Nitric OxideNitric Oxide

NO refers to nitrosyl radical (•NO) and its NO refers to nitrosyl radical (•NO) and its nitroxyl (NO–) and nitrosonium (NO+) ionsnitroxyl (NO–) and nitrosonium (NO+) ions

Freely diffusible, gaseous free radical.Freely diffusible, gaseous free radical. First described in 1979 as a potent relaxant First described in 1979 as a potent relaxant

of peripheral vasculature. of peripheral vasculature. Used by the body as a signaling molecule.Used by the body as a signaling molecule. Used as neurotransmitter, bactericide. Used as neurotransmitter, bactericide. Environmental PollutantEnvironmental Pollutant First gas known to act as a biological First gas known to act as a biological

messengermessenger

N O

Nitric Oxide in plantsNitric Oxide in plants

Affects aspects of plant Affects aspects of plant growth and growth and development.development.

Affects the responses to:Affects the responses to:

light, gravity, oxidative stress, light, gravity, oxidative stress, pathogenspathogens..

Can be a Can be a maturationmaturation and and senescencesenescence factorfactor

Has a concentration dependent Has a concentration dependent cytotoxiccytotoxic oror

protective protective (antioxidant)(antioxidant) e effects. ffects.

The source of NO synthesis in plants includes reduction of nitrite by nitrate reductase (NR) and the oxidation of arginine to citrulline by NOS. No gene similar to mammalian NOS has been found. suggesting different NOS. AtNOS1 encodes a protein similar to snail NOS

NO reversibly binds to ferrous iron within haemproteins, such as guanylate cyclase, haemoglobin and cytochrome oxidase. NO reacts at the diffusion limited rate with superoxide to produce peroxynitrite. NO or peroxynitrite, can react with thiols to produce nitrosothiols.

NO synthesis and reactions

NO, a simple gas, is able to diffuse across the membrane, and alters the activity of intracellular target enzymes.

Chemistry & signaling of NO in biological Chemistry & signaling of NO in biological environmentenvironment

NO signal transduction

H2O2 and NO might regulate the activity of TFs directly via nitrosylation (NO) or oxidation of cysteine residues (H2O2). Abbreviations: M–NO, iron–nitrosyl complexes;

M, heme- or iron-containing proteins; M–Fe(II)/Fe(III), Fe(II)/Fe(III)-containing metalloproteins; NOx, refers collectively to

NO.2, N2O3 and N2O4; R–NO, nitrosated

proteins

S-nitrosation–denitrosation is reversible and could represent a mechanism for regulating signal transduction.

Besides its half-life, a protein’s fate is manifest in its destination.

ROS – NO perturbations

-A large extent and diversity of NO-dependent modifications of biomolecules. Identification of these modifications has outraced our ability to gauge their physiological import. STKE 2004 (2004) 52

The Challenge

- A molecule in a biological system encodes information in its shape, charge, hydrophobicity, and reactivity.

Any change in a molecule’s composition encodes new information in the amount, rate, and duration of that change and where in the cell or organism the change takes place.

Feature ROS RNS

Primary catalyst Multisubunit flavocytochrome Multisubunit flavocytochrome

Substrates O2, NADPH O2, NADPH, L-arginine

Primary product Inorganic radical (O2) Inorganic radical (·NO)

Actions at low levels Activate or inhibit receptors, Activate or inhibit receptors, enzymes, enzymes, transcription

transcription factors factors

Actions at high levels Cause mutagenesis, apoptosis, necrosis Cause mutagenesis, apoptosis, necrosis

Basis of celllar resistance SODs; catalase; peroxiredoxins; Under study

redox cycles involving

glutathione, thioredoxin, glutaredoxin,

trypanothione,ovothione, mycothione;

methionine sulfoxide reductase;

ascorbate; -tocopherol; urate; -keto acids

Parallels between ROS and RNS in defenseParallels between ROS and RNS in defense

Nitric oxide-mediated signaling pathway in:

2001

plant defense against pathogens

Steady-state current-voltage curves derived from voltage clamp recordings (insets) from an intact guard cell before (○), during NO (■), and after washing (●). Curves are corrected for instantaneous current recorded at each voltage. Inset, Corresponding current traces cross-referenced by symbol with the voltage protocol

NO inactivates IK,out.

NONO--induced cell death in induced cell death in ArabidopsisArabidopsis occurs independently of ROSoccurs independently of ROS

(a) Cells were treated with methyl viologen (MV) to generate O2 · , NO donor (RBS), and/or the peroxynitrite scavenger and SOD-mimetic MnTBAP.

(b) Cells were treated with RBS, H2O2 and/or catalase (CAT).

cGMP in NOcGMP in NO--induced cell deathinduced cell death

Cells were pre-treated with ODQ (guanylate cyclase inhibitor) and/or 8Br-cGMP prior to RBS.

The effects of the caspase-1 inhibitor Ac-YVAD-CMK on NO- and H2O2-induced cell death

Main actions of nitric oxide (NO), peroxynitrite (ONOO−) and nitrosothiols (RSNO) on

mitochondria.

NO specifically and reversibly inhibits cytochrome oxidase (complex IV); nitrosothiols inactivate complex I; whereas peroxynitrite inhibits multiple respiratory complexes and aconitase, and activates the proton leak and permeability transition pore (ANT-PTP), which may contribute to NO-induced cell death. Inhibitions are indicated by dark arrows/bolts, while light ones indicate activations

NO + Fe2+↔Fe2+−NOReversible binding to haem a3. Responsible for inhibition at high ferrocytochrome c. concentrations−competitive with O2

NO + Cu2+ ↔ Cu+−NO+

Cu+−NO++H2O↔Cu++NO2−+2H+

Binding to Cu

The Role of OThe Role of O22

Work on the ryanodine receptor and mitochondrial

respiration points to a reciprocal, concentration-

dependent influence of O2 and NO on each other’s

physiologic actions. In each case, NO exerts more

control when the concentration of oxygen ([O2]) falls. A

related concept was originally articulated for hemoglobin,

where it was argued that S-nitrosylation controls O2

delivery, whereas [O2] controls S-nitrosylation.

Purification and Characterization of the Putative Tobacco NOS

In vitro, a GST-AtNOS1 fusion protein was capable of NOS activity and showed similarities to mammalian NOS isoforms, but was not stimulated by typical mammalian NOS cofactors.

ABA- and nitrite-induced NO generation correlate with stomatal closure

Stomatal apertures (black bars)

wt (white bars) or NR mutant nia1, nia2 (black)

wild type (gray bars), abi1-1 (black bars), or abi2-1 (white bars)

NO involvement in ABA-induced stomatal closure

Effect of c-PTIO on ABA-induced stomatal closure

NO regulates K+ and Cl- channels in guard cells through a subset of ABA-evoked signaling pathways

Voltage-clamp recordings from an intact guard cell (Inset) Steady-state I-V curves from voltage-clamp steps before (o), after 2 min of exposure to 10 µM SNAP (▲), and after washing in buffer-SNAP (●).

-NO

+NO

NO promotes INTRACELLULAR Ca2+ release and thereby regulates guard cell ion channels

ABA raises cytosolic-free [Ca2+] ([Ca2+]i) and cytosolic pH (pHi); these signals inactivate inward-rectifying K+ channels (IK,in) to prevent K+ uptake and activate outward-rectifying K+ channels (IK,out) and Cl- (anion) channels (ICl) at the plasma membrane to facilitate solute efflux

PNAS 2003 vol. 100 ; 11116

NO scavengers suppress ABA action in closing stomata NO donors promote it in the absence of ABA NO selectively inactivates IK+, in and Cl- out

NO scavenger cPTIO blocks inactivation of IKin

and activation of IClout by ABA and NO

but not ABA-mediated activation of IKout

Steady-state current for IK,out at +30 mV, IK,in at -200 mV, and ICl at -70 mV (shaded bars), effects of a 10-min exposure to ABA with or without 20 µM cPTIO (Left) and to 10 µM SNAP with or without 20 µM cPTIO (Right).

NOS activity and NO are reduced in Atnos1 mutants

A) NOS activity in leaf extracts of wild-type, Atnos1 mutant, and rescued Atnos1 plants. L-NAME inhibits the NOS activity in wild-type and rescued Atnos1 plants.

B) AtNOS1 mRNA levels in roots after treatment with ABA for 30 min in dark do not increase substantially.

C to H) NO production (shown as green fluorescence from the NO-sensitive dye DAF-2 DA) is increased by ABA in roots of wild-type seedlings and is reduced in Atnos1 mutants with or without 50 µM ABA. L-NAME (200 µM) inhibits ABA-induced NO production Science 302 (2003) 100 - 3.

AtNOS1 functions in ABA-induced stomatal closure

(A) ABA induces NO production in guard cells of wt and rescued Atnos1 plants but not Atnos1 mutants, as shown by the increase in fluorescence from the NO-sensitive dye DAF-

2 DA. L-NAME inhibits the ABA-induced NO production in all three genotypes. (B) Rel fluorescence signal from guard cells corresponding to treatments in (A) (n = 8) are shown. (C) ABA-induced stomatal closure is inhibited in Atnos1 mutants and is partially restored in rescued Atnos1. (D) ABA fails to inhibit light-induced stomatal opening in Atnos1 mutants but functions well in rescued Atnos1 mutant plants.

Phenotypes of the Atnos1 mutant and complementation with a 35S-AtNOS1. (A) Wt and (B) Atnos1 mutants with yellow first true leaves (C) Atnos1 containing a 35S-AtNOS1 transgene (rescued Atnos1) has green true leaves. (D) Atnos1 mutation inhibits shoot growth (E) Root development is inhibited in Atnos1 mutants grown on agarose. (F) Reproductive growth and fertility are reduced in Atnos1 mutants. (G) Western blots of leaf extracts show that the Atnos1 mutation reduces AtNOS1 protein levels. Equal amount of protein loaded (AtNRT1.1 control). (H to K) Treatment of Atnos1 mutants with NO restored greening of first true leaves, whereas treatment with sodium ferrocyanide (a SNP analog that does not produce NO) had no effect

The mutant is impaired in leaf greening, shoot growth, and fertility

Results- cell viabilityResults- cell viability

GA-induced PCD

ABA-inhibits PCD

Exogenous NO donor delays

PCD

cell viabilitycell viability

NO delays the GA-induced loss of NO delays the GA-induced loss of CAT and SOD(mRNA+protein)CAT and SOD(mRNA+protein)

Plant Physiol, August 2002, Vol. 129, pp. 1642-1650

NO and Cell DeathNO and Cell Death

NO and H2O2 cause cell death

NO and O2- react to form peroxynitrite

Peroxynitrite (ONOO -) does not cause cell death

Too much O2- ‘mops up NO’ – no death

Delladonne et al. (2001) PNAS 98:13454

+PBITUPsm (avrRpm 1)

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1mM H2O2 10mM H2O2 NO 1mMH2O2+NO

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BHT delays GA-induced PCD in BHT delays GA-induced PCD in barley aleurone layersbarley aleurone layers

Aleurone layers were incubated with GA alone or with GA plus 1 mM BHT, and were then loaded with the fluorescent probes FDA and FM 4-64 at 24, 36, and 48 h

SUMMARYSUMMARY

NO delays the onset of GA-induced PCD NO delays the onset of GA-induced PCD of barley aleurone cells.of barley aleurone cells.

NO may act to protect the cell from NO may act to protect the cell from oxidative stress as an oxidative stress as an antioxidantantioxidant..

NO has cytotoxic and protective effects NO has cytotoxic and protective effects in plants.in plants.

NO delays the GA-induced loss of CAT NO delays the GA-induced loss of CAT and SOD and canand SOD and can be an endogenous be an endogenous modulatormodulator of aleurone cell viability. of aleurone cell viability.

NO is produced in barley aleurone cells.NO is produced in barley aleurone cells. But, we can not specify the exact But, we can not specify the exact

mechanism by which NO exerts its mechanism by which NO exerts its protective effect.protective effect.

NO signalling in NO signalling in plantsplants

ArabidopsisArabidopsis flowering time flowering time

(A) Two Arabidopsis plants, sown at the same time. The left is already flowering, whereas on the right it is delayed because it carries active FRI.

(B) An electron micrograph of an extremely early flowering transgenic plant overexpressing the floral pathway integrators FT and LFY.

(C) The consequence of loss of floral meristem identity function. The lfy mutant (left) has leaf-like structures in place of the flowers ( right)

September 26, 2004

Multiple Pathways Control Flowering Multiple Pathways Control Flowering TimeTime

Integration of the Multiple Inputs

Depending on the wavelength of light, the light quality t the inputs promote or repress activation of genes termed floral pathway integrators. FRI repression prevents promotive photoperiod

(PP) accelerating flowering in late summer or autumn. Vernalization (VRN) antagonizes FRI repression, reducing FLC activity thus enabling long days in spring to upregulate the floral path integrators (FPI)

Smaller circles and thinner arrows indicate a lesser role

Science, Vol 296, 285-289 , 12 April 2002

Air pollutant nitric oxide acts as a Air pollutant nitric oxide acts as a plant hormone to delay flowering in plant hormone to delay flowering in plantsplants. . The scientists discovered that The scientists discovered that while plants produce their own while plants produce their own internal nitric oxide to regulate internal nitric oxide to regulate flowering, they are also influenced by flowering, they are also influenced by external concentrations of the external concentrations of the chemicalchemical..

Exogenous NO promotes vegetative growth but inhibits reproductive

development

The effects of an NO donor SNP on plant growth and development. Arabidopsis seedlings were grown in SNP during long days (16h light/8h dark) for 5 weeks. (B) [SNP] on growth

(C and D) The effect of SNP on flowering times. Fresh weight per shoot (B), the rosette leaf number (C), and days to bolting (D) from experiments as in (A) and fig. S1A plotted as a function of the concentrations of SNP that were applied, respectively

Science, Vol 305, 1968-1971 , 24 September 2004

Involvement of cGMP in the flowering Involvement of cGMP in the flowering regulationregulation

The involvement of cGMP in the regulation of the flowering of The involvement of cGMP in the regulation of the flowering of PharbitisPharbitis was was

investigated by exogenous applications of cGMP and chemicals that change investigated by exogenous applications of cGMP and chemicals that change

the cGMP level and analyses of endogenous cGMP level.the cGMP level and analyses of endogenous cGMP level.

There was a significant difference in the cyclic GMP level between 16-h-long There was a significant difference in the cyclic GMP level between 16-h-long

night conditions and a long night with a night-break. During a long inductive night conditions and a long night with a night-break. During a long inductive

night the oscillation of cGMP was observed with four main peaks in 4, 7, 11, night the oscillation of cGMP was observed with four main peaks in 4, 7, 11,

14 h, whereas a 10 min flash of red light in the middle of the night was able 14 h, whereas a 10 min flash of red light in the middle of the night was able

to modify these rhythmical changes in the second half of the long night. to modify these rhythmical changes in the second half of the long night.

These results have shown that there are oscillations in the concentration of These results have shown that there are oscillations in the concentration of

cGMP in the night and the biosynthesis and/or deactivation of cGMP is cGMP in the night and the biosynthesis and/or deactivation of cGMP is

affected by light treatment and therefore it may be involved in the affected by light treatment and therefore it may be involved in the

regulation of photoinduction processes in cotyledons. regulation of photoinduction processes in cotyledons.

From these combined results, we propose a hypothesis that cGMP is From these combined results, we propose a hypothesis that cGMP is

involved in the control of photoperiodic flower induction in involved in the control of photoperiodic flower induction in Pharbitis nilPharbitis nil..

((J Plant J Plant PhysiolPhysiol, 2004, March vol. 161. , 2004, March vol. 161.

277-284)277-284)

Endogenous NO represses the floral Endogenous NO represses the floral transitiontransition

A) The root growth phenotype in nox1 mutant. B) Endogenous NO levels in nox1 and WT. White-light images are shown at the bottom. C) The levels of L-Arg and NO in WT, nox1, and cue1-5. Plants grown under 12h light/12h dark cycles were harvested 6h after dawn. D) The nox1 mutant flowers late. WT and nox1 were grown in soil under 12-hour light/12-hour dark cycles and were photographed after 60 days of growth. (E and F) Flowering times of nox1 and cue1-5 mutants. The rosette leaf number (E) and days to flowering (F) from experiments as in (D) were scored. G) The NO synthase 1 (nos1) mutant that produces fewer NO flowers early under long days. (H) The nos1 mutant flowers earlier than WT under SNP treatments.

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NO affects the expression of genes NO affects the expression of genes that control the floral transitionthat control the floral transition. .

NO suppresses the photoperiod pathway