Tema 4.

RUTAS DE TRANSDUCCIÓN
DE SEÑALES EN EL SISTEMA DE
DEFENSA DE LAS PLANTAS

4.2. Reactive oxygen and nitrogen species

 The plant NADPH oxidase.

 Production of NO in plants.
 Reactive Oxygen Species (ROS)

Generation of different reactive oxygen species (ROS) via energy transfer or

sequential univalent reduction of basic state oxygen

Apel and Hirt, 2004

Reactive oxygen species in response to
different stimuli

O2-/H2O2

Sewelam et al., 2016

 H2O2 detection by DAB staining

Pseudomonas syringae pv Peronospora parasitica

tomato DC3000 isolate Emwa1
Diaminobenzidine
(DAB)

Trypan Blue
(TB)

5 hpi 3 dpi
DC3000 DC3000 MgCl2 cotyledon leaf
(avrRpm1)
O2- detection by NBT staining
 ROS quantification by luminol assay

ROS detection during PTI in response to Pto. Smith and Heese (2014)
 ROS quantification by luminol assay

ROS detection by a luminol assay in response to the bacterial PAMP flg22 in

different Arabidopsis mutants.

Torres et al. 2013

ROS production is a marker of the
activation of the defense response

Torres (2010)
Biphasic production of ROS in the apoplast in
response to avirulent pathogens

Piedras et al., (1998) MPMI

Balance and the double role of ROS

Production Scavenging

Level of H2O2

Del Rio, 2015, J. Exp. Bot.

ROS and the integration of the plant defense
responses

Defense

Activation of defense
Cross-linking Cell Death genes (PR, …)
of cell wall proteins
Lignification

MAPK
+ +
ROS SA synthesis
cascade
+ +
_
Scavenging and
antioxidants Systemic signals
Redox state
(GST, ASP)
GSG
GSSG
Adaptado de Desikan et al., 2004
 Sources of ROS in
plant defense

 Intracellular production:

•Metabolic origin: Chloroplasts,

Peroxisomes, mitochondria.

•Down regulation of
scavenging/antioxidant systems

 Enzymatic production of
apoplastic ROS:

•NADPH oxidase

•Cell wall peroxidases

Neill et al., 2002

ROS produced by cell-wall peroxidases
 The NADPH oxidase

 Functional domains of the NADPH oxidases

Heme
motifs

NOX2/gp91phox N-

DUOX1
N-
Peroxidase
homology
NOX5 N-

AtrbohD N-

EF-hands Transmembrane
domains

Torres & Dangl, 2005

 The plant NADPH oxidase

RBOH: Respiratory Burst Oxidase Homologues

e- 2H+
O22- H2O2

APOPLAST

CITOSOL

Cys
SNO

Adapted from Suzuki et al., (2011) Curr. Op in Plant Biol 14, 6: 691–699
 Phylogenetic relation between NADPH
oxidases from plants animals and fungi.
Fungi

Animal

Plant

Adapted from Torres & Dangl, 2005

Redundant plant Rboh-NAPDH oxidases

AtrbohD
AtrbohF

Suzuki et al., Cur. Op. Plant Biol. (2011)

 The NADPH oxidase is the main source of
ROS in response to pathogens
AtrbohD D3

F3 F4 F5
AtrbohF

1 kb

DC3000(avrRpm1) DC3000(avrRpt2)

WT
Col-0 atrbohD atrbohF atrbohD/F Col-0 atrbohD atrbohF atrbohD/F
2.5 x 107 cfu/mL, 5 hpi 2.5 x 107 cfu/mL, 8 hpi

Most ROS production is AtRBOHD dependent

Torres et al., 2003
 ROS produced by the NADPH oxidase is a
positive regulator of the HR
Bacteria
DC3000(avrRpm1)
Col-0 atrbohD atrbohF atrbohD/F

DAB

2.5 x 107 cfu/mL

6 hpi

TB

!! 106 cfu/mL
24 hpi

The rboh mutants display reduced HR in response to

an avirulent bacteria
ROS produced by the NADPH oxidase is a
positive regulator of the HR

Electrolyte leakage after inoculation with P. syringae

DC3000 (avrRpm1), 107 cfu/ml
80
conductivity (uS/cm)

Col-0
60
x atrbohD
40
atrbohF
20
atrbohD/F
0
0 2 4 6 8 10

tim e (hours)

The rboh mutants display reduced HR in response to

an avirulent bacteria
 ROS produced by the NADPH oxidase is a
negative regulator of the HR
Col-0 atrbohD atrbohF atrbohD/F
DAB

x100

x200

TB

x100

The rboh mutants display enhanced cell death in

response to an Hyaloperonospora parasítica isolate.
 ROS produced by the NADPH oxidase is a
negative regulator of the HR

100
90 Sporangiophores
80 per 1st leaf
70
60 H: >20
50
M: 10-20
40
30 L: 1> x <10
20
10 0: 0
0

H M L 0 H M L 0 H M L 0 H M L 0 H M L 0

WS Col-0 D F DF
Sporangiophores on the first leaf,
7 dpi; 5x104 spores/ml
3 different experiments, More than 200 leaves counted for each class.
 Role of NADPH oxidase-dependent
ROS on defense and cell death :

1. The NADPH oxidase is the main source of ROS in

response to pathogens as well as to other
environmental and developmental factors.

2. ROS produced by the NADPH oxidase is a positive

regulator of the HR in response to avirulent bacteria.

3. ROS produced by the NADPH oxidase acts as

negative regulator of the HR in response to an isolate
of Hyaloperonospora parasitica.

4. There is not a clear direct role for NADPH oxidase

dependent ROS in disease resistance.
Role of RBOH: an oversimplification

Marino et al., (2012)

Regulation of the NADPH oxidase activity
e- 2H+
O22- H2O2

APOPLAST

 RBOHD is mainly controlled posttraductionally at their N-term by:

CITOSOL

– Ca2+ via direct binding to EF hand motifs

– Phosphorylation by Ca2+-dependent Cys
protein kinases (CPKs).
– Phosphorilation by other Ca2+-independent
SNO kinases (BIK1)

– Binding of small Rac GTPases

– Binding of phospholipase Dα1-derived phosphatidic acid

 Transcriptional control is also important

Kadota et al., (2015)

 Ca2+-independent regulation of the
NADPH oxidase activity
e- 2H+
O22- H2O2

APOPLAST

CITOSOL

•Plasma membrane-associated cytoplasmic kinase

Cys BIK1 (BOTRYTIS-
INDUCED KINASE1), directly interacts with and SNO
phosphorylates RBOHD
upon PAMP perception.

•Phosphorilation by BIK1 is required for ROS production during PTI.

Kadota et al., (2015)

Cross-talk ROS and Ca2+ signaling

Kadota et al., (2015)

 Transcriptional activation of the RBOH
genes is important for their function

• RBOHD and RBOHF are differentially induced in response to pathogens.

-Trypan Blue –cell death

- Diaminobenzidine-H2O2
- RBOHD and RBOHF Promoter::GUS fusions-Expression

Morales et al., 2016.

 Transcriptional activation of the RBOH
genes is important for their function

Col-0 pD-HA-D pD-HA-F pF-HA-D pF-HA-F

rbohD

• RBOHD promoter is necessary to drive ROS production and to

complement rbohD deficient mmune response .

•RBOHD promoter is not sufficient to drive ROS production and to

complement the rbohD mutant, since pD-HA-F construct failed to restore
the wild-type response in this mutant (RBOHD gene is also required)

Morales et al., 2016.

Production and scavenging of ROS determines
their level and function
SOD, superoxide dismutase
Prx, peroxiredoxin
CAT, catalase
GR, glutathione reductase
GPX, glutathione peroxidase

APX, ascorbate peroxidase;

Redox modification MDAR, monodehydroascorbate
reductase
DHAR,dehydroascorbate reductase
controlled uncontrolled Srx, sulfiredoxin

Change in activity, localization,… Cell death

SIGNALING Waszczak et al. 2015 J Exp. Bot.

ROS cross-talk with other signals

Torres, 2010
 Sinergism between ROS and SA

Fase transitoria Fase persistente

ROS
SA

0h 1h 2h 3h 4h 5h 6h

Activación de
Patógenos Genes de Defensa

RBOH ROS
Respuesta
RBOH ROS
Hipersensible
+ +
+ Resistencia
Benzoic Benzoate-2- SA Sistémica
acid hydroxylase Adquirida
Benzoic Benzoate-2- SA
acid hydroxylase

Draper J: Salicylate, superoxide synthesis and cell suicide in plant defense. Trends
Plant Sci. 1997, 2:162-165.
ROS lies between the different stress
reponse pathways

Spinelli et al., (2011), www.intechopen.com

 ROS signaling interact with MAPK

In red, kinases induced by ROS.

RBOH activate a MAPK phosphorylation cascade, which can form a positive
amplification loop.
Kangasjarvi et al., (2010), Physiol Plant
ROS signaling interact calcium signaling

Xia et al., (2015), J Exp Bot

Model on how ROS and calcium mediate signal
propagation in the plants

Local stress stimuli induce an initial response involving ROS, calcium and other
metabolites. This local response initiate a rapid wave that involves ROS, calcium and
membrane depolarization that propagates fast and mediates the systimic defence or
acclimatory mechanisms.
Miller et al., (2009), Sci. Signal.
A rapid systemic signal dependent on RbohD
in response to abiotic stress
Cold water High light

Heat Stress

Hot water High salt

A-D) Zat12::Luc plants in WT and rbohD backgond 30 minutes after stress treatment.
(E) Time course protein blot analysis showing accumulation of heat response proteins
in systemic or local leaves of wild-type and rbohD plants subjected to heat stress.

Miller et al., (2009), Sci. Signal.

Production of NO in response to the elicitor
Criptogein

PTIO: NO scavenger

NMMA: NOS inhibitor

 DAF: Diaminofluorescein (specific stain)

Routes of NO Formation in Plants

Moreau et al., 2010

Intermediates in NO signaling

SIGNALING INTERMEDIATES:

• NO

• ONOO- peroxinitrite

• S-Nitrosoglutathione (GSNO)
and other S-nitrosothiols (SNO)
• NO2 and Nitrated derivatives
of fatty acids (NO2-FA)

EFFECT:
S-Nitrosilation
Tyr-Nitration

Metal-Nitrosilation

Oxidation
Illustration of NO signaling
 Regulation of SA signaling by protein S-nitrosylation

 NON-EXPRESSOR OF PATHOGENESIS-RELATED GENES

1 (NPR1, also called NIM1)
• NPR1 is a regulator of TF that
modulate PR expression:

• Oligomerization-Monomerization
diferent activity:
• Oligomer:
-Interacts with the repressor
protein NON-INDUCIBLE
IMMUNITY 1 (NIM1)-
INTERACTING (NIMIN)
• Monomer:
-Moves to the nucleus
-Active form: activates TGA TF
that promote PR expression
• Oligomerization is dependent on S-
nitosylation
 Regulation of SA signaling by protein S-nitrosylation

SNO S-nitrosothiol SA
signaling
AtGSNOR1 S-nitrosoglutathione reductase

S-nitrosylation NPR1 ↓ SA
↓ SA ↓ AtGSNOR1 of NPR1 at the signaling
oligomerization
Cys156

Absence of NPR1 ↑ SA
↑ SA ↑ AtGSNOR1 S-nitrosylation signaling
monomerization
of NPR1
 The efficient induction of cell death
requires a balance between NO and ROS
 Cell death soybean in cell cultures after different treatments

SNP: NO donor
SA: salicylic acid
CA: Cantharidin (Phosphatase inhibitor)
G/GO: H2O2 production
Psg: Pseudomonas syringae
YE: elicitor Delledonne et al. 2001
 Interaction between NO and ROS

 The efficient induction of Cell Death

requires a balance between NO and ROS

SOD
RBOH O2 - H2O2

ONOO- Cell Death

NOS NO

Delledonne M, Zeier J, Marocco A, J. LC: Signal interactions between nitric oxide and
reactive oxygen intermediates in the plant hypersensitive disease resistance
response. Proc. Natl. Acad. Sci., USA 2001, 98:13454-13459.
 Interaction between NO and ROS

 The efficient induction of Cell Death

requires a balance between NO and ROS

SOD
RBOH O2 - H2O2

ONOO- Cell Death

NOS NO

Rboh O2- H2O2

ONOO- Cell Death

NOS NO
 Interaction between NO and ROS

 The efficient induction of Cell Death

requires a balance between NO and ROS

SOD
RBOH O2 - H2O2

ONOO- Cell Death

NOS NO

SOD
RBOH O2 - H2O2
Cell Death
ONOO-

NOS NO
 Interaction between NO and ROS

 The efficient induction of Cell Death

requires a balance between NO and ROS

SOD
RBOH O2 - H2O2

ONOO- Cell Death

NOS NO

SOD
RBOH O2 - H2O2

Cell Death
ONOO-

NOS NO
Cross-talk ROS-NO

Wang et al., 2013

Cross talk between NO and ROS signaling

 S-nitrosylation of NADPH oxidase regulates cell

death in plant immunity
•S-nitrosilation of Cys 890 reduces the activity of RbohD.

d) NO donors dicrease NADPH oxidase activity.

e) Atgsnor1-1 (low SNO) shows increased NADPH oxidase activity in response to Pto DC3000(avrB).

Atgsnor1-3 (high SNO) shows dicrease NADPH oxidase activity in response to Pto DC3000(avrB).
Yun et al., 2011
 THE NITRIC OXIDE SYNTHASE (NOS)

Animals

Plant
iNOS
Controversy about the origin of NO in plants