber explants (112). Moreover,
explantsfrom wood specieswerealsoresponsivetoNOtreatmenttoinduceadven-
titious root formation (Figure 4A) (89). These results open a fascinating window
Annu. Rev. Plant Biol. 2003.54:109-136. Downloaded from www.annualreviews.org
by Universidad Nacional de Mar del Plata on 04/08/11. For personal use only.

9 Apr 2003 13:13 AR AR184-PP54-05.tex AR184-PP54-05.sgm LaTeX2e(2002/01/18) P1: FHD
122 LAMATTINA ET AL.
for further research in nursery programs aimed at improving the efficiency of
rooting.
In cucumber explants, IAA treatment induces a transient increase in the level of
endogenousNOinthebasal region ofthehypocotyl,wherethenewrootmeristems
develop (112). This localized NO bulk might stimulate the GC-catalyzed synthe-
sis of cGMP as occurs in mammalian systems (101). The GC inhibitor LY83583
reduced adventitious root formation both in IAA- and NO-treated cucumber ex-
plants. This effect was reversed when the permeable cGMP analog 8-Br-cGMP
was added together with LY83583 and NO or IAA (G. Pagnussat, L. Lanteri & L.
Lamattina, manuscript in preparation).
An earlier report in tobacco showed that the activation of defense genes by
NO was also induced by cGMP (42). cGMP can act via cADPR, which, in turn,
regulatesCa
2+
levelsas was reported in variousplant systems (2,90). Variations in
[Ca
2+
]mightparticipateinthesignaltransductionpathwayleadingtotheactivation
of mitotic processes and differentiation to initiate root development.
Nitric oxide can also act via a cGMP-independent pathway, activating phos-
phatases and protein kinases including MAPKs. NO donors and recombinant NOS
were shown to modulate two tobacco pathogen-activated MAPKs (83), indicating
that this signaling pathway is also present in plants. Interestingly, a rapid and tran-
sient increase of MAPK activity in response to low levels of auxins was reported
in Arabidopsis seedling roots (102a).
HowIAA induces the NO formation in the basal region of hypocotyl explants is
notknown.Recently, aplasma membrane-bound enzyme, thenitrite:NO reductase
(NI-NOR), was reported to be involved in NO formation in plant roots (147). The
highest activity was achieved at pH 6.1. It would be interesting to test whether
this activity could be enhanced by acidification of the milieu produced by IAA
treatmentand/orwhetherIAAisabletoregulatetranscriptionalorposttranslational
modifications of the NI-NOR expression leading to an increase in NO production.
Overall, the IAA-induced endogenous NO bulk in roots could result in a bifur-
cated signal transduction pathway in which NO mediates a cGMP-dependent or
-independent increase of cytosolic Ca
2+
, which in turn triggers changes in plant
gene expression leading the auxin response (Figure 5). It remains to be tested
whether NO-mediated MAPKs activation is also involved in such IAA-induced
process and whether NO is also acting in other IAA-mediated plant physiological
responses.
ABA, NO, and Water Deficit
Water deficit is associated with the accumulation of the plant hormone abscisic
acid (ABA) and the induction of ABA-regulated genes, which in turn are involved
in many physiological processes (54). ABA regulates various vital processes in-
cluding seed maturation, dormancy, and vegetative growth, and induces tolerance
to differentstresses including drought, salinity, and lowtemperatures(54). Among
all these processes, the control of stomatal movement is of great importance for
controlling the water loss through the transpiration stream while balancing the
Annu. Rev. Plant Biol. 2003.54:109-136. Downloaded from www.annualreviews.org
by Universidad Nacional de Mar del Plata on 04/08/11. For personal use only.

9 Apr 2003 13:13 AR AR184-PP54-05.tex AR184-PP54-05.sgm LaTeX2e(2002/01/18) P1: FHD
NITRIC OXIDE 123
Figure 5 Schematic representation of the signaling network involving plant hormones,
NO, and cellular messengers during plant responses to environmental stresses. Under stress
conditions, hormone signal transduction pathways are activated. The G-protein transduces
extracellular signals modulated by auxins, gibberellins (GA), abscicic acid (ABA), and cy-
tokinins (CKs). A special case is the NO-mediated inhibition of ethylene biosynthesis during
senescence-like processes. NO acts as a crossroad in hormone signaling to trigger metabolic
and physiological responses in at least three distinct ways: (a) a cGMP-dependent road in-
volving changes in cADPR-mediated [Ca
2+
]
cyt
,(b) a cGMP-independent road involving a
direct NO action on Ca
2+
channels via protein nitrosylation, and (c) an NO action on MAPK
activities,which areinvolvedin many physiologicaladaptationand developmentalprocesses.
NO might also act, via cGMP and Ca
2+
, on light-mediated processes, partially mimicking
the phytochrome signaling pathways.
requirement of gas exchange for photosynthesis (16,166). Once in the guard
cell, ABA first induces the depolarization of the plasma membrane potential
that leads to the generation of a driving force for K
+
efflux, inactivates K
+
in-
flux through inward-rectifying K
+
(K
+
in
) channels, and activates a current through
outward-rectifying (K
+
out
) channels. These changes together with both slow- and
fast-activatinganion channels facilitate the net loss of salt from the cells (17). Both
cytosolicfree Ca
2+
concentrations ([Ca
2+
]
cyt
) and cytosolicpH havebeen reported
Annu. Rev. Plant Biol. 2003.54:109-136. Downloaded from www.annualreviews.org
by Universidad Nacional de Mar del Plata on 04/08/11. For personal use only.

9 Apr 2003 13:13 AR AR184-PP54-05.tex AR184-PP54-05.sgm LaTeX2e(2002/01/18) P1: FHD
124 LAMATTINA ET AL.
to participate as second messengers of this response (16). ABA induces guard cell
[Ca
2+
]
cyt
elevation either by influx from the extracellular space or by release from
internalstores (139). Overall the long-term effluxof both anion and K
+
fromguard
cells contributes to the loss of guard cell turgor, leading to stomatal closing.
Inanimal systems,NOwasreportedto beawidespread regulatorof bothplasma
membrane and endomembrane Ca
2+
and K
+
channels (31,141). In plants, no data
are available on NO-mediated regulation of ion channels. Recently, it was shown
that exogenous addition of NO to both monocot and dicotyledonous epidermal
strips was sufficient to induce stomatal closure, through a Ca
2+
-dependent process
(51) (Figure 4D). Moreover, it was further reported that in Pisum sativum and
Vicia fava, ABA induces an increase of endogenous NO levels. This bulk of ABA-
induced NO production was reported to be sufficient and necessary for ABA
induction of stomatal closure (52, 106). Additionally, there is some convergent
evidence supporting the involvement of NR, through the production of NO, as a
putative keyenzyme bridging the gap between guard cell metabolism and stomatal
movement (52a). Therefore, considering both the NO-mediated ion regulation in
animals and NO involvement in stomatal closure response through changes in
[Ca
2+
]
cyt
, it is hyopothesized that NO can directly regulate endomembrane and
plasma membrane Ca
2+
channels, as well as other ion channels in plants.
Duringthestomatalclosureprocess,agreatproportionoftheionspassingacross
the plasma membrane must first be released to the cytosol from internal stores. At
leastfourdifferentCa
2+
channeltypeshavebeenidentifiedatvacuolarmembranes.
Two of these channels are ligand gated, one by inositol 1,4,5-triphosphate (IP3)
and the other by cADPR. The pharmacological properties of plant cADPR-gated
channels resemble those of ryanodine receptors in animal cells that, along with
IP3 receptors, are responsible for mobilization of ER-based Ca
2+
pools during sig-
naling (136). The role of NO in the control of Ca
2+
release from IP3-sensitive and
ryanodine-sensitive stores has been clearly ascertained in animals [for review see
(32)]. In plant cells,IP3 is rapidly producedwithin guard cells in response to ABA
and interacts with IP3-sensitive Ca
2+
channels to potentiate a rise in [Ca
2+
]
cyt
(18)
,
inhibiting K
+
in
channels at the same time (140). Cyclic ADPR was demonstrated
to function as a Ca
2+
releaser during ABA stimulation (139). Recently, Neill et al.
reported that nicotinamide, the antagonist of cADPR production, inhibits the ef-
fects of both ABA and NO on stomatal closure, suggesting that cADPR synthesis
is part of the NO signaling pathway (106).
The participation of NO as a signal molecule in guard cell movement is a
very recent topic, and hence no results have yet been reported from experiments
done with ABA-insensitive Arabidopsis mutants. Use of these mutants will help
to elucidate the role of NO in the process of guard cell signaling.
Cytokinins, Gibberelins, NO, and Phytochrome Pathways
There are many processes in which hormones and phytochrome interact or act
separately to give the same response. NO also triggers several of these responses.
Annu. Rev. Plant Biol. 2003.54:109-136. Downloaded from www.annualreviews.org
by Universidad Nacional de Mar del Plata on 04/08/11. For personal use only.

9 Apr 2003 13:13 AR AR184-PP54-05.tex AR184-PP54-05.sgm LaTeX2e(2002/01/18) P1: FHD
NITRIC OXIDE 125
These overlapping roles raise the question of whether light and hormones share
common components in signal transduction pathways to elicit the same response
and whether NO plays a role in this signaling cascade. Cytokinins (CKs) in par-
ticular can stimulate photomorphogenic responses, mainly those related with the
deetiolation process and pigment synthesis (153). In dark-grown seedlings, exo-
genous application of CKs inhibits hypocotyl elongation in a manner similar to
light treatment (30, 149). Likewise, NO significantly reduces hypocotyl elonga-
tion in Arabidopsis and lettuce seedlings grownin the dark (13). In cotyledons and
leaves growing under dark conditions, CK treatment cannot cause etiolation to
revert completely. However, the hormone can mimic the effect of red light pulses
because it abolishes the lag phase in chlorophyll production during subsequent il-
lumination (39). Furthermore, NO is also able to slightly increase the chlorophyll
level in wheat seedlings grown in the dark, but the NO effect is strongly poten-
tiated by short-term light pulses (13). In this sense, the NO effect is similar to
that of CKs, and probably, some components are induced only via phytochrome to
allowcomplete greening. Figure 5 showsa hypothetical model of NO involvement
in light-mediated processes and the cross-talk among hormones, NO, cGMP, and
Ca
2+
pathways.Bowleret al. havedemonstrated the involvementof heterotrimeric
G-protein,cGMP, and Ca/calmodulin in stimulating biological processes mediated
by phytochrome (21).
A classical biological assay to test the effects of CKs is the accumulation of
pigments such as anthocyanins and betacyanins. The synthesis of betacyanins
in Amaranthus spp. is promoted by CKs and red light (5,121). NO mimicked
CK action on betacyanin accumulation. Moreover, NOS inhibitors and an NO-
scavenger blocked the CK effect on betacyanin accumulation (137), suggesting
that NO acts downstream of CKs to promote that response, or that NO is necessary
to accomplish CK function. The first evidence suggesting a direct relationship
between CKs and NO production was that the exogenous application of CKs to
Arabidopsis, parsley, or tobacco cell cultures leads to a rapid stimulation of NO
release (156).
Some seeds are dependent on light for germination under certain conditions. In
these cases, GA was observed to act like the active form of phytochrome to induce
germination (170). CK treatment alone is generally ineffective in breaking dor-
mancy, but CKs act synergistically with light and GAs to allow germination (154).
Germination of the photoblastic lettuce seeds cv. Grand Rapids is a phytochrome-
dependent process above 26
◦
C, and it was demonstrated that NO donors are able
to promote germination in the dark to the same extent as both a GA treatment or a
5-min pulse of white light (13). However, seeds were also able to germinate in the
light,in the presence ofthe NO scavengercPTIO, suggestingthat light and NO can
stimulategerminationindifferentways(13).WhetherGAandNOactinpromoting
germination through the same or different pathways remains to be determined.
The proposed involvement of NO in CK and light signaling suggests that there
shouldbe aregulatedNO-synthesisthatdepends onthosesignals. Inthatsense, NR
is tightly regulatedby light and CKs (53,122). CKs effecton NR activityis mainly
Annu. Rev. Plant Biol. 2003.54:109-136. Downloaded from www.annualreviews.org
by Universidad Nacional de Mar del Plata on 04/08/11. For personal use only.

9 Apr 2003 13:13 AR AR184-PP54-05.tex AR184-PP54-05.sgm LaTeX2e(2002/01/18) P1: FHD
126 LAMATTINA ET AL.
accomplished at the transcriptional level (150). However, NR gene activation by
CKs seems not to be the only way to promote NO synthesis. As mentioned above,
NO could also be generated from nitrite by a nonenzymatic mechanism in the
presence of carotenoids in a process that requires light (35).
Ethylene, NO, and Senescence
Ethylene plays an active role in many plant responses to environmental and en-
dogenous signals (1). Ethylene profoundly influences every stage of plant growth
and development, from germination and cell expansion to stress responses and
fruit ripening.
Evidence of the interplay between NO and ethylene in the maturation and
senescence of plant tissues suggests an antagonistic effect of both gases during
these stages of plant development (92). Exogenous application of NO extended the
postharvest life of fresh horticultural produce by inhibiting ethylene production
(92). Moreover, the administration of sildenafil, an inhibitor of cGMP-degrading
phosphodiesterases, results in a greater inhibition of ethylene formation. This
treatment prevents wilting of flowers (142) and indicates that cGMP could be
involved in the NO-induced inhibition of ethylene metabolism. More recently,
it was demonstrated, by a noninvasive photoacoustic spectroscopic method, that
endogenous NO and ethylene content maintain an inverse correlation during the
ripening of strawberries and avocados (91). While unripe, green fruits contain
high-NO and low-ethylene concentrations; the maturation process is accompanied
by a marked decrease of NO concomitant with an increase of ethylene (91).
Senescence is a process characterized by water loss and desiccation of plant
tissues. As discussed above, NO can regulate stomatal closure by modulating
ion channels and Ca
2+
levels in guard cells. Therefore, it would be interesting
to test whether the decrease in NO level in senescent tissues determines the loss
of an important signal involved in the fine regulation of stomatal closure and a
concomitant water loss that contributes to an irreversible desiccation process.
CONCLUDING REMARKS AND FUTURE DIRECTIONS
As more data become available, it is evident that NO exerts an unprecedented di-
versity of biological effects. However, our understanding of the signaling function
of NO in plants remains very limited. In general, most of the results discussed
here were obtained by the use of NO donors, thus definitive answers as to the
role of endogenously produced NO, the way in which NO is transported among
the different cells or plant organs, the identity of the biologically active NO form,
and the target molecules and the chemical modifications induced by NO in vivo
must await further research. The relevance of (a) NO-mediated gene regulation,
probably best studied by extensive microarray analysis; (b) the redox-active prop-
erties of NO and cellular redox homeostasis regulation, through the analysis of
both redox events and coordinate interactions with metals; and (c) NO-dependent
Annu. Rev. Plant Biol. 2003.54:109-136. Downloaded from www.annualreviews.org
by Universidad Nacional de Mar del Plata on 04/08/11. For personal use only.

9 Apr 2003 13:13 AR AR184-PP54-05.tex AR184-PP54-05.sgm LaTeX2e(2002/01/18) P1: FHD
NITRIC OXIDE 127
posttranslational modifications, through chemical and genetical approaches, await
determination.
Is NO a Synchronizing Chemical Messenger?
In view of its extensive biological role, NO has been suggested to be a synchro-
nizing chemical messenger (3a). Due to its electrical neutrality, it was proposed
that NO can diffuse rapidly through the cytoplasm and biomembranes, thus affect-
ing many biochemical functions simultaneously [see (11) and references therein].
Therefore, a compilation and integration of results from existing data and ex-
periements as they become available will probably help the discussion about a
similar synchronizing role for NO in a wider number of biological systems.
In plants, it has been proposed that stomatal complexes (104), aleurone cells
(66,129), and pericycle (15,22) have all the properties of heterogeneous pop-
ulations in a single tissue. When the tissue receives an external or endogenous
stimulus transduced in a change in the concentration of a growth regulator, cells
respond progressively because each cell has its own sensing threshold, and when
this is reached, a response is initiated (110). The exogenous application of any of
the following plant hormones, IAA, ABA, or CKs, triggers endogenous NO pro-
duction (52,106, 112, 156). It is still not clear if NO functions as a synchronizing
messenger for the action of these hormones and, based on its high diffusible rate
across biological membranes, to simultaneously reach long distances and trigger
the biological response in discrete patch cell populations.
Theyear1999markedthe100-yearanniversaryofthediscoveryofAspirin.Like
Aspirin in animals, NO alleviates many stress symptoms in plants by contributing
to cellular homeostasis. Therefore, we feel confident that NO promises to be the
main protagonist in a major plant story that is just beginning to be written.
Toward a Regulated Endogenous Production of NO
The objective of engineering major crop plants to require less N fertilizer for op-
timal yield is fast becoming feasible. The demonstration that plant NR activity
modulates NO production from nitrite and NAD(P)H highlights the important is-
sue of how to genetically engineer NR to promote a more precise regulation of
its activity. NR is highly regulated by complex transcriptional and posttransla-
tional mechanisms. Its key role in N and C assimilation makes NR manipulation a
critical strategy in future approaches. Experiments on different plant species and
mathematical models on plant metabolic pathways will be required (100).
Because NO has been proposed to be an in vivo stress-sensing molecule in
animal systems (97), testing is recommended on the behavior of transgenic plants
containingarecombinantNRunderthecontrolofapromoterthatleadstoregulated
production of NO as a rapid response to environmental stress constraints.
Once the number of intracellular targetmoleculesfor NO are identified,a major
problemwillbehowtodiscriminateandlimit,withinasignaltransductionpathway,
the antagonistic modifications that NO could induce on various components at the
Annu. Rev. Plant Biol. 2003.54:109-136. Downloaded from www.annualreviews.org
by Universidad Nacional de Mar del Plata on 04/08/11. For personal use only.

9 Apr 2003 13:13 AR AR184-PP54-05.tex AR184-PP54-05.sgm LaTeX2e(2002/01/18) P1: FHD
128 LAMATTINA ET AL.
sametime.BecauseourknowledgeofbasicmechanismsoperatinginNO-mediated
effects in plants is still rudimentary, plant biologists must address the challenge of
elucidating the biochemical functions of such a simple and intriguing molecule.
ACKNOWLEDGMENTS
We aregratefulto RafaelPontLezica andClaudiaCasalongue forcommentsonthe
manuscript. We thank Russell L. Jones for communicating new and unpublished
findings.Researchintheauthors’laboratorywassupportedbyConsejoNacionalde
Investigaciones Cientificas y Tecnologicas (CONICET), Universidad Nacional de
Mardel Plata(UNMdP),AgenciaNacional dePromoci´onCient´ıficayTecnol´ogica
(ANPCyT), Fundaci´on Antorchas, Secretar´ıa para la Tecnolog´ıa, la Ciencia y la
Innovaci´on Productiva (Programa SETCIP-ECOS) (Argentina), and International
FoundationforScience(IFS,Sweden). Owingtospacelimitations, weregretbeing
unable to cite a number of important references.
The Annual Review of Plant Biology is online at http://plant.annualreviews.org
LITERATURE CITED
1. Abeles FB, Morgan PW, Saltveit ME
Jr. 1992. Ethylene in plant biology. San
Diego, CA: Academic
2. Allen GJ, Muir SR, Sanders D. 1995.
Release of Ca
2+
from individual plant
vacuoles by both InsP3 and cyclic ADP-
ribose. Science 268:735–37
3. Ames BN, Shigenage MK, Hagen TM.
1993.Oxidants,antioxidants,and thede-
generative diseases of aging. Proc. Natl.
Acad. Sci. USA 90:7915–22
3a. Anbar M. 1995. Nitric oxide: a synchro-
nizing chemical messenger. Experientia
51:545–50
4. Arnold WP, Mittal CK, Katsuki S,
Murad F. 1977. Nitric oxide activates
guanylate cyclase and increase guano-
sine3
0
,5
0
-cyclicmonophosphatelevelsin
various tissue preparations. Proc. Natl.
Acad. Sci. USA 74:3203–7
5. Bamberger E, Mayer AM. 1990. Effect
of kinetin on formation of red pigment
in seedlings of Amaranthus retroflexus.
Science 131:1094–95
6. Barroso JB, Corpas FJ, Carreras A, San-
dalio LM, Valderrama R, et al. 1999.
Localization of nitric oxide synthase
in plant peroxisomes. J. Biol. Chem.
274:36729–33
7. Beligni MV, Fath A, Bethke PC, Lamat-
tinaL,JonesR. 2002.Nitricoxideacts as
an antioxidant and delays programmed
cell death in barley aleurone cells. Plant
Physiol. 129:1642–50
7a. Beligni MV, Lamattina L. 1999. Is nitric
oxide toxic or protective? Trends Plant
Sci. 4:299–300
8. Beligni MV, Lamattina L. 1999. Nitric
oxide counteracts cytotoxic processes
mediated by reactive oxygen species in
plant tissues. Planta 208:337–44
9. Beligni MV, Lamattina L. 1999. Ni-
tric oxide protects against cellular dam-
age produced by methylviologen herbi-
cides in potato plants. Nitric Oxide Biol.
Chem.Arch.Biochem.Biophys.B3:199–
208
10. Beligni MV, Lamattina L. 2002. Ni-
tric oxide interferes with plant photo-
oxidative stress by detoxifying reac-
tive oxygen species. Plant Cell Environ.
25:737–48
Annu. Rev. Plant Biol. 2003.54:109-136. Downloaded from www.annualreviews.org
by Universidad Nacional de Mar del Plata on 04/08/11. For personal use only.

9 Apr 2003 13:13 AR AR184-PP54-05.tex AR184-PP54-05.sgm LaTeX2e(2002/01/18) P1: FHD
NITRIC OXIDE 129
11. Beligni MV, Lamattina L. 2001. Nitric
oxide in plants: the history is just begin-
ning. Plant Cell Environ. 24:267–78
12. Beligni MV, Lamattina L. 2001. Nitric
oxide:anon-traditionalregulatorofplant
growth. Trends Plant Sci. 6:508–9
13. Beligni MV, Lamattina L. 2000. Ni-
tric oxide stimulates seed germination
and de-etiolation, and inhibits hypocotyl
elongation, three light-inducible re-
sponses in plants. Planta 210:215–21
14. Benckiser G, Eilts R, Linn A, Lorch HJ,
Sumer E, et al. 1996. N
2
O emission from
differentcropping systems and from aer-
ated, nitrifying and denitrifying tank of
a municipal waste water treatment plant.
Biol. Fertil. Soil 32:257–65
15. Blakeley LM, Rodaway SJ, Hollen LB,
Croker SG. 1972. Control and kinetics
of branch rootformation in cultured root
segments of Haplopappus ravenii. Plant
Physiol. 50:35–42
16. Blatt M, Grabov A. 1997. Signalling
gatesinabscisicacid-mediatedcontrolof
guard cell ion channels. Physiol. Plant.
100:481–90
17. Blatt M. 2000. Ca
2+
signalling and
control of guard-cell volume in stom-
atal movements. Curr. Opin. Plant Biol.
3:196–204
18. Blatt MR. 2000. Cellular signaling and
volume control in stomatal movements
in plants. Annu. Rev. Cell Dev. Biol.
16:221–41
19. Bogdan C. 2001. Nitric oxide and the
regulation of gene expression. Trends
Cell Biol. 11:66–75
19a. Bonnard G, Gualberto J, Lamattina L,
GrienenbergerJM. 1992.RNAediting in
plant mitochondria. Crit. Rev. Plant Sci.
10:503–24
20. Botrel A, Magn´e C, Kaiser WM. 1996.
Nitrate reduction, nitrite reduction and
ammonium assimilation in barley roots
in response to anoxia. Plant Physiol.
Biochem. 34:645–52
21. Bowler C, Neuhaus G, Yamagata H,
Chua NH. 1994. Cyclic GMP and Cal-
cium mediate phytochrome phototrans-
duction. Cell 77:73–81
22. Bradford KJ, Trewavas AJ. 1994. Sensi-
tivitythresholds andvariable time scales
in plant hormone action. Plant Physiol.
105:1029–36
23. BredtDS, HwangPM, Glatt CE, Lowen-
stein C, Reed RR, Snyder SH. 1991.
Cloned and expressed nitric oxide syn-
thase structurally resembles cytochrome
P-450 reductase. Nature 351:714–21
24. Bremner JM. 1997. Sources of nitrous
oxide in soils. Nutr. Cycl. Agroecosys.
49:7–16
25. Brown EG, Newton RP. 1992. Analyti-
cal procedures for cyclic nucleotides and
their associated enzymes in plant tissue.
Phytochem. Anal. 3:1–13
26. Brown EG, Edwards MJ, Newton RP,
Smith CJ. 1980. The cyclic nucleotides
phosphodieterases of spinach chloro-
plasts and microsomes. Phytochemistry
19:23–30
27. Campbell WH. 1996. Nitrate reductase
biochemistry comes of age. Plant Phys-
iol. 111:355–61
28. Campbell WH. 1999. Nitrate reductase
structure, function and regulation:bridg-
ing the gap between biochemistry and
physiology. Annu. Rev. Plant Physiol.
Plant Mol. Biol. 50:277–303
29. Chen YJ, Rosazza JPN. 1995. Purifica-
tion and characterization of nitric oxide
synthase (NOS
Noc
) from a Nocar-
dia species. J. Bacteriol. 177:5122–
28
30. ChoryJ,ReineckeD,SimS,WashburnT,
BrennerM.1994.Aroleforcytokininsin
de-etiolationofArabidopsis.det mutants
have an altered response to cytokinins.
Plant Physiol. 104:339–47
31. Clementi E, Meldolesi J. 1997. The
cross-talkbetween nitric oxide andCa
2+
:
astorywithacomplexpastand a promis-
ing future.Trends Biochem.Sci. 18:266–
69
32. Clementi E. 1998. Role of nitric
oxide and its intracellular signaling
Annu. Rev. Plant Biol. 2003.54:109-136. Downloaded from www.annualreviews.org
by Universidad Nacional de Mar del Plata on 04/08/11. For personal use only.

9 Apr 2003 13:13 AR AR184-PP54-05.tex AR184-PP54-05.sgm LaTeX2e(2002/01/18) P1: FHD
130 LAMATTINA ET AL.
pathwayinthecontrolofCa
2+
homeosta-
sis. Biochem. Pharmacol. 55:713–18
33. Colliver BB, Stephenson T. 2000. Pro-
duction of nitrogen oxide and dinitrogen
oxide by autotrophic nitrifiers. Biotech-
nol. Adv. 18:219–32
34. Conrad R. 1995. Soil microbial pro-
cesses involved in production and con-
sumption of atmospheric trace gases.
Adv. Microb. Ecol. 14:207–50
35. Cooney RV, Harwood PJ, Custer LJ,
Franke, AA. 1994. Light mediated con-
version of nitrogen dioxide to nitric
oxide by carotenoids. Environ. Health
Persp. 102:460–62
36. Correll CC, Ludwig ML, Bruns C,
Karplus PA. 1993. Structural pro-
totypes for an extended family of
flavoproteins reductases: comparison of
phthalate dioxygenase reductase with
ferredoxin reductase and ferredoxin.
Protein Sci. 2:2112–33
37. Crawford NM. 1995. Nitrate: nutrient
and signal for plant growth. Plant Cell
7:859–68
38. Davidson EA, Kingerlee W. 1997. A
global inventory of nitric oxide emis-
sions from soils. Nutr. Cycl. Agroecosys.
48:37–50
39. Dei M. 1982. A two-fold action of ben-
zyladenine in chlorophyll formation in
etiolated cucumber cotyledons. Physiol.
Plant. 64:153–60
40. Delledonne M, Xia Y, Dixon RA, Lamb
C. 1998. Nitric oxide functions as a sig-
nal in plant defense resistance. Nature
394:585–88
41. Dobashi K, Pahan K, Chahal A, Singh I.
1997.Modulationofendogenous antiox-
idant enzymes by nitric oxide in rat C6
glial cells. J. Neurochem. 68:1896–903
41a. Drozdowics YM, Jones RL. 1995.
Hormonal regulation of organic and
phosphoric acid release by barley aleu-
rone layers and scutella. Plant Physiol.
108:769–76
42. Durner J, Wendehenne D, Klessig DF.
1998. Defense gene induction in tobacco
by nitric oxide, cyclic GMP, and cyclic
ADP ribose. Proc. Natl. Acad. Sci. USA
95:10328–33
43. Durner J, Klessig DF. 1999. Nitric oxide
as a signal in plants. Current Opin. Plant
Biol. 2:369–74
44. Elphick MR, Melarange R. 1998. Nitric
oxide function in an echinoderm. Biol.
Bull. 194:260–66
45. Ermler U, Siddiqui RA, Cramm R,
Friedrich B. 1995. Crystal structure of
the flavohemoglobin from Alcaligenes
eutrophus at 1.75
˚
A resolution. EMBO J.
14:6067–77
46. Fang FC. 1999. Nitric Oxide and Infec-
tion. New York: Kluwer/Plenum
47. FAO, Fertilizer Yearbook. 1990. United
Nations. Rome: FAO
48. Fehsenfeld F, Meagher J, Cowling E.
1993. Southern Oxidants Study Annual
Report, pp. 47–61. Raleigh, North Car-
olina
49. FoissnerI, Wendehenne D, Langebartels
C, Durner J. 2000. In vivo imaging of
an elicitor-induced nitric oxide burst in
tobacco. Plant J. 23:817–24
50. Furchgott RF, Zawadzki JV. 1980. The
obligatory role of endothelial cell in the
relaxation of arterial smooth muscle by
acetylcholine. Nature 288:373–76
51. Garc´ıa Mata C, Lamattina L. 2001. Ni-
tric oxide induces stomatal closure and
enhances the adaptive plant responses
against drought stress. Plant Physiol.
126:1196–204
52. Garc´ıa-Mata C, Lamattina L. 2002. Ni-
tric oxide and abscisic acid cross talk in
guard cells. Plant Physiol. 128:790–92
52a. Garc´ıa-Mata C, Lamattina L. 2003.
Abscisic acid, nitric oxide and stomatal
closure. Is nitrate reductase one of the
missing links? Trends Plant Sci. 8:20–26
53. Gaudinova A. 1990. Effect of cytokinin
on nitrate reductase activity. In Molecu-
lar Aspects of Hormonal Regulation of
Plant Development, ed. M Kutacek, MC
Elliot, M Machackova, pp. 225–31. The
Hague: SPB Acad.
Annu. Rev. Plant Biol. 2003.54:109-136. Downloaded from www.annualreviews.org
by Universidad Nacional de Mar del Plata on 04/08/11. For personal use only.

9 Apr 2003 13:13 AR AR184-PP54-05.tex AR184-PP54-05.sgm LaTeX2e(2002/01/18) P1: FHD
NITRIC OXIDE 131
54. GiraudatJ,ParcyF,BertaucheN,GostiF,
Leung J, et al. 1994.Current advances in
abscisic acid action and signaling. Plant.
Mol. Biol. 26:1557–77
55. Goretski J, Hollocher TC. 1988. Trap-
pingofnitricoxideproducedduringden-
itrification by extracellular hemoglobin.
J. Biol. Chem. 263:2316–23
56. Gouvˆea, CMCP, Souza JF, Magalh˜aes
ACN, Martins IS. 1997. NO-releasing
substancesthatinducegrowthelongation
in maize root segments. Plant Growth
Regul. 21:183–87
57. Gow AJ, Ischiropoulos HJ. 2001. Nitric
oxidechemistryandcellular signaling.J.
Cell Physiol. 187:277–82
58. Graziano M, Beligni MV, Lamattina L.
2002. Nitric oxide improves internal
iron availability in plants. Plant Physiol.
130:1852–59
59. Gross SS. 2001. Targeted delivery of ni-
tric oxide. Nature 409:577–78
60. Grubusic D, Konjevic R. 1990. Light
and nitrate interaction in phytochrome-
controlled germination of Paulownia
tormentosa seeds. Planta 181:239–
43
61. Grubusic D, Giba Z, Konjevic R.
1992. The effect of organic nitrates
in phytochrome-controlled germination
of Paulownia tormentosa seeds. Pho-
tochem. Photobiol. 56:629–32
62. Halliwell B, Gutteridge JMC. 1984.
Oxygen toxicity, oxygen radicals, tran-
sition metals, and disease. Biochem. J.
219:1–14
63. Harper JE. 1981. Evolution of nitrogen
oxide(s) during in vivo nitrate reductase
assay of soybean leaves. Plant Physiol.
68:1488–93
64. Henry YA, Ducastel B, Guissani A.
1997.Basicchemistryof nitricoxideand
related nitrogen oxides. In Nitric Oxide
Research from Chemistry to Biology, ed.
YA Henry, A Guissani, B Ducastel, pp.
15–46. Austin, TX: Landes Co. Biomed.
Publ.
65. HibbsJB, Bastian NR. 1999. The discov-
ery of the biological synthesis of nitric
oxide. See Ref. 46, pp.13–33
66. Hillmer S, Gilroy S, Jones RL. 1993.
Visualizing enzyme secretion from indi-
vidual barley protoplasts. Plant Physiol.
102:279–86
67. Hmadcha A, Bedoya FJ, Sobrino F,
PintadoE. 1999.Methylation-dependent
genesilencing induced byinterleukin 1B
via nitric oxide production. J. Exp. Med.
190:1595–603
68. Hofmann F, Ammendola A, Schloss-
mannJ.2000.RisingbehindNO:cGMP-
dependent protein kinases. J. Cell Sci.
113:1671–76
69. Hogg N, Kalyanaraman B, Joseph J,
Struck A, Parthasarathy S. 1993. Inhibi-
tion of low-densitylipoprotein oxidation
by nitric oxide. Potential role in athero-
genesis. FEBS Lett. 334:170–74
70. Horemans N, Foyer CH, Asard H. 2000.
Transport and action of ascorbate at
the plasma membrane. Trends Plant Sci.
5:263–67
71. Huber SC, Bachmann M, Huber JL.
1996. Post-translation regulation of ni-
trate reductase activity: a role for Ca
2+
and 14-3-3 proteins. Trends Plant Sci.
1:432–38
72. Huie RE, Padmaja S. 1993. The reaction
of NO with O
−
2
. Free Radical Res. Com.
18:195–99
73. Ignarro LJ, Byrns RE, Buga GM, Wood
KS.1987. Endothelium-derivedrelaxing
factor from pulmonary artery and vein
possess pharmacological and chemical
properties identical to those of nitric ox-
ide radical. Circ. Res. 61:866–79
74. Intergov. Panel Climate Change Tech.
Analyses Impacts, Adapt. Mitigation.
1992. Contribution of Working Group II
to the Second Assessment Report of
the Intergovernmental Panel on Climate
Change.London:CambridgeUniv.Press
75. Jeffrey SR, Snyder SH. 1995. Nitric ox-
ide: a neural messenger. Annu. Rev. Cell
Dev. Biol. 11:417–40
76. Kaiser WM, Weiner H, Huber SC. 1999.
Annu. Rev. Plant Biol. 2003.54:109-136. Downloaded from www.annualreviews.org
by Universidad Nacional de Mar del Plata on 04/08/11. For personal use only.

9 Apr 2003 13:13 AR AR184-PP54-05.tex AR184-PP54-05.sgm LaTeX2e(2002/01/18) P1: FHD
132 LAMATTINA ET AL.
Nitrate reductase in higher plants: a case
study for transduction of environmental
stimuli into control of catalytic activity.
Physiol. Plant. 105:385–90
77. Kanner J, Harel S, Granit R. 1991. Nitric
oxide as an antioxidant. Arch. Biochem.
Biophys. 289:130–36
78. Karplus PA, Daniels MJ, Herriot JR.
1991. Atomic structure of ferredoxin-
Nadp
+
reductase, prototype for a struc-
turally novel flavoenzyme family. Sci-
ence 251:60–66
79. Kikuchi K, Nagano T, Hayakawa H, Hi-
rata Y, Hirobe M. 1993. Detection of
nitric oxide production from perfused
organ by luminol-H
2
O
2
system. Anal.
Chem. 65:1794–99
80. Kikuchi K, Nagano T, Hayakawa H,
Hirata Y, Hirobe M. 1993. Real time
measurements of nitric oxide produced
ex vivo by luminol-H
2
O
2
chemilu-
miniscence method. J. Biol. Chem.
268:23106–10
81. Kikuchi K, Nagano T, Hirobe M. 1996.
Novel detection method of nitric ox-
ide using horseradish peroxidase. Biol.
Pharm. Bull. 19:649–51
82. Klepper L. 1979. Nitric oxide (NO) and
nitrogen dioxide (NO
2
) emissions from
herbicide-treated soybean plants. Atmos.
Environ. 13:537–42
83. Klessig DF, Durner J, Noad R, Navarre
DA, Wendehenne D, et al. 2000. Nitric
oxide and salicylic acid signaling in
plantdefense.Proc.Natl.Acad.Sci. USA
97:8849–55
84. Kojima H, Nakatsubo N, Kikuchi K,
Urano Y, Higuchi T, et al. 1998. Di-
rect evidence of NO production in rat
hippocampus andcortex using anew flu-
orescent indicator: DAF-2 DA. Neurore-
port 9:3345–48
85. Kozlovb AV, Bini A, Iannone A, Zini
I, Tomasi A. 1996. Electron Param-
agnetic Resonance characterization of
rat neuronal nitric oxide production
ex vivo. Methods Enzymol. 268:229–
36
86. Kramlich JC, Linak WP. 1994. Nitrous
oxide behaviour in the atmosphere, and
in combustion and industrial systems.
Prog. Energ. Combust. 20:149–202
87. Kuo RC, Baxter GT, Thompson SH,
Stricker SA, Patton C, et al. 2000. NO is
necessary and sufficient for egg activa-
tion at fertilization. Nature 406:633–36
88. Deleted in proof
89. Lamattina L, Beligni MV, Garc´ıa-Mata
C, Laxalt AM. 2001. Method of enhanc-
ing the metabolic function and the grow-
ing conditions of plants and seeds. US
Patent No. 6 242 384 B1
90. Leckie CP, McAisnsh MR, Hetherington
AM.1998.cADPRand stomatalclosure.
Plant Physiol. 114(Suppl.):272
91. Leshem YY, Pinchasov Y. 2002. Non-
invasive photoacustic spectroscopic de-
termination of relativeendogenous nitric
oxide and ethylene content stoichiome-
try during the ripening of strawberries
Fragariaanannasa(Duch)andavocados
Persea americana (Mill.). J. Exp. Bot.
51:1471–73
92. Leshem YY, WillsRBH, Ku VVV. 1998.
Evidence for the function of the free
radical gas nitric oxide (NO) as an en-
dogenous regulating factor in higher
plants. Plant Physiol. Biochem. 36:825–
33
93. Lipton SA, Choi Y-B, Pan Z-H, Lei SZ,
Chen H-SV, et al. 1993. A redox-based
mechanism for the neuroprotective and
neurodestructive effects of nitric oxide
and related nitroso compounds. Nature
364:626–32
94. Lloyd-Jones DM, Bloch KD. 1996. The
vascular biology of nitric oxide and its
role in atherogenesis. Annu. Rev. Med.
47:365–75
95. Lucas KA, Pitari GM, Kazerounian S,
Ruiz-Stewart I, Park J, et al. 2000.
Guanylyl cyclases and signaling by
cyclic GMP. Pharmacol. Rev. 52:375–
414
96. Malinski T, Taha Z. 1992. Nitric oxide
release from a single cell measured in
Annu. Rev. Plant Biol. 2003.54:109-136. Downloaded from www.annualreviews.org
by Universidad Nacional de Mar del Plata on 04/08/11. For personal use only.

9 Apr 2003 13:13 AR AR184-PP54-05.tex AR184-PP54-05.sgm LaTeX2e(2002/01/18) P1: FHD
NITRIC OXIDE 133
situ by a porphyrinic-based microsensor.
Nature 358:676–78
97. Malyshev I, Manukhina E, Golubeva L,
Zenina T, Aymasheva N, et al. 1999. NO
paradox in adaptation of the organism.
Acta Physiol. Scand. 167:43
97a. Martin CT, Morse RH, Kanne RM, Gray
HB, Malmstrom BG, Chan SI. 1981. Re-
actionofnitricoxidewithtree and fungal
lacasse. Biochemistry 20:5147–55
98. Martinez A. 1995. Nitric oxide synthase
in invertebrates. Histochem. J. 27:770–
76
99. Mayer B. 2000. Nitric Oxide. Heidel-
berg, Ger.: Springer-Verlag. 667 pp.
100. McAdams HH, Arkin A. 1999. Genetic
regulationat thenanomolarscale.Trends
Genet. 15:65–69
101. McDonald LJ, Murad F. 1995. Nitric ox-
ide and cGMP signaling. Adv. Pharma-
col. 34:263–76
102. Misko TP, Schilling RJ, Salvemini D,
Moore WM, Currie MG. 1993. A flu-
orescent assay for the measurement of
nitric oxide in biological samples. Anal.
Biochem. 214:11–16
102a. Mockaitis K, Howell SH. 2000. Auxin
induces mitogenic activated protein ki-
nase (MAPK) activation in roots of Ara-
bidopsis seedlings. Plant J. 24:785–96
103. Moncada S, Palmer RM, Higgs EA.
1991. Nitric oxide: physiology, patho-
physiology and pharmacology. Pharma-
col. Rev. 43:109–42
104. Mott KA, Buckley TN. 2000. Patchy
stomatal conductance emergent collec-
tive behavior. Trends Plant Sci. 5:258–
62
105. Mott HR, Carpenter JW, Campbell SL.
1997. Structural and functional analysis
ofa mutantRas proteinthat isinsensitive
to nitric oxide activation. Biochemistry
36:3640–44
105a. Nathan C, Xie Q-W. 1994. Nitric oxide
synthases: roles, tolls and controls. Cell
78:915–18
106. Neill SJ, Desikan R, Clarke A, Hancock
JT. 2002. Nitric oxide is a novel compo-
nent of abscisic acid signaling in stom-
atal guard cells. Plant Physiol. 128:13–
16
107. Nelson RS, Ryan SA, Harper JE. 1983.
Soybeanmutants lacking constitutive ni-
trate reductase activity. I. Selection and
initialplantcharacterization.PlantPhys-
iol. 72:503–9
108. Nims RW, Cook JC, Krishna MC,
Christodoulou D, Poore CMB, et al.
1996. Colorimetric assays for nitric ox-
ide stock solutions and donor com-
pounds. Methods Enzymol. 268:93–105
109. Ninnemann H, Maier J. 1996. Indica-
tions for the occurrence of nitric oxide
synthases in fungi and plants and the in-
volvement in photoconidiation of Neu-
rospora crassa. Photochem. Photobiol.
64:393–98
110. Novick A, Weiner M. 1957. Enzyme in-
duction as an all or none phenomenon.
Proc. Natl. Acad. Sci. USA 43:553–66
111. Nunoshiba T, de-Rojas-Walker T, Wish-
nok JS, Tannenbaum SR, Demple B.
1993. Activation by nitric oxide of
an oxidative-stress response that de-
fends Escherichia coli against activated
macrophages.Proc.Natl.Acad.Sci.USA
90:9993–97
112. Pagnussat G, Simontachi M, Puntarulo
S, Lamattina L. 2002. Nitric oxide is
required for root organogenesis. Plant
Physiol. 129: 954–56
113. Palevitz BA, Lewis R. 1998. Erectile
dysfunction: serious research for a seri-
ous problem. Scientist 12:1
114. Palmer R, Ferrige A, Moncada S. 1987.
Nitric oxide release accounts for the bi-
ological activity of endothelium-derived
relaxing factor. Nature 327:524–26
115. Palmer RMJ, Ashton DS, Moncada S.
1998. Vascular endothelial cell synthe-
size nitric oxide from
L-arginine. Nature
333:664–66
116. Paul JW, Beauchamp EG, Zhang X.
1993. Nitrous and nitric oxide emis-
sion during nitrification and denitrifica-
tion from manure-amended soil in the
Annu. Rev. Plant Biol. 2003.54:109-136. Downloaded from www.annualreviews.org
by Universidad Nacional de Mar del Plata on 04/08/11. For personal use only.

9 Apr 2003 13:13 AR AR184-PP54-05.tex AR184-PP54-05.sgm LaTeX2e(2002/01/18) P1: FHD
134 LAMATTINA ET AL.
laboratory. Can. J. Soil Sci. 73:539–
53
117. Paveto C, Pereira C, Espinosa J, Mon-
tagna AE, Farber M, et al. 1995.
The nitric oxide transduction pathway
in Trypanosoma cruzi. J. Biol. Chem.
270:16576–79
118. Pawloski JR, Hess D, Stamler JS. 2001.
Export by red blood cells of nitric oxide
bioactivity. Nature 409:622–26
119. Penson SP, Schuurink RC, Fath A,
GublerF,Jacobsen JV,etal.1996.cGMP
is required for gibberellic acid-induced
geneexpressioninbarleyaleurone.Plant
Cell 8:2325–33
120. Pfeiffer S, Janistyn B, Jessner G, Pi-
chornerH,EbermannRE.1994.Gaseous
nitric oxide stimulates guanosine-3,5-
cyclic monophosphate formation is
spruce needles. Photochemistry 36:259–
62
121. Piatelli M, Giudici de Nicola M, Castro-
giovanni V. 1969. Photocontrol of ama-
ranthinsynthesisinAmaranthustricolor.
Photochemistry 8:731–36
122. Pilgrim ML, Caspar T, Quail PH, Mc-
Clung CR. 1993. Circadian and light-
regulated expression of nitrate reductase
in Arabidopsis.Plant Mol. Biol. 23:349–
64
123. Ramamurthi A, Lewis RS. 1997. Mea-
surements and modeling of nitric ox-
ide release rates for nitric oxide donors.
Chem. Res. Toxicol. 10:408–13
124. Reggiani R. 1997. Alteration of levels of
cyclic nucleotides in responseto anaero-
biosis in rice seedlings. Plant Cell Phys-
iol. 38:740–42
125. Regulski M, Tully T. 1995. Molecu-
lar and biochemical characterization of
dNOS:aDrosophila Ca
2+
/calmodulin-
dependent nitric oxide synthase. Proc.
Acad. Natl. Sci. USA 92:9072–76
126. Reiter CD, Teng RJ, Beckman JS. 2000.
Superoxide reacts with nitric oxide to ni-
tratetyrosineatphysiologicalpHviaper-
oxinitrite. J. Biol. Chem. 275:32460–66
127. Ribeiro EA Jr, Cunha FQ, Tamashiro
WMSC, Martins IS. 1999. Growth
phase-dependent subcellular localiza-
tion of nitric oxide synthase in maize
cells. FEBS Lett. 445:283–86
128. Richter C. 1997. Reactive oxygen and
nitrogen species regulate mitochon-
drial Ca
2+
homeostasis and respiration.
Biosci. Rep. 17:53–66
129. Ritchie S, McCubbin A, Ambrosse G,
Kao T-H, Gilroy S. 1999. The sensitivity
of barley aleurone tissue to gibberellin is
heterogeneous and may be spatially de-
termined. Plant Physiol. 120:361–70
130. Rockel P, Strube F, Rockel A, Wildt J,
Kaiser WM. 2002. Regulation of nitric
oxide (NO) production by plant nitrate
reductase in vivo and in vitro. J. Exp.
Bot. 53:1–8
131. Roelle PA, Aneja VP, Gay B, Geron C,
Pierce T. 2001. Biogenic nitric oxide
emissions from cropland soils. Atmos.
Environ. 35:115–24
132. Rotzinger S, Aragon CMG, Rogan F,
Amir S, Amit Z. 1995. The nitric oxide
synthase inhibitor NW-nitro-
L-arginine
methylester attenuates brain catalase ac-
tivity in vitro. Life Sci. 56:1321–24
133. Rowland A, Murray AJ, Welburn AR.
1985. Oxides of nitrogen and their
impact upon vegetation. Rev. Environ.
Health 5:295–342
134. Rubbo H, Radi R, Trujillo M, Telleri
R, Kalyanaraman B, et al. 1994. Nitric
oxide regulation of superoxide and per-
oxinitrite dependent lipid peroxidation.
Formation of novel nitrogen containing
oxidized lipid derivatives. J. Biol. Chem.
269:26066–75
135. Ruser R, Flessa H, Schilling R, Steindl
H, Beese F. 1998. Soil compactation and
fertilization effects on nitrous oxide and
methane fluxes in potato fields. Soil Sci.
Soc. Am. J. 62:1587–95
136. SandersD, BrownleeC,HarperJF.1999.
Communicating with calcium. Plant
Cell 11:691–706
137. Scherer GFE, Holk A. 2000. NO
donors mimic and NO inhibitors inhibit
Annu. Rev. Plant Biol. 2003.54:109-136. Downloaded from www.annualreviews.org
by Universidad Nacional de Mar del Plata on 04/08/11. For personal use only.

9 Apr 2003 13:13 AR AR184-PP54-05.tex AR184-PP54-05.sgm LaTeX2e(2002/01/18) P1: FHD
NITRIC OXIDE 135
cytokinin action in betalain accumu-
lation in Amaranthus caudatus. Plant
Growth Regul. 32:345–50
138. Schober A, Malz CR, Chober W, Meyer
DL. 1994. NADPH-diaphorase in the
central nervous system of thelarval lam-
prey (Lampetra planeri). J. Comp. Neu-
rol. 345:94–104
139. Schroeder JI, Kwak JM, Allen GJ. 2001.
Guard cell abscisic acid signalling and
engineering drought hardiness in plants.
Nature 410:327–30
140. Schroeder JI, Allen GJ, Hugouvieux V,
Kwak JM, Waner D. 2001. Guard cell
signal transduction. Annu. Rev. Plant
Physiol. Plant Mol. Biol. 52:627–58
141. Shin JH, Chung S, Park EJ, Uhm DY,
Suh CK. 1997. Nitric oxide directly acti-
vates calcium-activated potassium chan-
nels from ratbrain reconstituted lipid bi-
layer. FEBS Lett. 415:299–302
142. Siegel-Itzcovich J. 1999. Viagra makes
flowers stand up straight. West J. Med.
171:38
143. Sinclair J. 1987. Changes in spinach thy-
lakoid activity due to nitrite ions. Photo-
synth. Res. 12:255–63
144. Solomonson LP, Barber MJ. 1990. As-
similatory nitrate reductase: functional
properties and regulation. Annu. Rev.
Plant Physiol. Plant Mol. Biol. 41:225–
53
145. Stamler JS, Singel DJ, Loscalzo J.
1992. Biochemistry of nitric oxide
and its redox-activated forms. Science
258:1898–902
146. Stamler JS. 1994. Redox signaling: ni-
trosylationandrelatedtargetinteractions
of nitric oxide. Cell 78:931–36
147. Stohr C, Strule F, Marx G, Ullrich WR,
Rockel P. 2001. A plasma membrane-
bound enzyme of tobacco roots catalyse
the formation of nitric oxide from nitrite.
Planta 212:835–41
148. Stuehr DJ. 1997. Structure-function as-
pects in the nitric oxide synthases.
Annu. Rev. Pharmacol. Toxicol. 37:339–
59
149. Su W, Howell SH. 1995. The effects of
cytokinin and light on hypocotyl elon-
gation in Arabidopsis seedlings are in-
dependent and additive. Plant Physiol.
108:1423–30
150. Suty L, Moureaux T, Leydecker MT,
Teyssentier de la Serve B. 1993. Cy-
tokinins affects nitrate reductase ex-
pression through the modulation of
polyadenylation of the nitrate reductase
mRNA transcript. Plant Sci. 90:11–19
151. Tanno M, Sueyoshi S, Miyata N, Naki-
agewa S. 1996. Nitric oxide generation
from aromatic N-nitrosoureas at ambi-
ent temperature. Chem. Pharm. Bull.
44:1849–52
152. Thoiron S, Pascal N, Briat J-F. 1997. Im-
pactofirondeficiencyandironre-supply
during the early stages of vegetative de-
velopmentin maize (Zea mays L.). Plant
Cell Environ. 20:1051–60
153. Thomas TH, Hare PD, van Staden J.
1997. Phytochrome and cytokinin re-
sponses.PlantGrowthRegul.23:105–22
154. Thomas TH. 1984. Some reflections
on the relationship between endogenous
hormones and light-mediated seed dor-
mancy. Plant Growth Regul. 11:239–
48
155. Trewavas AJ. 1986. Resource allocation
under poor growth conditions. A major
role for growth substances in develop-
mental plasticity. Symp. Soc. Exp. Biol.
Med. 40:31–76
156. Tun NN, Holk A, Scherer FE. 2001.
Rapid increase of NO release in plant
cellcultures induced bycytokinin.FEBS
Lett. 509:174–76
157. Van Camp W, Van Montagu M, Inz´eD.
1998.H
2
O
2
andNO:redoxsignals indis-
ease resistanse. Trends Plant Sci. 3:330–
34
158. Vaucheret H, Kronenberger J, Lepingle
A, Vilaine F, Boutin J-P, Caboche M.
1992. Inhibition of tobacco nitrite reduc-
tase activity by expression of antisense
RNA. Plant J. 2:559–69
159. Wang M, Roberts DL, Paschke R, Shea
Annu. Rev. Plant Biol. 2003.54:109-136. Downloaded from www.annualreviews.org
by Universidad Nacional de Mar del Plata on 04/08/11. For personal use only.

9 Apr 2003 13:13 AR AR184-PP54-05.tex AR184-PP54-05.sgm LaTeX2e(2002/01/18) P1: FHD
136 LAMATTINA ET AL.
TM, Masters BS, Kim JJ. 1997. Three-
dimensional structure of NADPH-cyto-
chrome P-450 reductase: prototype for
FMN- and FAD-containing enzymes.
Proc. Natl. Acad. Sci. USA 94:8411–16
160. Welburn AR. 1990. Why are atmo-
sphericoxides ofnitrogenusually phyto-
toxic and not alternative fertilizers? New
Phytol. 115:395–429
161. Wendehenne D, Pugin A, Klessig DF,
Durner J. 2001. Nitric oxide: compara-
tivesynthesisandsignalinginanimaland
plant cells. Trends Plant Sci. 6:177–83
162. Werner-FelmayerG,GoldererG,Werner
ER, Grobner P, Wachter H. 1994. Pteri-
dine biosynthesis and nitric oxide
synthase in Physarum polycephalum.
Biochem. J. 304:105–11
163. White AC, Maloney EK, Bostani MR,
Hassoun PM, Fanburg BL. 1995. Nitric
oxide increases cellular glutathione lev-
els in rat lung fibroblasts. Am. J. Respir.
Cell Mol. Biol. 13:442–48
164. Wildt J, Kley D, Rockel A, Rockel P,
Segschneider HJ. 1997. Emission of NO
from several higher plant species. J.
Geol. Res. 102:5919–27
165. WinkDA,Cook JA, PacelliR, Liebmann
J, Krishne MC, Mitchell JB. 1995. Ni-
tric oxide (NO) protects against cellular
damage by reactive oxygen species. Tox-
icol. Lett. 82/83:221–26
166. Xiong L, Zhu JK. 2001. Abiotic stress
signal transduction in plants: molecular
and genetic perspectives. Physiol. Plant.
112:152–66
167. Xu L, Eu JP, Meissner G, Stamler JS.
1998. Activation of the cardiac calcium
release channel (ryanodine receptor) by
poly-S-nitrosylation. Science 279:234–
37
168. Yamasaki H, Sakihama Y, Takahashi S.
1999. An alternative pathway for ni-
tric oxide production in plants: new fea-
tures of an old enzyme. Trends Plant Sci.
4:128–29
169. Yamasaki H, Sakihama Y. 2000. Simul-
taneous production of nitric oxide and
peroxinitritebyplantnitratereductase:in
vitroevidencefor the NR-dependentfor-
mation of active nitrogen species. FEBS
Lett. 468:89–92
170. Yang YY, Nagatani A, Zhao YJ, Kang
BJ, Kendrick RE, Kamiya Y. 1995. Ef-
fects of gibberellins on seed germina-
tionofphytochrome-deficientmutantsof
Arabidopsis thaliana. Plant Cell Phys-
iol. 36:1205–11
171. Zielinski BS, Osahan JK, Hara TJ, Hos-
seini M, Wong E. 1996. Nitric ox-
ide synthase in the olfactory mucosa
of the larval sea lamprey (Petromy-
zon marinus). J. Comp. Neurol. 365:18–
26
172. Zottini M, Formentin E, Scattolin M,
Carimi F, LoSchiavo F, Terzi M. 2002.
Nitric oxide affects plant mitochondrial
functionalityinvivo.FEBSLett.515:75–
78
173. Zumft WG. 1997. Cell biology and
molecular basis of denitrification. Mi-
crob. Mol. Biol. Rev. 61:533–616
Annu. Rev. Plant Biol. 2003.54:109-136. Downloaded from www.annualreviews.org
by Universidad Nacional de Mar del Plata on 04/08/11. For personal use only.

17 Apr 2003 16:49 AR AR184-05-COLOR.tex AR184-05-COLOR.SGM LaTeX2e(2002/01/18) P1: GDL
Figure 2 Scheme showing the structure and domain organization of NOS from mammals and NR from plants. NOS and
NR, with their respective cofactors, are presented as homodimers displaying NO biosynthetic activity. For details see the text.
Mo-MPT, Mo-Molybdopterin; FAD, flavin adenine dinucleotide; FMN, flavin mononucleotide; H
4
Bip, tetrahydrobiopterin.
Annu. Rev. Plant Biol. 2003.54:109-136. Downloaded from www.annualreviews.org
by Universidad Nacional de Mar del Plata on 04/08/11. For personal use only.

17 Apr 2003 16:49 AR AR184-05-COLOR.tex AR184-05-COLOR.SGM LaTeX2e(2002/01/18) P1: GDL
Figure 3 NO regulates growth, developmental, and adaptive processes in plants.
Plants produce NO by NR and a putative NOS-like activity in both aerial organs and
roots. NO is also produced during the nitrification process by microorganisms in the
rhizosphere (33). Exchange of NO between the atmosphere and aerial plant organs as
well as between rhizosphere and roots occurs. Plants are sensing external and internal
signals and integrate them into a signal-transduction system operated by hormones
and cellular messengers, in which NO is involved. Then, plants act through metabolic
or gene expression changes in order to assess a balanced response during growth and
developmental processes. The circuitry of the complex network driving the chemical
signalspossessescontrolandsafeguardpointsandisthoughttobepermanentlyworking
to successfully adapt plants to a changing environment (89). Where no references are
given, examples are reviewed in References 11 and 171 or within the text. (
1
)L.
Lamattina et al. unpublished data; (?) No direct experimental evidence on the NO
involvement is available yet.
Annu. Rev. Plant Biol. 2003.54:109-136. Downloaded from www.annualreviews.org
by Universidad Nacional de Mar del Plata on 04/08/11. For personal use only.

17 Apr 2003 16:49 AR AR184-05-COLOR.tex AR184-05-COLOR.SGM LaTeX2e(2002/01/18) P1: GDL
Figure 4 Plant physiological responses to NO treatment. (A) NO induces adventi-
tious root formation in stem cuttings (SC) of lavender (Lavanta dentata). SC were put
in vermiculite and kept in a greenhouse under natural light conditions for 45 days. SC
weretreated with 100 µMSNP (NO) orH
2
O(control) every15days(89) (Bar= 1 cm).
(B)NOrevertsthe phenotype of the ys1 mutantofmaize (Zea mays), which is defective
inironuptakeand/or transport. Maize plants were grownhydroponically in thenutrient
solution alone (control) or nutrient solution supplemented with 100 µM SNP (NO).
Pictures were taken after 30 days of growing (58) (Bar = 2.5 cm). (C) NO protects
against oxidative damage. Potato (Solanum tuberosum) leaves were pretreated with
H
2
O (control) or 100 µM SNP (NO) for 24 h and added 0.4 mg L
−1
of the herbicide
diquat. Leaflets were keptfor36 h in these conditions and then incubated in 1 mg mL
−1
diaminobenzidine at 25
◦
C and complete darkness for 18 h for ROS-presence visualiza-
tion (Bar= 1 cm). (D) NO induces stomatal closure. Bright-field image of Vicia faba
guard cells. Epidermal strips were incubated in opening buffer (control) or in opening
buffer plus 100 µM SNP (NO) and loaded with the fluorescent probe DAF-2DA. Insets
show the same stomata under UV light. Green fluorescence corresponds to DAF-2DA
specific reaction with NO (Bar= 5 µm).
Annu. Rev. Plant Biol. 2003.54:109-136. Downloaded from www.annualreviews.org
by Universidad Nacional de Mar del Plata on 04/08/11. For personal use only.

P1: FRK
April 3, 2003 12:24 Annual Reviews AR184-FM
Annual Review of Plant Biology
Volume 54, 2003
CONTENTS
Frontispiece—Lloyd T. Evans xii
CONJECTURES
,REFUTATIONS, AND EXTRAPOLATIONS, Lloyd T. Evans 1
UNDERSTANDING THE
FUNCTIONS OF PLANT DISEASE RESISTANCE
PROTEINS, Gregory B. Martin, Adam J. Bogdanove, and Guido Sessa 23
PROTEIN PHOSPHATASES IN PLANTS, Sheng Luan 63
PLANT PEROXIREDOXINS, Karl-Josef Dietz 93
N
ITRIC
OXIDE:THE VERSATILITY OF AN EXTENSIVE SIGNAL
MOLECULE, Lorenzo Lamattina, Carlos Garc
´
ıa-Mata,
Magdalena Graziano, and Gabriela Pagnussat 109
BIOSYNTHESIS AND METABOLISM OF BRASSINOSTEROIDS,
Shozo Fujioka and Takao Yokota 137
THE COP9 SIGNALOSOME:REGULATING PLANT DEVELOPMENT
THROUGH THE CONTROL OF PROTEOLYSIS, Giovanna Serino
and Xing-Wang Deng 165
IRON TRANSPORT AND SIGNALING IN PLANTS, Catherine Curie
and Jean-Franc¸ois Briat 183
FROM BACTERIAL GLYCOGEN TO STARCH:UNDERSTANDING THE
BIOGENESIS OF THE
PLANT STARCH GRANULE, Steven G. Ball
and Matthew K. Morell 207
THE
PLANT CELL CYCLE, Walter Dewitte and James A.H. Murray 235
PHOSPHOLIPID-BASED SIGNALING IN PLANTS, Harold J.G. Meijer
and Teun Munnik 265
GIBBERELLINS AND
FLOWERING OF GRASSES AND CEREALS:PRIZING
OPEN THE LID OF THE “FLORIGEN”BLACK BOX, Rod W. King and
Lloyd T. Evans 307
PHOTOSYNTHESIS OF OVERWINTERING EVERGREEN PLANTS,
Gunnar
¨
Oquist and Norman P.A. Huner 329
STRUCTURE OF LINKAGE DISEQUILIBRIUM IN PLANTS,
Sherry A. Flint-Garcia, Jeffry M. Thornsberry, and Edward S. Buckler IV 357
SINGLE-NUCLEOTIDE MUTATIONS FOR PLANT FUNCTIONAL
GENOMICS, Steven Henikoff and Luca Comai 375
v
Annu. Rev. Plant Biol. 2003.54:109-136. Downloaded from www.annualreviews.org
by Universidad Nacional de Mar del Plata on 04/08/11. For personal use only.

P1: FRK
April 3, 2003 12:24 Annual Reviews AR184-FM
vi
CONTENTS
HOW DO CELLS KNOW WHAT THEY WANT TO BE WHEN THEY GROW
U
P?L
ESSONS FROM
EPIDERMAL PATTERNING IN ARABIDOPSIS,
John C. Larkin, Matt L. Brown, and John Schiefelbein 403
T
RANSFER
CELLS:CELLS SPECIALIZED FOR A SPECIAL PURPOSE,
Christina E. Offler, David W. McCurdy, John W. Patrick, and Mark J. Talbot 431
CHLOROPLAST
MOVEMENT, Masamitsu Wada, Takatoshi Kagawa,
and Yoshikatsu Sato 455
C
RYPTOCHROME STRUCTURE AND SIGNAL TRANSDUCTION,
Chentao Lin and Dror Shalitin 469
MEMBRANE-BOUND DIIRON CARBOXYLATE PROTEINS,
Deborah A. Berthold and P
˚
al Stenmark 497
LIGNIN
BIOSYNTHESIS
, Wout Boerjan, John Ralph, and Marie Baucher 519
APOMIXIS:ADEVELOPMENTAL PERSPECTIVE, Anna M. Koltunow
and Ueli Grossniklaus 547
MOLECULAR
MECHANISMS AND REGULATION OF K
+
TRANSPORT
IN
HIGHER PLANTS, Anne-Ali
´
enor V
´
ery and Herv
´
e Sentenac 575
P
ERCEPTION AND SIGNAL TRANSDUCTION OF CYTOKININS,
Tatsuo Kakimoto 605
FUNCTIONAL
GENOMICS OF P450S, Mary A. Schuler and
Daniele Werck-Reichhart 629
METABOLOMICS IN SYSTEMS BIOLOGY, Wolfram Weckwerth 669
R
EMODELING THE CYTOSKELTON FOR GROWTH AND FORM:
A
N OVERVIEW WITH SOME NEW VIEWS, Geoffrey O. Wasteneys
and Moira E. Galway 691
I
NDEXES
Subject Index 723
Cumulative Index of Contributing Authors, Volumes 44–54 753
Cumulative Index of Chapter Titles, Volumes 44–54 758
ERRATA
An online log of corrections to Annual Review of Plant Biology
chapters (if any, 1997 to the present) may be found at
http://plant.annualreviews.org/
Annu. Rev. Plant Biol. 2003.54:109-136. Downloaded from www.annualreviews.org
by Universidad Nacional de Mar del Plata on 04/08/11. For personal use only.