Download Signal transduction networks and the biology of plant cells*

Biol Res 32: 3 5 - 6 0 , 1999
Signal transduction networks and
the biology of plant cells*
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MAARTEN J C H R I S P E E L S L O R E T O HOLUIGUE , RAMON LATORRE , SHENG
LUAN , ARIEL O R E L L A N A H U G O PENA-CORTES , NATASHA V RAIKHEL , PAME­
LA C RONALD & ANTHONY TREWAVAS
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'Department of Biology, University of California San Diego, La Jolla, CA 92093-0116, USA
department of Molecular Genetics and Microbiology, Catholic University of Chile, Santiago, Chile
'Department of Biology, Faculty of Science, University of Chile, Santiago, Chile
department of Plant and Microbial Biology, University of California Berkeley, Berkeley, CA 94720, USA
department of Biology, Faculty of Chemistry and Biology, University of Santiago de Chile, Santiago, Chile
d O E Plant Research Lab, Michigan State University, East Lansing, MI 48824-1312
Plant Pathology Department, University of California Davis, Davis, CA 95616-8680
"Institute of Cell and Molecular Biology, University of Edinburgh, Edinburgh, United Kingdom
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ABSTRACT
The development of plant transformation in the mid-1980s and of many new tools for cell biology, molecular
genetics, and biochemistry has resulted in enormous progress in plant biology in the past decade. With the
completion of the genome sequence of Arabidopsis
thaliana just around the corner, we can expect even faster
progress in the next decade. The interface between cell biology and signal transduction is emerging as a new and
important field of research. In the past we thought of cell biology strictly in terms of organelles and their biogenesis
and function, and researchers focused on questions such as, how do proteins enter chloroplasts? or, what is the
structure of the macromolecules of the cell wall and how are these molecules secreted? Signal transduction dealt
primarily with the perception of light (photomorphogenesis) or hormones and with the effect such signals have on
enhancing the activity of specific genes. Now we see that the fields of cell biology and signal transduction are
merging because signals pass between organelles and a single signal transduction pathway usually involves multiple
organelles or cellular structures.
Here are some examples to illustrate this new paradigm. How does abscisic acid (ABA) regulate stomatal closure?
This pathway involves not only ABA receptors whose location is not yet known, but cation and anion channels in
the plasma membrane, changes in the cytoskeleton, movement of water through water channels in the tonoplast and
the plasma membrane, proteins with a farnesyl tail that can be located either in the cytosol or attached to a
membrane, and probably unidentified ion channels in the tonoplast. In addition there are highly localized calcium
oscillations in the cytoplasm resulting from the release of calcium stored in various compartments. The activities of
all these cellular structures need to be coordinated during ABA-induced stomatal closure.
For another example of the interplay between the proteins of signal transduction pathways and cytoplasmic
structures, consider how plants mount defense responses against pathogens. Elicitors produced by pathogens bind to
receptors on the plant plasma membrane or in the cytosol and eventually activate a large number of genes. This
results in the coordination of activities at the plasma membrane (production of reactive oxygen species), in the
cytoskeleton, localized calcium oscillations, and the modulation of protein kinases and protein phosphatases whose
locations remain to be determined. The movement of transcription factors into the nucleus to activate the defense
genes requires their release from cytosolic anchors and passage through the nuclear pore complexes of the nuclear
envelope.
This review does not cover all the recent progress in plant signal transduction and cell biology; it is confined to the
topics that were discussed at a recent (November 1998) workshop held in Santiago at which lecturers from Chile,
the USA and the UK presented recent results from their laboratories.
* This paper contains a summary of research results and relevant background information that was presented
at a US-Chile workshop on Plant Cell Biology and Signal Transduction, held in Santiago de Chile November
14-16, 1998. The workshop was organized by Maarten Chrispeels and Ariel Orellana. Support for the
workshop was provided by the National Science Foundation (USA), the Fundación Andes, Conicyt.
Fundación para Biología Celular, and the University of Chile
The authors are listed alphabetically.
** Corresponding author: Dr. Maarten J. Chrispeels, Department of Biology, University of California San
Diego, La Jolla, CA 92093-0116, USA, e-mail: [email protected], or Dr. Ariel Orellana, Departamento
de Biología, Facultad de Ciencias, Las Palmeras 3425 - Casilla 653, Universidad de Chile, Santiago, Chile,
e-mail: [email protected]
Received 26.3.99. Accepted 14.4.99
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C H R I S P E E L et al. Biol Res 32, 1999, 3 5 - 6 0
I. THE EXTRACELLULAR MATRIX: STRUCTURE,
BIOGENESIS AND ROLE IN SIGNALING
/. The extracellular
matrix or cell wall of
the plant cell is a multicomponent
struc­
ture.
We currently view the cell wall as a threed i m e n s i o n a l n e t w o r k of c e l l u l o s e mi­
crofibrils and cross-linking polysaccharides
(hemicellulose) embedded in a gel matrix
of acidic polysaccharides rich in galacturonic acid (pectins) and held together by
calcium bridges. The two major groups of
flowering plants, the monocots and the dicots, differ in their hemicellulosic poly­
mers; monocots have arabinoxylans, and
dicots have xyloglucans (see Carpita and
Gibeaut, 1993). This network is enmeshed
with a variety of structural proteins and
glycoproteins and partially cross-linked by
aromatic substances (monolignols). En­
zymes needed for the biosynthesis or modi­
fication of the network {e.g., peroxidase,
xyloglucan endotransglycosylase, afucosidase, glucanase) or for the modifi­
cation of metabolites (e.g., invertase, phos­
phatase, ascorbic acid oxidase) may be
ionically or covalently bound to the hemi­
cellulose network or the pectin matrix.
Assembly of the cell wall requires the
coordination of the activities of large cel­
lulose synthase complexes on the outer face
of the plasma membrane with those of many
different glycosyltransferases in the Golgi
that assemble the pectin and hemicellu­
losic polymers. These polymers are trans­
ported to the plasma membrane in secre­
tory vesicles, as are the cell wall proteins
and glycoproteins, and their cross-linking
is catalyzed by cell wall enzymes. In spite
of this cross-linking, the walls of young
cells remain flexible and can extend through
breaking and reforming of cross-links to
a c c o m m o d a t e the e n o r m o u s volume
changes that characterize the growth of
plant cells. In highly differentiated cells
such as xylem vessels or fibers, the walls
become thick, stiff and incompressible af­
ter impregnation with lignin, a polymer
that is formed extracellularly by polymer­
ization of secreted monolignol subunits (see
Boudet, 1998).
Cell differentiation is accompanied by
the synthesis and secretion of different com­
ponents of the ECM of plant cells, as visu­
alized by the interaction of monoclonal
antibodies against cell wall epitopes. This
process has recently been studied in Zinnia
cells. When exposed to the hormones auxin
and c y t o k i n i n , Z i n n i a m e s o p h y l l cells
transdifferentiate into tracheary elements.
This transdifferentiation is accompanied
by a new pattern of gene expression and
changes in the repertoire of proteins and
polysaccharides at the cell surface (Stacey
etal, 1995).
2. The extracellular
matrix is a source
signals for development and defense.
of
Two types of cell wall macromolecules
appear to generate signals for plant cells:
the arabinogalactan proteins and the hemicelluloses. Arabinogalactan proteins
(AGPs) are extracellular or plasma mem­
brane-anchored proteins that are typically
more than 90% carbohydrate. The polypep­
tide backbone has a domain that is rich in
Ala, Ser, Thr, and Hyp, and the carbohy­
drate moiety consists of short arabinosides
attached to Hyp and arabinogalactans Olinked to Hyp or Ser. The arabinogalactan
sidechains have 30 to 150 sugar residues,
and most have a pure B(l->3)-D galactan
backbone with terminal L-arabinofuranosyl
residues on short sidechains (Du et al,
1996). AGPs have been shown to promote
the differentiation of single, densely cyto­
plasmic carrot cells into somatic embryos.
The AGPs are not produced by the cells
that differentiate into embryos, but need to
be present in the culture medium or pro­
duced by neighboring cells (McCabe et al,
1997).
Completely different signals, small oli­
gosaccharides of 5-15 sugar residues, are
involved in defense responses and in the
loosening of the hemicellulosic network
that permits plant cells to grow in size as a
result of their internal osmotic pressure.
Oligosaccharides involved in defense re­
a c t i o n s r e s u l t from the b r e a k d o w n of
polysaccharides by plant or fungal enzymes.
These oligosaccharides bind to receptors
C H R I S P E E L et al. Biol Res 32, 1999, 3 5 - 6 0
Xyloglucan
PGA junction
zone
RGI with
arabinogalactan
side-chains
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Extensin
Figure I. Model of a type 1 cell wall in the process of expanding. A model of possible alterations in structure
that first permit microfibril separation and then lock them into place. Microfibrils of a single layer align
parallel in a helical arrangement around elongating cells. Xyloglucans can separate from the microfibrils
under the action of expansins and be cleaved by glycosidases or endo-transglycosylases. When displacement
caused by turgor pressure within the cell is complete, extensin molecules, inserted radially, interlock the
separated microfibrils to arrest further stretching. Additional proteins may also be inserted to cross-link
extensin, forming a heteropeptide network. Formation of intramolecular covalent bonds among the indivi­
dual wall proteins signals the end of elongation. From Carpita and Gibeaut, 1993, reprinted by permission
of the publisher, Blackwell Science.
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C H R I S P E E L et al. Biol Res 3 2 , 1 9 9 9 , 3 5 - 6 0
on the plasma membrane and this signal is
transduced to the nucleus, where a battery
of plant defense genes are induced (see
below) (Hahn, 1996). Oligosaccharides in­
volved in growth are the result of the action
of e n d o g l u c a n a s e s o n t h e c e l l w a l l
xyloglucans. They are broken down by
exoglucanases and may provide substrates
for xyloglucan endo-transglycosidases that
are thought to modify the xyloglucan net­
work.
3. A gene that encodes a subunit of cellu­
lose synthase has at last been
identified.
Cellulose is a linear polymer made of 6-1,4
linked glucose units. In the primary cell
wall the degree of polymerization reaches
over 2,000. Fifty to 80 of these linear poly­
mers lie in parallel and bind tightly to each
other through hydrogen bonds, creating a
microfibril. Electron microscope studies
of freeze-fractured membranes provided the
first suggestion that a rosette-shaped pro­
tein complex located in the plasma mem­
brane may be involved in cellulose biosyn­
thesis. Purification of the protein(s) in­
volved in the synthesis of cellulose proved
to be difficult and was unsuccessful at least
for plants. However cellulose synthetase
was purified successfully from extracts of
Acetobacter xylinum, a bacterium that syn­
thesizes cellulose. This allowed the cloning
of the gene that encodes the catalytic subunit of cellulose synthase. Use of this gene
as a probe to search for homologous genes
led to the identification of putative cellu­
lose synthases in plants. Analyzing the
Acetobacter sequence and the structure of
the gene and hydrophobic cluster analysis
allowed the identification of a signature
s e q u e n c e ( D . . . D . . . D . . . Q x x R W ) that is
p r e s e n t in a g r o u p of p r o c e s s i v e 8glycosyltransferases, which could be
present in cellulose synthases from other
organisms as well (Saxena et al, 1995).
This information helped Delmer's group to
identify two ESTs from cotton, obtained by
random sequencing of a cottonhair cDNA
library, as the first two potential genes
encoding cellulose synthase from higher
plants (Pear et al, 1996). Although direct
evidence that these two genes indeed en­
code cellulose synthase was not provided,
all the circumstantial evidence strongly
supported this conclusion.
The identification of a gene that encodes
a cellulose synthase of higher plants came
from studies with Arabidopsis
thaliana.
Arioli et al (1998) identified after exten­
sive screening an Arabidopsis mutant that
has impaired cellulose synthesis. The phenotype of this mutant, radial swelling (rsw)
of the roots mimics the response of wild
type roots to cellulose synthesis inhibitors
such as dichlorobenzonitrile. Genetic map­
ping allowed the identification of the locus
RSW1 and the further cloning of the gene.
Analysis of the RSW1 amino acid sequence
showed that it contains the D...D...D...QxxRW
motif and is homologous to the cel-A genes
from cotton. The deficiency of cellulose in
the R S W mutant could be complemented
by transformation with the RSW1 cDNA.
The sequences encoded by these cellu­
lose synthase genes have been used to search
for homologous genes through out plant
databases, and a number of different genes
potentially involved in cellulose biosyn­
thesis have been identified. Thus, a vast
field of research to understand the molecu-
UDP-Glc
UDP-Glc
transporter
Glucosyl
transferase
Figure 2. Proteins needed for the biosynthesis of a
glucan in the Golgi. The cycle begins with the uptake
of UDP-Glc by a transporter that also permits uridine
monophosphate to leave. UDP-Glc is the substrate
of a membrane-anchored glucosyltransferase that
adds a glucose (G) to a growing chain, and produces
UDP, which is cleaved by UDPase to produce P. and
UMP. Whether the glucan is anchored to a protein
(as shown) is not known.
C H R I S P E E L et al. Biol Res 32, 1999, 3 5 - 6 0
lar basis of cellulose biosynthesis has just
emerged.
4. The biosynthesis
of
hemicellulosic
polysaccharides
and the carbohydrate
moieties of proteoglycans
and
glycoproteins
occurs in the Golgi
apparatus.
In contrast to cellulose biosynthesis, which
takes place in the plasma membrane, hemicelluloses, pectin, and the glycosidic chains
of proteoglycans are synthesized intracell u l a r ^ in the Golgi apparatus (Driouch et
al, 1993). The formation of polysaccharides in the lumen of the Golgi requires the
following enzymes and transporters: (1)
glycosyltransferases that use sugar nucleotide diphosphate as substrate and cause
the p o l y s a c c h a r i d e chain to g r o w ; (2)
UDPases that degrade nucleotide diphosphate produced by the glycosyltransferases;
(3) a transporter that removes inorganic
phosphate; (4) a transporter that exchanges
sugar nucleotide diphosphate for nucleotide
monophosphate (see Fig 2). Little is known
about the glycosyltransferases involved in
this process, and so far the only enzyme
that has been purified and cloned is the
xyloglucan oc-l,2-fucosyltransferase, an
enzyme involved in the fucosylation of
xyloglucan, one of the most abundant hemicelluloses in dicots (Perrin et al, 1999).
The sequence of this enzyme predicts a
single transmembrane domain close to the
amino terminus, resembling the structure
of mammalian glycosyltransferases that
participate in protein glycosylation in the
Golgi.
T o p o l o g i c a l analysis of glucan synthase
I, an e n z y m e involved in x y l o g l u c a n bios y n t h e s i s , indicates that its active site is
located in the lumen of the Golgi cisternae ( M u ñ o z et al, 1996). U n p u b l i s h e d
r e s u l t s from O r e l l a n a ' s and M o h n e n ' s
g r o u p s i n d i c a t e that the active site of
x y l o g l u c a n a - 1 , 2 - f u c o s y l t r a n s f e r a s e and
polygalacturonate a-4-galacturonosyltransferase, e n z y m e s i n v o l v e d in xyloglucan ( h e m i c e l l u l o s e ) and h o m o g a l a c turonan (pectin) biosynthesis respectively, would also be p r e s e n t in the lumen
of Golgi c i s t e r n a e .
39
The question is then, how do the nucleotide sugars get into the lumen of the Golgi
cisternae? Studies using pea Golgi vesicles
show that UDP-Glc can be transported into
the lumen of Golgi vesicles by a protein
mediated process (Munoz et al, 1996). This
membrane transporter seems to be specific
for UDP-Glc; therefore it seems possible
that different transporters would be responsible for the incorporation of the substrates
used by the different Golgi glycosyltransferases involved in both polysaccharides
and proteoglycans biosynthesis, as well as
protein glycosylation.
After the transfer of the sugar into the
oligosaccharide, the nucleoside diphosphate
(UDP) is quickly transformed into UMP and
i n o r g a n i c p h o s p h a t e by a N u c l e o s i d e
Diphosphatase present in the lumen of the
Golgi apparatus (Neckelmann and Orellana,
1998). The hydrolysis of UDP eliminates a
potential inhibitor for the transferases and
also drives the transfer reaction toward the
polymerization of sugars. U M P leaves the
Golgi cisternae by a process stimulated by
UDP-Glc, suggesting that the UDP-Glct r a n s p o r t e r would be a U D P - G l c / U M P
antiporter. On the other hand, inorganic
phosphate is not accumulated in the cisternae and is transported out of the cisternae.
5. The ECM-plasma
membrane-cytoskeleton continuum plays a role in signal transduction.
Recent evidence indicates that receptor kinases are important players in signal transduction pathways. Receptor kinases are proteins that have an extracellular domain that
receives the signal and a cytoplasmic domain that transmits the signal. Some other
plasma membrane anchored proteins also
have large extracellular domains. For example, some AGPs are anchored by a GPI
anchor. Recently a m e m b r a n e - a n c h o r e d
glucanase was discovered that plays a major
role in regulating cell elongation (Nicol et
al, 1998). When plant cells are plasmolyzed, the plasma membrane does not pull
evenly away from the cell wall, but the two
remain in close contact at very specific adhesion points called Hechtian strands. These
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C H R I S P E E L et al. Biol Res 32, 1999, 3 5 - 6 0
Hechtian strands are not found when cells
are incubated with short peptides that con­
tain an Arg-Gly-Asp (RGD) motif. Isolated
protoplasts of plant cells readily agglutinate
when they are incubated with a genetically
engineered protein that contains numerous
RGD motifs, suggesting that plasma mem­
branes contain RGD receptors. In animal
cells, adhesion is mediated by plasma mem­
brane receptors called integrins that interact
with extracellular adhesion proteins (e.g.,
fibronectin and vitronectin) via the tripeptide Arg-Gly-Asp (RGD), a motif that is
conserved in adhesive proteins. On the cy­
toplasmic side of the membrane integrins
interact with proteins of the cytoskeleton,
thereby establishing the ECM-plasma membrane-cytoskeleton continuum. There is as
yet very little evidence that in plants transplasma membrane proteins interact with the
cytoskeleton, but we know that a large num­
ber of signals induce rapid cytoskeleton re­
organization, leading to the suggestion that
a similar continuum exists in plant cells.
II. THE SECRETORY SYSTEM PLAYS AN IMPORTANT
ROLE IN SORTING PROTEINS FOR DIFFERENT CEL­
LULAR DESTINATIONS
/. Vacuolar targeting requires
vacuolar sorting
signal.
a
specific
The secretory system is a series of or­
ganelles that includes the e n d o p l a s m i c
reticulum (ER), Golgi apparatus, plasma
membrane and vacuole. Soluble proteins
containing an N-terminal signal sequence
are co-translationally inserted into the ER
lumen, and after their synthesis is complete
they are transported through the secretory
system in transport vesicles that bud from
one compartment and fuse with the next.
F r o m the ER, proteins are transported
through the Golgi cisternae to the transGolgi network, where a major sorting event
occurs. Proteins are either secreted to the
outside of the cell (the default pathway) or
they are targeted to the vacuole via a prevacuolar compartment, a route that is be­
lieved to be mediated by membrane-bound
receptors to which the cargo proteins bind
so that they can be properly sorted.
For a soluble protein to reach the vacu­
ole, it must contain a specific vacuolar
sorting signal. Three types of vacuolar
sorting signals have been identified (Fig
3). Some proteins contain a propeptide,
which comprises the sorting signal and
which is removed from the protein upon
deposition in the vacuole. Several pro­
teins are known to contain a propeptide at
their N - t e r m i n u s , including sweet potato
sporamin and barley aleurain (reviewed in
Chrispeels and Raikhel, 1992). Deletion
and mutagenesis studies have shown that
the N - t e r m i n a l propeptide is required for
transport to the vacuole and have defined
a conserved motif within the propeptides,
which is essential for proper sorting and
Signal Peptide
Sporamin,
Aleurain
NTPP
CTPP
Tobacco Chitinase,
Barley Lectin
Phytohemaglutinin
Part of mature protein
Figure 3. Location of vacuolar targeting signals in plant proteins. Vacuolar targeting signals can be
propeptides at the C-terminus (CTPP) or the N-terminus (NTPP) of a protein, or be part of the mature
protein.
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C H R I S P E E L et al. Biol Res 32, 1999, 3 5 - 6 0
thus is thought to be recognized by the
sorting machinery.
The second type of propeptide signal is
found at the C-terminus of several vacuolar
proteins, including barley lectin and to­
bacco chitinase (reviewed in Chrispeels
and Raikhel, 1992). No common motif can
be identified between these sorting signals,
and while the propeptides have been shown
to be both necessary and sufficient for vacu­
olar transport, extensive mutagenesis studies
have determined that there is no sequence
motif that is required for their function
(reviewed in Bar-Peled et al, 1996). It is
still not known whether a receptor protein
is present at the trans-Golgi network that
recognizes this type of signal.
Finally, a region of the mature protein
can also serve as a sorting signal. Bean
phytohemagglutinin contains this type of
signal (reviewed in Chrispeels and Raikhel,
1992). although the nature of the signal
means that it is very difficult to precisely
identify the residues responsible for tar­
geting information. Legumin also appears
to contain vacuolar sorting information within
the mature protein, and the entire legumin a
chain is required for efficient sorting (BarPeled et al, 1996). The mechanism by which
these signals work is not known.
2. Cells have more than one type of vacuole
and probably have multiple paths for pro­
teins to reach the vacuole.
Studies using the fungal m e t a b o l i t e
wortmannin, an inhibitor of phospholipid
metabolism in tobacco cells, have shown
that proteins containing an N-terminal sig­
nal are transported to the vacuole by a
different mechanism than proteins contain­
ing a C - t e r m i n a l signal ( r e v i e w e d in
Bassham and Raikhel, 1997). In the pres­
ence of wortmannin, proteins containing
the barley lectin C-terminal propeptide were
secreted, whereas those containing the
sporamin N-terminal signal were correctly
sorted to the vacuole. In addition, mem­
brane proteins are transported by a diffe­
rent pathway compared to soluble proteins
(reviewed in Bassham and Raikhel, 1997).
Therefore, it appears that multiple path-
ways to the vacuole exist within a single
plant cell. In fact, some specialized cell
types have been shown to contain two dis­
tinct types of vacuoles, which have their
own d i s c r e t e c o m p l e m e n t of p r o t e i n s
(Neuhaus and Rogers, 1998). In mature
vegetative cells, these vacuoles are thought
to fuse to form a single large central vacu­
ole upon which all the vacuolar targeting
pathways converge (reviewed in Bassham
and Raikhel, 1997).
3. Vacuolar targeting requires
transmem­
brane cargo receptors that function in traf­
fic between the trans-Golgi
network and
the prevacuolar
compartment.
Using an affinity column with immobilized
peptide corresponding to the aleurain Nterminal propeptide, a protein was isolated
from pea clathrin-coated vesicles that is
thought to represent a sorting receptor for
vacuolar proteins that have this signal (re­
viewed in Beevers and Raikhel, 1998). This
protein (BP-80) is an integral membrane
protein that can also bind to other N-terminal signals, but not to the barley lectin Cterminal propeptide. An Arabidopsis homologue of BP-80 (named AtELP) was
identified by searching the DNA sequence
AtVTIla
(v-SNARF.)
AtELP
(Vacuolar
Cargo Receptor)
AtPKPUp
(t-SNARK)
Prevacuolar
Compartment
Figure 4. Schematic diagram of targeting and fusion
of Golgi-derived clathrin-coated vesicles with the
prevacuolar compartment. Only three components
of the machinery are depicted in this figure: a putative
vacuolar sorting receptor, AtELP, a vSNARE,
AtVTI 1 A, and a tSNARE, AtPEP 12p. Other members
of the machinery that are important for vesicle
targeting and target membrane fusion, including
soluble and membrane receptors, and proteins that
constitute the clathrin coat, are not shown on this
diagram.
42
C H R I S P E E L etal.
Biol Res 32, 1999, 3 5 - 6 0
database using motifs common to sorting
receptors from various eukaryotic species
(reviewed in Beevers and Raikhel, 1998).
Both the pea and Arabidopsis proteins have
been localized by electron microscopy to
the Golgi apparatus, and in addition AtELP
was found in the pre-vacuolar compart­
ment (Sanderfoot et al, 1998) (Fig 4). These
receptor-like proteins are proposed to bind
to their cargos (proteins containing an Nterminal vacuolar sorting signal) at the
trans-Golgi network and package them into
clathrin-coated vesicles for transport to the
prevacuolar compartment.
After the formation of vesicles contain­
ing vacuolar cargo proteins from the transGolgi network, the vesicles have to be di­
rected to the prevacuolar compartment and
fuse with that organelle. A number of pro­
teins have been identified from various
organisms that are required for the correct
targeting, docking and fusion of a transport
vesicle with its target membrane. As some
components for vesicle targeting and fu­
sion appear to be similar between many
different organisms and different stages of
the secretory pathway, the basic mecha­
nisms for these processes are probably con­
served. Information from the better studied
yeast and mammalian systems can there­
fore be very useful in the study of vesicle
trafficking in plants.
4. The SNARE hypothesis for vesicle fusion
and the discovery of plant homologues
that
function in this process.
A c c o r d i n g to the S N A R E h y p o t h e s i s ,
vesicle docking requires the interaction of
a vesicle SNARE (vSNARE) with a SNARE
on the target membrane (tSNARE) (Beevers
and Raikhel, 1998). The first v S N A R E
(syntaxin 1) was isolated from the synaptic
plasma membrane of neurons, where it is
involved in neurotransmitter release. It has
been shown to form a complex with several
soluble and integral membrane proteins that
arc all believed to function in vesicle dock­
ing and fusion by mechanism as yet un­
known. A number of syntaxin homologues
have now been identified in non-nerve cells
and in various organisms. They have simi­
lar sequences and predicted secondary
structures to syntaxin 1, and therefore may
function in a similar way in a variety of
vesicle fusion events. It has been hypoth­
esized that a different syntaxin isoform
may be required for each transport step of
the s e c r e t o r y p a t h w a y ( R o t h m a n and
Sollner, 1997).
Typical syntaxin homologues are type II
transmembrane proteins. Most of the pro­
tein is hydrophilic and faces the cytosol,
except for a hydrophobic domain about 2030 amino acids long at the C-terminus of
the protein. The hydrophilic part is thought
to interact with other factors involved in
vesicle trafficking, whereas the hydropho­
bic domain is responsible for anchoring the
protein in the membrane.
Several syntaxin homologues have now
been identified in plants that are also pre­
sumably involved in transport through the
secretory pathway. By functional comple­
mentation of the yeast
(Saccharomyces
cerevisiae)
secretory pathway mutant
pep 12. researchers have isolated a cDNA
clone (AtPEPl 2) from Arabidopsis
thaliana
ecotype Columbia that encodes a protein
homologous to yeast P e p l 2 p and other
members of the syntaxin family (reviewed
in Bassham and Raikhel, 1997). The yeast
pep 12 mutant is defective in transport of
some soluble proteins to the vacuole and is
thought to function in transport between
the trans-Golgi network and the prevacuolar
compartment (PVC). AtPEPl 2p may there­
fore play a role in transport to the vacuole
in plants. A t P E P l 2 p has been localized by
electron microscopy to a PVC in Ara­
bidopsis roots and may act as a receptor for
trans-Golgi network-derived transport
vesicles en route to the vacuole (Fig 4). By
genetic analysis, another Arabidopsis
syntaxin homologue, KNOLLE (reviewed
in Beevers and Raikhel, 1998), was identi­
fied. It is presumably involved in cytokin­
esis in the embryo, and is located at the
forming cell plate during cell division (re­
viewed in Beevers and Raikhel, 1998). Sev­
eral syntaxin homologues therefore exist
in Arabidopsis, and each of them may be
involved in separate steps of the secretory
system or function at different develop­
mental stages, or in different tissues. Re-
43
C H R I S P E E L et al. Biol Res 32, 1999, 3 5 - 6 0
cently, a v S N A R E protein, A t V T I l a , that
most likely interacts with AtPEP12 has
been identified (H. Zheng and N. Raikhel,
unpublished data) (Fig 4).
III. THE PLASMA MEMBRANE AND THE TONOPLAST
CONTAIN CHANNELS AND TRANSPORTERS WITH
IMPORTANT SIGNALING FUNCTIONS
1. Calcium is an important second mes­
senger in many signal transduction
networks.
2+
The concentration of free C a in the cyto­
plasm is very low (10 to 200 nM) and most
of the cellular calcium is sequestered in
calcium stores in cytoplasmic organelles
or in the cell wall (Fig 5). The movement of
C a from the cytoplasm into the stores
requires energy in the form of ATP and is
carried out by calcium dependent C a 2+
2+
ATPases. Movement in the other direction
(release into the cytosol) occurs via cal­
cium channels down the electrochemical
calcium gradient. Both types of proteins,
pumps and channels, are particularly abun­
dant in the plasma membrane and the tonoplast, but also occur in other membranes
such as the ER and the mitochondria. In
1957, Hodgkin and Keynes performed a
simple but seminal experiment. They in­
jected radioactive calcium and potassium
into the cytoplasm of an axon. After sev­
eral hours they measured the distribution
of the two radioactive ions and found that
potassium had diffused throughout the cy­
toplasm, but the radioactive calcium had
barely moved from the site of injection.
The inescapable conclusion was that cal­
cium is immobile in the cytoplasm. When
calcium is artificially elevated in the cyto­
plasm, the extra calcium will bind to pro-
extracellular Ca
2+
fi
wall binding sites. lmM
(pectins, proteins)
membrane phospholipid
binding sites
ATPase and
Ca /H antiport
2+
+
Figure 5. Summary of the major calcium relationships in a plant cell as presently understood. The figure indicates
the presence of channels and calcium-ATPases and estimates of concentration in the main organelles. The
presence of cytoplasmic binding sites inhibits direct calcium diffusion in the cytoplasm and necessitates the
production of calcium waves using IP -calcium sensitive channels in the ER and vacuole.
3
44
C H R I S P E E L et al. Biol Res 3 2 , 1 9 9 9 , 3 5 - 6 0
2+
teins and is sequestered into C a stores.
These stores are in the cell wall, the vacu­
ole, the ER and the mitochondria. Calcium
channel proteins permit the flow of cal­
cium from the stores back into the cyto­
plasm. These channels open when cells are
signaled in some way, and calcium then
enters the cytoplasm down in its electro­
chemical gradient. Families of calcium
channels are known to exist: some are activ­
ated by changes in membrane potential,
some by membrane stretch and yet others
by second messengers. A single channel
can transmit 10 atoms of C a per second.
Elevation of calcium at the channel mouth
can be rapid, and concentration here can
reach 100 m M . The channels rapidly close
when cytosolic calcium is elevated and
calcium is then pumped back into the intra­
cellular stores or into the wall. Transient
elevations of calcium, sometimes called
spikes, may last anywhere from a few sec­
onds to many minutes, depending on the
characteristics of the stimulating signal.
6
2+
2. How does calcium make waves, and how
do the waves move?
Calcium itself does not move through the
cytoplasm; however, this calcium spike can
propagate itself as a wave (Malho et al,
1998). This propagation depends on the
second messenger, inositol 1,4,5 triphos­
phate (IP ). This molecule is generated
through the action of the plasma membrane
bound enzyme, phospholipase C. When
phospholipase C is activated it hydrolyzes
phosphatidyl inositol diphosphate (PIP ) to
yield diacylglycerol and I P . Unlike cal­
cium, I P is freely mobile in the cytoplasm,
and I P - s e n s i t i v e calcium channels are
found in organelles throughout the cyto­
plasm. When I P is present, the calcium
channels open and calcium enters the cyto­
plasm. The role of I P in mobilizing cal­
cium was demonstrated in plant cells a
decade ago (Alexandre et al, 1990; Gilroy
et al, 1990), and I P binding to the channels
was discovered more recently (Allen et al,
1995). These observations help us under­
stand how calcium makes a wave. The I P sensitive calcium channels need both I P
3
2
3
3
3
3
3
3
3
3
and calcium to open (Marchant and Taylor,
1997). When I P binds to a channel, a cal­
cium binding site is briefly exposed. If
calcium is absent, the channel rapidly inac­
tivates. If calcium is present, the calcium
binding site is occupied and the channel
opens. However, the binding of calcium
only serves to delay the eventual IP3-induced channel inactivation. Mobilization
of calcium through a single IP3-dependent
calcium channel is therefore brief and selflimiting. So how does the wave move?
When an IP3-dependent calcium channel
opens, the calcium concentration near ad­
jacent channels will increase. The calciumbinding sites of these channels will be oc­
cupied and they will briefly open, in turn
enabling the opening of others. Calcium is
therefore responsible for inducing further
calcium release and this release underpins
wave movement. The wave is not a forward
movement of calcium, but a forward move­
ment of calcium release. IP3-induced inac­
tivation of channels generates direction in
calcium wave movement away from the
point of origin. Because these channels are
located in membranes, the wave moves
across the face of the membrane.
3
3. Plasma membrane potassium
play fundamental
roles in signal
tion processes in plants.
channels
transduc­
Potassium transport processes play a fun­
damental role in plant cell physiology. Po­
tassium ion m o v e m e n t s across the cell
membrane are involved in leaf movement,
cell elongation, plant growth and develop­
ment and in the regulation of stomatal ap­
erture (for reviews see Schroeder et al,
1994; Cherel et al, 1996; Fox and Guerinot,
1998). Potassium is the dominant cation
within the vacuole, and accumulation of K
inside this organelle is essential for gene­
rating the turgor pressures that drive cell
expansion. Potassium transport in plants is
mediated by carriers and channels and is
customarily divided into high- and lowaffinity K uptake (Schroeder et al, 1994).
High-affinity K transport is defined as the
K influx that occurs when the external K
concentration is in the range of 1 to 200
+
+
+
+
+
45
C H R I S P E E L et al. Biol Res 32, 1999, 3 5 - 6 0
lnM (e.g., Fox and Guerinot, 1998) and was
thought to be mediated exclusively by car­
riers. Low-affinity K uptake is mediated
by voltage-dependent inward rectifier K
channels when the external concentration
of K is > 0.3 mM. These channels belong
to a superfamily of ion channels designated
the S4 superfamily, which includes Na ,
C a , and K voltage-dependent channels
(Jan and Jan, 1992). In all of these chan­
nels, the fourth transmembrane domain (S4)
contains several positively charged amino
acids in every third position (Fig 6B).
KAT1 and AKT1 (Fig 6A) were the first
inward rectifier K channels cloned from
the plant Arabidopsis
thaliana (Schroeder
et al, 1994). The KAT1 channel is ex­
pressed in guard cells and the vascular
system of the stem and plays a crucial role
in stomatal opening (Fox and Guerinot,
1998). AKT1 is present in roots and plays
an important role in K uptake from soil.
Mutants from Arabidopsis thaliana in which
+
+
+
+
2+
+
+
+
the AKT1 channel gene was disrupted show
reduced K uptake and grow poorly on
media containing < 100 mM K (Hirsch et
al, 1998). This result suggests that inward
rectifier channel activity might also medi­
ate high-affinity uptake (Schroeder et al,
1994). K S T 1 , a gene-encoding a guard cell
K channel, was cloned from a Solanum
tuberosum cDNA library based on its simi­
larity to K A T 1 . KST1 is also present in
flowers, albeit at a low level. Four more
inward rectifier K channels genes have
been identified in Arabidopsis thaliana and
Solanum tuberosum: AKT2 and AKT3 and
SKT2 and S K T 3 . Two outward rectifier K
channel genes have also been cloned from
Arabidopsis
thaliana: SKOR (Gaymard et
al, 1998) and K C O l . SKOR is present in
root stelar tissues and its role is to release
K into the xylem sap. K C O l is expressed
in seedlings and at low levels in leaves and
flowers. For original references see Fox
and Guerinot (1998).
+
+
+
+
+
+
B
Pore
Plant K channels subunits
Gene
KAT1
KST1
Spedes
A. Thelma
S. tuberosum
K Transport
Subunit structure
Inward
SKT2/3
S. tuberosum
Inward
W
WA V
I S
MBWS
s
B *w
V 1?M
T Pr V
WR№'5!'
• T13 ¥ S,t0
1 T | [:LT|
A LJJWS
1' T i • L
a vv
I
Shaker l_ V I L V
RATI
LSM L L»
AKT1
F N M l LU
SKOR L L L I LT
Inward
A. thaliana
D A F
•
TI
1
'•v, ft ?
" 1
|à y
VF
L
L
VH
I F L S HS G LO I .
¥ S SLFA L E DI F
V G A L F Ä LE D N F
V I L F F H IKE D I I
'
SKOR
A. thaliana
Outward
KCOl
A. thaliana
Outward
90
S4
Inward
AKT1/2/3
Shaker
KÄT1
AKT1
SKOR
Bum
a
Nucleotide binding
Ankyrin lepejt
domain
Figure 6 A. Cloned plant potassium channels and their subunit structure. B. Primary sequence alignment of
the pore region (top) and S4 segment (bottom) for three plant channels in comparison to Shaker. Potassium
channel fingerprint GYGD is highlighted. Conserved positively-charged residues in the S4 segment are
indicated in white by the amino acid number. C. Proposed transmembrane structure of one potassium channel
subunit indicating membrane spanning segments SI through S6 and the pore region (top), and tetrameric
subunit assembly to form one channel (bottom).
46
C H R I S P E E L et al. Biol Res 32, 1999, 3 5 - 6 0
4. Inward and outward rectifying
potas­
sium channels of plants and animals have
the same basic
structure.
As can be appreciated in Figure 6, inward
rectifiers and SKOR channels share the
same subunit structure consisting of six
transmembrane domains (S1-S6) and a pore
domain (P) linking segments S5 and S6.
The S4 domain contains conserved, regu­
larly-spaced, positively-charged amino acid
residues (Fig 6B). Many different K c h a n ­
nel genes in metazoans have this same ba­
sic structure, and Shaker, the first K chan­
nel gene cloned from the fly
Drosophila
melanogaster
(Tempel et al, 1987), is a
classic example. In Shaker K channels,
the S4 domain forms part of the voltage
sensor and the P region is part of the ion
conductive pore (Papazian and Bezanilla,
1997; Doyle et al, 1998). The pore region
of K channels is extremely well conserved
(Fig 6B). The Shaker K channel is a tetrameric structure formed by four identical
subunits (Fig 6C), and KAT1 and AKT1
are also likely to form tetramers (Cherel et
al, 1996). Additionally, a putative cyclic
nucleotide-binding domain is present in
the carboxyl terminus in both KAT1 and
A K T 1 . In AKT1 an ankyrin domain is
present downstream of the cyclic nucle­
otide-binding domain (Fig 6C). It is worth
noting here that KAT1 has a great simi­
larity to the ether a-go-go (eag)
Droso­
phila channel. The eag channel forms part
of a large class of K channels containing a
cyclical nucleotide binding domain and is
activated by hyperpolarizing voltages (e.g.,
Trudeau et al, 1995).
+
+
+
+
+
+
5. Aquaporins, or water channel
occur in the plasma membrane
vacuolar
membrane.
proteins,
and the
How does water flow through a biological
membrane? Does it simply diffuse through
the lipid bilayer, or does it pass more or
less unimpeded through specialized pores?
Recent discoveries show that water-flow
t h r o u g h m e m b r a n e s is f a c i l i t a t e d by
aquaporins (AQPs), integral membrane pro­
teins that can increase the water permea­
bility of a membrane 10-20 fold (Preston et
al, 1992; Maurel et al, 1993). Aquaporins
are not pumps, but simply facilitate the
passage of water through the membrane
when the osmotic or hydrostatic pressure is
higher on one side than on the other.
Aquaporins belong to the MIP family of
membrane proteins, which also includes
glycerol channels and possibly some ion
channels. These 26-28 kDa proteins have
six membrane-spanning domains and the
signature sequence N P A V T .
The activity of water channel proteins is
most conveniently assayed by expressing
them in Xenopus oocytes and then mea­
suring the swelling rate when the oocytes
are shifted to a hypotonic condition. AQPs
form tetramers in the membrane, but the
monomer is probably the unit that trans­
ports water. A molecular model suggests
that two cytoplasmic loops are half-buried
in the lipid membrane to form the aqueous
pore.
Both the abundance and the activity of
aquaporins can be regulated. Some aqua­
porins are expressed in a tissue-specific or
organ-specific pattern. This is true both in
mammals and in plants, where aquaporins
have been most studied. In
Arabidopsis
there are at least 23 different cDNAs that
fall into two large groups on a cladogram,
TIPs (tonoplast integral proteins) and PIPs
(plasma membrane integral proteins). In
addition, there is a small group of more
distantly related aquaporins (Weig et al,
1997). It is important to note that all of
these genes have not yet been shown to
encode active aquaporins. Indeed, some of
the proteins appear not to be active as
aquaporins when their mRNAs are intro­
duced in Xenopus oocytes. This could mean
that they do not encode aquaporins or that
the polypeptides made in the oocytes do
not behave as active aquaporins. Perhaps
they do not arrive at the plasma membrane,
or they need to be post-translationally mod­
ified or they need to interact with other
proteins not present in oocytes. In any
case, we cannot rule out the idea that the
these m e m b e r s of the M I P family have
completely different functions and may be
involved in the transport of other sub­
stances.
47
C H R I S P E E L et al. Biol Res 32, 1999, 3 5 - 6 0
Aquaporin expression can be altered by
hormones or environmental signals, such
as light or drought. Phosphorylation may
regulate the activity of AQP as has been
shown for Arabidopsis
a - T I P and spinach
PM28A. When spinach plants experience
water deficit, the plasma m e m b r a n e
aquaporin PM28A is less phosphorylated
than when the leaves are turgid and this
inhibition of phosphorylation may decrease
the activity of the water channel. When
PM28A is expressed in Xenopus oocytes,
its water channel activity is inhibited by
agents that prevent phosphorylation
(Johansson et al, 1998).
6. Aquaporins have multiple roles in cel­
lular and whole plant
physiology.
What is the role of aquaporins in cellular
physiology or in the whole plant? Deter­
mining the role of aquaporins is likely to
take many years of research, but some hy­
potheses are beginning to emerge as a re­
sult of studying the expression patterns of
aquaporins. Expression has so far been
studied mostly at the mRNA level and not at
the protein level, simply because it is much
easier to make gene-specific probes than to
make protein-specific a n t i b o d i e s when
dealing with a family of homologous pro­
teins. Analysis of the expression pattern of
the active aquaporin y-TIP from Arabi­
dopsis and Z m T I P l from Zea mays shows
that expression is highest in meristematic
and expanding cells, young root epidermis,
the endodermis, the parenchyma cells that
surround functional xylem vessels, and in
the pedicel (Barrieu et al, 1998; Chaumont
et al, 1998). What does this tell us about the
possible functions of aquaporins? The high
expression in the meristem and expanding
cells may be related to the biogenesis of
new vacuoles and the rapid expansion of
vacuolar volume that accompanies cell en­
largement. TIPs may be needed to sustain
the rapid water influx into the vacuole that
sustains cell growth in size. The high ex­
pression in the epidermis, endodermis and
pedicel suggests that TIPs may be present
at high levels in cells that can undergo
sudden fluxes in ions or metabolites. If
ions and metabolites suddenly enter the
cytoplasm, then the cell has to adjust its
osmotic pressure, and the most readily
available source of water is in the vacuole
(Fig 7). However, it is only readily avail­
able if the tonoplast is truly permeable to
water. High expression in the parenchyma
cells that surround the active xylem vessels
c o u l d i n d i c a t e an i m p o r t a n t r o l e in
transcellular water flow. Water flows radi­
ally in and out of vessels, and this flow will
be less impeded if the tonoplast is perme­
able to water. In this case the water perme­
ability of the tissue would reside in and be
r e g u l a t e d by t h e p l a s m a m e m b r a n e .
Whereas these are all interesting hypoth­
eses, they need to be supported by evidence
and such evidence will probably have to
c o m e from m u t a n t s that lack specific
aquaporins. A recent report indicates that a
A
• Metabolites Channel/Transporter
© T o n o p l a s t Aquaporin
B
• Tonoplast Aquaporin
Figure 7. Schematic representation of two roles for
tonoplast aquaporins. A. Tonoplast aquaporins are
needed for cytoplasmic osmotic equilibration in cells
that can experience rapid fluxes of metabolites or
mineral nutrients. B. Tonoplast aquaporins permit
rapid transcellular flow and increase the effective
cross-section of the cytoplasm for symplastic flow.
From Barrieu etal, 1998. Reprinted with permission
of the publisher.
48
C H R I S P E E L et al. Biol Res 32, 1999, 35-60
PIP antisense mutant of Arabidopsis has an
unusually large root system as if the plant
were trying to compensate for slower water
loss from the shoot.
IV. SIGNAL TRANSDUCTION NETWORKS IN ABIOTIC
STRESSES
7. Plants have both rapid
sponses to water deficit.
and slow
re-
An important difference between plants and
animals is that plants cannot move around
at will. This leads to a big difference when
it comes to the way they interact with their
environment. During evolution, animals
developed mechanisms to respond to the
environment by their behavior. Plants, on
the other hand, developed mechanisms to
respond to their e n v i r o n m e n t d e v e l o p menially. For plant biologists, it has always
been important to understand how plants
respond to their environment, especially
the stressful environments, because this is
critical for agriculture. Perhaps the most
common problem for farmers worldwide is
water stress, which can result from drought,
salinity, or cold temperature. Plants respond to water stress by both rapid and
slow pathways. The rapid pathway involves the regulation of ion channels that
close the stomatal pores in the leaves by
reducing guard cell turgor. Potassium fluxes
through potassium channels (see below)
play an important role in this process. The
slow response involves cascades of gene
expression that shape the developmental
program of the plant.
Ion channel regulation in guard cells provides a nice model for studying the early
steps in the water stress pathway. Studies
have shown that water stress elicits at least
two early p a t h w a y s ; one involves production of a plant hormone abscisic acid
(ABA), and the other is ABA-independent
(Fig 8). A number of reports suggest that
the A B A - d e p e n d e n t pathway further
branches out to contain calcium-dependent
and calcium-independent pathways, both
of which regulate the activity of several ion
channels in the plasma membrane and tonoplast (Assmann, 1993; Ward et al, 1995).
Calcium-dependent pathways inactivate the
inward K channel, and calcium-independent pathways activate the outward K channel. Both pathways may be involved in the
activation of outward anion channels. The
combination of these effects causes a net
efflux of ions and decline in guard cell
turgor, a prelude to stomatal closure.
An ABA-independent pathway directly
utilizes the osmotic stress as a signal that
regulates ion channels through a volume or
osmo-sensing mechanism (Liu and Luan,
1998). If a guard cell is in a swelling state
during stomatal opening, the inward K
channel is activated, while the outward K
channel is inactivated. This leads to increase in the turgor pressure and accelerates the opening process. If a guard cell is
in a "shrinking state" during hyperosmotic
shock caused by drought or darkness, the
inward K channel will be inactivated, and
the outward K channel will be activated.
This leads to further efflux of K and speeds
up the closure process. The volume, or
osmo-sensing mechanism, provides a way
for both positive feedback of stomatal
movements and for response to water stress.
In the slow response, a water stress signal
is perceived at the plasma membrane and
transduced all the way to the nucleus where
+
+
+
+
+
+
+
Osmotic Sensor
two component system
shol-like protein
oxidative burst
mechanical force
DgpaD fe8l[¡Í)©d][LD©ÍÍB®DT)
second messengers
CDPK/MAPK cascade
ABA
transcriptional factors
Gene Expression
Biochemical Processes
Figure 8. Regulation of stomatal closure resulting
from water deficit or osmotic stress. The three or
perhaps four pathways all lead to the opening of K
channels. The outflux of K causes a drop in guard
cell turgor and subsequent stomate closure.
+
+
49
C H R I S P E E L et al. Biol Res 32, 1999, 3 5 - 6 0
gene expression is initiated. It is not hard to
see the rationale because most develop­
mental and physiological changes in a plant
start with gene expression. Examples of
how scientists approach this problem are
given below.
Hyperosmotic Stress (drought, low humidity etc.)
ABA dependent pathway
/
2+
Ca -dependent
A B A - i n d e p e n d e n t pathway
\
/
2
Ca +-independent
Cytoskeleton?
\
*"
2. Molecular,
genetic and cellular
ap­
proaches have elucidated the signal trans­
duction pathways
involved in stress re­
sponses.
When plants are exposed to water deficit or
osmotic stress, they respond physiologi­
cally with adaptive processes. These pro­
cesses have a biochemical basis that in turn
depends on the expression of new genes.
How the water deficit signal is perceived
by the hypothesized osmotic sensor and
transduced to the nucleus (Fig 9) is pres­
ently being investigated with molecular,
genetic, and cellular techniques. The first
approach uses molecular biology. A gen­
eral strategy here is to first look for genes
that are induced by water stress conditions,
which can be done by a number of methods
such as differential screening of cDNA li­
braries. The next step is to define the cis
elements that are responsible for the stress
induction. These cis-acting elements are
usually located in the promoter region of
the gene. Using this information, one can
find the transcriptional factors that interact
with the cis-acting elements and go up­
stream of the signaling pathway by finding
components that regulate the transcriptional
factors. One of the leaders who use this
approach is Kazuo Shinozaki's group in
Japan. Here are two reports that precisely
d e s c r i b e the p o w e r of this a p p r o a c h .
Y a m a g u c h i - S h i n o z a k i and S h i n o z a k i
(1994) describe how the initial cis-acting
elements were identified. Liu et al (1998)
continued to identify the transcriptional
factors that act on the cis-acting elements.
More recently, a genetic approach was
applied to the dissection of the water stresspathway. This approach starts by fusing a
water stress-responsive promoter to the
luciferase reporter and then asking: Can we
select for mutants that have abnormal ex­
pression pattern of this reporter gene in
???
|
/
Ion channels
\
"*
G u a r d cell t u r g o r
Stomatal aperture
Figure 9. Components in water stress signaling that
lead to the expression of new genes and the
physiological response of the plants.
ransgenic Arabidopsisl
A chemical mu­
tagenesis procedure was used to mutagenize
the seeds, and seedlings were monitored
for luciferase expression under a fluores­
cent camera. Plants with aberrant expres­
sion patterns of reporter genes under var­
ious conditions (drought, cold, salinity)
were isolated and tested further to identify
those with true defects in their response to
water stress. A number of mutants have
been identified that are categorized into
many types. Some are constitutive response
mutants that express the reporter gene even
under normal conditions, suggesting a mu­
tation in negative regulators of the path­
way. Some are less responsive to the stress
condition, suggesting a mutation in posi­
tive regulators. Some are hypersensitive to
the stress condition, also suggesting mu­
tation in negative regulators (Ishitani et al,
1997).
A good example for a cellular approach
that utilized a transient assay system was
given by Sheen (1996). An ABA-responsive
promoter was fused to the gene-encoding
green fluorescent protein (GFP) and trans­
formed into maize mesophyll protoplast by
electroporation. The expression of G F P
was monitored under fluorescence micro­
scope after the cells were treated by ABA,
cold, and high salt. All these treatments
caused expression of GFP in the trans­
formed cells. With the idea that calcium
may serve as the second messenger for
A B A , c a l c i u m i o n o p h o r e was used to
mimic the treatments. Indeed calcium in­
creases in the cells led to expression of
GFP. Then the author went on to show that
50
C H R I S P E E L et al. Biol Res 32, 1999, 3 5 - 6 0
a calcium-dependent protein kinase
(CDPK) may mediate calcium messenger
and a protein phosphatase may antagonize
the CDPK function.
Biochemical approaches were also used
to tackle the early steps in the water stress
pathway. Most noteworthy might be the
finding of mitogen-activated protein kinase (MAPK) cascade in the early stage of
the water stress signaling (Jonak et al,
1996). This approach involves a technique
called in-gel kinase assay that can detect
the activity of a protein kinase in the protein extract isolated from plant cells treated
by various stress conditions. Using this
technique, M A P K is found to activate rapidly and transiently following water stress.
Activation of MAPK involves phosphorylation at both threonine and tyrosine residues and inactivation requires these residues to be dephosphorylated. Protein kinases and phosphatases involved in these
processes have recently been identified
and have opened up a new window to understanding stress-signaling pathways
(Meskiene et al, 1998; Xu et al, 1998;
Gupta et al, 1998; Luan, 1998).
In s u m m a r y , studies using these approaches suggest that water stress signaling
may have ABA-dependent and independent pathways. ABA-dependent pathways
may have calcium-dependent and calciumindependent branches. ABA-independent
pathways may involve osmosensing pathways that activate M A P K cascades. All
pathways may play a role in rapid response, such as stomatal regulation and
slow response involving gene expression.
A combination of these approaches will be
fruitful in dissecting the details of these
p a t h w a y s ( S h i n o z a k i and Y a m a g u c h i Shinozaki, 1997).
3. Abscisic acid and jasmonic acid
ate wound signal
transduction.
medi-
Wounding of plants, as often occurs when
they are attacked by insects, causes the
plants to mount a defense response. New
genes are expressed both at the site of
wounding and after the transmission of a
systemic signal throughout the plant. When
this phenomenon first began to be investigated, C. A. Ryan and his group established that wounding results in the induction
of proteinase inhibitors (PINs), and the
induction of the genes that encode these
inhibitors (pin genes) has been used as a
diagnostic to investigate the signal transduction pathway that leads from wounding
to gene expression. The plant hormones
abscisic acid (ABA) and jasmonic acid (JA)
play an important role in the conversion of
many environmental signals into changes
in plant gene expression. The in vivo involvement of ABA and JA in the gene
activation processes that follow mechanical damage of the plant tissue is supported
by the finding that endogenous ABA and
JA levels rise three- to six-fold upon
wounding. This increase is, moreover, not
restricted to the tissue damaged directly
but can also be detected in non-wounded,
systemically induced tissues (Peña-Cortés
et al, 1995). This phenomenon is common
to several plant species including potato,
tomato and tobacco (Sánchez-Serrano et
al, 1991). Furthermore, in all three plant
species, a correlation has been established
between the ABA and JA increase and either
the expression of the pin2 gene family (in
potato and tomato) or the activity of an
introduced reporter gene driven by the pin2
promoter in transgenic potato or tobacco
plants (Peña-Cortés and Willmitzer, 1995).
Further evidence for the involvement of
ABA and JA in wound-induced pin2 gene
expression was provided by a series of
experiments in which potato plants were
sprayed with ABA or JA and pin2 mRNA
accumulated in the absence of any wounding
(Peña-Cortés et al, 1991; Hildmann et al,
1992). Both n o n - s p r a y e d and directlysprayed l e a v e s s h o w e d i n c r e a s e d pin2
mRNA levels with a pattern identical to the
one described for wounded plants. Conclusive evidence for the involvement of ABA
in wound-induced activation was obtained
from mutants impaired in ABA biosynthesis. In those mutants, wound induction
of pin2 was not observed (Peña-Cortés et
al, 1989). However, in these mutants treatment with ABA caused a return of the accumulation of pin2 mRNA to levels normally
found in wild-type plants upon wounding
C H R I S P E E L et al. Biol Res 32, 1999, 3 5 - 6 0
(Peña-Cortés et al, 1995). Unexpectedly,
exogenous application of JA to these mutants also lead to the accumulation of pin2
mRNA, suggesting that the JA site of action
is located downstream from the site affected by ABA in the signal transduction
pathway mediating wound response (PeñaCortés et al, 1996).
4. An electrical current may mediate the
systemic
activation
of
wound-induced
genes.
The local and systemic activation of pin2
genes following wounding suggest the existence of a signal that moves from the
injured tissue to the upper or lower leaves
and leads to the systemic induction of pin2
gene expression. Several mediators, both
chemical and physical, have been suggested
as the putative systemic signal. Phytohormones such as ABA (Peña-Cortés et al
1989; Hildmann et al, 1992), JA (Farmer
and Ryan, 1990, 1992; Peña-Cortés et al,
1993), ethylene (O'Donnell et al, 1996),
the peptide systemin (Pearce et al, 1991),
and oligosaccharides (Ryan, 1987) all have
been demonstrated to be chemical signals.
Moreover, hydraulic and electrical signals,
classified as physical signals, have been
implicated in wound-induced gene expression.
The a p p e a r a n c e of variation potentials
throughout most of the shoot has been reported following localized wounding by
heating or burning in several plants (Wildon
et al, 1989; Malone and Stankovic, 1991).
Furthermore, Wildon et al (1992) reported
that mechanical wounding and localized
burning generate electrical signals that are
propagated through the plant, thereby inducing pin2 expression systemically.
Like wounding, the application of electrical current was able to initiate ABA and
JA accumulation in wild-type plants but
not in ABA-deficient plants (Herde et al,
1996). These results suggested that, like
wounding, electrical current requires the
presence of ABA for the induction of pin2
gene expression. In contrast to wounding
and electrical current, the burning of leaves
activated pin2 gene expression in ABAdeficient plants by directly triggering the
51
biosynthesis of JA via an alternative pathway that is independent of endogenous ABA
levels (Herde et al, 1996). Analyzing tomato ABA mutants that had progressively
lower ABA levels it was found that there is
a t h r e s h o l d l e v e l of A B A for s t r e s s induced gene expression (Herde et al, 1999).
Since pin2 mRNA was not detected upon
wounding or current application in mutants
with progressive attenuation in ABA content, it was suggested that a threshold value
of ABA exists and functions as an on/off
switch for wound-induced pin2 gene expression. Therefore, the ABA level functioning as the threshold value for the ABAdependent pathway that leads to pin2 gene
expression upon wounding and current application was between 21 % and 4 7 % of that
found in wild-type tomato plants.
The A B A level that a c c u m u l a t e d in
wounded and current-treated plants was
roughly reflected by the respective JA content of wild-type and mutant plants after
these treatments (Peña-Cortés et al, 1995).
Unwounded wild-type and ABA-deficient
plants had almost identical levels of JA,
suggesting that JA biosynthesis is not influenced by endogenous levels of ABA.
Harms et al (1995) reported that high endogenous level of JA in transgenic plants
overexpressing a flax aliene oxide synthase (AOS) cDNA did not induce constitutive pin2 gene expression. One possible
explanation for these phenomena is that
endogenous ABA and JA are stored in cellular compartments different from those
w h e r e A B A and JA a c c u m u l a t e upon
wounding. JA biosynthesis or at least part
of it, is suggested to occur in the chloroplast (Herde et al, 1995), which implies
that accumulation of JA in this organelle is
not sufficient to lead to changes in the
expression of JA-responsive genes such as
pin2.
5. The biosynthesis of J A during stress may
involve two different
cellular
compartments, the chloroplast and the peroxisomes.
The biosynthetic pathway for JA, called
the octadecanoic pathway, was first elucidated by Vick and Zimmerman (1987). The
52
C H R I S P E E L et al. Biol Res 3 2 , 1999, 3 5 - 6 0
synthesis of JA starts with linoleic acid and
i n v o l v e s t h e s e q u e n t i a l a c t i v i t i e s of
lipoxygenase to make fatty acid hydroper­
oxide, hydroperoxide dehydratase or allene
oxide synthase (AOS) to produce allene
oxide, and allene oxidase to produce 12oxo-phytodienoic acid (PDA). 12-oxo-PDA
t h e n u n d e r g o e s c h a i n s h o r t e n i n g via 6oxidation. It is assumed that these final steps
of the pathway take place in the peroxi­
somes, because that is where the 8-oxidation
e n z y m e s a r e l o c a t e d in p l a n t c e l l s .
Kleiter and G e r h a r d t (1998) r e c e n t l y p r o ­
v i d e d evidence that long-chain fatty acids
can be degraded completely into their con­
stituent acetyl units by higher-plant per­
oxisomes. But where are the first steps in
the pathway carried out? When Herde et al
(1995) examined the location of the flax
AOS in the transformed tobacco plants (see
above) they found it to be located in the
chloroplasts. Subsequently, Blee and Joyard
(1996) showed that the first three enzymes
in the pathway, up to the production of
DPA, are located in the chloroplast enve­
lope (Fig 10).
The failure of the increased levels of JA
in the AOS transgenic plants to activate the
Local and
systemic
signals
Pathogen or
Insect Attack
or Wounding
\1
PLASMA MEMBRANE
-Lipase
I
1
1
LOX
AOS
AOC
PEROXISOME
Reductase
ß-Oxidase
-12-oxo-PDA
X
Reductase
^
ß-Oxidase
I
Jasmonic
Acid
Gene activation
T
Phosphatase
Figure 10. Alternative pathway for jasmonic acid biosynthesis in plants. Over-expression of the flax allene
oxide synthase (AOS) cDNA leads to an increase of endogenous jasmonic acid (JA) levels in transgenic
potato plants, the transgenic protein being accumulated in the chloroplast. Blee and Joyard (1996) showed
the presence of this protein in the plastidial envelope allowing the formation of 12-oxo-PDA in this
organelle. The last steps of JA biosynthesis involve three B-oxidation reactions that may take place in the
peroxisomes. Acyl-CoA oxidase activity involved in the decarboxylation of fatty acids generates H O
Plastid and cytosolic phosphatases are involved in the signaling pathway mediating JA biosynthesis and
subsequently gene activation. Kinase activity seems also to be required for the wound-induced accumulation
of J A but not for gene activation. Salicylic acid inhibits JA biosynthesis by preventing the wound-induced
AOS gene expression (Harms et al, 1998). LOX, lipoxygenase; AOC, allene oxide cyclase; 12-HPLA,
hydroperoxylinolenic acid.
2
r
53
C H R I S P E E L et at. Biol Res 32, 1999, 3 5 - 6 0
pin2 genes could be explained by an accu­
mulation of JA in a compartment (possibly
the p e r o x i s o m e s ) that avoids the inter­
action of JA with its putative receptor and
subsequent gene activation. Although sixweek old AOS plants show JA levels 6-10
times higher than those found in untransformed plants, they do not show phenotypic changes. However, in older plants,
the older leaves of the transgenic lines
show symptoms that resemble the leaf le­
sion found in pathogen infected leaves.
The lesions become necrotic and expand on
the surface leading to leaf-death and ab­
scission. This phenotype expands to the
upper part of the plant, and after 13-weeks
the transgenic lines have only a few healthy
leaves at the top of the plant (the youngest
leaves). At this time untransformed plants
show the first senescence symptoms in the
older leaves. The phenotype observed in
the transgenic plants correlates with an
even higher JA content, reaching levels 1220 higher than those observed in normal
plants. In these older leaves pin2 gene ex­
pression has been turned on and other JAresponsive genes such as A O S , lipoxy­
genase have been activated as well. In ad­
dition, the expression of pathogen-related
genes (PRs) is also upregulated in the
transgenic plants (Pena-Cortes unpublished
results). More interestingly, the transgenic
plants with increased levels of JA also show
elevated levels of H , O This increase cor­
relates with the appearance of the symp­
toms and with the activation of the JAresponsive genes. Treatment of control
plants with H O also results in the accu­
mulation of pin2-, AOS-, LOX- and PRmRNA. Furthermore, mechanical wounding
or JA application leads to an increase of
endogenous levels of H 0 in potato leaves.
There are at least two p o s s i b l e expla­
nations for this observed increase in H 0 .
The rise in H O could simply be a byproduct
of the increased JA biosynthesis because
the reaction in the pathway catalyzed by
acyl-CoA oxidase generates H 0 . It is also
possible the JA activates other reactions
that lead to the production of H 0 or in­
hibits reactions that scavenge H 0 Further
work is clearly needed to determine how
the wound signal is transmitted in plants.
r
2
z
2
2
2
2
z
2
2
2
2
2
2
2
V. PLANTS RESPOND TO BIOTIC STRESSES FROM
PATHOGENS AND PESTS WITH A MULTITUDE OF
DEFENSE RESPONSES THAT INVOLVE COMPLEX
SIGNALING NETWORKS
1. Plant pathogen interactions
are gov­
erned by single genes in a gene for the gene
system.
Genetic analysis of many plant pathogen
interactions has demonstrated that plants
contain single resistance genes (or R genes)
that confer resistance against a specific
race of the pathogens that expresses a spe­
cific and complementary avirulence (avr)
gene. If either the plant R gene mutates or
the pathogen avr gene mutates, then resist­
ance of the plant to the pathogen is lost.
This is generally referred to as the gene for
gene system of plant pathogen resistance.
Resistance of a plant to a pathogen mani­
fests itself in a variety of ways, but is often
accompanied by a hypersensitive response
(HR). HR is the localized induction of cell
death at the site of infection, often re­
sulting in numerous visible necrotic spots on
the infected plant organ. Pathogenicity oc­
curs when the plant does not detect the
presence of the pathogen soon enough to
mount an effective defense response. Re­
sistance of a plant to a pathogen sets off a
series of reactions that include a rapid oxi­
dative burst at the plasma membrane, ion
fluxes characterized by H - K exchange,
crosslinking of cell wall proteins and syn­
thesis of phenolics, production of antimi­
crobial compounds (phytoalexins), and in­
duction of the pathogen related (PR) genes
that encode proteins such as chitinases,
glucanases, osmotin, etc. These responses
of the plant occur in many different plant
pathogen interactions (bacteria, viruses,
fungi) suggesting that there is a common
signal transduction network for the re­
sponse. In addition to this local necrotic
response, the pathogen also induces a sys­
temic response that results in a reduction of
the severity of disease symptoms after in­
fection by any pathogen, including a nor­
mally virulent one. This phenomenon is
referred to as systemic acquired resistance
(SAR).
+
+
54
C H R I S P E E L et al. Biol Res 32, 1999, 3 5 - 6 0
2. Molecular
identification
sistance (R)
genetic approaches led to the
and cloning of pathogen re­
genes.
tion of the proteins. Song et al (1995)
cloned the rice Xa21 gene which confers
r e s i s t a n c e to Xanthomonas
campestris
oryzae pv. oryzae, the organism that causes
rice bacterial blight. Xa21 e n c o d e s a re­
c e p t o r - l i k e kinase with an LRR in its
e x t r a c e l l u l a r d o m a i n . The d o m a i n struc­
tures of different R genes are shown in
Figure 11. This domain is thought to trans­
duce an e x t r a c e l l u l a r signal through acti­
vation of the intracellular kinase domain
(Ronald, 1997). Yet another class is rep­
resented by the rice gene Xa21D, encod­
ing a p r e s u m e d secreted LRR ( W a n g et
al, 1998). A l t h o u g h these genes have
highly c o n s e r v e d structural motifs, vir­
tually nothing is known about the subcel­
lular location of the p r o t e i n s , the identity
of the ligands or the d o m a i n s essential
for p a t h o g e n r e c o g n i t i o n . Once the mo­
lecular d e t e r m i n a n t s g o v e r n i n g these in­
t e r a c t i o n s are known we can begin to
d e v e l o p novel strategies for disease con­
trol.
In the past several years, disease resist­
ance (R) genes have been isolated from
diverse species such as tomato, Arabidopsis,
t o b a c c o , flax, b a r l e y and r i c e . Inter­
estingly, the proteins they encode share cer­
tain motifs that all suggest a role in signal
transduction. For example, the tomato Pto
gene that confers resistance to a bacterial
disease encodes a serine/threonine kinase,
suggesting a role in a phosphorylation cas­
cade. The largest group of R proteins car­
ries leucine-rich repeats (LLR), putative
cytoplasmic signaling domains and nucle­
otide binding sites. These proteins are
thought to reside entirely within the cell.
The tomato disease R gene Cf9, on the
other hand, is an extracytoplasmic glyco­
protein with an LRR motif in the extracel­
lular domain. Such LRR domains may be
instrumental in bringing about dimeriza
Cf
:
ii 3
J
Xa21D
Xa21
1
Pto
NBS-LRR
Figure II. Domain structure of different resistance (R) gene products involved in plant disease resistance.
55
C H R I S P E E L et al. Biol Res 32, 1999. 3 5 - 6 0
diffusion to
surrounding cells
Figure 12. Signals in plant defense response against pathogens. The defense response is triggered by
specific recognition between the product of the avirulence gene from the pathogen (Avr), and the product
of the resistance gene from the plant (R). After this recognition, several plant metabolites like H,0,.
ethylene, salicylic acid (SA), jasmonates (JA) and nitric oxide (NO) are accumulated. These metabolites
act as secondary signals for the transcriptional activation of defense genes.
3. Signaling for transcriptional
activation
of genes in the defense response
against
pathogens.
A number of genes that encode for defense
proteins are activated at different times
after infection ( H a m m o n d - K o s a c k and
Jones, 1996). These defense proteins have
different functions in the defense reaction,
such as antimicrobial activity, cell wall
reinforcement, synthesis of phenolic com­
pounds, and protection against oxidative
stress. Temporal coordination in the tran­
scription of these defense genes is con­
trolled by a complex network of signaling
pathways (Yang et al, 1997; Blumwald et
al, 1998). In recent years, significant ad­
vances have been obtained in the identifi­
cation of components involved in this net­
work. Thus, several plant metabolites have
been identified as secondary signals, which
are transiently accumulated in the plant
cell at different times after the pathogen
signal is perceived. These metabolites are
reactive oxygen species (ROS), such as
H , 0 „ salicylic acid (SA), jasmonates (JA),
ethylene, and nitric oxide (NO) (Fig 12)
(Durner et al, 1997; Van Camp and Van
Montagu, 1998). Furthermore, classical
components for intracellular signal trans­
duction, such as ion fluxes, G-proteins,
protein phosphorylation cascades, cyclic
AMP and cyclic ADP-ribose, have been
also identified (Zhu et al, 1996; Blumwald
et al, 1998; Durner et al, 1998).
One of the signaling pathways that has been
most studied in recent years is the SAmediated pathway (Durner et al, 1997). This
pathway leads to the transcriptional activation
of genes encoding for acidic pathogenesisrelated proteins (PR proteins) and for oxidative
stress-detoxifying enzymes (like glutathione­
s-transferase, GST). The biosynthesis of SA
occurs via the phenylpropanoid pathway,
through benzoic acid (BA). Other signals such
as ethylene, jasmonates, NO and H 0 activate
this biosynthetic pathway, through the transcrip­
tional activation or the enzymatic activation of
two key enzymes (phenylalanine ammonia-ly ase,
PAL, and benzoic acid 2-hydroxylase, BA2H).
SA is accumulated in the vacuole as a glucoside
(SAG), which is inactive (Fig 13).
2
2
56
C H R I S P E E L et al. Biol Res 32, 1999, 3 5 - 6 0
oxidative burst
metabolism
2
2
I
->- detoxification
catalases
AsPx
SA-
A
lipid peroxides?
(+)
(-)
SAI,
PA
-*-BA
PAL
NPR
SA
BH2H
A
/
(+)
SAG
tx defense genes
(GST, GPx, acidic PR)
\ ^ ^ A B P )
S A
I
ethylene
jasmonates
NO
Figure 13. Perception and transmission of'SA signal. Salicylic acid (SA) is synthesized from phenylalanine
(PA) and benzoic acid (BA). This biosynthetic pathway is activated by ethylene, jasmonates and NO (which
induce the expression of the gene for the enzyme phenylalanine ammonia-lyase, PAL), and by H , 0 , (which
activates the enzyme benzoic acid 2-hydroxylase, BA2H). SA can be accumulated as the inactive glucoside
form (SAG). SA activates transcription of genes encoding for acidic pathogenesis-related proteins (acidic
PR) and for the oxidative stress-detoxifying enzymes glutathione-S-transferase (GST), and glutathione
peroxidase (GPx). This effect of SA can be mediated by free radicals (S A)produced by inhibition of catalases
and ascorbate peroxidase (AsPx)by the product of genes nprl/sail/niml,
or by a SA-binding protein
(SABP2).
Different approaches have been used to
elucidate the mechanism by which SA is
able to transduce the signal through the
nucleus and activate transcription of genes.
Although some of the components involved
have been identified, the mechanism is still
unclear. Genetic approaches have led to the
isolation of the n p r l / s a i l / n i m l class of
Arabidopsis mutants (npr, nonexpresser of
PR gene; sai, salicylic acid insensitive;
nim, noninducible immunity). Recently, the
NPR1 protein was identified as a 60kDasoluble protein containing ankyrin repeats,
that are thought to function in proteinprotein interactions (Cao et al, 1997). Overexpression of NPR1 leads to enhanced re­
sistance to diverse pathogens (Cao et al,
1998).
Biochemical and molecular approaches
have lead to the characterization of SAbinding proteins in tobacco (Durner et al,
1 997). One group of these SA-binding pro­
teins are h e m e - c o n t a i n i n g e n z y m e s in­
volved in detoxification of H 0 , such as
catalases and ascorbate peroxidase (AsPx).
Binding of SA to these proteins inhibits
2
2
their enzymatic activity, leading to an in­
crease in the endogenous concentration of
H O The hypothesis that H 0 and other
ROS could act as second messengers down­
stream of SA in the transcriptional acti­
vation of genes is now being debated. It has
been also suggested that the radical SA,
produced by the interaction of SA with
catalases and peroxidases, could partici­
pate as a secondary signal through lipid
peroxidation. Recently, a new soluble SAbinding protein was identified in tobacco,
although its nature and function remain to
be elucidated (Du and Klessig, 1997). In
the nucleus, the SA signal is able to acti­
vate transcription of immediate early and
late genes. Several immediate early genes,
which transcription does not require de
novo protein synthesis, have been isolated.
A h o m o l o g u e to myb gene (Yang and
Klessig, 1996), a gene with homology to
glucosyltransferase genes (Horvath and
Chua, 1996), and GST genes ( U l m a s o v et
al, 1994) are a m o n g t h i s c l a s s . C i s elements that respond to SA with an imme­
diate early kinetics have been identified in
2
r
2
2
57
C H R I S P E E L et al. Biol Res 32, 1999, 3 5 - 6 0
the promoter of some of these and other
viral and bacterial genes (Qin et al, 1994).
In vitro DNA-protein binding assays and in
vivo transcriptional assays using transgenic
plants have given some information about
the mechanism by which these elements
are activated by SA. Results obtained from
these studies indicate that SA increases the
binding of transcription factors (from the
TGA family of bzip factors) to these cis
elements, mediated by protein phosphory­
lation (Jupin and Chua, 1996; Stange et al,
1997). Interestingly, other signals like
MeJA and auxins are also able to activate
transcription of GST genes, using the same
cis-elements and the same m e c h a n i s m .
Therefore, it seems that these nuclear events
of transcriptional activation of oxidative
stress-protective genes are the convergence
poi nt of different signal transduction path­
ways.
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