Elicitor Signal Transduction Leading To Production of Plant Secondary Metabolites

Biotechnology Advances 23 (2005) 283 – 333
www.elsevier.com/locate/biotechadv
Research review paper
Elicitor signal transduction leading to production

of plant secondary metabolites
Jian Zhaoa,T, Lawrence C. Davisb, Robert Verpoortec
a
Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine,
Houston, TX 77030, USA
b
Department of Biochemistry, Kansas State University, Manhattan, KS 66506, USA
c
Division of Pharmacognosy, Section Metabolomics, Institute of Biology, Leiden University, NL-2300 RA,
Leiden, The Netherlands
Received 28 November 2004; received in revised form 27 January 2005; accepted 31 January 2005
Available online 13 March 2005
Abstract
Plant secondary metabolites are unique sources for pharmaceuticals, food additives, flavors, and
other industrial materials. Accumulation of such metabolites often occurs in plants subjected to stresses
including various elicitors or signal molecules. Understanding signal transduction paths underlying
elicitor-induced production of secondary metabolites is important for optimizing their commercial
production. This paper summarizes progress made on several aspects of elicitor signal transduction
leading to production of plant secondary metabolites, including: elicitor signal perception by various
receptors of plants; avirulence determinants and corresponding plant R proteins; heterotrimeric and
small GTP binding proteins; ion fluxes, especially Ca2+ influx, and Ca2+ signaling; medium
alkalinization and cytoplasmic acidification; oxidative burst and reactive oxygen species; inositol
Abbreviations: ACC, 1-aminocyclopropane-1-carboxylic acid; ADPR, ADP-ribose; AFLP, amplified

fragment length polymorphism; AOS, allene oxide synthase; CDPK, Ca2+-dependent protein kinases; CHS,
chalcone synthase; DAG, diacylglycerol; ERF, ethylene response factors; EST, expressed sequence tags; G-
proteins, GTP-binding proteins; GSH, glutathione; HPL, hydroperoxide lyase; IP3, Inositol-1,4,5-trisphosphate;
JA, jasmonic acid; MAPK, mitogen-activated protein kinase; lysoPC, lysophosphatidylcholine; MeJA, methyl
jasmonate; OPDA, 12-oxo-PDA; PA, phosphatidic acid; PAL, phenylalanine ammonia lyase; PC, phosphati-
dylcholine; PKC, protein kinase C; PLA, phospholipase A; PLC, phospholipase C; PLD, phospholipase D; PMT,
pinosylvin-O-methyltransferase gene; ROS, reactive oxygen species; SA, salicylic acid; STS, stilbene synthase.
T Corresponding author. Tel.: +1 713 798 7039; fax: +1 713 798 7817.
E-mail addresses: jzhao1@bcm.tmc.edu (J. Zhao)8 ldavis@ksu.edu (L.C. Davis)8
verpoort@chem.leidenuniv.nl (R. Verpoorte).
0734-9750/$ - see front matter D 2005 Elsevier Inc. All rights reserved.
doi:10.1016/j.biotechadv.2005.01.003
284 J. Zhao et al. / Biotechnology Advances 23 (2005) 283–333
trisphosphates and cyclic nucleotides (cAMP and cGMP); salicylic acid and nitric oxide; jasmonate,
ethylene, and abscisic acid signaling; oxylipin signals such as allene oxide synthase-dependent
jasmonate and hydroperoxide lyase-dependent C12 and C6 volatiles; as well as other lipid messengers
such as lysophosphatidylcholine, phosphatidic acid, and diacylglycerol. All these signal components
are employed directly or indirectly by elicitors for induction of plant secondary metabolite
accumulation. Cross-talk between different signaling pathways is very common in plant defense
response, thus the cross-talk amongst these signaling pathways, such as elicitor and jasmonate,
jasmonate and ethylene, and each of these with reactive oxygen species, is discussed separately. This
review also highlights the integration of multiple signaling pathways into or by transcription factors, as
well as the linkage of the above signal components in elicitor signaling network through protein
phosphorylation and dephosphorylation. Some perspectives on elicitor signal transduction and plant
secondary metabolism at the transcriptome and metabolome levels are also presented.
D 2005 Elsevier Inc. All rights reserved.
Keywords: G-protein and Ca2+; Cytoplasmic acidification; Reactive oxygen species; Cyclic nucleotides and
inositol trisphosphate; Nitric oxide and salicylic acid; Jasmonate and ethylene; Transcription factors; Protein
phosphorylation
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285
2. Elicitors and signal transduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 287
3. Elicitor signal perception. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289
3.1. Elicitor and elicitor binding sites . . . . . . . . . . . . . . . . . . . . . . . . 289
3.2. Avirulence determinants and R proteins . . . . . . . . . . . . . . . . . . . . 292
4. Elicitor signal transduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293
4.1. GTP binding proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 294
4.2. Ion fluxes and Ca2+ signaling. . . . . . . . . . . . . . . . . . . . . . . . . . 295
4.3. Cytoplasmic acidification . . . . . . . . . . . . . . . . . . . . . . . . . . . . 296
4.4. Oxidative burst and reactive oxygen species . . . . . . . . . . . . . . . . . . 297
4.5. Inositol 1,4,5-trisphosphates and cyclic nucleotides. . . . . . . . . . . . . . . 298
4.6. Salicylic acid and nitric oxide . . . . . . . . . . . . . . . . . . . . . . . . . 299
4.7. Jasmonate pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 300
4.8. Ethylene pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301
4.9. ABA signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305
5. Lipid signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306
5.1. Oxylipins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306
5.2. Lipid messengers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307
5.3. Other lipid elicitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309
6. Cross-talk of signaling pathways. . . . . . . . . . . . . . . . . . . . . . . . . . . . 309
6.1. Elicitor and JA pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . 310
6.2. JA and ethylene pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . 311
6.3. JA and other pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312
6.4. ROS and other pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312
6.5. Abiotic elicitors, ROS and oxylipin pathways . . . . . . . . . . . . . . . . . 313
7. Integration of multiple signaling pathways by transcription factors . . . . . . . . . . 314
7.1. Transcription factors involved in regulation of plant secondary metabolism . . 314
7.2. Integration of various signals in transcription factors . . . . . . . . . . . . . . 315
J. Zhao et al. / Biotechnology Advances 23 (2005) 283–333 285
8. Linkage of signaling components by protein phosphorylation and dephosphorylation 317

9. General features of elicitor induction of plant secondary metabolism . . . . . . . . 318
10. Perspectives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 320
Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322
1. Introduction
Plant secondary metabolites are unique resources for pharmaceuticals, food

additives, and fine chemicals. They also provide original materials used in other
areas. Besides direct extraction from plants, and chemical synthesis to provide those
compounds or derivatives with similar uses, plant cell culture has been developed as a
promising alternative for producing metabolites that are difficult to be obtained by
chemical synthesis or plant extraction. However, in spite of four decades of efforts,
production of plant secondary metabolites by plant cell culture technology is still
facing many biological and biotechnological limitations. One of the major obstacles is
the low yield of plant secondary metabolites in plant cell cultures. Since the major
roles of plant secondary metabolites are to protect plants from attack by insect,
herbivores and pathogens, or to survive other biotic and abiotic stresses, some
strategies for culture production of the metabolites based on this principle have been
developed to improve the yield of such plant secondary metabolites. These include
treatment with various elicitors, signal compounds, and abiotic stresses (Yukimune et
al., 1996; Zhao et al., 2000, 2001a,b,c; Zhang et al., 2004). Many such treatments
indeed effectively promote the production of a wide range of plant secondary
metabolites, both in vivo and in vitro. However, the productivity is still rarely
competitive for industrial application. Only the production of shikonin by Lithosper-
mum erythrorthizon cell cultures and of taxol by Taxus cell cultures is successfully
industrialized for commercial application. Considerable efforts are continuously being
put in these studies for production of many other highly valuable compounds (Fig. 1).
A recent work for improving production of plant secondary metabolites was mainly
focused on the following aspects: 1) manipulation of plant cell cultures to improve
productivity of target compounds, through improving chemical processing and
bioreactor performance or employing elicitors, abiotic stresses, and other approaches,
regardless of their mechanisms (Zhong, 2001); 2) studying signal transduction
pathways underlying various effective strategies leading to biosynthesis of target
secondary metabolites; 3) studying transcription factors and their regulation mecha-
nisms, including genetic manipulation of regulator genes to improve production of
target secondary metabolites (Memelink et al., 2001); 4) cloning of secondary
metabolite biosynthetic genes, and genetic modification of key genes to engineer the
metabolic flux to target compounds (Verpoorte and Memelink, 2002); 5) studying
metabolic flux and profiling metabolic intermediates to understand whole pathways and
overall regulation of target compound accumulation (Sumner et al., 2003); and 6)
studying gene transcripts for plant secondary metabolism by profiling and analyzing
OH O OH
O
Me
+ O
CMe2 N
O
OH O O
I. Shikonin, the only secondary metabolite that is II. Sanguinarine, a benzylisoquinoline alkaloid
successfully produced by large-scale plant cell culture. produced by some poppy cell cultures.
AcO
O OH
OH Me
H
Ph N O Me Me
Me H
O Ph O H O
OH
O AcO
O
Ph
III. Taxol, a diterpene compound from Taxus spp. has very strong anticancer activity.
Studies on biosynthetic pathway and cell culture for its production are in process.
H
N
HO Me = CH3
N
H H
O Ac = CH3CO
Me
H Ph =
MeO O
V. β-Thujaplicin, an antifungi tropolone from

IV. Ajmalicine, an extensively studied terpenoid indole Cupressus lusitanica cell culture, and the
alkaloid from Catharanthus roseus cell culture. explanation for symbols in chemical structures.
Fig. 1. Several typical plant secondary metabolites with special interests.
global gene expression under different conditions to understand the regulation of plant
secondary metabolism in a whole sense (Goossens et al., 2003).
The general cellular process and regulatory principle for activation of plant secondary
metabolite biosynthesis is that, an extracellular or intracellular signal is perceived by a
receptor on the surface of the plasma membrane or endomembrane; the elicitor signal
perception initiates a signal transduction network that leads to activation or de novo
biosynthesis of transcription factors, which regulate the expression of biosynthetic genes
involved in plant secondary metabolism. The resulting enzymes catalyze the biosynthesis
of target secondary metabolites. Much effort has been put onto cloning biosynthetic
genes, identifying transcription factors, revealing the signal transduction steps underlying
elicitor activation of plant secondary metabolism, and also genetic manipulation of
transcription factors or biosynthetic genes to engineer the production of target secondary
metabolites (Sato et al., 2001). These efforts are expected to substantially improve
production of interesting secondary metabolites and eventually to be applied in industry.
However, due to lack of detailed information about biosynthetic genes and their
regulation, such as the signal transduction pathways and transcription factors involved,
success in metabolic engineering of plant secondary metabolites is so far very limited.
Studying upstream signal transduction pathways that regulate biosynthetic genes and
transcription factors is as important as cloning of biosynthetic genes and identification of
transcription factors. It is expected that a better understanding of signal transduction will
significantly improve specificity and efficiency of genetic modification using tran-
scription factors or biosynthetic genes, and it will further improve the performance of
manipulation of the metabolic flux towards certain secondary metabolites and lead to
improved yields of these important products. This may also help to develop strategies to
modify the production of target compounds by either activation or suppression of certain
metabolic pathways.
This review is to highlight advances in understanding of the elicitor signal transduction
that lead to production of plant secondary metabolites either in vivo or in vitro. The
signaling pathways presented here include a wide range of elicitors and plant hormones.
The signal components and cross-talk among these signaling pathways, integration of
cross-talking pathways by transcription factors, linkages of all components by protein
phosphorylation and dephosphorylation, are reviewed.
2. Elicitors and signal transduction
Elicitors are chemicals or biofactors from various sources that can induce physiological
changes of the target living organism. In a broad sense, delicitorsT, for a plant refer to
chemicals from various sources that can trigger physiological and morphological
responses and phytoalexin accumulation. It may include abiotic elicitors such as metal
ions and inorganic compounds, and biotic elicitors from fungi, bacteria, viruses or
herbivores, plant cell wall components, as well as chemicals that are released at the attack
site by plants upon pathogen or herbivore attack. It is well known that treatment of plants
with elicitors, or attack by incompatible pathogens, causes an array of defense reactions,
including the accumulation of a range of plant defensive secondary metabolites such as
phytoalexins in intact plants or in cell cultures (Fig. 2). However, with regard to signal
transduction from elicitor perception to defense reactions in plants, the term elicitor as
used here only refers to fragments from fungal or plant cell wall components, bacterial,
virus or herbivore constituents, or synthetic analogs with elicitor activity (see Fig. 2).
Elicitor/receptor
interaction
G-protein
Biphasic Ca2+ spiking
Cytoplasmic acidification
Biphasic H2O2
Multiphasic protein phosphorylation

Rapid IP3, NO, or cAMP production
Early response genes

ethylene
Jasmonic acid
Phenylpropanoid
Production of secondary
1 2 3 4 5 10 20 30 h
Fig. 2. Schematic illustration of the sequential defense reactions in plants induced by elicitors. In many particular
cases, only some of these events were detected.
From a pathogenesis point of view, many elicitors may act as avirulence

determinants of a plant genetic system that refers to a general response of gene-for-
gene resistance in plant innate immunity, in which plant resistance genes (R genes)
confer resistance to pathogens with a matching avirulence (avr) gene by specific
recognition events. Elicitors or avirulence determinants must be recognized by plant
receptors or R proteins localized to the plasma membrane or the cytoplasm before
initiating signaling pathways, which lead to defense reactions such as synthesis of PR
(pathogenesis-related proteins), or defense secondary metabolites. Molecular recognition
and physical interaction between elicitor signal molecules and specific plant receptors
are complex processes but are required for specific elicitor signal transduction. By
causing changes in receptor conformation or activation of receptor kinases, elicitors
subsequently or indirectly activate their corresponding effectors, such as ion channels,

G-proteins, lipases, and kinases, which then transduce the elicitor signal to downstream
defense responses.
3. Elicitor signal perception
3.1. Elicitor and elicitor binding sites
Signal perception is the first committed step of the elicitor signal transduction
pathway and much effort has been put into isolation of effective elicitor signal molecules
from fungal and plant cell extracts or other sources, and identification of the
corresponding receptors from plant plasma membranes. It is now clear that there are
several different classes of components that can completely substitute for fungal elicitors
in the elicitation effect. These include poly- or oligosaccharides such as chitin, and
chitosan and their fragments (e.g., chitooligosacharides), xyloglucans, laminarin and
other h-glucans and their fragments and oligogalacturonides, proteins (e.g., harpin or
elicitins such as cryptogein) or peptides (e.g., 13-pep, systemin, and flg22), as well as
lipid derivatives such as syringolide, Nod factors and lipopolysaccharides. Poly- or
oligosaccharides are the most well studied signal molecules in elicitor signal
transduction. Many elicitors, such as chitin, xyloglucans, chitosan, h-glucan and
oligogalacturonide, exhibit elicitor activity across different plant species and highly
induce phytoalexins in plants, suggesting that different plants possess some common
receptors to sense these signals. Binding tests with specific saccharide elicitors, using
membrane-enriched fractions have led to the discovery of a number of specific receptors.
Interaction of most of these saccharide and proteinaceous or lipid elicitor and their
receptors show high affinity, specific, reversible, and saturable binding, which indicate
they are receptor-ligand interactions. Saccharide elicitor binding sites have been studied
in various plant cells, as shown in Table 1 (Shibuya et al., 2002). One oligosaccharide
elicitor can be recognized by several plants through their receptors with similar binding
properties (Ito et al., 1997; Okada et al., 2002).
The binding sites for protein or peptides elicitors were also characterized in several
plants. In parsley suspension culture, a 13-amino acid peptide elicitor has been well
studied and its interaction with a receptor on parsley cell membrane has also been
elucidated (Nennstiel et al., 1998). Harpin protein from Pseudomonas syringae pv
phaseolicola elicits the hypersensitive response and the accumulation of pathogenesis-
related gene transcripts in tobacco. A binding site for harpin in tobacco plasma membranes
was identified with specific, reversible, and saturable binding activity (Lee et al., 2001). A
bacterial flagellin is a protein elicitor containing a 22-amino acid peptide (flg22) as the
elicitor activity domain. Flg22 can be recognized by some plant species and a receptor-like
kinase with a high binding affinity to flg22 was identified from Arabidopsis (Bauer et al.,
2001). Some proteins secreted by fungal pathogens were characterized as elicitors
(elicitins); a 193 kD receptor for an elicitin, cryptogein, has been isolated from tobacco
plasma membranes (Bourque et al., 1999). All these elicitor-binding sites were identified
in plant plasma membranes (Table 1).
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J. Zhao et al. / Biotechnology Advances 23 (2005) 283–333
Table 1
Elicitors identified with their binding sites or receptors in plants
Elicitor Elicitor source Elicitor properties Plant receptors Induced plant defense References
responses
h-glucan; Phytophthora sojae, 1,6-h-linked and A receptor with h-glucan Ca2+/ion fluxes; ROS Fliegmann et al.,
h-glucan; Phytophthora 1,3-h-branched binding site and generation, medium 2004; Umemoto
megasperma heptaglucoside 1,3-h-glucanase activity alkalinization, defense et al., 1997; Day
Chitin oligosaccharide in soybean cell cultures; gene expression, et al., 2000
74 kD, gene cloned in phytoalexin biosynthesis
soybean root cells;
85 kD membrane protein
in soybean cell cultures
N-Acetylchitooligosaccharide Pyricularia oryzae Oligosaccharide 75 kDa in rice cells, Ca2+/ion fluxes; Ito et al., 1997;
66 kDa in barley cells, ROS generation, Okada et al., 2002
68 kDa in carrot cells, defense gene expression,
60 kDa in wheat leaves phytoalexin biosynthesis
Flagellin/flg22 Bacteria Flg22, 22 amino acids Toll-like LRR receptor- Ca2+/ion fluxes, Bauer et al., 2001;
forming elicitor active kinase in Arabidopsis, ROS generation, Meindl et al., 2000
domain of flagellin 115 kD protein in medium alkalinization
microsomal, tomato
Harpins Pseudomonas spp 10–12 kDa protein 115 kD protein in ROS generation, Lee et al., 2001
microsomal tobacco or hypersensitive response
Arabidopsis
Systemin Tomato leaves 18-amino acid peptide 160 kDa membrane JA and protease inhibitor Scheer and Ryan,
protein, a LRR kinase biosynthesis, ROS 2002
receptor in tomato. production, Ca2+ flux.
13-pep Phytophthora sojae 13-amino acid peptide 19 kDa plasma Ca2+/influx and K+/ Nennstiel et al.,
from 42 kDa membrane-bound H+exchange, ROS, JA, 1998
glycoprotein protein with high and phytoalexin
binding affinity in accumulation
parsley cell cultures
Cryptogeins Phytophthora cryptogea; Glycoprotein 193 kD plasma Membrane depolarization Bourque et al.,
A number of 10–12 kD membrane protein from Ca2+, K+/Cl-efflux, H2O2, 1999
elicitins tobacco MAPK, and capsidiol

accumulation
Lipopolysaccharides; Gram-negative bacteria Glycolipids elicitor Unknown tobacco, Ion flux and oxidative burst, Meyer et al., 2001;
Nod factors Rhizobia Dolichos biflorus G-protein activation, Ca2+ Etzler et al., 1999;
(lipochitooligosaccharide) (46-kD lectin), soybean root hair formation in host Day et al., 2000
(52 kD lectin), all have legume plants
apyrase activity
Syringolides Escherichia coli with A glycolipid elicitor 34 kD protein in soluble Ca2+, K+/Cl-efflux, Ji et al., 1998
avir gene fraction from soybean phytoalexin biosynthesis,
cell cultures defense genes expression
Volicitin Fatty acid amide Secreted to attack site in A plasma membrane JA/ethylene biosynthesis, Lait et al., 2003;
N-linolenoyl-l-glutamine synthesized by insects defense against insects protein in maize and indole and /or terpene Truitt et al., 2004
tobacco emission during
plant–insect interaction
291
On the other hand, a glycolipid elicitor molecule such as syringolide is perceived by a

soluble protein (Ji et al., 1998) and the Nod factor is specifically bound to a 46 or 52 kD
lectin in Dolichos biflorus or soybean, respectively (Etzler et al., 1999). Recently, a
putative Nod-factor receptor kinase gene (NFR5) was isolated from Lotus japonicus.
NFR5 encodes an unusual transmembrane serine/threonine receptor-like kinase and is
shown to be essential for Nod-factor perception and the earliest detectable plant responses
to bacteria and Nod-factor (Madsen et al., 2003). All these studies demonstrated the first
essential step of plant–microbe interactions, indicating that specific recognition between
elicitor and plant receptors may determine plant defense response and elicitor signal
transduction.
Studies on another special group of elicitors involved in plant–herbivore interactions
have recently made good progress (Schmelz et al., 2003a,b)(Table 1). The herbivore-
specific elicitors, including fatty-acid–amino-acid conjugates from Manduca spp, volicitin
from Spodoptera exigua, h-glucosidase from Pieris brassicae oral secretions, and
bruchins from cowpea weevil oviposition fluid, can elicit direct defense responses such as
accumulation of antinutritive toxic compounds to deter herbivores or indirect defense
responses such as producing volatile sesquiterpene and indole compounds to attract
predators and parasitoids of the herbivores and to repel herbivores away from plants
(Turlings et al., 1990; Schmelz et al., 2003a,b). The herbivore elicitor volicitin (a fatty acid
amide) that induces accumulation of secondary metabolites and emission of indole or
sesquiterpene volatiles in plants is recognized by a plasma membrane protein in corn (Lait
et al., 2003; Truitt et al., 2004). Systemin, a plant-derived elicitor increases upon herbivore
action. It is an 18-amino acid peptide produced at wound sites of tomato plants in response
to wounding or attack of herbivorous insects, which activates complicated signaling
pathways leading to defense responses. A systemin receptor SR160 was purified and the
gene for it was cloned from tomato. Surprisingly it is very similar to brassinosteroid
receptor in Arabidopsis (Scheer and Ryan, 2002).
3.2. Avirulence determinants and R proteins
Certain elicitors from plant pathogens, from a plant pathogenic point of view, can be
avirulence determinants delivered by incompatible pathogens, while the receptors that
recognize these avirulence determinants are plant R proteins. This so-called gene-for-gene
resistance in plant–microbe interactions typically reflects the importance of race-specific
recognition between plant R genes and pathogen avr genes. Each plant genome has a large
number of structurally different R genes encoding several diverse classes of R proteins,
which confer plant resistance to corresponding avirulence determinants in plant pathogens
(Tang et al., 1996; Nimchuk et al., 2003). The most abundant types of R proteins belong to
the group containing the nucleotide-binding site and leucine-rich repeat domains (NBS-
LRR). The NBS-LRR genes contain two distinct groups of genes with either an N-terminal
domain with Toll-like receptor homology (TIR-NBS-LRR) or an N-terminal coiled-coil
motif (CC-NBS-LRR) (Dixon et al., 2000; Meyers et al., 2003). The NBS-LRR proteins
such as Rps2, Rpm1, N, L6, M, Rpp5, Prf, and I2C-1 have a conserved LRR region of
variable length, which may be responsible for recognizing particular avr gene products.
Another group comprises proteins with an extracellular LRR domain, a transmembrane
domain, and a small cytoplasmic domain, such as Cf-2, Cf-4, Cf-9 proteins. The tomato
Pto protein, which is a cytoplasmic serine/threonine protein kinase, represents the third
group of R genes. The rice R gene Xa-21 combines the features of the second and third
groups with an extracellular LRR domain and a cytoplasmic serine/threonine protein
kinase (Dangl and Jones, 2001).
Avr determinants such as hydrophilic proteins are delivered inside plant cells during
infection by a specialized type III secretion system of plant pathogenic bacteria (Galán and
Collmer, 1999) or outside plant cells (between cell wall and the plasma membrane)
through other types of secretion systems (type I–IV). This is consistent with the
intracellular localization of the R proteins (LRR regions) for perceiving the corresponding
Avr determinants. An increasing body of evidence also suggests that many R proteins and
Avr proteins may not directly interact. Instead, R protein serves as a guard that detects the
attack on a third host protein by the bacterial Avr protein and subsequently initiates the
plant defense responses (Nimchuk et al., 2003). It appears that the highly variable LRRs in
both groups of R proteins may be involved in pathogen recognition, either intracellularly
or extracellularly, so R gene–avr gene interaction provides a structural basis that has been
subjected to evolutionary modification in response to variation in Avr gene products
(Dangl and Jones, 2001). The specific recognition and diverse interactions between plants
and elicitors or avirulent pathogens may explain why some pathogens or elicitors induce
the accumulation of plant phytoalexin or other defensive compounds, while others cannot,
or why phytoalexins generally confers the plant resistance to avirulent pathogens instead
of virulent ones (Hammerschmidt, 1999).
4. Elicitor signal transduction
Following perception of elicitor signals, plant receptors are activated, and then in turn
activate their effectors, such as ion channels, GTP binding proteins (G-proteins), and
protein kinases. Activated effectors transfer the elicitor signals to second messengers,
which further amplify the elicitor signal to other downstream reactions (Ebel and
Mithoefer, 1998; Blume et al., 2000). The sequentially occurring events in elicitor-
induced defense responses can be organized as follows (Fig. 2): perception of elicitor by
receptor, reversible phosphorylation and dephosphorylation of plasma membrane
proteins and cytosolic proteins, cytosolic [Ca2+]cyt spiking, plasma membrane
depolarization, Cl and K+ efflux/H+ influx, extracellular alkalinization and cytoplasmic
acidification, mitogen-activated protein kinase (MAPK) activation, NADPH oxidase
activation and reactive oxygen species (ROS) production, early defense gene expression,
ethylene and jasmonate production, late defense response gene expression, and
secondary metabolite accumulation (Fig. 2). Elicitor signal transduction is a multiple
component network with various sequential reactions to establish an efficient defense
(Boller, 1995). These multiple components consist of some parallel or cross-linking
signaling pathways leading to different target responses. An elicitor-signaling pathway
may vary with perception of different elicitor signals or with target defense responses.
The following is a list of components that ever are found to work in an elicitor signal
transduction network.
4.1. GTP binding proteins
G-proteins constitute a class of ancient eukaryotic proteins, including heterotrimeric

complexes consisting of a-, h-, and g-subunits, and monomeric small G-proteins. Because
heterotrimeric G-proteins occur in plants with limited gene copy number, study of them is
much less advanced than that in mammals (Assmann, 2002). On the other hand, plants
have many copies of small G-proteins, such as the Rop family (homologues of Rho
GTPases), Rac-like proteins, and the Rab/Ypt family, which have been cloned from
various plants (Yang, 2002). An increasing body of evidence shows that G-proteins
regulate various cellular processes related to growth, development, hormone signaling, and
defense responses (Assmann, 2002; Yang, 2002). Some studies have shown that G-
proteins may participate in plant defense responses, probably by coupling with receptors to
mediate the elicitor signal to yield further effectors, such as ion channels and
phospholipases, for signal transduction to downstream reactions, such as production of
plant secondary metabolites (Legendre et al., 1992; Gelli et al., 1997; Aharon et al., 1998;
Park et al., 2000). In tomato cell culture in response to pathogen Cladosporium fulvum
extracts containing Avr5 (Vera-Estrella et al., 1994), binding of Avr5 to the receptor results
in dephosphorylation of H+-ATPase and activation of G-proteins, which further transduce
elicitor signals to downstream reactions, such as activation of NADPH oxidase.
Analyses using various G-protein activators and inhibitors suggest that G-proteins are
involved in elicitor-induced biosynthesis of various secondary metabolites in plants such
as benzophenanthridine alkaloids in bloodroot cell culture (Mahady et al., 1998), 6-
methoxymellein in carrot cell culture (Kurosaki et al., 2001), isoflavonoid phytoalexin in
soybean culture (Rajasekhar et al., 1999), benzophenanthridine alkaloids in California
poppy cell culture (Roos et al., 1999), scoparone in lemon cell culture (Ortega et al.,
2002), and h-thujaplicin in Cupressus lusitanica cell culture (Zhao and Sakai, 2003a).
Speculations have been made that there is a link between the activation of ion channels
(such as K+/Ca2+ channels), phospholipase A (PLA), phospholipase C (PLC) or
phospholipase D (PLD) with G-proteins in plants (Roos et al., 1999; Meijer and Munnik,
2003). A direct physical interaction between the G-protein a-subunit and PLDa in tobacco
and Arabidopsis was demonstrated recently, and was suggested to play a role in
transmembrane signaling pathways (Lein and Saalbach, 2001; Zhao and Wang, 2004).
Also the coupling between the G-protein a-subunit and PLA2 in fungal elicitor-induced
benzophenanthridine production was demonstrated in California poppy cell culture (Roos
et al., 1999). Antisense suppression of the G-protein a-subunit expression almost
abolished elicitor activation of PLA and downstream reactions, including phytoalexin
accumulation (Roos et al., unpublished results, personal communication). The only
identified G-protein coupled receptor in Arabidopsis GCR1 is suggested to affect PLC and
inositol trisphosphate (IP3) signaling (Apone et al., 2003). Biochemical and genetic
evidence show that heterotrimeric G-proteins or small G-proteins are involved in cation
ion fluxes and oxidative burst (Aharon et al., 1998; Saalbach et al., 1999; Wang et al.,
2001; Assmann, 2002). However, because of disputable specificity of pharmacological
methods used in plant G-protein studies, further genetic evidence is needed.
The Rac-like small G-protein OsRac1, a homologue of mammalian Rac GTPase,
regulates the elicitor-induced ROS production in rice cell culture through a NADPH
oxidase. Over expression of OcRac1 in rice plants causes an increased ROS and the
phytoalexin momilactone production, and defense gene expression in rice, which thereby
shows more resistance to pathogens (Kawasaki et al., 1999). A rice dwarf1 mutant
(deficient in a single copy heterotrimeric G-protein a-subunit gene) is characterized as a
pathogen sensitive mutant showing reduced hypersensitive response (Suharsono et al.,
2002). Sphingolipid elicitor-induced H2O2 production and PR-protein gene expression are
significantly reduced in dwarf1 mutant cell cultures. These results show that the rice small
G-protein OcRac1 gene expression is also suppressed in dwarf1 mutant cells. However,
engineered constitutive expression of OcRac1 in dwarf1 mutant cell culture restores its
responses to sphingolipid elicitors. It is thus proposed that the heterotrimeric G-protein a-
subunit acts upstream of the small GTPase Rac in disease resistance of rice (Suharsono et
al., 2002). Recently, more small G-proteins, as well as the G-protein a-subunit, were
identified to be involved in reactive oxygen species generation and cell death, as well as
pathogen resistance (Yang, 2002). NtRac5, a Rac GTPase in tobacco, negatively regulates
NADPH oxidase activity by the modulating membrane trafficking process, through which
NADPH oxidase is translocated to the plasma membrane and is recruited for the plant
hypersensitive response induced by cryptogein treatment (Morel et al., 2004). Also
recently, Arabidopsis G-protein a- and h-subunits were shown to be necessary for ozone-
induced ROS generation and cell death, suggesting ozone reception may involve G-
proteins (Booker et al., 2004; Fedoroff N.V., personal communication). Clearly, both
heterotrimeric and small G-proteins are involved in plant defense responses and
production of some secondary metabolites.
4.2. Ion fluxes and Ca2+ signaling
Elicitor-induced ion fluxes are immediate responses of plant cells, occurring within 5
min after elicitor treatment. Even though it is still not well understood how an elicitor
stimulates ion transport in plant cells, ion fluxes such as K+/H+ exchange, Cl effluxes and
Ca2+ influx, are generally observed as the earliest responses of plant cells to avirulent
pathogens or elicitors (Trewavas and Malhó, 1998; White and Broadley, 2003). Among
these ion fluxes, Ca2+ influx is regarded as one of the most significant events, since Ca2+ is
a key second messenger for many diverse physiological changes and cellular processes
(Trewavas and Malhó, 1998). Various techniques, such as membrane patch clamp, uptake
of [45Ca2+], Ca2+ binding to fluorescent dye or a Ca2+-sensor protein indicator, have been
used to address this early elicitor response (White and Broadley, 2003; Chandra et al.,
1997; Cessna et al., 2001). Measurements show that different elicitors induce diverse
cytosolic [Ca2+]cyt spiking from the resting level of 50–100 nM to 1–5 AM in 2–5 min. For
a typical example in tobacco cells, a biphasic [Ca2+]cyt increase is seen after treatment with
oligogalacturonide, laminarin (a linear h-1, 3-glucan), chitooligosaccharide, lipopolysac-
charide, or cryptogein (Lecourieux et al., 2002). After the first rapid response, a weak and
long-lasting second peak is observed upon cryptogein treatment while oligosaccharide and
lipopolysaccharide elicitors induce a strong and transient second peak. It was shown that
the first [Ca2+]cyt peak induced by oligosaccharides is caused by the influx of extracellular
Ca2+, and the second [Ca2+]cyt pulse may be due to PLC activation and IP3-released Ca2+
from intracellular calcium stores, such as the vacuoles, Golgi apparatus, and endoplasmic
reticulum (Lecourieux et al., 2002). Some reports show that elicitor-induced Ca2+ spiking
not only mediates subsequent events, but also further amplifies Ca2+ signaling through
Ca2+-dependent production of H2O2, which is able to induce Ca2+ influx from extracellular
sources (Price et al., 1994; Lecourieux et al., 2002).
Elicitor-induced Ca2+ flux is important for elicitor-induced accumulation of plant
secondary metabolites (Smith, 1994). This dramatic elicitor-induced [Ca2+]cyt spiking
activates many intracellular processes directly, or through Ca2+ sensors. A ubiquitous Ca2+
sensor is calmodulin, which can be activated by Ca2+ binding. Ca2+ and activated
calmodulin further activate Ca2+/calmodulin-dependent protein kinase and protein
phosphatase, membrane-bound enzymes, or transcription factors. For example, NAD (P)
H oxidase, a major source of ROS production (such as H2O2 and O2 ) is induced by
elicitors (Keller et al., 1998; Bolwell and Wojtaszek, 1997), while Ca2+-dependent protein
kinases (CDPK), a large kinase family with essential roles in plant defense responses, are
regulated by Ca2+ binding. Phospholipases responsible for biosynthesis of jasmonate or
other messengers, such as inositol 1,4,5-trisphosphate (IP3), phosphatidic acid (PA) and
diacylglycerol (DAG), are also regulated by Ca2+ (Wang, 2002; Meijer and Munnik, 2003;
de Jong et al., 2004). These ROS, protein kinase cascades, and lipid signaling messengers
further transfer and amplify elicitor signals to downstream reactions. Another significant
effect of [Ca2+]cyt spiking is differential activation of transcription factors, which directly
regulate all defense gene expression (Dolmetsch et al., 1997; Yang and Poovaiah, 2002).
Therefore, elicitor-induced Ca2+ spiking is one of the earliest events that act as a master
messenger for almost all downstream response reactions.
4.3. Cytoplasmic acidification
Medium (or apoplast) alkalinization is one of the very early events occurring in elicitor-
treated plant cell cultures. It has been used as a marker of elicitor responses for studying
elicitor-binding sites in plant cells (Boller, 1995). Medium alkalinization is thought to
result from elicitor-induced depolarization of the plasma membrane and subsequent K+/H+
exchange, with Ca2+ influx/Cl efflux (Boller, 1995). A corresponding cytoplasmic
acidification was observed accompanying the medium alkalinization. Cytoplasmic
acidification is regarded as an essential step in signal transduction, leading to the
oxidative burst and biosynthesis of plant secondary metabolites (Sakano, 2001).
For example, artificial acidification of the cytoplasm in tobacco cell culture increases
phenylalanine ammonia-lyase (PAL) and 3-hydroxy-3-methylglutaryl-CoA reductase
(HMGR) gene transcription, both of which are essential to the phenylpropanoid and
isoprenoid pathways (Lapous et al., 1998). Acidification increases phenylalanine
ammonia lyase gene transcription in rice (He et al., 1998). Armero and Tena (2001)
showed that inhibition of the plasma membrane H+-ATPase or, more specifically, the
proton efflux, may start a signaling pathway leading to the activation of phytoalexin and
isoflavone excretion in chickpea seedlings. These results suggest that elicitor-induced
cytoplasmic acidification is not only a result but also a regulatory process, which
mediates other cellular responses. The biosynthesis of benzophenanthridine alkaloids in
elicitor-induced California poppy cell cultures is also regulated by acidification of the
cytoplasm. Such acidification seems to be necessary and sufficient for biosynthesis of
benzophenanthridines (Roos et al., 1998). Further studies have demonstrated that the
vacuolar proton pool is very important for elicitor-induced cytoplasmic acidification and
alkaloid biosynthesis. The yeast elicitor-activated G-protein-coupled phospholipase A2
hydrolyzes phospholipids and causes a transient increase of endogenous lysophospha-
tidylcholine, which in turn activates the tonoplast H+/Na+ antiporter and causes
subsequent cytoplasmatic acidification (Viehveger et al., 2002). At high dosage of
elicitor, this G-protein-PLA2-lysophosphatidylcholine-H+-ATPase will not work, instead,
jasmonate signaling plays a dominant role in induction of benzophenanthridines (Roos,
personal communication).
4.4. Oxidative burst and reactive oxygen species
Another significant event in plant defense responses is the oxidative burst, a common
early response of plant cells to pathogen attack and elicitor treatment. The ROS,
predominantly superoxide anion (O2 ) and hydrogen peroxide (H2O2) in plant–pathogen
interactions, are toxic intermediates resulting from successive steps in the reduction of
molecular O2. There are several sources for generation of ROS in plants, including
NADPH oxidase, apoplastic peroxidase, and other oxidases in mitochondria, chloroplasts,
and peroxisomes. ROS generation in elicitor-treated plant cell cultures mainly depends on
NADPH oxidase, and apoplastic peroxidases in some species (Bolwell and Wojtaszek,
1997). In many plant systems, biphasic ROS generation is observed; the first phase occurs
at about 10–30 min and the second at 1–3 h after fungal elicitation (Bolwell and
Wojtaszek, 1997; Zhao et al., 2001b). Cytosolic free Ca2+ spiking is a prerequisite in many
elicited cells for activation of ROS generation (Zhao et al., 2001b; White and Broadley,
2003). Two different NADPH oxidase genes were characterized in potato as being
responsible for the elicitor-induced two-phase oxidative burst; one of them can be
regulated by Ca2+-independent protein phosphorylation (Yoshioka et al., 2001). ROS
generation may be differentially regulated by Ca2+ and protein phosphorylation/
dephosphorylation, and a Ca2+-binding NADPH oxidase was cloned earlier. It is likely
that elicitor-induced biphasic ROS generation occurs either by elicitor directly binding and
thus activating NAD(P)H oxidase or via Ca2+/calmodulin-dependent protein kinases.
The ROS exerts various effects on plant defense responses, including cell wall
reinforcement (cross-linking of structural protein and lignin polymers), hypersensitive cell
death, defensive gene activation, as well as defensive compound induction (Levine et al.,
1994). In some plant cell cultures, ROS is shown to be sufficient for induction of plant
secondary metabolite accumulation, while in others it is not. In some plants, H2O2
mediates the elicitor-induced accumulation of secondary metabolites, such as the
isoflavonoid glyceollin in soybean (Degousee et al., 1994; Guo et al., 1998), p-
coumaroyloctopamine in potato tubers (Matsuda et al., 2001), indole alkaloids in
Catharanthus roseus (Zhao et al., 2001b), tcibulin 1 and 2 in Allium cepa cell cultures
(Kravchuk et al., 2003), saponin in ginseng (Hu et al., 2003a), h-thujaplicin in Mexican
cypress (Zhao and Sakai, 2003b), or momilactones in rice cell cultures (Yamaguchi et al.,
2004). In other plants, O2 is the mediator for the elicitor-induced production of plant
secondary metabolites, such as phytoalexin (furanocoumarin) accumulation in parsley cell
cultures (Jabs et al., 1997), diterpene rishitin in potato tuber (Mehdy, 1994), medicarpin in
lucerne (alfalfa) (Tang and Smith, 2001), and capsidiol in tobacco (Perrone et al., 2003).
The hydroxyl radical is also implicated in the induction of other phytoalexins (Mehdy,
1994; Kuchitsu et al., 1995).
How ROS mediates elicitor-induced production of secondary metabolites is still not
clear, but it is known that ROS induces expression of many defense genes and
secondary metabolite biosynthetic genes, such as sesquiterpene cyclases and PAL. It is
reported that ROS-caused oxidative stress may regulate the stability of some defense
gene transcripts in plant cells (Mehdy, 1994). Certainly, H2O2-mediated non-enzymatic
or enzymatic lipid peroxidation can initiate the octadecanoid pathway leading to
biosynthesis of JA and related compounds, and other oxylipins, which have been
reported to function in the induction of plant secondary metabolites (Thoma et al.,
2003). Low-energy ultrasound can transiently cause reactive oxygen species, JA, and
taxol production, just like wounding or elicitor treatment. Studies showed that ROS is
upstream of JA accumulation while both ROS and JA are key signal components in the
stimulation of taxol production in Taxus chinensis cells induced by low-energy
ultrasound (Wu and Ge, 2004).
4.5. Inositol 1,4,5-trisphosphates and cyclic nucleotides
Elicitor-induced phosphoinositide breakdown has been reported to occur in many

different plants. One of the significant phosphatidylinositol turnover paths is phosphatidyl
inositol 4,5-diphosphate (PIP2) hydrolysis by PLC. This enzyme can be activated by
[Ca2+]cyt spiking to cleave PIP2 to yield two secondary messengers, IP3 and DAG. IP3 can
mobilize Ca2+ from intracellular calcium stores like the endoplasmic reticulum, Golgi
body, or vacuole in plants (Berridge, 1993). IP3 mobilization of Ca2+ from intracellular
calcium stores to the cytosol has been reported in many plant systems (Allen et al., 1995).
Thus the IP3–Ca2+ signaling pathway is regarded to mediate the elicitor-induced
phytoalexin production. An increased PLC activity or IP3 level in the cytosol of plant
cells in response to elicitor treatment is necessary for accumulation of some defensive
secondary metabolites. For example, IP3 signaling is involved in elicitor-induced
accumulation of furanocoumarins in parsley (Renelt et al., 1993), pisatin in Pisum
sativum (Toyoda et al., 1992), 6-methoxymellein in carrot cell (Kurosaki et al., 1987),
medicarpin in lucerne cell culture (Walton et al., 1993), scoparone in lemon seedlings
(Ortega and Perez, 2001), and h-thujaplicin in the Mexican cypress (Zhao et al., 2004b).
Another group of second messengers, the cyclic nucleotides cAMP or cGMP, is also
significantly accumulated in some plant cells in response to an elicitor. Cyclic AMP has
been reported to accumulate in response to elicitor treatment and to mediate elicitor
induction of phytoalexin biosynthesis in several species, such as carrot (Kurosaki, 1997),
lucerne (Smith, 1994, and the literature cited therein), French bean (Bolwell and Cyclic,
1995), and Mexican cypress cell culture (Zhao et al., 2004a). Analogues of cAMP
including dibutyryl-cAMP also can stimulate accumulation of these phytoalexins in the
absence of elicitor. The cAMP stimulation of phytoalexin accumulation is Ca2+/K+-
dependent (Smith, 1994; Kurosaki, 1997; Zhao et al., 2004a). A growing body of
physiological, biochemical, and genetic evidence suggests that cAMP or cGMP plays an
important role in regulation of cation channels on the plasma membrane of plant cells in
response to elicitor or other stresses (Li et al., 1994; Volotovski et al., 1998; Bolwell and
Cyclic, 1995), and that cyclic nucleotide-gated cation channels play a very important role
in plant defense responses (Clough et al., 2000).
Both cyclic GMP and cyclic ADP ribose are potent Ca2+ mobilizing agents. Cyclic
GMP acts through cyclic nucleotide-gated ion channels in the plasma membrane while
cyclic ADP ribose binds to intracellular Ca2+ channels localized in endomembrane
systems and activates Ca2+ release. NO signaling (see below), which involves cyclic
nucleotides, may cross-talk with Ca2+ signaling. Cyclic GMP has been shown to stimulate
chalcone synthase gene expression and initiate anthocyanin pigment biosynthesis in
soybean (Bowler et al., 1994). Like the IP3–Ca2+ signaling pathway, cyclic nucleotides–
Ca2+ signaling may also work in stimulation of accumulation of plant secondary
metabolites.
4.6. Salicylic acid and nitric oxide
Salicylic acid (SA) is a well-known inducer of plant systematic acquired resistance

(SAR) in plant–pathogen interaction, but it is not a universal inducer for production of
plant defensive metabolites. SA quickly accumulates at the site of infection during
pathogen attack and plant hypersensitive reaction, and it spreads to other parts of the
plant to induce a wide range of defense responses. Fungal elicitors also stimulate SA
accumulation in some plant cell cultures, however, induction of most secondary
metabolites by fungal elicitors is SA-independent. This may suggest that accumulation
of plant secondary metabolites is a local response but not a systemic one. However, SA
indeed induces gene expression related to biosynthesis and production of some classes
of secondary metabolites in plants (Taguchi et al., 2001). For example, indole alkaloids
in C. roseus cell cultures can be induced by acetylsalicylic acid, an analogue of SA
(Zhao et al., 2000). Pilocarpine in jaborandi leaves (Avancini et al., 2003), alkaloids in
hairy root cultures of Brugmansis candida (Lee et al., 2001), as well as indole
glucosinolates in many plants such as oilseed rape and Arabidopsis, can be induced by
SA, MeJA and elicitors (Bennett and Wallsgrove, 1994; Kiddle et al., 1994; Brader et
al., 2001; Mikkelsen et al., 2003). In Rubia cordifolia cultures, both MeJA and SA
strongly induced anthraquinone phytoalexin production (Bulgakov et al., 2002). In hairy
root cultures of Brugmansia candida that produce the tropane alkaloids scopolamine and
hyoscyamine, SA significantly stimulates the release of both alkaloids (Pitta-Alvarez et
al., 2000). It also stimulates related biosynthetic gene expression in Scopolia parviflora
(Kang et al., 2004). Even though both indole glucosinolates and the typical phytoalexin
camalexin in Arabidopsis are toxic compounds beneficial for plant defense, and all are
derived from the same biosynthetic pathway (Brader et al., 2001), the production of
camalexin is SA-independent.
Nitric oxide (NO) has well-known biological functions in mammals, but it is only
recently recognized as a signal compound in plants. NO can be generated in plants either
non-enzymatically via light-mediated conversion of NO2 by carotenoids, or enzymati-
cally from NO2 by NADPH nitrate reductases (Foissner et al., 2000; Neill et al., 2002).
A mammalian-type NO synthase (NOS) was found in several plants (Neill et al., 2002),
and it is apparent that NO serves as a signal for plant growth, development, and defense
(Delledonne et al., 1998; Klessig et al., 2000; Neill et al., 2002). The Avr factors from
pathogens stimulate NO production, and this NO promotes disease resistance of the
plants, collaborating with ROS in the oxidative burst (Delledonne et al., 1998).
Transcriptional profiling showed that NO treatment induces some stress- and disease-
related signal transduction component genes and defense genes, implying that the NO
signal pathway(s) may be related to secondary metabolism (Aziz et al., 2003). Indeed,
some studies have shown that NO is involved in production of secondary metabolites. In
ginseng cell cultures, fungal elicitors stimulate saponin production, and this is partially
mediated by NO, with NO biosynthesis being induced by the fungal elicitor (Hu et al.,
2003b).
It was also shown that phytoalexin production in soybean is partially mediated by NO
synthase (Modolo et al., 2002). Production of rishitin, a sesquiterpene phytoalexin in
potato tuber, is induced by NO treatment, but unlike other cases, a cell wall component
elicitor effectively stimulates the phytoalexin biosynthesis without being affected by a NO
biosynthesis inhibitor. This shows that elicitor-induced O2 triggers phytoalexin biosyn-
thesis independent of the NO signal (Noritake et al., 1996). Victorin-induced
avenanthramide A production involves the NO signal, Ca2+-calmodulin, and a protein
kinase (Yang et al., 2004). A fungal elicitor-induced accumulation of avenanthramides in
Avena sativa involves NO (Yang et al., 2004). However, in C. lusitanica cell culture, NO
does not induce h-thujaplicin accumulation, even though the elicitor induces NO
production (Zhao et al. unpublished results). It is recently shown that elicitor-induced
NO production is essential for triggering catharanthine production in C. roseus cell culture
(Xu and Dong, in press).
4.7. Jasmonate pathway
Jasmonic acid and its related compounds (all called JA signals here) have long been
observed to be transducers of elicitor signals for the production of plant secondary
metabolites (Farmer et al., 2003). Exogenous application of JA signaling compounds,
including jasmonic acid, methyl jasmonate, as well as their conjugated compounds to the
plant cell culture or intact plant stimulates biosynthesis of secondary metabolites (Fig. 3)
(Gundlach et al., 1992; Mueller et al., 1993; Tamogami et al., 1997). Induction of plant
secondary metabolite accumulation by the JA signaling pathway is not limited to certain
types of metabolites, but includes a wide variety of plant secondary products including
terpenoids, flavonoids, alkaloids, and phenylpropanoids plus many other types of
secondary metabolites in most plants, as shown in Table 2. Therefore, the JA signaling
pathway is generally regarded as an integral signal for biosynthesis of many plant
secondary products. Also because many elicitors stimulate endogenous JA biosynthesis in
plants, the JA signaling pathway is regarded as a transducer or mediator for elicitor
signaling, leading to accumulation of plant secondary metabolites (Gundlach et al., 1992;
Mueller et al., 1993). Elicitor-induced indole alkaloids in C. roseus (Menke et al., 1999),
phytoalexin biosynthesis in rice (Nojiri et al., 1996), indole glucosinolates biosynthesis in
Arabidopsis (Brader et al., 2001), and h-thujaplicin in Mexican cypress cell culture (Zhao
et al., 2004c), all support the idea that JA signaling is a mediator of secondary product
accumulation.
Apoplastic Elicitors or avirulence determinants
O2 - • O2
H2 O2 Ion channel Plasma membrane
NADPH oxidase Lipases (PLA) receptor

receptor
H2 O2 Ca 2+
G-proteins
α-Linolenic acid Lysophospholipids

Cytosol Reactive oxygen species
Lipoxygenase HPL
13(S)-Hydroperoxyoctadecatrienoic acid 12-oxo-9- 3(Z )-hexenal
dodecenoic acid +
Lipid peroxidation Allene oxide synthase
12,13(S)Epoxyoctadecatrienoic acid
G1-phytoprostanes
Allene oxide cyclase
E1-phytoprostanes
A1-phytoprostanes Traumatin 2(E )-hexenal
12-Oxophytodienoic acid
B1-phytoprostanes 12-Oxo- PDA reductase
F1-phytoprostanes
Dihydro-12-oxophytodienoic acid
Cyclic oxylipins 3 x β -oxidation
Defense gene expression
Jasmonic acid
Gene expression
Carboxyl methyltransferase
Methyl jasmonate Biosynthesis of
Biosynthesis of defensive defensive secondary
secondary metabolites metabolites
Fig. 3. Schematic illustration of biosynthetic pathway of JA related and other oxylipins, and their mediation of
elicitor signal transduction leading to accumulation of plant secondary metabolites. Elicitors or avirulent
determinants are perceived by specific receptors localized to the plasma membrane (or in the cytoplasm). The
activated receptors may then activate ion channels and G-proteins and then activate phospholipases (such as
PLA2), through Ca2+ signaling or by G-protein coupling. Phospholipases hydrolyze phospholipids, such as PC,
into fatty acid and lysoPC; the former can be a precursor for biosynthesis of JA and related oxylipins via the
octadecanoid pathway, or peroxidized by reactive oxygen species to produce another class of pentacyclic oxylipin
phytoprostanes. All these pentacyclic oxylipins can activate biosynthesis of plant secondary metabolites. In
addition, the oxidized fatty acids HPOD (13S-hydroperoxy-9(Z),11(E)-octadecadienoic acid.) and HPOT (13S-
hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid) by LOX can also be used by HPL-pathway for C6-aldehyde
and C12 oxoacid generation. Hexenal (from HPOD) or cis-3-hexenal (from HPOT), as well as traumatin (from
C12 oxoacid), can act as defense signals to induce accumulation of plant defensive secondary metabolites.
4.8. Ethylene pathway
Ethylene is a phytohormone that regulates a wide range of plant processes, from growth
and development to defense responses. Ethylene production can be induced by various
stresses, such as wounding, ozone, microbial pathogen and insect attack, as well as small
molecule elicitors. While both JA and ethylene signaling pathways are essential for plant
defense responses against those stresses, ethylene is not a common signal for induction of
plant secondary metabolism. There are only a limited number of instances where
accumulation of plant secondary metabolites can be affected by ethylene, and the effects
can be either positive or negative (Table 2). For example, ethylene treatment increases
flavonoid, anthocyanin and stilbenoid production via up-regulating their biosynthetic
genes in grape (Vitis vinifera) cell cultures (Chung et al., 2001; El-Kereamy et al., 2003).
Elicitor-induced production of some secondary metabolites requires ethylene as an integral
signal, but the concentration of ethylene in the culture is critical for its effects. Ethylene at
high concentrations may inhibit the biosynthesis of secondary metabolites, whereas at low
302
Table 2
Elicitor signal transduction involved in production of plant secondary metabolites
Plants Elicitors Signaling components Targeted biosynthetic Targeted secondary References
enzyme or gene metabolites
Allium cepa Polysaccharide Ca2+, cAMP, ROS Tcibulin1, tcibulin2 Kravchuk et al.,
2003

Arabidopsis thaliana Fungal elicitor, MeJA, JA, SA AS, CYP79, CYP83, Indole glucosinolates, Brader et al.,
SA; JA-independent AS, PAT, PAI, InGPS, camalexin 2001; Mikkelsen
TSA et al., 2003
Avena sativa (Pc-2) Elicitor (Puccinia Ca2+, NO, protein PAL, C4H, 4CL, HHT, Avenanthramides Yang et al., 2004
cornoata), phytotoxin kinases CCoAMT
victorin
Catharanthus roseus Yeast elicitor, MeJA Ca2+, H2O2, JA, protein TDC, STR, SGD, D4H, Ajmalicine, Menke et al.,
kinases DAT catharanthine, vindoline 1999; Zhao et al.,
2001b
Citrus limon Alternaria altenata G-protein, Ca2+, IP3, PAL Umbelliferone; Ortega and Perez,
protein kinases scoparone 2001; Ortega
et al., 2002
Coleus blumei Yeast elicitor MeJA 4CL, PAL, C4H Rosmarinic acid Szabo et al., 1999;
Petersen and
Simmonds, 2003
Cupressus lusitanica Oligosaccharide, MeJA Ca2+, H2O2, JA, IP3, IPP isomerase, GPP h-thujaplicin Zhao et al., 2001c,
G-proteins synthase, monoterpene 2004a; Zhao and
synthase Sakai, 2003a,b
Daucus carota Fungal elicitor, from cAMP, Ca2+, IP3, 6-hydroxymellein 6-methoxymellein; Kurosaki, 1997;
Pythium G-proteins, ethylene, JA synthase, PAL 4-hydroxybenzoic acid Kurosaki et al.,
aphanidermatum 1987, 2001
Eschscholtzia MeJA, Fungal elicitor JA, cyclopentenone BBE, CFS, SPS, MSH, Benzophenanthridines, Blechert et al.,
californica isoprostanes PPH sanguinarine 1995; Thoma
et al., 2003
Glycine max, h-glucan, MeJA, Ca2+, H2O2, MAP CHS, CHR, DP6H, F6H Glyceollins, apigenin, Graham and
Glycine max 12-oxo-PDA; Gluta- Kinase, NO daidzein, genistein, Graham, 1996;
thione luteolin Modolo et al.,
2002
Glycyrrhiza glabra MeJA JA bAS, SQS, UGSGT, Soyasaponin Hayashi et al.,
PKR 5-deoxyflavonoid 2003
Hyoscyamus niger, Fungal elicitor; MeJA, Ca2+, MeJA, SA PMT Hyoscyamine, Singh et al., 1998;
Hyoscyamus muticus SA solavetivone, rishitin, Kang et al., 2004
lubimin, scopolamine
Hypericum perforatum Mannan, Colletotrichum MeJA SA PKS Hypericin, Kirakosyan et al.,
gloeosporioides pseudohypericin, 2000; Walker
infection hyperforin et al., 2002

Lithospermum Polysaccharides, fungal JA HBG Shikonin, rosmarinic Mizukami et al.,
erythrorthizon elicitor, MeJA acid 1993; Yazaki
et al., 2002
Lycopersicon Fungal elicitor, systemin, JA, H2O2, PAL Scopoletin Thoma et al., 2003
esculentum MeJA cyclopentenones
Medicago sativa Fungal elicitor cAMP, Ca2+, IP3, O-2 PAL, IFR, DMID, VR Medicarpin sativan Walton et al.,
Vericillium alboatrum 1993; Smith,
1994; Tang and
Smith, 2001
Nicotiana tabacum, Cryptogein, cellulase, Ca2+, H2O2, JA, FPPS, 5eAS, ADH, SGT Capsidiol, debneyol, Taguchi et al.,
Suspension culture, MeJA, marine brown cyclopentenones, SA, scopoletin and its 2001; Lecourieux
Nicotiana attenuata algae auxin, and ethylene glucoconjugate scopolin, et al., 2002;
nicotine Thoma et al., 2003
Oryza sativa N-acetylachitoheptaose, G-protiens, Ca2+ influx, PAL, N7M Momilactones, Nojiri et al., 1996;
MeJA, sphingolipid H2O2, JA, ethylene sakuranetin, Suharsono et al.,
cerebrosides phytocassans 2002; Umemura
et al., 2002
Panax ginseng Oligogalacturonic acid H2O2, JA, Ca2+, NO AS, SQS, SQE Saponin Hu et al., 2003a,b
Petroselinum crispum 13-pep glycoprotein, H2O2, Ca2+, O2 , protein PAL, 4CL, CHS, BMT Furanocoumarin, Dittrich et al.,
12-oxo-phytodienoic kinases butylidene phthalides, 1992; Hahlbrock
acid (OPDA) flavonoid apiin et al., 2003
Pisum sativum Fungal IP3, DAG PAL Pisatin Toyoda et al.,
1992, 2000
Rauvolfia canescens Yeast elicitor, MeJA JA, 12-oxo-PDA SGD Raucaffricine Gundlach et al.,
1992; Parchmann
et al., 1997
303
(continued on next page)
304
Table 2 (continued)
Plants Elicitors Signaling components Targeted biosynthetic Targeted secondary References
enzyme or gene metabolites
Sanguinaria canadensis Elicitor, acetylsalicylic G-proteins, Ca2+, protein DHBO, THB-NMT, Sanguinarine, Mahady et al.,
acid, MeJA, ABA kinase THC-NMT chelerythrine 1998

Solanum tuberosum MeJA, arachidonic acid Ca2+, H2O2, JA, protein HMGR1, HMGR2 Rishitin, lubimin, Choi et al., 1994;
from Phytophythora kinase, O2 , NO phytuberin and Noritake et al.,
infestans phytuberol 1996
Taxus brevifolia, Taxus Fungal elicitor, MeJA, Ca2+ PAL, GGDPS, Taxol, baccatin III, Yukimune et al.,
cuspidata ABA taxadiene synthase 10-deacetylbaccatin III, 1996; Hefner
cephalomannine et al., 1998; Luo
et al., 2001
Vitis vinifera suspension MeJA, ethylene, Ca2+, medium CHS, STS PAL, STS Stilbene, piceids, Puig et al., 1995;
cells laminarin from alkalinization, ROS, CHS, F3H resveratrol, and Aziz et al., 2003;
Phytophthora sojae MAPK e-viniferin, anthocyanins El-Kereamy et al.,
(delphinidin, cyanidin, 2003
etc.)
Abbreviations for genes and enzymes: 4CL, 4-coumarate: CoA ligase; 5eAS, 5-epi-aristolochene synthase; ADH, aristolochene/deoxy-capsidiol hydroxylase; AS,
anthranilate synthase; bAS, h-amyrin synthase; BBE, berberine bridge enzyme; BMT, SAM-bergaptol-O-methyltransferase; C4H, cinnamate 4-hydroxylase; CCoAMT,
caffeoyl-CoA 3-O-methyltransferase; CFS, (S)-cheilanthifoline synthase; CHR, chalcone reductase; CHS, chalcone synthase; CYP79, cytochrome P450 CYP79; CYP83,
cytochrome P450 CYP83;. D4H, desacetoxyvindoline 4-hydroxylase; DAT, deacetylvindoline 4-O-acetyltransferase; DHBO, dihydrobenzophenanthridine oxidase;
DMID, 7,2V-dihydroxy-4V-methoxyisoflavanol dehydratase; DP6H, dihydroxypterocarpan 6a-hydroxylase; F3H, flavanone 3-hydroxylase; F6H, flavonoid 6-hydoxylase;
FPPS, farnesyl pyrophosphate synthase; GGDPS, geranylgeranyl diphosphate synthase; GPP, geranyl pyrophosphate synthase; HBG, 4-hydroxybenzoate 3-
geranyltransferase; HHT, hydroxyanthranilate hydroxycinnamoyltransferase; HMGR, 3-hydroxy-3-methylglutaryl coenzyme A reductase; IFR, isoflavone reductase;
IPP isomerase, isoprepenyl pyrophosphate isomerase; InGPS, indole-3-glycerolphosphate synthase; MSH, (S)-cis-N-methylstylopine 14-hydroxylase; N7M, naringenin 7-
o-methyltransferase; N8DT, naringenin 8-dimethylallyltransferase; PAI, phosphoribosyl-anthranilate isomerase; PAL, phenylalanine ammonia lyase; PAT, phosphor-
ibosylanthranilate transferase; PKR, polyketide reductase; PKS, polyketide synthase; PMT, putrescine: SAM N-methyltransferase, PPH, protopine 6-hydroxylase; SGD,
strictosidine h-d-glucosidase; SGT: scopoletin glucocyltransferase SPS, (S)-stylopine synthase; SQE, squalene epoxidase SQS, squalene synthase; STR, strictosidine
synthase; STS, stilbene synthase; TDC, tryptophan decarboxylase; THB-NMT, tetrahydroberberine-N-methyltransferase THC-NMT, tetrahydrocortisine-N-methyl-
transferase TSA, tryptophan synthase UGSGT, UDP-glucuronic acid: soyasapogenol h-glucuronosyltransferase; VR, vestitone reductase.
concentration it promotes the elicitor-induced production (Pan et al., 2000; Zhao et al.,
2004a). Oligosaccharides can potentiate methyl jasmonate-induced production of taxol, an
important anticancer medicine, in Taxus canadensis (Linden and Phisalaphong, 2000).
Their synergistic effects on taxol production are further regulated by ethylene, which is
produced by the cell cultures in response to elicitation. Ethylene is required for
phytoalexin production in carrot (Fan et al., 2000). It also plays essential roles in
herbivore-induced emission of sesquiterpene and indole volatiles in many herbivore-
attacked plants, such as corn and tomato (Schmelz et al., 2003a,b; Paré et al., 1998).
Increased ethylene supply (18 ppm) in bioreactor can significantly improve taxane
taxuyunnanine C production in T. chinensis suspension cells (Pan et al., 2000), suggesting
a key role of ethylene in scaleup of taxane production.
However, ethylene in the headspace of culture vessels negatively affects anthocyanin,
anthocyanidin, and carotenoid accumulation in Vaccinium pahalae cell culture (Shibli et
al., 1997). Also, in Salvia miltiorrhiza hairy root culture, treatment with Ag+, an ethylene
action inhibitor, induces production of three diterpenoid tanshinones (Zhang et al., 2004).
Similarly, Ag+ significantly increases scopolamine release and accumulation of scopol-
amine and hyoscyamine in the roots (Pitta-Alvarez et al., 2000). This implies that ethylene
acts as an inhibitor of accumulation of the above substances. Clearly, ethylene interacts
with JA or elicitor signaling to modulate the production of secondary metabolites induced
by JA or elicitor.
4.9. ABA signaling
ABA is a well-known senescence-triggering plant hormone. ABA plays important

roles throughout plant life and development, affecting seed germination, plant growth,
senescence and plant stress responses. Especially under some stresses like hyper- or
hypo-osmotic stress, salt, cold and drought stress, ABA acts as an important signal
molecule to regulate expression of sets of defense genes. ABA is also reported to
regulate biosynthesis of secondary metabolites in some plant cell cultures. Indole
alkaloids in C. roseus cell culture can be stimulated by ABA (Zhao et al., 2000). ABA
also stimulated benzophenanthridine alkaloids production in bloodroot cell culture
(Mahady et al., 1998). ABA may also be a signal for osmotic stress-induced
accumulation of plant secondary metabolites, because production of many plant
secondary metabolites can be stimulated by osmotic stresses, such as sorbitol, mannitol,
sucrose, and salt stress in a culture medium, while those stresses generally stimulate
ABA biosynthesis. For example, both osmotic stress and ABA can stimulate production
of indole alkaloids in C. roseus cell culture (Zhao et al., 2000). ABA also stimulates
taxol production in Taxus spp cell culture (Luo et al., 2001); so do high concentrations
of sucrose and mannitol. The osmotic stress induces hydroxycinnamic acid amides and
tryptophan accumulation in barley (Hordeum vulgare L.) (Ogura et al., 2001). JA and
ABA also induce the production of these compounds, suggesting that ABA and JA may
mediate osmotic stress-induced secondary metabolism. However, the profiles of
compounds induced by application of JA and ABA show that JA exhibits stronger
activity for hydroxycinnamic acid amides than ABA, while ABA is more active in
tryptophan accumulation. Morever, the major hydroxycinnamic acid amide in JA-treated
leaves was p-coumaroylagmatine but in ABA-treated leaves was p-coumaroyl-3-

hydroxyagmatine, suggesting that JA and ABA work in distinct ways (Ogura et al.,
2001). On the other hand, ABA negatively regulates the accumulation of some
secondary metabolites. ABA inhibits soybean flavonoid accumulation (Graham and
Graham, 1996), most probably by inhibiting flavonoid biosynthetic gene expression
(Kim et al., 2003).
5. Lipid signaling
5.1. Oxylipins
Oxylipins are classes of biologically active compounds that are generated by oxidative
catabolism of polyunsaturated fatty acids (adding oxygen to the 9 or 13 position of the C18
chain of linoleic and linolenic acids) by the coordinated action of lipases, lipoxygenase,
and a group of cytochromes P450 (CYP74 family), including allene oxide synthase
(AOS), and hydroperoxide lyase (HPL). Biosynthesis of oxylipins in plants most often
results from mechanical injury, herbivore and pathogen attack, and other environmental
stresses, and thus confers a wide range of defense responses of plants. The best studied
oxylipin compounds are jasmonic acid (JA), and its methyl ester (MeJA), or biologically
active cyclooxylipins such as OPDA (12-oxo-PDA), from the AOS-dependent octadeca-
noid pathway (Creelman and Mullet, 1997). A non-enzymatic oxidation of 9- or 13-
linoleic and linolenic acids also leads to the production of another group of cyclo-
pentenone oxylipins, which also show biological activities, such as induction of defense
gene expression and phytoalexin biosynthesis (Fig. 3) (Thoma et al., 2003). The
production of this group of cyclopentenone oxylipins can be triggered by ROS.
Peroxidation products of linolenic acid were isolated from tobacco leaves and cell
cultures; these molecules proved able to stimulate defense response genes and phytoalexin
accumulation in tobacco and bloodroot cell cultures (Thoma et al., 2003). This is the first
evidence for lipid peroxide signals from ROS-induced lipid peroxidation showing
physiological functions. In parallel to the AOS-dependent octadecanoid pathway leading
to biosynthesis of cyclopentenone oxylipins, the HPL-dependent formation of C-6
aldehydes or their alcohols and C-12 omega-keto-fatty acids leads to other groups of
universal oxylipins. These widespread oxylipins play various other roles in plants (Bate et
al., 1998; Blée, 1998). For instance, a C12 product derived from linolenic acid is the
precursor of traumatin, which triggers cell division and plays a role in wound healing. The
C6 aldehyde products also play an important role in defenses against microbial pathogens
and insects (Croft et al., 1993) and C6 volatiles of the HPL pathway can induce
phytoalexin accumulation (Zeringue, 1992).
The biosynthetic pathway of JA-related cyclopentenone oxylipins via the octadeca-
noid pathway or non-enzymatic pathway, as well as that of HPL-derived oxylipins is
shown in Fig. 3. These pathways can be activated by elicitors (caused by pathogens, or
insect attacks), wounding, ozone and other biotic or abiotic stresses. Glycoprotein and
cryptogein elicitors down-regulate phosphatidylcholine (PC)–PLC and up-regulate PLA,
resulting in a rapid accumulation or metabolism of lipid molecules, such as DAG, PA
and lysoPC, in parsley and tobacco cell cultures in the response to elicitor (Scherer et
al., 2002). Biosynthesis of the JA family oxylipins in plants has been extensively
studied, and most enzymes that catalyze each biosynthetic step have been characterized
(Schaller, 2001). However, the enzymes for the first step are still not completely
understood.
Several forms of stress and developmental cues regulate key first steps of oxylipin
biosynthesis, such as phospholipases, especially PLA. These enzymes release fatty-acid
precursors from membrane lipids as part of the pathogen attack inducing a hypersensitive
response. Recent studies showed that the enzymes in JA biosynthesis may use these
phospholipid derived fatty acids as substrates. PLA is the most likely lipase involved in JA
biosynthesis, and elicitor activation of PLA2 activity has been observed in tomato, potato,
soybean and California poppy cell culture (Meijer and Munnik, 2003, and literature cited
therein). A good correlation between the elicitor-induced PLA activation and induction of
benzophenanthridine alkaloid by PLA enzymatic products (lysoPC and linolenic acid) is
well documented in elicitor-induced alkaloid accumulation by California poppy cell
cultures (Roos et al., 1999).
Besides JA and MeJA, other pentacyclic oxylipin intermediates also have biological
activity, and may serve equivalent roles. For example, 12-oxo-PDA, even more strongly
than JA, induces phytoalexin biosynthesis in soybean and parsley cell cultures (Dittrich et
al., 1992; Fliegmann et al., 2003). Elicitor-induced 12-oxo-PDA levels in some plant
species are even higher than JA levels, and analogues of 12-oxo-PDA also induce alkaloid
production, suggesting that these pentacyclic oxylipins have the same biological function
as JA or MeJA (Blechert et al., 1995; Parchmann et al., 1997). An Arabidopsis JA-
deficient mutant opr3 can express all JA-dependent genes in response to wounding
because the mutant plants can accumulate pentacyclic oxylipin intermediates (Stintzi et al.,
2001).
5.2. Lipid messengers
As a ubiquitous enzyme family, phospholipases play various roles in stress responses

(Wang, 2002; Meijer and Munnik, 2003). Three main classes of phospholipases, PLA2,
PLC, and PLD, not only participate in or regulate oxylipin signaling pathways, but also
generate other lipid products that can act as messengers influencing multiple cellular
processes (Fig. 4). PLA2 hydrolyzes phospholipids (such as PC) into a fatty acid and
lysophospholipid (such as lysoPC). The fatty acid can be a precursor for oxylipin
biosynthesis, and lysoPC may be involved in multiple cellular processes (Meijer and
Munnik, 2003). One important finding on functions of lysoPC is that it can activate
H+-ATPase in the tonoplast and cause cytoplasmic acidification, which is shown to
activate defense responses and phytoalexin production (Viehveger et al., 2002). Besides
producing IP3 that acts as an important second messenger to trigger Ca2+ release from
internal Ca2+ stores, PLC when activated by many sorts of stresses also produces DAG.
In mammalian cells, DAG can activate protein kinase C (PKC) and other kinase cascades.
There are few reports on the existence and functions of PKC homologues in plants. In the
Arabidopsis genome there are no PKC homologs. However, DAG-dependent PKC
homologous genes have been isolated from other plants, and shown to function in plant
Elicitors or avirulent determinants

K+
receptor plasma membrane lipids receptor receptor
PC PC
G-protein Ca2+ PLA Ca2+ PLD G-protein PLC Ca2+

or effector or effector Ca2+
Choline IP3 PIP2

Fatty acid + Lyso PC + PAP +
PA DAG Ca2+ release
from inner
DAGK
H+ pump Ca2+ stores
Protein kinases
Cytoplasmic Ion channels
Oxylipins Protein kinases Ca2+ spikes
acidification ROS
Defensive gene expression
Biosynthesis of defensive plant secondary metabolites

Fig. 4. Lipid messengers derived from hydrolysis of the plasma membrane. Proteins in the plasma membrane and
the plasma membrane itself can perceive various stresses at first. Phospholipase A (PLA), Phospholipase C
(PLC), and Phospholipase D (PLD) are most often activated by various environmental and hormonal stresses, and
hydrolyze membrane phospholipids, such as phosphatidylcholine (PC) and PIP2, to produce various lipid signal
molecules. Free fatty acid products from PLA action can be used for oxylipin signaling pathway, and another
hydrolysis product of PC by PLA, lysophosphatidylcholine (lyso PC), has multiple roles in cellular processes,
including activation of proton pumping from endomembrane systems to the cytosol that will induce cytoplasmic
acidification and activate biosynthesis of secondary metabolites. PLC hydrolyzes PIP2 into DAG and IP3, which
can activate protein kinases and mobilize Ca2+ from intracellular Ca stores. As an emerging messenger, PA can be
produced either by PLD hydrolyzing phospholipids or through DAG phosphorylation by DAG kinase (DAGK),
while PA can also be converted into DAG by PA phosphatase (PAP). PA may directly or indirectly affect plant
secondary metabolism.
defense responses including production of defensive secondary metabolites (Subrama-

niam et al., 1997; Chandok and Sopory, 1998; Vasconsuelo et al., 2003, 2004).
PLD hydrolyzes phospholipids into PA and a free head group (Fig. 4); for example,
PLD hydrolyzes phosphatidylcholine into PA and choline, or phosphatidylserine into PA
and serine (Wang, 2002). PA has emerged as a messenger involved in many cellular
processes, such as ROS generation and protein kinase cascade (Wang, 2002; Munnik,
2001). Elicitor N-acetylchitooligosaccharide induced phytoalexins in rice suspension cell
cultures may be mediated by H2O2 and PLD (Yamaguchi et al., 2004). It is also suggested
that H2O2-inducible PLD activation enhances signal transduction leading to phytoalexin
biosynthesis in rice cells (Yamaguchi et al., 2004). PA can be dephosphorylated into DAG
by PA phosphatases while DAG can be converted into PA by DAG kinase (Munnik,
2001). Elicitor or pathogen-triggered PLC and PLD activation as well as conversions

between PA and DAG have been observed in some plant systems (Meijer and Munnik,
2003; de Jong et al., 2004). In pea, pathogen-induced DAG accumulation seems to be
necessary for pisatin accumulation because inhibition of DAG conversion into PA can
potentiate the production of pisatin (Toyoda et al., 2000). There is clearly a complex cross-
talk between these pathways that use PA and DAG.
5.3. Other lipid elicitors
In addition to the above plant-derived lipid signals listed in Table 1, some lipophilic
substances such as amphotericin or other lipophilic products from bacteria or fungal
pathogens could stimulate phytoalexin accumulation (Jabs et al., 1997). Arachidonic acid
from potato pathogen Phytophthora infestans elicited sesquiterpene phytoalexin accumu-
lation in potato tuber. It is thought that a metabolite of arachidonic acid generated by
lipoxygenase, but not arachidonic acid itself, is the elicitor of phytoalexin production
(Boller, 1995). Arachidonic acid also induced indole alkaloid accumulation in C. roseus
cell culture (Zhao et al., 2000). Comparison of a lipid elicitor ergosterol and a
proteinaceous elicitor cryptogein showed that, even though both ergosterol and cryptogein
stimulated medium alkalinization, oxidative burst, and phytoalexin synthesis in tobacco
(Nicotiana tabacum) cell cultures, ergosterol induced a weaker oxidative burst and pH
change but a higher phytoalexin accumulation than cryptogein (Kasparovsky et al., 2003).
Both cryptogein and ergosterol elicit a Ca2+ spiking, but cryptogein stimulates mainly an
apparent external Ca2+ influx whereas ergosterol stimulates mainly the mobilization of
internal calcium stores mediated by IP3 and serine/threonine protein kinases (Kasparovsky
et al., 2003). Obviously, cryptogein is perceived at the plasma membrane, whereas
ergosterol is most probably perceived inside of the cells, which may contribute to these
differences.
Some other complex lipid compounds derived from metabolism in plants also act as
signal molecules for various cellular processes. Pathogen or plant derived sphingolipid
cerebroside A and C are emerging as signals in plants; they have been reported to stimulate
phytoalexin in rice suspension culture, via a Ca2+ signaling pathway (Koga et al., 1998).
Lipid elicitors, such as syringolides, can pass through the plasma membrane and be
perceived by cytoplasmic receptors (Ji et al., 1998). As shown in Fig. 4, the lipid elicitor
signaling pathway may function through Ca2+ signaling as well as the other common
pathways.
6. Cross-talk of signaling pathways
Although above signaling components are reported to play roles in elicitor signaling
pathway leading to production of secondary metabolites, it is likely that only some of them
are involved in the process in a particular plant or for production of a particular secondary
metabolite. More and more studies show that one defensive cellular process usually is not
regulated by only one signaling pathway, but typically involves two or more signaling
pathways, which collaboratively regulate the process. Cross-talk among multiple signaling
pathways is an important mechanism in the plant signal transduction networks (Fig. 4). It
is believed that cross-talk among multiple signal transduction pathways enables plants to
activate different sets of genes temporally and spatially in different situations against a
wide variety of stresses. Some examples follow.
6.1. Elicitor and JA pathways
Even though JA is a major signal for plant secondary metabolism, interactions of JA

paths with other signals are often observed. The most significant and complicated is the
relationship between elicitor and JA signaling pathways. Though the JA signaling pathway
is usually regarded as a transducer for elicitor signal transduction leading to biosynthesis
of plant secondary compounds (Gundlach et al., 1992; Mueller et al., 1993), more recent
studies show that JA stimulated accumulation of plant secondary metabolites does not
fully overlap with elicitor-induced production of secondary metabolites, either in species
of metabolites, or in stimulation strength and patterns. Moreover, some data reject the idea
that JA signaling is an obligatory mediator of elicitor-induced accumulation of plant
secondary metabolism, since production of some secondary metabolites is not stimulated
by JA signaling (Chen and Chen, 2000).
For example, phytoalexin induction by h-glucan in parsley cell culture does not require
JA signaling but involves ROS, even though h-glucan indeed induces JA biosynthesis
(Hahlbrock et al., 2003). In soybean cell culture, an elicitor induces phytoalexin but does
not induce JA biosynthesis. Therefore, JA signaling does not mediate elicitor signal
transduction leading to phytoalexin production, even though MeJA or its precursor OPDA
indeed induce the phytoalexin accumulation (Fliegmann et al., 2003). In Glycyrrhiza
glabra cell culture, 5-deoxyflavonoid biosynthesis and the related genes for polyketide
reductase enzymes are stimulated by both yeast elicitor and MeJA. However, for
soyasaponin biosynthesis and its biosynthetic genes, h-amyrin synthase, squalene
synthase, and UDP-glucuronic acid: soyasapogenol B glucuronosyltransferase, there is
up-regulation by MeJA and down-regulation by yeast elicitor (Hayashi et al., 2003). These
results show that yeast elicitor and MeJA differentially regulate biosynthesis of different
target compounds in the same plant. In tobacco cell cultures, both MeJA and cellulase
elicitor stimulated capsidiol accumulation and its biosynthetic gene expression, but elicitor
induced a strong and transient expression of 5-epi-aristolochene synthase, the aristolo-
chene/deoxy-capsidiol hydroxylase gene, and enzyme activity, while MeJA only induced a
moderate and much delayed gene expression and enzyme activity. Moreover, MeJA
induced expression of at least two sesquiterpene cyclase genes, including one that the
elicitor did not induce (Mandujano-Chavez et al., 2000).
The differences between JA and elicitor signaling-induced secondary metabolite
accumulation are also observed in Salvia miltiorrhiza cell cultures. MeJA effectively
induced phenolic acids such as lithospermic acid B, and showed little effect on tanshinone
biosynthesis. Yeast elicitor induced more tanshinone, such as cryptotanshinone, than
phenolic compounds such as rosmarinic acid. But in crown gall cultures, MeJA inhibited
elicitor-induced tanshinone formation whereas in hairy root cultures yeast elicitor and
MeJA synergistically induced tanshinone production (Chen and Chen, 2000). Also, in
Hyoscyamus muticus root culture, MeJA alone could not induce sesquiterpene production,
but MeJA induced high levels of sesquiterpenes in wounded cultures. Also, MeJA and
fungal elicitor both induced sesquiterpenes but with different quantitative and qualitative
compositions (Singh et al., 1998). Fungal elicitation induced more lubimin and less
solavetivone production whereas MeJA induced predominantly solavetivone and a little
lubimin production, suggesting that MeJA and elicitor favor activation of different
enzymes, resulting in earlier metabolite solavetivone and subsequent metabolite lubimin
accumulation (Singh et al., 1998). All these observations suggest that exogenous
application of MeJA or JA induced a subset of secondary metabolite biosynthetic genes,
which may modulate expression of genes and accumulation of compounds induced by
elicitors.
6.2. JA and ethylene pathways
Even though ethylene itself induces production of a few plant secondary metabolites,
it most often interacts with the JA signaling pathway, leading to secondary metabolite
production. Interaction of JA and ethylene signaling pathways is one of the most
significant interactions known so far, which influences many aspects of plant develop-
ment and defense responses, like in plant–microbe interaction, plant–insect interaction,
wounding or after exposure to ozone. It also affects accumulation of plant secondary
metabolites. JA signaling can lead to ethylene production in many plants such as tomato,
Arabidopsis, tobacco, and suspension cells of Taxus spp and C. lusitanica, while
ethylene apparently stimulates JA production only in some plants such as tomato and
Arabidopsis (Xu et al., 1994; Mirjalili and Linden, 1996; Zhao et al., 2004c). In many
cases, JA and ethylene cooperatively regulate defense responses. Ethylene enhances JA-
induced production of taxol in Taxus spp cell cultures (Mirjalili and Linden, 1996) and of
h-thujaplicin in C. lusitanica cell cultures (Zhao et al., 2004c). Ethylene enhances the JA
induced gum production in tulip shoots (Saniewski et al., 1998). In corn, synergistic
stimulation of volatile emission was also observed with JA and ethylene (Schmelz et al.,
2003c).
In other cases, however, ethylene and MeJA stimulate different sets of defense genes,
and their effects antagonize each other. Ethylene suppresses JA induction of gene
expression for nicotine biosynthesis in tobacco (Shoji et al., 2000). Even in the same
plant, interaction patterns of JA and ethylene signaling can vary with different target
responses. In tobacco, JA and ethylene act antagonistically in nicotine biosynthesis,
whilst for insect-induced sesquiterpene volatile emission, no significant interaction can
be observed even though both JA and ethylene pathways are required (Kahl et al., 2000;
Shoji et al., 2000). MeJA or elicitor-induced JA biosynthesis is required for indole
glucosinolates in Arabidopsis but is not affected by the ethylene signaling pathway
(Brader et al., 2001). An insect-derived elicitor, volicitin, stimulates volatile emission,
and exogenous ethylene can synergize volicitin-induced volatile emission from maize
(Schmelz et al., 2003b). The evidence suggests that interactions between insect-derived
elicitors and insect-induced ethylene are critical in the stimulation of volatile emission
(Schmelz et al., 2003a,b).
One of the practical reasons for understanding the influence of JA and ethylene
signaling on production of plant secondary metabolites, is that both MeJA and ethylene are
volatile components in the headspace of plant cell culture vessels. Thus there may be some
uncertainty of the comparability of in vitro and in vivo conditions. In an intact plant air
exchange generally will be greater than in a culture vessel and effective concentrations in
the plant are difficult to determine.
6.3. JA and other pathways
Cross-talk among some signaling pathways is species-specific or case-specific. SA and

related compounds are potent inhibitors of JA biosynthesis or signaling pathways. In many
cases, SA inhibits JA signaling pathway for secondary metabolite production (Maleck et
al., 2000; Zhao et al., unpublished results). A rice isoflavone reductase-like gene (OsIRL)
is induced by fungal elicitor or MeJA, but is strongly suppressed by SA and ABA (Kim et
al., 2003). This antagonistic interaction may act through a group of transcription factors
(Li et al., 2004). For production of indole glucosinolates in many plants, both the JA and
SA signaling play a role (Bennett and Wallsgrove, 1994; Kiddle et al., 1994; Brader et al.,
2001; Taguchi et al., 2001). Profiling this group of secondary metabolites and their
biosynthetic genes shows that some glucosinolates are JA-dependent while others are SA-
dependent (Mikkelsen et al., 2003).
The JA signaling pathway seems to be counteracted by cytokinin signals; JA signaling-
induced sakuranetin production and its biosynthetic enzyme activities in rice are inhibited
by cytokinin and low concentration of ascorbic acid, but enhanced by high dosage of
ascorbic acid (Tamogami et al., 1997). Kinetin also inhibits elicitor-induced hypersensitive
response and phytoalexin production in potato tubers, probably by inhibition of ROS
generation (Tamogami et al., 1997).
Elicitors of defense responses can modulate pathways other than those related to
pathogen attacks. Loreti et al. (2002) demonstrated that chitosan and oligogalacturonides
as well as sugars, can repress the gibberellin signaling pathway that leads to the induction
of a-amylase in barley embryos. Furthermore, two gibberellin-responsive genes CIGR1
and CIGR2 are induced either by gibberellin or by elicitor N-acetylchitooligosaccharide in
rice cells (Day et al., 2000), strongly suggesting a cross-talk between elicitor and
gibberellin signaling pathways. These results suggest a complex cross-talk between the
defense, hormonal and metabolic signaling pathways. In C. roseus cell culture, JA is a
positive signal for accumulation of indole alkaloids whereas auxin is a negative regulator.
Auxin-inhibition of indole alkaloid accumulation can be reversed by addition of MeJA or
removal of auxin (Gantet et al., 1998), suggesting that auxins may suppress endogenous
JA biosynthesis in C. roseus cell cultures.
6.4. ROS and other pathways
ROS are very important signals for defense responses and phytoalexin accumulation
in some plants. In most cases, such as in C. roseus or parsley cell cultures, Ca2+ influx
is required for elicitor-induced ROS production (Zhao et al., 2001b). In h-glucan treated
soybean cell culture, it was shown that ROS generation is independent from the Ca2+
flux (Mithofer et al., 2001). In the systemin-induced tomato defense response, systemin-
induced JA biosynthesis and signaling induced ROS production and Ca2+ flux, and then
protease inhibitors (Scheer and Ryan, 2002). By contrast, in parsley cell culture, Pep-13
induced JA biosynthesis is Ca2+-dependent but ROS-independent. There are some
controversial reports on NO cross-talk with ROS in plant defense responses to pathogen
and elicitors (Delledonne et al., 1998): it appears that NO works collaboratively in many
cases of plant defense response, while antagonistically in some cases (Neill et al., 2002,
and literature cited therein). In tobacco cell cultures, it was found that the NO signal can
stimulate cyclic GMP and cyclic ADP-ribose (ADPR) signals, as well as SA
accumulation. This cyclic GMP and cyclic ADPR-dependent NO signaling pathway
leads to PAL activation (Durner et al., 1998). Both cyclic GMP and cyclic ADPR are
potent Ca2+ mobilizing agents. Cyclic GMP acts through cyclic nucleotide-gated ion
channels in the plasma membrane while ADPR binds to intracellular Ca2+ channels
localized to endomembrane systems and activates Ca2+ release. Therefore, NO signaling
may cross-talk with Ca2+ signaling. It was reported that cryptogein-induced NO
production is nitric oxide synthase-dependent and involved in Ca2+ signaling by
promoting intercellular Ca2+ release (Lamotte et al., 2004).
6.5. Abiotic elicitors, ROS and oxylipin pathways
For elicitation of biosynthesis of plant secondary metabolites in many plants, the

oligopeptide glutathione (GSH) is a very special elicitor. GSH plays a very important
role in the plant redox system, for example to scavenge excessive ROS in oxidative
stress-challenged plants. GSH or its oxidized form GSSG both obviously induce
accumulation of phytoalexins or other defensive secondary metabolites in many plants,
yet the mechanism involved is not understood. Examples include isoflavonoids in
soybean, capsidiol, debneyol, and nicotine in tobacco, numerous compounds in
chickpeas, and various other compounds in other species (Edwards et al., 1991; Neill
et al., 1994; Zhao et al., 2000; Armero et al., 2001). A recent study shows that GSH
elicits production of pterocarpan phytoalexins medicarpin and maackiain, production of
isoflavones biochanin A, formononetin, and isoflavanones homoferreirin and cicerin in
chickpea (Cicer arietinum L.) seedlings (Armero et al., 2001). However, the GSH-
induced phytoalexins and isoflavonoids are synthesized de novo from early phenyl-
propanoid pathway precursors rather than being derived from pre-existing conjugate
forms or immediate precursors. Most of them are released into the surrounding external
medium.
Metals, such as Co2+, Ni2+, Fe2+, Ag+, and V, all stimulate the biosynthesis of a wide
range of plant secondary metabolites in different plant species and systems (Neill et al.,
1994; Tumova et al., 2001; Zhao et al., 2001a,b,c). Even though the mechanisms by which
these metals promote accumulation of plant secondary metabolites are not clear, induction
of oxidative stress or ROS generation by these metals is often observed (Mithofer et al.,
2004; Kandlbinder et al., 2004; Zhao et al., 2005).
It is well recognized that both biotic elicitors such as herbivore or pathogen attack and
abiotic elicitors such as heavy metals and inorganic compounds, as well as osmotic and
salt stresses, often induce the synthesis and accumulation of the same defense-related
secondary metabolites. The explanation for the common features of the both elicitor signal
transduction pathways is not well established. Likely mechanisms for this may be through
ROS signaling, lipid peroxidation, and ultimately the generation of oxylipins since the
generation of ROS is a common event in abiotic and biotic elicitation (Thoma et al., 2003;
Mithofer et al., 2004; Zhao et al., 2005), although ROS may be initiated from quite
different pathways in abiotic and biotic elicitation. It is worth noting that ROS in the
oxidative stress may not always be the mediator of signaling. Oxidative stress induced
redox status changes in the cells may be also a very important signal for induction of a
wide spectrum of genes being expressed and defense responses (Kandlbinder et al., 2004).
It is likely that the role of GSH in the secondary metabolism may be partially through GSH
changing the overall redox status.
7. Integration of multiple signaling pathways by transcription factors
Cross-talk of multiple signaling pathways is very common and important for

accumulation of plant secondary metabolites and other defense responses, but how
different signaling pathways are integrated into the single cellular process (such as
phytoalexin biosynthesis) is still not very clear. The regulation of cellular processes
commonly occurs at several different levels including transcription, RNA processing, and
translation, as well as by post-translational modification such as protein phosphorylation.
However, a majority of studies show that transcriptional modulation of related genes is a
common response to pathogens or elicitor signals. Studies on DNA sequences of elicitor-,
JA-, and ethylene-regulated genes have demonstrated that several elicitor-, JA-, and
ethylene-response elements in these genes are involved with biosynthesis of secondary
metabolites. Such cis-acting elements in promoter regions are sequence-specifically bound
by a class of DNA-binding proteins designated as transcription factors (TFs). Elicitor
activation of defense gene transcription requires action of TFs, which can recruit the
general transcription machinery to stimulate gene expression. TFs have several conserved
structures such as nuclear localization signal and DNA-binding domain, which direct the
TFs to nuclei and promote binding of TFs to specific sequences in the vicinity of a gene.
There are many types of TFs with different DNA binding domains, such as helix-loop-
helix, zinc-finger, or basic zipper, which determine what DNA sequence they can bind to
(Liu et al., 1999).
7.1. Transcription factors involved in regulation of plant secondary metabolism
Essential roles of TFs in plant secondary metabolism recently have been

demonstrated in several systems (Memelink et al., 2001). For instance, fungal elicitor
or MeJA treatment induced rapid accumulation of AP2/ERF-domain TFs, which bind to
a JA- and elicitor-responsive element (GCC box like element) in the promoter region of
the Strictosidine synthase gene (Str) and activate several genes of the indole alkaloid
biosynthesis (van der Fits and Memelink, 2000). Another P-box element is present in the
Str gene which binds with the transcription factor CrBPF-1, which is induced only by
elicitor, in a JA-independent manner, suggesting that specificity of elicitor induction of
secondary metabolite accumulation may be also determined by TFs (Memelink et al.,
2001).
The phenylpropanoid pathway has been a model for studying plant secondary
metabolism regulation by TFs. The phenylpropanoid pathway leads to biosynthesis of
flavonoids and pigments, as well as lignin and phenolic compounds. The enzymes
chalcone synthase (CHS), phenylalanine ammonia lyase (PAL) and 4-coumarate: CoA
ligase (4-CL) are brate-limitingQ enzymes in these pathways to various products. The
particular DNA sequence motifs known as H-box and L-box-related sequences are highly
conserved in promoter regions of genes involved in the phenylpropanoid pathway, such as
PAL and CHS genes. These highly conserved cis-acting elements, such as G-box, H-box,
L-box, and P-box, in CHS, PAL and 4-CL genes, can be bound by TFs such as Myb and
bHLH (basic helix-loop-helix) in response to light, UV irradiation, elicitor, wounding, and
other stresses (Davies and Schwinn, 2003). Successful genetic manipulation of Myb and
bHLH genes leads to increased pigmentation of plants or accumulation of flavonoids and
anthocyanins (Mooney et al., 1995; Bradley et al., 1998; Grotewold et al., 1998),
indicating the essential roles of TFs in regulation of plant secondary metabolism. Indole
alkaloid production was also improved by overexpression of the transcription factor
ORCA3, a member of the AP2/ERF-domain TFs family, upon terpene precursor feeding
(van der Fits and Memelink, 2000).
Since TFs play such an important role in plant secondary metabolism, analysis of the
DNA sequence of a known secondary metabolite gene may help to predict the possible
regulation of the gene. An example shows that the pinosylvin-O-methyltransferase gene
(PMT), and stilbene synthase gene (STS) from Scots pine (Pinus sylvestris) contain
several cis-elements such as Myb-responsive elements, G-, H-, and GC-boxes, as well as
W-boxes. The gene expression profiles showed that STS and PMT indeed were induced
by ozone, wounding, and fungal elicitor, and expression of these genes resulted in
stilbene formation (Chiron et al., 2000). The cotton (+)-y-cadinene synthase, a
sesquiterpene cyclase, catalyzes a branch-point step leading to biosynthesis of
sesquiterpene phytoalexins including gossypol in Gossypium arboreum (Xu et al.,
2004). A recent study with the use of the yeast one-hybrid system demonstrated that a
WRKY transcription factor regulates fungal elicitor-or MeJA-induced (+)-y-cadinene
synthase gene expression.
7.2. Integration of various signals in transcription factors
It is believed that all signal transduction pathways finally converge on TFs, and almost
all genes for secondary metabolite biosynthesis are regulated by specific TFs. Synthesis
and activation of TFs are complicated. They can be: 1) stimulated directly by elicitor or
various signaling pathways, or; 2) regulated by other TFs or by a positive feedback
control, or; 3) be constitutively synthesized as an inactive form and activated by other TFs
through protein–protein interaction and phosphorylation or dephosphorylation. For
example, WRKY1,-3,-4, and-5 transcription factor gene products appear to accumulate
rapidly after elicitation, without the requirement of de novo protein synthesis (Hahlbrock
et al., 2003). Myb and bHLH, essential for regulating phenylpropanoid and flavonoid
biosynthesis pathway, can physically interact with each other (Memelink et al., 2001).
Binding of TFs to DNA can be regulated via protein phosphorylation and dephospho-
rylation to in turn regulate expression of many target genes, including TF genes (Liu et al.,
1999). Therefore, regulation of TF biosynthesis, activation, and inactivation provides a

flexible network for regulation of plant secondary metabolites.
Cross-talk of signaling pathways via TFs has been implicated in different stresses,
such as wounding, drought, cold, pathogen/fungal elicitor, high-salt, and hormones
(Liu et al., 1999; Eulgem et al., 2000; Maleck et al., 2000; Denekamp and Smeekens,
2003). For example, wounding induces expression of TF genes belonging to AP2,
WRKY, and Myb families. These can also be induced by SA, JA, ethylene, and
pathogen attack (Maleck et al., 2000). WRKY proteins are a family of zinc-finger type
TFs, which bind specifically to W-box type cis-elements in many elicitor response
genes. When WRKY-type TFs are induced, whether by wounding, SA or by ethylene,
W-box-containing-genes such as chitinase, and strictosidine synthase are expressed
(Eulgem et al., 1999, 2000). In this case, TFs focus multiple signals to a single
defense response. WRKY70, a common component in SA- and JA-mediated signal
pathways, acts as an activator of SA-induced genes and a repressor of JA-responsive
genes and serves to integrate these mutually antagonistic signaling pathways (Li et al.,
2004).
A GCC box that is bound by ethylene response factors (ERF) has been found in the
promoter region of many ethylene-regulated genes (Fujimoto et al., 2000; Wang et al.,
2002). In addition, a GCC box was also found in the promoter region of a JA-regulated
PDF1.2 gene, suggesting that besides regulating ethylene-mediated gene expression,
ethylene response factors may also play important roles in regulating JA-responsive gene
expression via interaction with the GCC box (Brown et al., 2003). A transcription factor
ERF1, that can integrate JA and ethylene signaling in plant defense responses was found to
work as a downstream component of both ethylene and JA signaling pathways (Lorenzo et
al., 2003). A family of calmodulin-binding or CGCG box-binding proteins, integrates
different hormonal or environmental signals to some common signaling pathways and
responses (Yang and Poovaiah, 2002).
The genes or enzymes involved in different steps to produce a certain plant secondary
metabolite pathway are often found in a gene cluster or as a protein complex in the cells.
For example, the oat h-amyrin synthase gene AsbAS1 is clustered together with the
genes required for distinct steps (such as 2,3-oxidosqualene cyclization, beta-amyrin
oxidation, glycosylation, and acylation) in biosynthesis of avenacin, an antibacterial
triterpenoid (Qi et al., 2004). It has been demonstrated that flavonoid metabolism is
catalyzed by an enzyme complex localized to the endoplasmic reticulum (Burbulis and
Winkel-Shirley, 1999). Yeast two-hybrid assays, affinity chromatography, and immuno-
precipitation assays indicated that chalcone synthase, chalcone isomerase, dihydro-
flavonol 4-reductase, and flavonol 3-hydroxylase interact and assemble as a
macromolecular complex. For rapid multiple-step-biosynthesis of flavonoids and
triterpenoids, the expression of gene clusters and formation of enzyme complexes are
effective means of regulation. Many genes in the phenylpropanoid pathway possess the
same TF-binding cis-elements and are regulated by the same TF (Memelink et al., 2001;
Davies and Schwinn, 2003). Such gene clusters and protein complexes facilitate the
spatial and temporal regulation of metabolic fluxes, which in turn indicates the essential
role of TFs in signal transduction leading to the production of plant secondary
metabolites.
8. Linkage of signaling components by protein phosphorylation and

dephosphorylation
Even though signaling components are identified to mediate elicitor or pathogen

signaling transduction to defense responses, including production of defensive secondary
metabolites, how these events are connected and mechanisms by which elicitor signal is
transduced and amplified have yet to be defined. For example, how receptor-mediated
activation of ion channels and G-proteins, how G-proteins regulate ion channels, how Ca2+
and ROS activate defense and secondary metabolism genes, as well as how JA, SA, NO,
and ethylene cross-talk and activate downstream reactions, are largely unknown. However,
protein kinases and protein phosphatases are assumed to be links between various
signaling components and cross-talk among various signaling pathways (Lebrun-Garcia et
al., 1998; Lecourieux et al., 2000). Work using various inhibitors or activators of protein
kinases or protein phosphatases has shown that both protein phosphorylation and
dephosphorylation play essential roles in elicitor-, JA-, ethylene-, ABA-or other stress-
induced production of plant secondary metabolites (Felix et al., 1991). Recent studies
using genomic and proteomic techniques including two-dimensional electrophoresis and
mass spectrometry further showed the important role of dynamic protein phosphorylation
and dephosphorylation in regulating protein or enzyme activity and consequent cellular
processes. From two-dimensional gel electrophoresis during elicitation in cell cultures it is
evident that immediately after elicitation, plasma membrane proteins show a reversible
phosphorylation that is essential for ion flux and subsequent signal transduction and
defense responses (Dietrich et al., 1990). Later, cytoplasmic proteins are phosphorylated
and some previously phosphorylated proteins are dephosphorylated.
The importance of protein phosphorylation and dephosphorylation can be conceived
from the activation and inactivation of so many important proteins or enzymes critical to
the cellular process by phosphorylation and dephosphorylation. Many MAPKs and
CDPKs have been identified to play a role in defense responses, including secondary
metabolite production (Morris, 2001). A MAPK in parsley cells regulating elicitor
activation of the oxidative burst is regulated by Ca2+ influx (Ligterink et al., 1997). MAPK
cascades are involved in ethylene signal transduction and JA biosynthesis pathway and
many other cellular signaling pathways, including phytoalexin biosynthesis in parsley cell
cultures (Kwak and Lee, 1997; Lebrun-Garcia et al., 1998; Ouaked et al., 2003). Protein
phosphorylation and dephosphorylation processes are involved in elicitor signal trans-
duction throughout, from elicitor perception, elicitor-induced Ca2+/ion fluxes (Dietrich et
al., 1990), medium alkalinization /cytoplasmic acidification, ROS production, ethylene
and JA biosynthesis and signaling, transcription factor binding to DNA, NO biosynthesis,
and other downstream components, including biosynthesis of secondary metabolites
(Dietrich et al., 1990; Kwak and Lee, 1997; Liu et al., 1999; Morris, 2001). On the other
hand, almost all signaling components could initiate protein kinase cascades: elicitor
binding activates some receptor-kinase activity; ion channels may be regulated by
phosphorylation or dephosphorylation; Ca2+ is one of the core regulators of multiple
kinase cascades; H2O2, SA, JA, ethylene, NO, all can activate certain protein kinase or
phosphates activity, and TE are also regulated by phosphorylation or dephosphorylation
(Dietrich et al., 1990; Liu et al., 1999; Morris, 2001).
Protein phosphorylation and dephosphorylation are not only in signaling pathways;

they are also involved more directly in regulation of biosynthesis of some secondary
metabolites. PAL is the first enzyme of phenylpropanoid biosynthesis and plays an
important role in the biosynthesis of flavonoids, lignins, and many other compounds, and
possibly also including the well-known signaling molecule salicylic acid. CDPKs have
been identified from French bean and Arabidopsis with capability to phosphorylate PAL
(Allwood et al., 1999; Cheng et al., 2001), and elicitors regulate this phosphorylation.
These results present a link in elicitor signal transduction between calcium flux, protein
kinases, PAL post-translational regulation, or salicylate and secondary metabolite
production.
9. General features of elicitor induction of plant secondary metabolism
Studying elicitor signal transduction while focusing on only one secondary

metabolite usually gives very limited information about metabolic fluxes. Investigations
of the signal transduction involved in the production of multiple secondary metabolites,
for example, from two branches of a common biosynthetic pathway, have led to more
significant findings. It is observed that elicitors induce rearrangement of metabolic
fluxes between a constitutively expressed pathway and an elicitor-inducible pathway.
These differential regulations of branch compound biosynthesis reflect an important
feature of elicitor induction of plant secondary metabolism. Elicitors selectively induce
the accumulation of phytoalexin-like compounds that act as antimicrobial or repellent
agents to kill microbes or repel insects. One cost of the biosynthesis of phytoalexins is
the decreased accumulation of other primary or secondary compounds. For example,
tobacco suspension cultures can constitutively synthesize sterols by steadily expressing
the isoprenoid pathway, especially the rate-limiting squalene synthase gene. A fungal
elicitor stimulates sesquiterpene phytoalexin production, but inhibits sterol biosynthesis,
by suppressing the squalene synthase gene and activating the sesquiterpene cyclase
genes, which are localized at a branch point of the isoprenoid pathway (see review,
Chappell, 1995). The same things also occur in potato tuber tissue, in which the
elicitor arachidonic acid inhibits sterol biosynthesis but activates sesquiterpene
biosynthesis by regulating the same set of genes as in tobacco. Expression of another
gene family for the HMGR gene leads to comparable metabolic channeling, with
accumulation of sterols being enhanced by overexpressing HMGR1, and sesquiterpene
phytoalexin accumulation by overexpressing HMGR2. Elicitors differentially regulate
these genes (Choi et al., 1994).
Light induces accumulation of the anthocyanin, cyanidin 3-dimalonyl glucoside, in
etiolated sorghum (Sorghum bicolor) by inducing anthocyanin biosynthesis genes
flavanone 3-hydroxylase, dihydroflavonol 4-reductase, and anthocyanidin synthase
(Clive Lo and Nicholson, 1998). However, fungal inoculation suppresses the
expression of these three genes and anthocyanin accumulation, but fungal elicitation
stimulates accumulation of a phytoalexin, 3-deoxyanthocyanidin, and also activation of
PAL and CHS genes. Similar phenomena may also occur in other plants and other
synthetic pathways. Switching metabolic flux from one branch to another branch may
by controlled by TFs. Different isoforms of TFs or a set of similar TFs in differently

combined ways can activate some genes but inactivate other genes in branched
pathways (Hahlbrock et al., 2003). Clarifying the signaling pathways and regulation
mechanisms involved in these switching processes will greatly help us to selectively
produce certain desired natural products.
Almost all fungal elicitors stimulate the phenylpropanoid pathway because this
pathway leads to accumulation of phenolic compounds required for cell wall and SA
biosynthesis; cell wall reinforcement is observed to be a basic defense strategy for plant
cells against pathogen invasion (Fig. 5). Because H2O2 is required for cross-linking of cell
wall lignin, the oxidative burst is also a common response of plant cells to elicitors or
pathogens (Meyer et al., 2001). Non-pathogenic, symbiotic bacteria may evade or
Elicitor and avirulence determinants

Cell wall Cell wall reinforcement
Apoplast peroxidase antimicrobe /insect-repellence
Lipid + -
H2 O2 K /Cl
elicitor
receptor O2 O2 - Channel receptor O2 O2 - H2 O2
PLA
Ca2+ NADPH AC PLC NADPH
P Ca2+ oxidase
G-protein oxidase G-protein
cAMP
P 2+
Ca / H +
DAG + IP3
NOS/NR NO
receptor
ROS P GC
LysoPC Oxylipin signaling cGMP
pathway [Ca2+]cyt
Ca2+ Ca2+
Ethylene
H+-ATPase signaling Protein kinase cADP ribose
IP3
H+ P phosphatase
P
P SA
P
Cytoplasmic Intracellular
acidification Ca2+ stores
activation of TFs
Vacuole
Nuclei
Activation of TF genes Biosynthesis of signal molecules such
as JA and SA, or cell wall precursors
P-box H-box W-box early response genes
ERE AP2 TATA secondary metabolism genes
Cytosol
Production of plant
Transfer to and localize to cellular
Translation to enzymes secondary metabolites
apartments, such as chloroplasts
Fig. 5. A comprehensive schematic illustration of elicitor signal transduction network. Proteinaceous or

carbohydrate elicitor molecules are recognized by specific receptors on the plasma membrane. G-proteins may be
coupled to receptors and mediate elicitor-induced ion channel activation. Ion fluxes, especially Ca2+ influx, cause
cytosolic free Ca2+ spiking which causes activation of protein kinases, peroxidases, NADPH oxidases, and
phospholipases, which further generate other signaling messengers, such as reactive oxygen species, DAG, IP3,
cAMP, lysoPC, JA, ethylene, NO, cADP ribose, and SA. All these messengers compose paralleling or cross-
linking pathways to integrate these signals onto regulation of transcription factors (TFs). Various transcription
factors integrate these signaling to activate gene expression by transcription machinery. Phenylpropanoid pathway
is one of earlier induced pathways for cell wall reinforcement. Most genes for secondary metabolite synthesis are
later response genes. The circled bPQ shows protein phosphorylation and dephosphorylation-dependent regulation
while the circled bCaQ shows Ca2+-dependent regulation. Lipid elicitor such as syringolide or cerebroside could be
perceived by receptors localized to the cytoplasm. Lipid elicitors also induce Ca2+ signaling and oxidative burst,
and eventually stimulate the accumulation of plant secondary metabolites. NOS: nitric oxide synthase; NR:
Nitrate reductase; AC: AMP cyclase; GC: GMP cyclase. It is must be noted that a plant may only use parts of this
elicitor signaling network to fulfill their responses to elicitors.
attenuate this response. For example, the Nod factor of rhizobial cells, stimulates a
response of the legume host, such as production of genistein or another flavonoid, but does
not induce any oxidative burst in symbiotic host cells, indeed it inhibits it (Shaw and Long,
2003). Such an oxidative burst might kill the bacteria. On the other hand, Nod factor does
stimulate an oxidative burst in non-host plant cells, such as tomato (Shaw and Long,
2003). This is an example for highly evolved adaptation between particular host plants and
bacteria in natural evolution. In this instance the Nod factor is a very specific elicitor of
legume secondary metabolites.
A synergistic effect of multiple elicitors on production of secondary metabolites in
plant or cell cultures is often observed. This synergistic stimulation effect is most often
seen when a plant-derived (endogenous) elicitor and a microbe-derived (exogenous)
elicitor are applied together, or two microbe-derived elicitors are perceived by different
plant receptors. For example, oligosaccharides (chitin or chitosan) can potentiate
methyl jasmonate-induced production of paclitaxel in T. canadensis (Linden and
Phisalaphong, 2000). Plant derived cerebroside sphingolipid and N-acetylchitohexaose
synergistically stimulate phytoalexin production in rice cell cultures (Umemura et al.,
2002). Also, two purified oligosaccharide elicitors derived from fungal cell walls, N-
acetylchitoheptaose and a tetraglucosyl glucitol from rice blast fungus (Magnaporthe
grisea), synergistically activate phytoalexin biosynthesis in cultured rice cells
(Yamaguchi et al., 2002). Studies on inhibition of the binding of radiolabeled N-
acetylchitooligosaccharide elicitor to the rice plasma membrane indicates that the two
elicitors are recognized by different receptors, suggesting that interaction between
different signal transduction downstream of each elicitor/receptor may enhance the
resistance against pathogens.
10. Perspectives
Despite the increasing demands and tremendous importance of plant secondary

products for human beings, as well as decades of research on the area, much of plant
secondary metabolism is still poorly understood. Different levels of research on plant
secondary metabolite production, such as chemical processing, signal transduction
networks, molecular regulatory mechanisms, and engineering metabolic fluxes, will be
of great interest and active topics of plant secondary metabolism in the near future. A
large body of evidence suggests that signal transduction leading to biosynthesis of plant
secondary metabolites is a complicated network that is closely related to the regulatory
machinery of plant defense responses. Dissection of the signaling network will lead to
discoveries of more secondary metabolite biosynthetic genes and regulatory factors, and
will facilitate a specific and efficient engineering of the production of target secondary
metabolites. Taking advantages of advanced metabolic engineering, researchers can
construct new secondary metabolic pathways in one species by transferring heterologous
genes from various other species for production of novel compounds. However,
traditional approaches using non-specific pharmacological methods, or targeting at only
one or two signaling pathways, enzymes and genes, or secondary metabolites, is far
from enough to understand the whole pictures of plant secondary metabolism. Given the
complicated metabolic networks of plant secondary metabolism, cross-talk of signaling

pathways, interaction of the complex primary and secondary metabolic fluxes, and
various levels of regulatory mechanisms of protein and gene activity, it is difficult to
obtain a complete view of plant secondary metabolism by only using traditional
techniques.
With the development of functional genomics, proteomics and metabolomics, many
new and powerful tools could be applied to plant secondary metabolism study, and
improve overall understanding and practical manipulation of plant secondary metabolite
production. Transcript-profiling analysis of plants under different treatments could display
differential regulation of plant secondary metabolism-related genes in an individual plant.
Expressed sequence tags (EST) and microarrays provide a powerful approach for this
purpose. There are EST profiles from glandular cells producing essential oils (Lange et al.,
2000), EST profiles across the trunk wood of Robinia pseudoacacia tree for heartwood
chemicals (Yang et al., 2003) and EST profiles from Stevia rebaudiana for diterpene
steviol glycosides metabolism (Brandle et al., 2002). The cDNA-amplified fragment
length polymorphism (AFLP) technology offers an alternative to identify genes involved
in plant secondary metabolism (Goossens et al., 2003). Transcription profiling helps to
identify more genes involved in biosynthesis of secondary metabolites, but also facilitates
isolation of some possible signal transduction components such as TFs.
Meanwhile, profiling the whole or a group of secondary metabolites from plants
under various treatments helps to understand metabolic flux and the related regulatory
mechanisms. Because of the high energetic cost of the biosynthesis of secondary
metabolites for defense or other biological purposes, plant cells have various strategies
to control intermediate pool size and metabolic flow directions. Although this regulation
is mainly controlled at enzyme and gene expression levels, a quantitative analysis of
metabolic intermediates will provide useful information about exact metabolic fluxes and
metabolic flux shifts due to stresses, just as checking gene expression patterns or
enzyme activity does. Especially, in many pathways with only a limited number of genes
or enzymes having been characterized, profiling metabolic intermediates is a very direct
and important way to understand plant secondary metabolism and regulation.
Particularly, the time dependent profiling of metabolites would provide essential
information.
Monitoring a whole set of related secondary metabolites and related compounds in
plants under different conditions is an especially powerful way to learn the effects of
various stresses and signaling transduction pathways. Various high resolution spectrometry
techniques, such as GC-MS, LC-MS, GC/EI-TOF-MS, EI-MS/MS, and NMR, make this
possible (Sumner et al., 2003). These metabolomic tools have been proved to be useful for
profiling targeted groups of secondary metabolites in particular plant organs. As an
example we may consider a plant root. Because of the special environments a root is
surrounded by, secretion of secondary metabolites is significantly affected by various
biotic and biotic factors. Walker et al. profiled the Arabidopsis root-secreted secondary
metabolites after elicitation with MeJA, SA, and fungal elicitors (Walker et al., 2003).
Targeting taxol production, elicitor-induced Taxus were profiled by Ketchum et al. (2003)
showing that MeJA treated-Taxus cell suspension cultures accumulate taxols oxygenated
at C position 13, but not 14.
Since plants employ multiple signaling pathways, such as JA, ethylene, ABA, and SA
signaling pathways, to regulate defense response against elicitor or various stresses, each
defense response, such as biosynthesis of defensive compounds, should be a combined
result from multiple signaling actions. Quantitatively profiling the signal components such
as stress hormones and their changes in response to various abiotic or biotic elicitors may
provide important information for elicitor signal transduction. As an example, Schmelz et
al. (2003c) analyzed hormones, signal compounds, pathogen phytotoxins and defensive
secondary metabolites produced by plants, and signal compounds such as salicylic acid,
jasmonic acid, indole-3-acetic acid, and abscisic acid, in response to elicitation by
pathogens. Their results show that the bacterial pathogens induce JA, SA, ABA, and auxin
accumulation, while herbivory induces JA and volatile organic compounds, but inhibits
IAA production, wounding induces changes in JA, IAA, and ABA levels whereas drought
only induces formation of ABA (Schmelz et al., 2003a,b,c). Even though these results are
what might be expected, their established methods and techniques lay useful groundwork
for future study.
Combining the approaches of transcription profiling proteomics and secondary
metabolite profiling, offers the most powerful tool ever in studying all aspects of plant
secondary metabolism as a whole. Goossens et al. (2003) successfully used the cDNA-
AFLP transcript profiling technique to analyze MeJA-induced signal transduction and
differential gene expression, which is well confirmed by the metabolic flux shift as
indicated by secondary metabolite analysis. Their informative results suggest that MeJA
treatment reprograms transcription and shifts metabolic fluxes by involving many cellular
processes of signal transduction. In summary more studies on details of a single signal
transduction pathway leading to secondary metabolites, high throughput analysis of gene
transcripts and proteins, and measurement of secondary metabolic intermediates will all be
essential for better understanding of the signal transduction pathways and control of plant
secondary metabolism. Through such a holistic approach, in fact a systemic biology
approach, in which as many parameters as possible are measured and in which biostatistic
tools are used for identifying correlations, similarities and differences, we may expect new
insights in the complex and dynamic processes in the elicitor signal transduction pathways.
Acknowledgements
We regret not being able to cite many other excellent publications in these areas due to
the space limitation. We are grateful to Dr. Roos from Martin-Luther-University, Germany
for an invaluable discussion on his most recent results. We also thank Dr. XiaoYan Tang
for her critical reading of this manuscript and helpful suggestions. This is contribution #
05-88-j of the KAES.
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Elicitor Signal Transduction Leading To Production of Plant Secondary Metabolites

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Elicitor Signal Transduction Leading To Production of Plant Secondary Metabolites

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Biotechnology Advances 23 (2005) 283 – 333

Research review paper

Elicitor signal transduction leading to production

Abbreviations: ACC, 1-aminocyclopropane-1-carboxylic acid; ADPR, ADP-ribose; AFLP, amplified

8. Linkage of signaling components by protein phosphorylation and dephosphorylation 317

Plant secondary metabolites are unique resources for pharmaceuticals, food

V. β-Thujaplicin, an antifungi tropolone from

Fig. 1. Several typical plant secondary metabolites with special interests.

2. Elicitors and signal transduction

Biphasic Ca2+ spiking

Multiphasic protein phosphorylation

Early response genes

From a pathogenesis point of view, many elicitors may act as avirulence

subsequently or indirectly activate their corresponding effectors, such as ion channels,

3. Elicitor signal perception

3.1. Elicitor and elicitor binding sites

J. Zhao et al. / Biotechnology Advances 23 (2005) 283–333

On the other hand, a glycolipid elicitor molecule such as syringolide is perceived by a

3.2. Avirulence determinants and R proteins

4. Elicitor signal transduction

4.1. GTP binding proteins

G-proteins constitute a class of ancient eukaryotic proteins, including heterotrimeric

4.2. Ion fluxes and Ca2+ signaling

4.3. Cytoplasmic acidification

4.4. Oxidative burst and reactive oxygen species

4.5. Inositol 1,4,5-trisphosphates and cyclic nucleotides

Elicitor-induced phosphoinositide breakdown has been reported to occur in many

4.6. Salicylic acid and nitric oxide

Salicylic acid (SA) is a well-known inducer of plant systematic acquired resistance

4.7. Jasmonate pathway

Apoplastic Elicitors or avirulence determinants

NADPH oxidase Lipases (PLA) receptor

α-Linolenic acid Lysophospholipids

4.8. Ethylene pathway

J. Zhao et al. / Biotechnology Advances 23 (2005) 283–333

J. Zhao et al. / Biotechnology Advances 23 (2005) 283–333

J. Zhao et al. / Biotechnology Advances 23 (2005) 283–333

4.9. ABA signaling

ABA is a well-known senescence-triggering plant hormone. ABA plays important

leaves was p-coumaroylagmatine but in ABA-treated leaves was p-coumaroyl-3-

5.2. Lipid messengers

As a ubiquitous enzyme family, phospholipases play various roles in stress responses

Elicitors or avirulent determinants

G-protein Ca2+ PLA Ca2+ PLD G-protein PLC Ca2+

Choline IP3 PIP2

Defensive gene expression

Biosynthesis of defensive plant secondary metabolites

defense responses including production of defensive secondary metabolites (Subrama-

2001). Elicitor or pathogen-triggered PLC and PLD activation as well as conversions

5.3. Other lipid elicitors

6. Cross-talk of signaling pathways

6.1. Elicitor and JA pathways

Even though JA is a major signal for plant secondary metabolism, interactions of JA

6.2. JA and ethylene pathways

6.3. JA and other pathways

Cross-talk among some signaling pathways is species-specific or case-specific. SA and

6.4. ROS and other pathways

6.5. Abiotic elicitors, ROS and oxylipin pathways

For elicitation of biosynthesis of plant secondary metabolites in many plants, the

7. Integration of multiple signaling pathways by transcription factors

Cross-talk of multiple signaling pathways is very common and important for

7.1. Transcription factors involved in regulation of plant secondary metabolism