Download Hydrogen peroxide plays multiple roles in plant

Hydrogen Peroxide in Plants: A Versatile Molecule of Reactive
Oxygen Species Network
Li-Juan Quan1, Bo Zhang2 , Wei-Wei Shi2 and Hong-Yu Li*
(1，2. MOE Key Laboratory of Arid and Grassland Ecology, School of Life sciences, Lanzhou University ，
Lanzhou，730000)
*Author for correspondence.
Tel: +86 (0)13519640428;
Fax : +86(0)931 891 2561;
E-mail: lihy @lzu.edu.cn
Supported by the National Natural Science Foundation of China (30170238; 30670070)
Abstract
Plants often face the challenge of severe environmental conditions, which include various biotic and abiotic
stresses, all of which exert adverse effects on plant growth and development. With the evolution of plants,
Plants have evolved complex regulatory mechanisms in adapting to various environmental stressors, One of
the consequences of much stress is an increase in the cellular concentration of reactive oxygen species（ROS）,
which is subsequently converted to hydrogen peroxide（H2O2）. Even under normal conditions, higher plants
produce ROS during the metabolic process. Excess concentrations of ROS results in oxidative damage to or
the apoptotic death of cells, Development of an antioxidant defense system in plants protects them against
oxidative stress damage. ROS and, more particularly, H2O2 plays versatile roles in plant normal
physiological processes and resistance to stresses. Recently, H2O2 has been regarded as a signaling molecule
and regulator of the expression of some genes in cells. This review describes various aspects of H2O2 function,
generation and scavenging, genes regulation and the crosslink with other physiological functional molecules
during plant growth, development and resistance responses.
Key words: antioxidant system; gene regulation; hydrogen peroxide (H2O2); reactive oxygen species (ROS);
signaling molecule
Abbreviation: ABA, abscisic acid; APX, ascorbate peroxidase; CaM, calmodulin; CAT, catalase; CDPKs,
cacium-dependent protein kinases; DHA, dehydroascorbate; DHAR, dehydroascorbate reductase; GPX,
glutathione peroxidase; GR, glutathione reductase; GSH, glutathione; GSSG, glutathione reductase; H2O2,
hydrogen peroxide; HR, hypersensitive reaction; iNOS, inducible nitric oxide synthase; .OH, hydroxyl radical; JA,
Jasmonic acid; MAPKs, mitogen-activated protein kinases; MAPKKKs, mitogen-activated protein kanase kinase
kinases; MDHA, mondehydroascorbate reductase; NO, nitric oxide; NOS, nitric oxide synthase; O2-, superoxide
radical; ROS, reactive oxygen species; SA, salicylic acid; SAR, systemic acquired resistance; SOD, superoxide
dismutase; UV, ultra-violet.
As a kind of reactive oxygen species (ROS), hydrogen peroxide (H2O2) has been given much
attention during the last decades. Ample evidence has proven that H2O2 plays an important role in
plants under severe environmental conditions, which include various biotic and abiotic stresses
(Dat et al. 2000). H2O2 participates in many resistance mechanisms, including reinforcement of
the plant cell wall, phytoalexin production, and enhancement of resistance to various stresses
(Dempsey and klessig 1995). Recently, H2O2 has also been shown to act as a key regulator in a
broad range of physiological processes such as senescence (Peng et al. 2005), photorespiration
and photosynthesis (Noctor and Foyer 1998a), stomatal movement (Bright et al. 2006), cell cycle
(Mittler et al. 2004), and growth and development (Foreman et al. 2003). To some extent, excess
H2O2 accumulation can lead to oxidative stress in plants, which then triggers cell death. The
evolution of all aerobic organisms is dependent upon the development of efficient
H2O2-scavenging mechanisms (Arora et al. 2002), Enzymes, including superoxide dismutase
(SOD), catalase (CAT), peroxidase (POD), ascorbate peroxide (APX) and glutathione reductase
(GR) (Zhang et al. 1995; Lee and Lee 2000), and nonenzymatic antioxidants such as tocopherols,
ascorbic acid (AsA), and glutathione (GSH) (Wingsle and Hallgren 1993; Kocsy et al. 1996;
Noctor et al. 1998) work in concert to detoxify H2O2. Sustaining the H2O2 concentration at an
appropriate level can promote plant development and reinforce resistance to environment stressors.
H2O2 modulates the expression of various genes (Neill et al. 2002). The H2O2 induced transcripts
encoded proteins with functions such as metabolism, energy, protein destination and transport,
cellular organization and biogenesis, cell rescue of defense, and transcription ( Desikan et al.
2001a).Among these genes, the genes encoding potential transcription factors should be
emphasized due to their capacity for activating the expression of downstream target genes
( Desikan et al. 2001a). Using cDNA microarray technology, A large-scale analysis of gene
transcription has been undertaken looking in Arabidopsis and tobacco during oxidative stress
(Desikan et al. 2001a; Vandenabeele et al.2003；Vanderauwera et al. 2005).More studies have
provided evidence that H2O2 itself is a key signal molecule mediating a series of responses
(Desikan et al. 2003) and activating many other important signal molecules (Ca2+, SA, ABA, JA,
ethylene, NO) of plants (Gundlach et al. 1992; Dempsey and Klessig 1995; Liu et al. 2004;
Desikan et al.2004; Wendehenne et al. 2004). These signal molecules function together and play a
complex role in signal transduction of resistance responses, and growth and development in plant.
This article describes various aspects of H2O2 function, generation and scavenging, gene
regulation and crosslinks with those physiological functional molecules during plant growth,
development and resistance responses.
Origin of H2O2
Since the oxygen molecule (O2) emerged in earth, it is usually said to be the final electron receptor
during the biology respiration. Recently, studies have estimated that 1% of O2 consumed by plants
is diverted to produce reactive oxygen species (ROS) in various sub-cellular loci (Bhattacharjee
2005).
Reactive oxygen species (ROS), a collective term for radicals and other non-radical but reactive
species derived from the oxygen molecule (O2), has been implicated in numerous developmental
and adaptive responses in both animal and plant cells (Dypbukt et al. 1994; De Marco and
Roubelasis-Angelakis 1996; Lamb and Dixon 1997). The earliest report about ROS production in
plants is that challenged potato with incompatible P. infestant lead to reduction of cytochrome C
that induces a hypersensitive reaction (HR) involving active defense reaction, and the reaction can
be inhibited by SOD (Doke 1983a,b). The kinds of ROS have been investigated in plant including
hydrogen peroxide (H2O2), superoxide anion (O2-), hydroxyl radicals (. OH), singlet oxygen (1O2)
and nitric oxide (NO.) by far, H2O2, O2- , .OH can transform themselves into each other (figure 1).
H+
2e
oxygen molecule
(O2)
superoxide anion
(O2-)
SOD
Hydrogen peroxide
(H2O2)
Hydrogen peroxide
(H2O2)
(cell wall)
(cytosol
mitochondria
chloroplast)
PKC
Hydrogen peroxide
(H2O2)
Fe2+
Hydroxyl radicals (Feton reaction)
(.OH)
Figure 1.
Transition between the oxygen molecule (O2), superoxide anion (O2-), hydrogen peroxide (H2O2) and
hydroxyl radicals (.OH).
During oxidative burst, O2 is reduced to O2-, and then the O2- undergoes spontaneous dismutation at a higher rate
and at acidic pH, which is also found in the cell wall (Sutherland 1991). O2- is also catalyzed by superoxide
dismutase (SOD) enzymes, which occur in the cytosol, chloroplasts, and mitochondria (Scandalios 1993), O2 can
also be reduced to H2O2 by protein kinase C (PKC) (Juan et al. 2004), PKC exists in all organelle of plants (Juan et
al. 2004). H2O2 reacts with Fe2+ leading to the H2O2-dependent formation of .OH (Arora et al. 2002).
It has been estimated that both resistance responses to stresses and normal physiological
metabolism can lead to ROS production (Van Breusegem et al. 2001). By comparison, O2- and
H2O2 are weaker oxidizing agents. Under normal condition, the half-life of H2O2 is probably 1ms,
and other forms of ROS, including superoxide anion (O2-), hydroxyl radicals (.OH) and singlet
oxygen (1O2), their half-life are very short, about 2-4 µs (Bhattacharjee 2005). Excess H2O2 leads
to oxidative stress and is capable of injuring cells. During the course of evolution, plants were able
to achieve a high degree of control over H2O2 accumulation (Droge 2002). Recent investigations
revealed that ROS, especially H2O2 is a central component of the signal transduction cascade
involved in plant adaptation to the changing environment (Neill et al. 2002). H2O2 participates in
the physiological metabolism of plant and activate defense responses to various stresses. H2O2 is
beginning to be accepted as a second messenger for signals generated by means of ROS because
of its relatively long life and high permeability across membranes (Neill et al. 2002; Huang et al.
2002; Yang and Poovaiah 2002).
Versatile roles of H2O2
Hydrogen peroxide (H2O2) plays a dual role in plants: at low concentrations, it acts as a signal
molecule involved in acclimatory signaling triggering tolerance against various abiotic and biotic
stresses (Laloi et al. 2004; Fukao and Bailey-Serres 2004; Mittler et al. 2004). And, at high
concentrations, it orchestrates programmed cell death (Dat et al. 2000)
H2O2 takes part in resistance mechanism, reinforcement of plant cell wall (lignification,
cross-linking of cell wall structural proteins) phytoalexin production and resistance enhancement
(Dempscy and Klessig 1995). In plant-microbe interaction, H2O2 production in plants can kill
the pathogen directly or induces defense genes to limit infection by the microbe. H2O2 can be used
as a marker in tobacco leaves for testing the occurrence of plant basal defense reactions (Bozso et
al. 2005). Under other stress conditions, which include UV-radiation, salt stress, drought stresses,
light stress, metal stress, high or low temperature and so on. H2O2 production in plants induces
resistance to various stresses and protects itself from being hurt. Recently, it’s been suggested that
H2O2 is not only a defensive signal molecule but it also functions as a signal molecule during plant
growth and development. Evidence suggested that H2O2 production plays a key role in separating
and culturing of protoplast during reproduction of tobacco protoplast (Papadakis and
Roubelasis-Angelakis 2002). Using a luminescence probe one can check H2O2 accumulation in
the germinating of radish seeds (Schopfer et al. 2001). H2O2 can also regulate the plant cell cycle.
Treated tobacco with fungi elicitor produced H2O2 and activated MAPK protein (Suzuki et al.
1999). MAPK as a key signal protein regulates the cell cycle. The links between the H2O2 cell
cycle are orthologous protein of MAPKKK, ANP1 and NPK1 (Suzuki et al. 1999) In addition,
H2O2 is also a signal molecule related to senescence (Bhattacharjee 2005). It has been proven that
there is more H2O2 accumulation in old leaf than young leaf. Hence, H2O2 also takes part in
ABA-induced stomatal opening and closing (Pei et al. 2000; Neill et al. 2002).
Distribution of H2O2
pH-dependent cell wall peroxidase is able to oxidize NADH and in the process catalyze the
formation of superoxide anion (O2-); and cell wall oxidase catalyzes the oxidation of NADH to
NAD+, which in turn reduces O2 to O2-, consequently is dismutated to produce O2 and H2O2
(Bhattacharjee 2005). In addition, germin-like oxalate oxidases and amine oxidases have been
proposed to generate H2O2 at the apoplast (Bolwell and Wojtaszek 1997; Hu et al. 2003; Walters
2003). Cell membrane NADPH-dependent oxidase (NADPH oxidase) has recently received a lot
of attention as a source of H2O2 for the oxidative burst; In addition, there are other enzymes at the
surface of plasma membranes capable of generating H2O2 (cell wall polyamine oxidase) (Vianello
and Macri 1991). It has been identified that respiratory burst oxidase homologues (rboh), plant
homologues of the catalytic subunit of phagocyte NADPH oxidase (gp91phox), as a source of ROS
during the apoplastic oxidative burst (Agrawal et al. 2003). ROPs (Rho-related Gtpases from plant)
closely related to the mammalian Rac family, triggering H2O2 production and then the oxidative
burst, most likely by activating the NADPH oxidase (Agrawal et al. 2003).
Plant mitochondria as an“energy factory” is believed to be a major site of H2O2 production
related to continuous physiological processes under aerobic conditions (Rasmusson et al. 1998).
The mitochondria electron transport chain (ETC) is comprised of four complex NADH
dehydrogenase(C Ⅰ ), succinate dehydrogenase(C Ⅱ ), ubiquinol-cytochrome bc1(C Ⅲ ), and
cytochrome c oxidase (C Ⅳ) (Rasmusson et al. 1998). There are also five enzymes existing only
in plants: they are one alternative oxidase (AOX), four NAD(P)H dehydrogenase assembled to
flavoproteins, so they are a potential source of ROS production (Mller 2001). During respiration,
O2 may undergo an univalent reduction at the sites of H2O2 generation in complexes Ⅰand Ⅲ of
the respiratory chain (Figure 2). The ubiquinone site in complex Ⅲ appears as the major site of
mitochondrial H2O2 production (Braidot et al. 1999), this site catalyzes the conversion of O2 into
the O2- by a single electron. Of some substrates along respiratory chain. Flavoproteins, Quinols,
especially semiquinols, its energy barrier of redox is very low, the electron before transporting to
final oxidase reacts with O2 to form O2- (Elstner 1991). In aqueous solution, O2- is moderately
reactive but can generate H2O2 by dismutation (Rasmusson et al. 1998). About 1-5% of
mitochondria O2 consumption leads to H2O2 production (Mller 2001). The activity of CⅠ can be
inhibited by rotenone and diphenyleneiodo (DPI) (Meloamp et al. 1996); and the activity of CⅢ
can be inhibited by KCN, KCN interdicts the Q cycle, so inhibits the semi-quinone production
(Rasmusson et al. 1998).
O2-
INTERMEMBRANE
SPACE
Cyc C
O2
e
INNER
MITOCHONDRIA
Ⅱ
MATRIX
e
Ⅳ
Ⅲ
Ⅰ
UQ
MEMBRANE
UQ
O2
e
superoxide anion
(O2- )
O2
MnSOD
hydrogen peroxide
(H2O2)
Figure 2. Sites of hydrogen peroxide formation in mitochondria electron transfer system.
H2O2 production is at the two main sites, Complex I and III. The ubiquinone site (UQ) in complex Ⅲ catalyzes
the conversion of O2 to O2- by a single electron transfer (Rasmusson et al. 1998). Since UQ is bound to two sites in
complex III, one close to the inner surface of the inner mitochondria membrane, the other close to the out surface,
ROS might be found on either side of the membranes (Rasmusson et al. 1998). O2- is converted into H2O2 by
Mn-SOD (Mller 2001). CI NADH dehydrogenase; CII succinate dehydrogenase; CIII ubiquinol-cytochrome bc1; c
Ⅳ cytochrome C oxidase
Chloroplasts are also a major source for H2O2 production. Chloroplasts consist of pigment and
protein, two photo reaction systems: photo-system Ⅰ(PSⅠ) and photo-system Ⅱ(PS Ⅱ)
(Asada and Takahashi 1987). There is a photosynthesis electron transport, calling ‘Z’-scheme.
Recently the electron transport chains (ETC) in photo-system Ⅰ(PSⅠ) have been considered to
be the source of O2- in chloroplasts(figure 3). Normally, the electron flow from the excited PS
centers is directed to NADP+, which is reduced to NADPH. It then enters the Calvin cycle and
reduces the final electron acceptor, CO2. In situations of overloading of the ETC, a part of the
electron flow is diverted from ferredoxin to O2, reducing it to superoxide anion via a Mehler
reaction (Wise and Naylor 1987; Elstner 1991). Later studies have revealed that the acceptor side
of ETC in PS Ⅱalso provides sides (QA, QB) with electron leakage to O2 producing O2（Takahashi and Asada 1988）
（Figure 3）.On the external, “stromal” membrane surface O2- is
enzymatically by CuZn-SOD or spontaneously dismutated to H2O2（Takahashi and Asada 1988）
P680
P680* PSⅡ
P700* PSⅠ
QA
QB
e
O2
e
O 2-
CuZnSOD
e
e
H2O2
FeS
e
PQ
Fd
acceptor side
NADP+
P700
NADPH
Calvin cycle
Figure 3. Production of hydrogen peroxide in chloroplast at the site of PSI and PSII.. 680*, P700*:photo reaction
center Ⅱ and Ⅰ,electron flows from PSⅡ to PSⅠ. QA: quinone A. QB: quinone B. PQ: proton quinone. FeS:
ironsulfur protein. Fd:ferredoxin. At these sites of electron leakage provides electrons for O2 producing O2-, O2- is
dismutated to H2O2 by CuZn-SOD（Takahashi and Asada 1988）.
Peroxisomes are subcellular organelles with an essentially oxidative type of metabolism. It is
also called glyoxysome. peroxisomes produce superoxide radicals (O2-) as a consequence of their
normal metabolism. At least, two sites of O2- generation are demonstrated (Figure 4) (refer to Del
Río et al. 2002). One is in the organelle matrix, in which the generating system is identified as
Xanthine oxidase (XOD), Xanthine oxidase (XOD) catalyzes the oxidation of Xanthine and
hypoxanthine to uric acid and is a well-known producer of O2- (Corpas et al. 2001). Another site is
in the peroxisome membranes dependent on NAD (P) H. Peroxisome membrane, a small electron
transport chain, is composed of a flavoprotein NADH and cytochrome b, and O2- is produced by
the peroxisome electron-transport chain. Monodehydroascorbate reductase (MDHAR)
participating in O2- production by peroxisome membranes (Del Río et al. 1989). O2- radicals are
rapidly converted into H2O2 and O2 by CuZn-SOD (Del Río et al. 2002).
H2O2
Xanthine
O2
MATRIX
XOD
CuZnSOD
O2-
Uric acid
peroxisomal
metabolism
NAD+
NADH
e
MEMBRANE
MDHAR
Ctyb
CYTOSOL
O2
e
e
O2
O 2-
O 2-
H2O2
Figure 4. Production of hydrogen peroxide in peroxisomes.
The model is based on results recently described (Jimenez et al. 1997) monodehydroascorbate reductase(MDHAR)
is an NADH-dependent enzyme. Matrix and membrane are two sites of O2- generation. XOD oxidizes Xanthine to
Uric acid, providing electrons for O2 to product O2-, cyt
b also provides electrons for O2 to produce O2-; O2- then
is converted into H2O2 by SOD, XOD (Xanthine oxidase) and cyt b (cytochrome b).
Localization of H2O2 scavenging enzymes
The accumulation of H2O2 increases the probability of hydroxyl radical formation via Teton-type
reaction. This leads to the phenomenon known as oxidative stress (Bartosz 1997; Foyer and
Noctor 2000). In plant cells, enzymes and redox metabolites act in synergy to carry out H2O2
scavenging (Table 1)
TABLE 1. H2O2 scavenging enzymes
Enzyme
EC number
Reaction catalyzed
-+
O2- +2 H+ <=> 2 H2O + O2
Superoxide dismutase
1.15.1.1
O2
Catalase
1.11.1.6
2 H2O2 <=> 2 H2O +O2
Glutathione peroxidase
1.11.1.12
2GSH+PUFA-OOH<=>GSSG+PUFA+2 H2O
Glutathine reductase
1.6.4.2
NADPH+GSSG <=> NADP +2GSH
Ascorbate peroxidase
1.11.1.11
AA+ H2O2 <=> DHA+2 H2O
Guaiacol type peroxidase
1.11.1.7
Donor + H2O2<=>Oxidized donor +2 H2O
Major ROS-scavenging enzymes of plants include superoxide dismutase (SOD), ascorbate
peroxidase (APX), catalase (CAT), and glutathione peroxidase (GPX) (Table1). These enzymes
provide cells with highly efficient machinery for detoxifying O2- and H2O2. The balance between
SOD and the different H2O2-scavenging enzymes in cells is considered to be crucial in
determining the steady state level of O2- and H2O2 (Asada and Takahashi. 1987；Bowler et al.
1991)
In plants, the main enzymatic H2O2 scavenger of photosynthetic cells is CAT, which convert
H2O2 into H2O and O2 (Scandalios 1987). CAT scavenges H2O2 generated during mitochondrial
electron transport, β-oxidation of the fatty acids, and most importantly in photorespiratory
oxidation (Scandalios et al. 1997). In perxoxisomes/ glyoxysomes, CAT predominates. CAT
isoforms are distinguished on the basis of organ specificity and responses to environmental stress
(Willekens et al 1994a). A CAT isoform has been reported to be present in maize mitochondria
(Scandalios et al 1980), but no mitochondrial form has been reported in C3 species (Foyer and
Noctor 2000). Peroxisomes contain a large amount of CAT, but its properties suggest that the
enzyme is inefficient in removing low concentrations of H2O2 (Willekens et al. 1994a)
Peroxidase (POD) is a heme-containing glycoprotein encoded by a large mutigene family in
plants and involved in various physiological processes. Studies have suggested that POD plays a
role in lignification, cross-linking of cell wall structure proteins and defense against pathogen
(Kawano 2003). POD exists as isoenzymes in individual plant species (Hiraga et al. 2001).
Ascorbate peroxidase（APX） is the main enzyme responsible for H2O2 removal in the chloroplast,
peroxisomes and mitochondria. APX utilizes ascorbate as its specific electron donor to reduce
H2O2 to water (Asada 1992). Glutathione peroxidase (GPX) is a family of isoenzymes that uses
glutathione to reduce H2O2 and organic and lipid hydro-peroxides, thereby protecting cells against
oxidative damage. GPX is an important H2O2 scavenging enzyme in mammals. In plants, GPX
exists in the cytosol to reduce H2O2 to water. But the ability of plant GPX to scavenge H2O2
decreases largely due to its Cys residue without selenium. Hence, the major functions of GPX in
plants are lignin biosynthesis, degradation of indole-3-acetic acid and resistance to pathogens
(Asada 1992). However, except for the donor specific peroxidase mentioned above, there is a
group of non-donor specific peroxidase in plant cells, for which guaiacol is a common donor,
named guaiacol peroxidase (Mika and Luthje 2003). Recently, two distinct guaiacol peroxidases
(pm POD1 and pm POD2) have been separated from the plasma membrane. However more
functions of guaiacol peroxidase are still unclear (Mika and Luthje 2003).
Balance between H2O2 and cell redox
An appropriate intracellular balance between H2O2 generation and scavenging exists in all cells
(figure 5) (refer to Mittler et al 2004). This “redox homeostasis” requires the efficient coordination
of reactions in different cell compartments and is governed by a complex network of prooxidant
and antioxidant systems. The latter include nonenzymatic scavengers such as ascorbate,
glutathione, hydrophobic molecules, tocopherols and detoxifying enzymes (Noctor and Foyer
1998a).
INNER
MEMBRANE
Ascorbate
MATRIX
DHA
H 2O
FD
MDA
O2-
Ascorbate
MDA
H2O2
O 2-
CuZnSOD
H2O2
H 2O
H2O2
PSⅠ
PSⅡ
e
O2-
e
APX
CuZnSOD
e
Complexes
ubiquinone
CuZnSOD
Chloroplast
Mitochondria
Catalase
GSSG
H2O2
H 2O
Ascorbate
O 2DHAR
MDAR
APX
H2O2
Ascorbate
APX
H2O
MDA
H2O
CuZnSOD
GPX GR
DHA
2GSH
H 2O
Cytosol
MDA
Peroxisome
Figure 5. Localization of hydrogen peroxide (H2O2) scavenging pathways in plant cells(chloroplast, peroxisome,
cytosol and mitochondria).
The enzymatic pathways responsible for H2O2 detoxification are shown. The water-water cycle detoxifies O2- and
H2O2. H2O2 distributes in peroxisomes, mitochondria, chloroplast and cytosol.
Catalase (CAT), ascorbate
peroxidase(APX). SOD and other components of the Ascorbate-glutathione cycle are also present in mitochondria
and peroxisomal. Glutathione peroxidase(GPX) is involved in H2O2 removal in the cytosol. H2O2 can easily
diffuse through membranes and antioxidants such as glutathione and ascorbic acid (reduced or oxidized) can be
transported between the different compartments. Abbreviations: DHA, dehydroascrobate; DHAR,DHA reductase;
FD, ferredoxin; GLR, glutaredoxin; GR, glutathione reductase; GSH, reduced glutathione; GSSG, Oxidized
glutathione; IM, inner membrane; MDA, monodehydroascorbate; MDAR, MDA reductase; PSⅠ,photosystemⅠ;
PSⅡ, photosystemⅡ.
Ascorbate is present in chloroplasts, cytol, and vacuole and apoplastic spaces of leaf cells in
high concentration (Foyer et al. 1991). It is perhaps the most important antioxidant in plants, with
a fundamental role in the removal of H2O2 (Polle et al. 1990). The ascorbate/glutathione cycle is
the most important H2O2 – detoxifying system in the chloroplasts. But it also has been an
identifying system in the cytosol (Nakano and Asada 1981), peroxisomes, and mitochondria
(Jimenez et al. 1997). Two enzymes are involved in the regeneration of reduced ascorbate, namely
mono-dehydro-ascorbate reductase (MDHR) which uses NAD (P) H directly to recycle ascorbate
and dehydro-ascorbate reductase (DHR). Mono-dehydro-ascorbate is reduced directly to ascorbate
by using electrons derived from the photosynthetic electrons transport chain as follows (Arora et
al. 2002):
4 Mono-dehydro- ascorbate(MDHR) + 2 H2O→ 4 Ascorbate + O2
H 2O 2
AsA
GSSG
H 2O
GR
DHAR
APX
MDHA
DHR
NADPH
NADP+
GSH
Figure 6. Ascorbate-glutathione cycle (Halliwell-Asada pathway) of H2O2 scavenging. AsA, ascorbate; APX,
ascorbate-peroxidase;
MDHAR,
mondehydroascorbate
reductase;
DHA,
dehydroascorbate;
DHAR,
dehydroascorbate reductase; GR, glutathione reductase; GSH, Glutathione.
Regulation of genes expression related to H2O2
Hydrogen peroxide (H2O2) has been regarded as the second messenger for gene activation in
mammalian systems as well as in plant. In plants, increased H2O2 level induces the expressing not
only of defense genes, but also other resistance genes (Mitter et al. 2004).
Temperature-independent induction of smHSPs has been observed in response to high light (HL)
(Pnueli et al. 2003; Yamamoto et al. 2004) and to various other abiotic stress conditions
(Zimmermann et al. 2004). A subset of genes within the heat shock response might be triggered by
increased levels of H2O2 (Larkindale and Knight 2002). H2O2 is clearly able to induce the smHSPs
17.6 class. In the different assessed abiotic stresses, these smHSPs are coexpressed with AtHsf 2A;
recently, this class of cytoplasmic smHSPs has been shown not to be under transcriptional control
of HsfA1a/HsfA1b during heat shock (Busch et al. 2005). And recently a role of AtHsfA4a in the
early sensing of H2O2 stress has been demonstrated in Arabidopsis (Davletova et al. 2005)
Recent studies of knockout and antisense lines for Cat2, Apx1, chlAOX, mitAOX, CSD2,
2-cysteine PrxR and various NADPH oxidases have revealed a strong link between H2O2 and
processes such as growth, development, stomatal responses and biotic and abiotic stress responses
(Mittler 2004). Based on the analysis of the different mutants. Cat2, Apx1, ChlAOX, CSD2 and
2-cysteine PrxR are essential for the protection of chloroplasts against oxidative damage.
Suppression of CSD2, for example, results in the induction of a High-light (HL) stress response in
Arabidopsis plants grown under a low light intensity (Rizhksy et al. 2003). Catalase deficiency
triggers growth retardation and high sensitivity to ozone and high light stress (Vandenabeele et al.
2004).the absence of Apx1 results in reduced photosynthetic activity, augmented induction of heat
shock proteins during light stress and altered stomatal responses (Pnueli et al. 2003). Catalase
deficiency triggers growth retardation and high sensitivity to Ozone and high light stress
(Vandenabeele et al. 2004). By contrast, the absence of the NADPH oxidase genes AtrbohD and
AtrbohF suppresses H2O2 production and the defense responses of Arabidopsis against pathogen
attack (Torres et al. 2002). And knockout of atrbohC has an altered root phenotype (Foreman et al.
2003). AtrbohD and AtrbohF are also essential for abscisic acid signaling in guard cells (Kwak et
al. 2003).
Figure 7 (refer to Vanderauwera et al. 2005) presents the overlap of the H2O2-induced genes
with each of the environmental stresses. Twenty genes were induced in response to H2O2 and
under at least two stress conditions. Within these 20 commonly induced genes, two transcription
factors, DREB2A and ZAT12, could be identified, which have already been linked to H2O2
responses (Rizhksy et al. 2004). DREB2A is known to be a key regulator of drought response
(Shinozaki and Yamaguchi-Shinozaki 2000), whereas ZAT12 participates in regulation of
cold-responsive genes and contributes to an increase in freezing tolerance (Rizhsky et al. 2004).
The up-regulation of other H2O2-responsive transcription factors was restricted to one specific
environmental stress. AtWRKY48 was also induced by cold, whereas two NAC family proteins
were responsive to drought. These NAC proteins belong to the ATAF suvfamily and have been
shown to respond upon abscisic acid, dehydration, and salt treatments (Fujita et al. 2004)
At4g12400 Stress-induced protein sti1
At1g62510 Lipid transfer protein
At2g24100 Unknown
At5g10695 HSP70
At5g11090 Unknown
At5g05410 DREB2A
At1g66500 Zn finger protein
At1g68440 Unknown
At3g12580 Hsp70
Cold
Heat
6
At5g49520AtWRKY48
16(1)
59(3)
3(1)
0
At2g26150 At Hsf 2A
At3g51910 At HsfA7a
At4g11660 At HsfB2b
11(1)
8(2)
At5g59820 ZAT12
At1g19020 Unknown
At2g32210 Unknown
Drought
At5g63790 NAC-family protein
At1g77450 NAC-family protein
At5g65300 Unknown
At3g02840 Unknown
At1g66160 Unknown
At5g57220 Cytochrome P450
At4g20830 reticuline oxidase
At5g64310 Arabinogalactan-protein
At1g66290 Disease resistance protein
At1g02400 Dioxygenase
Figure 7. H2O2-up-regulated genes within three principal environmental stresses.
The different stress conditions (cold, heat, and drought) are indicated together with the number of genes within the
overlap with the H2O2-responsive genes and the total number of gene input of each of the stresses as well. In the
Venn diagram, numbers of genes are given that are unique to that gene set or in the common sections between sets,
with the amount of transcription factors present within a specific gene set in parentheses. Transcription factors are
indicated in blue.
In Arabidopsis, a network of at least 152 genes is involved in managing the level of H2O2
(Mittler et al 2004). Expression profiles were compared between control and catalase-deficient
Arabidopsis plants (CAT2HP1) by high light (HL) exposure. In the CAT2HP1 plants, HL
irradiation results in elevated level of H2O2 positively regulate genes involved in the defense
response, hypersensitive response, protease, transcription and translation, and mitochondrial
metabolism (Vandenabeele et al. 2003, 2004). In total 349 transcripts were significantly
up-regulated by H2O2 in catalase-deficient plants and 88 were down-regulated (Vanderauwera et
al. 2005).
Transgenic Catalase-deficient tobacco plant (CAT1AS) were exposing to high light (HL).
Because the CAT1AS plants only maintain 10% of their residual catalase activity, H2O2 cannot be
scavenged efficiently. During HL exposure, H2O2 accumulated early and sustained over time. The
expression kinetics of >14,000 genes were monitored by using transcript profiling technology
based on cDNA-amplified fragment length polymorphism. Clustering and sequence analysis of
713differentially expressed transcript fragments revealed a transcriptional response that mimicked
that reported during both biotic and abiotic stresses (Vandenabeele et al. 2003). Including the
up-regulation of genes involved in hypersensitive response, vesicular transport, posttranscriptional
processes, biosynthesis of ethylene and jasmonic acid, mitochondrial metabolism, and cell death
(Figure 8)(refer to Vandenabeele et al. 2003).
Nox
DAGK
PLC
Px
oxidative burst
HL
AmOx
ROS
C 2 H2
SA
SAMs
UGSG
JA
Lox
ACCs
Photorespiration
H2O2
ACCo
ARRM
MYB
ARC1
PLK
Shaggy AP-2
Grp
WAK-1
Pti
12-OPDR
signal interplay
WRKY
WIPK
PAS
DES
ARF
Kinase SCRC
PP2C
HBF-1
vesicular transport
ADL2b
defense response
protein degradation
Ub E2
E3 BAG
mitochondria
CYT-C PHB
26s
cell death
AOX
BCS1 Peptidase
Figure 8. Model for the role of H2O2 in the induction of defense and cell death and the relation of the genes
identified in this expression analysis.
HL intensities provoke an increase in photorespiratory H2O2 in CAT1AS plants. Signal transduction components
in close interaction with hormone signals, vesicular transport, protein degradation, and mitochondrial responses
regulate the induction of the defense response and cell death. 12-OPDR, 12-oxophytodienoatereductase; 26S, 26S
proteasome non-ATPase regulatory subunit; DYN, dynamin; ACCo, 1-aminocyclopropane-1-carboxylic acid
oxidase;ACCs,1-aminocyclopropane-1-carboxylic acid synthase; ADL2b, ADL2b dynamin; AmOx,amine oxidase;
AOX, alternative oxidase; AP2, APETALA2 domain-containingprotein; ARF, ADP-ribosylation factor; ARRM,
two-component
cytokinine
response
regulator;
BAG,
BAG-domain-containing
protein;
BCS1,
ubiquinolcytochromereductase synthase; CYT-C, cytochrome C; DAGK, diaglycerolkinase; DES, divinyl ether
synthase; E2, ubiquitin-conjugating enzyme; E3,ubiquitin-protein ligase; Grp, glycine-rich protein; HBF1, bZIP
DNA-bindingprotein, HBF1; HL; LOX, lipoxygenase; MYB, MYB transcription factor; Nox,NADPH oxidase;
PAS, PAS-domain containing protein; PHB, prohibitin; PLC,phospholipase C; PP2C, protein phosphatase 2C; Pti,
Pto-interacting
protein;
Px,
peroxidase;
RLK,
receptor-like
kinase;
ROS;
SAMs,
S-adenosyl-Lmethioninesynthetase; SCRC, SCARECROW transcription factor; Shaggy, SHAGGY-like kinase;
Ub, ubiquitin; UGSG, UDP-glucose:SA:glucosyltransferase;WAK1; WIPK, wound-induced kinase.
H2O2：Part of signaling network
Hydrogen peroxide (H2O2) acts as a signaling molecule, the second messenger, mediating the
acquisition of tolerance to both biotic and abiotic stresses (Desikan et al. 2003). H2O2 plays a
signaling role in various adaptive processes. Plant can sense, transport and induce cellular
responses. These responses include defense reactions against pathogens, ABA-mediated stomatal
closure (Neill 2002a), and regulation of cell expansion (Suzuki et al., 1999), plant senescence
(Bhattacharjee 2005) and programmed cell death (Mittler 2002). H2O2 can modulate the activities
of many components in signaling, such as protein phosphatases, protein kinases and transcription
factors (TFs) (Cheng and Song 2006). H2O2 is also found to communicate with other signal
molecules and the pathway forming part of the signaling network that controls response
downstream of H2O2 (Neill 2002a).
H2O2 and Ca2+、K+
Calcuim as a ubiquitous internal- second messenger can regulate diverse cellular processes in
plant. The earlier reactions of plant cells to stresses are changes in plasma membrane permeability
leading to calcium and proton influx appears to be necessary and sufficient for induction of the
hydrogen peroxide (H2O2) (Pei et al. 2000). More lines of evidence concerning the relationship
between H2O2 and Ca2+ signals were provided by the study of H2O2 homeostasis in Arabidopsis
(Yang and Poovaiah. 2002). H2O2 production requires a continuous Ca2+ influx, which activates
the plasma membrane-localized NADPH oxidase (Lamb and Dixon 1997), subsequently
calcium-dependent cellular responses referred to anion and K+ efflux (Blein 1991). Challenge
Abrabidopsis with H2O2 triggered a biphasic Ca2+ elevation (Rentel and Knignt 2004). A recent
study demonstrated that aequorin-expressing tobacco cell cultures also displayed a biphasic [Ca2+]
2+ inhibitors, preventing
cyt signature in response to H2O2 challenge (Lecourieux 2002). Ca
increases of cytosolic Ca2+ concentration, also delayed the accumulation of endogenous H2O2.
Further evidence indicated that H2O2 and Ca2+ were both involved in a signaling cascade leading
to the closure of stomata in Arabidopsis (Pei et al., 2000). In this study, H2O2-activated Ca2+
channels Mediated both the influx of Ca2+ in protoplasts and increases in [Ca2+]cyt in intact guard
cells (Pei et al. 2000)
Calmodulin(CaM), ubiquitous calcium-binding protein, bound and activated some plant
catalases in the presence of calcium. Ca2+/CaM has been supposed to increase H2O2.generation
through Ca2+/CaM dependent NAD kinase that affects the concentration of available NADPH
during activation of NADPH oxidase (Harding et al. 1997). Ca2+/CaM can down-regulate H2O2
levels in plants by stimulating the catalytic activity of plant catalase (Yang and Poovaiah 2002),
controlling H2O2 homeostasis in plants. Cacium-dependent protein kinases (CDPKs) are
implicated as major primary Ca2+ sensors in plants. CDPKs activation, like activation of
mitogen-activated protein kinases (MAPKs), is triggered by biotic and abiotic stresses. N-terminal
CDPK 2 signaling triggered enhanced levels of the phytohormones jasmonic acid and ethylene but
not salicylic acid. Elevated CDPK signaling compromises stress-induced mitogen-activatied
protein kinases (MAPKs) activation and this inhibition requires ethylene synthesis and perception
(Andrea et al. 2005).
Potassium (K+) is essential to plants and required in large quantities by plant (Shin and
Schachtman 2004). Changes in the kinetics of Rb+ uptake in Arabidopsis roots occur within 6h
after K+ deprivation. H2O2 increases when the plants are deprived of K+. H2O2 accumulates in a
discrete region of roots that has been shown to be active in K+ uptake and translocation (Shin and
Schachtman 2004). It might play a role in cellular signaling of K+ deprivation (Shin and
Schachtman 2004). During stomatal closure, in addition to regulating calcium channels, H2O2 also
inhibits K+ channel activity and induces cytosolic alkalinzation in guard cells (Zhang et al. 2001a,
b)
H2O2 and salicylic acid
H2O2 has also been supposed to play a critical role in the activation of hypersensitive reaction (HR)
(Bestwick et al. 1997). Following activation of localized resistance associated with HR, plants
display systemic acquired resistance (SAR). Salicylic acid (SA) has emerged as a key signal in the
establishment of SAR (Dempser and Klessig 1995). Benzoic acid is immediated-prescursor of SA.
H2O2 is further implicated in SA synthesis as the conversion of benzoic acid into SA is catalyzed
by the H2O2 – mediated activating of benzoic-acid-2 hydroxylase (Dempser and Klessig 1995).
Nah G gene encoded salicylate hydroxylase converts SA into catechol. That appears to have little
effect on plant response-to infection (Bi et al. 1995; Friedrich et al.1995; Mur et a l.1997). PR-1
gene induction by H2O2 is suppressed in NahG plants, suggesting that SA acts downstream of
H2O2 induction. Van camp suggested a H2O2 –SA interaction pattern: H2O2 and SA constitute a
self-amplifying system; H2O2 induces SA accumulation and SA enhances H2O2 level (Van camp et
al. 1998). Challenge transgenic tobacco Samsun NN expressing salicylate hydroxylase (35S-SH-L)
with avirulent strains of pseudomonas syringae delays H2O2 accumulation by 2-3h. That indicates
an early transient rise in SA potentate the oxidative burst with resultant effects on accumulation of
H2O2 (Del Río et al. 2002). Soybean CaM (ScaM)-4 and ScaM-5 genes specifically depend on the
increase of intracellular Ca2+ level. Expression of ScaM-4 and ScaM-5 in transgenic
tobacco-plants triggered spontaneous induces an array of systemic acquired resistance
(SAR)-associated genes. But have normal level of endogenous salicylic acid (SA). Indicating that
SA is not involved in the SAR gene induction mediated by SCaM-4or SCaM-5 (Wayne et al.
2001). Transgenic tobacco expressing an antisense copy of SABP (salicylic acid-binding protein)
catalase exhibits not only a reduction in catalase activity and but also constitutive expression of
PR –1 genes (Sanchez-Casas and Klessig.1994; Chen et al. 1995)
H2O2 and nitric oxide
In mammals, the generator of NO by inducible Nitric oxide synthetase (iNOS) plays an important
role in inflammation and host defense responses (Nathan and Shiloh 2000). Anmounting body of
evidence suggests that NO is a novel effecter of plant growth, development and defense. For
example, NO was shown to be involved in photo-morphogenesis, leaf expansion, root growth,
senescence, and phytoalexin production ( Noritake et al. 1996；Beligni and Lamattina 2000). An
increasing number of reports suggest that H2O2 emerges following synthesis of Nitric Oxide (NO)
and that NO collaborates with H2O2 in plant disease resistance (Delledonne et al. 2001; Hancock
et al. 2002; Wendehenne et al. 2004). NO is indispensable to salicylic acid (SA) function as a
SAR inducer (Durner and Wendehenne 1998; Song and Goodman 2001). Nitric Oxide as a
bioactive molecule displays pro-oxidant and anti-oxidant properties in plant as well. The role of
NO may be explained by its relative timing and intensity, NO and H2O2 released in plants cells
may be different in different plant-pathogen systems (Urszula and Rozalska 2005). Two main
potential roles for NO produced during plant-pathogen interactions have been postulated in
relation with H2O2: NO can act as an antioxidant, scavenging excess H2O2, ending
radical-mediated lipid peroxidation and inhibiting H2O2 signaling pathways, which leads to cell
death; NO can act synergistically with H2O2 to induce SAR (Urszula and Rozalska 2005). Recent
demonstrations of nitric oxide synthase (NOS) in plants peroxisomes also has a function in plant
cells as a source of signal molecules like nitric oxide (NO.) and hydrogen peroxide (H2O2)
(Barroso et al. 1999).
H2O2 and abscisic acid
Abscisic acid (ABA) is an endogenous anti-transpirant that reduces water loss through stomatal
pores on the leaf surface (Tardieu et al. 1992). The regulation of stomatal closure involves various
controls that help the plant adapt to a variety of environmental changes (Hetherington and
Woodward 2003). H2O2 is an essential signal in mediating stomatal closure induced by abscisic
acid (ABA) via the activation of calcium-permeable channels in the plasma membrane (Pei et al.
2000; Neill 2002a). The phenotype of the open stomatal 1(ost1) protein kinase mutant, which is
disrupted in ABA-induced ROS production but able to close stomatal in response to H2O2. The
following discovered that H2O2 is also an essential signal mediating ABA-induced stomatal
closure (Neill et al. 2002a).
H2O2 and ethylene
The plant hormone ethylene is involved in regulation of a wide variety of development, and
physiological events, such as seed germination, pathogen and stress responses, fruit ripening,
senescence and regulation of Legumes (Abeles et al. 1992; O’Donnell et al. 1996; Penninckx et al.
1996).The role of ethylene as a signal of defense response is supported by various studies (Liu et
al. 2004). Treatment of plants with ethylene was shown to induce the synthesis of basic
chitinases(PR-3) and β -1-3-glucanases(PR-2)(BoIIer et al. 1983; Mauch et al. 1984).
Exogenous H2O2 challenge CAT lose mutant pine needle induces ethylene production. And the
production peak emerged before endogenous H2O2 accumulation in plant. H2O2 may be an
up-stream signal molecular (Ievinsh and Tiberg 1995). Ozone challenged tobacco, 1h later induced
H2O2 production in apoplast and ethylene accumulation (Schraudner et al. 1998). As for
ozone-sensitive mutant Arabidopis challenged by ozone, studies suggested it is necessary for
ethylene biosynthesis and signal transduction to O2- accumulation (Overmyer et al. 2000). MAPKs
have also been shown to regulate ethylene signal transduction in Medicago and Arabidopsis,
implying the involvement of a MAPKKK pathway (Fatma et al. 2003)
H2O2 and jasmonate
The rapid accumulation of jasmonate (JA) has been observed in many cultured plant cells in
response to various elicitor treatments (Gundlach et al. 1992). In suspension-cultured rice cells, an
N-acetylchitoheptaose elicitor led to synthesis of the phytoalexin, momilactone A, which is
preceded by the accumulation of jasmonate (Nojiri et al. 1996). Methyl jasmonate (MeJA), a
methyl ester of jasmonic acid (JA), is a well-established signal molecule in plant defense
responses and an effective inducer of secondary metabolite accumulation in plant cell cultures
such as hydrogen peroxide (Wang and Wu 2005). SA has been supposed to antagonize jasmonic
acid (JA) biosynthesis and signaling. SA levels were reduced in salicylate hydroxylase-expressing
of tobacco plants, while JA levels were not elevated when challenged by Pseudomonas syringae
pv. Phaseolicola (Takahashi et al. 2004). Contreatment with various concentrations of SA and JA
was assessed in tobacco and Arabidopsis. There was a transient synergistic enhancement in the
expression of genes associated with either JA (PDF 1.2 and Thi 1.2) or SA (PR1) signaling when
both signals were applied at low concentrations. Antagonism was observed at prolonged treatment
times or at higher concentrations (Takahashi et al. 2004). Transduction of the SA signal requires
the function of NPR1, a regulatory protein that was identified in Arabidopsis through genetic
screens for SAR- compromised mutants (Cao et al. 1997). SA and JA are modulated through a
novel function of NPR1. NPR1 is essential for SA-mediated defense gene expression, and is not
required for the suppression of JA signaling (Steven et al. 2003).
Concluding remarks
In plant metabolism, reductive activation of molecule oxygen produces an array of reactive
oxygen species (ROS), such as singlet oxygen (1O2), superoxide anion (O2-), hydrogen peroxide
(H2O2) and hydroxyl radicals (.OH). For many years, research has focused on the detrimental
effects of ROS, which were considered as undesirable, harmful byproducts in an oxygenic
atmosphere. In the last decade, increasing evidences have suggested that ROS, especially H2O2
play an important role as signaling molecules, and is produced both accidentally and deliberately
by plant controls and fine tuned metabolic networks. It is safe to say that ROS is “two-faced”,
being “harmful” when produced in excess and “beneficial” at lower concentrations. ROS at these
“beneficial” levels plays a part in sensing the environment and regulating development, growth,
and environmental accumulation.
H2O2 is a byproduct of cellular metabolism, and in plants it is produced by relatively large
amounts in mitochondria, chloroplasts, peroxisomes/glyoxysomes, and at the plasma membrane
and cell wall. Growing evidence suggests that H2O2 plays a versatile role in plant defense and
physiological reaction. There is also H2O2 accumulation during the normal plant metabolism
condition. It functions as an important signal molecular during plant growth and development.
H2O2 accumulation is maintained at the very low level because of the existence of an antioxidant
system in plant, for eliminating excess H2O2 production, and maintaining the level of H2O2 at a
normal dynamic balance.
For the continuous production of H2O2, an unavoidable consequence of aerobic metabolic
processes such as respiration and photosynthesis sensing changes of H2O2 concentrations that
result from metabolic disturbances is used by plants to activate stress responses that help the plant
cope with environmental changes. H2O2 is the reactive oxygen species (ROS) whose physiological
functions are the most extensively and long-term. And H2O2 is a less oxidant among other kinds of
ROS. It can also act as a balance point in plant between oxidative and oxidative stress. The effect
of H2O2 in plants varies according to different conditions. In a word, the merit of H2O2 outweighs
its demerit to plants. Genetically, some of the H2O2 –sensitive genes could also be involved in
plant resistance and hormone signaling. In addition, H2O2 is apparently used as an intracellular
signal that often works together with other molecules to controls a variety of processes of plant. In
a word, H2O2 plays a key role in regulating plant growth, development, resistance responses and
signal transduction (Figure 9).
H2O2 metabolism in plants
physiological
stress perception
metabolism
pathogen recognition
photosythesis
respiration
H2O 2
low
accumulation
high
normal
oxidative stress
ion flux
SA
signal
NO
transduction
genes
regulation
redox
balance
JA
antioxidant
system
ethylene
ABA
regulating plant
growth and development
Figure 9. Plant endogenic H2O2 accumulation during normal metabolism (photosynthesis, respiration, growth,
senescence, stomatal close) and various abiotic and biotic stress conditions, which include UV-C radiation, low
and high temperature, salt stress and pathogenic stress).
Excess H2O2 can induce oxidative stress, injuring plant cells. When the amount of H2O2 accumulation is
maintained at normal level by a series of antioxidant molecular and enzyme, H2O2 acts as a second messenger and
functions with other important signal molecules. They work together to protect plants from stresses and in
regulating plant growth and development.
H2O2 production is indispensable during plant growth, development and resistance responses.
As a stable ROS, H2O2 has higher stability and longer half-life, which allows H2O2 to have great
capability to buffer other ROS molecules. In another view, the balance between H2O2 and cell
redox under oxygen scavenging enzymes plays a versatile role in changing oxygen relative impact
on cells and altering cell resistance mechanism. Thus we can get better understanding of the roles
of H2O2 in plants using physiological and genetic analysis in the future research.
Acknowledgement:
The author thank professor Hong-Yu Li (College of Life Sciences at Lanzhou University) for
reviewing and improving early drafts of the manuscript and Bo Zhang (College of Life Sciences at
Lanzhou University) for helping to develop many of the ideas.
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