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Die and let live — programmed cell death in plants
Eric Lam*, Dominique Pontier and Olga del Pozo
Cysteine and serine proteases are prominent players in the
control of developmental and pathogen-activated cell deaths in
plants. Ethylene, salicylic acid, the small G-protein Rac,
calcium and reactive oxygen species are recurring mediators of
death signaling. Lastly, the mitochondrion has emerged in both
plant and animal systems as a ‘central depot’ that interprets
multiple signals and in some instances determines the fate of
the cell.
Addresses
Biotech Center, Foran Hall, Cook College, 59 Dudley Road, Rutgers
University, New Brunswick, NJ 08903, USA
*e-mail: [email protected]
Current Opinion in Plant Biology 1999, 2:502–507
1369-5266/99/$ — see front matter © 1999 Elsevier Science Ltd.
All rights reserved.
Abbreviations
AA
antimycin A
AEBSF
4-(2-aminoethyl)-benzenesulfonyl-flonyl-fluoride
AOX
Alternative oxidase
BI-1
Bax inhibitor-1
HR
hypersensitive response
NO
nitric oxide
PCD
programmed cell death
PR
pathogenesis-related
ROS
reactive oxygen species
SA
salicylic acid
SHAM
salicylhydroxamic acid
TE
tracheary element
VDAC
voltage dependent anion channel
Introduction
The study of programmed cell death (PCD) has seen a
remarkable boom in the past 10 years since the early recognition in the 1970’s by Andrew Wyllie and his coworkers
that a variety of animal cells die with similar morphological
features which appeared to be the result of an active suicidal process (for review see [1]). This renaissance of PCD
research was brought about by the convergence of work on
PCD with studies of animal genes involved in oncogenesis
such as Bcl-2, and genetic research on cell death mutants
by Bob Horvitz and his coworkers in the nematode
Caenorhabditis elegans (reviewed in [1]). The realization that
molecules with similar biochemical functions and structural domains are conserved in diverse organisms to regulate
PCD convinced most skeptics that this process can indeed
be studied at the molecular level with defined players.
Remarkably, although many signaling pathways and regulators of animal PCD have been defined in the past
10 years, they revolved around only a small number of central players and their modes of action appeared to be
variations on a few themes. The central enzymatic activity
that has been most well-characterized is a growing family
of cysteine proteases called caspases. They are synthesized
as zymogens that require specific cleavage and subsequent
oligomerization in order to reveal their enzymatic activities. Once activated via proteolysis, they appear to activate
a cascade of proteases in a serial fashion that is reminescent
of the classic blood clotting factor cascade.
PCD research in plants has also begun to blossom in the
past five years [2,3]. There are three major reasons for our
interest in this process in plants. First, it is a way of life for
plants, just as it is for animals. Essential processes such as
xylem differentiation and tapetal cell degeneration all
involve PCD. Second, comparison of the mechanisms and
molecules that are involved in the two kingdoms should
enlighten us about the evolution of PCD and may help to
determine the indispensible players in the chain of events
that lead to cell death in eukaryotes. Third, from an
applied perspective, the ability to regulate cell death in
plants may have important applications in agriculture and
post-harvest industries. For example, suppression of PCD
induced by biotrophic pathogens could minimize disease
symptoms and inhibition of cell death during senescence
may prolong the shelf-life of crops and vegetables. In this
review, we discuss work published in the past year that has
begun to reveal some of the players that are involved in
controling PCD in plant cells.
The demise of a cell — the phenomenon
What actually occurs when a plant cell commits suicide? In
contrast to apoptosis in animals, the presence of a rigid wall
in plant cells poses a distinct problem: because the dying
cell cannot be packaged into small apoptotic bodies and
then engulfed by its neighbors, as in the case for their animal counterparts, plants must deal with suicidal cells and
their contents in fundamentally different ways. A distinguishing feature of plant cells is the prominant vacuole,
which contains many catabolic enzymes such as proteases
and nucleases. It is thus not surprising that this organelle
appears to play an important role in tracheary element
(TE) differentiation [4••] as well as degeneration of the
aleurone in barley [5•]. The disruption of the vacuole during TE development appears to catalyze the autolysis of
the cell’s content in a systematic fashion after the synthesis of the reticulated secondary cell wall [4••,6].
The self-ingestion approach to the dismantling of cells
destined for PCD is likely to be a common strategy for
plants. However, the sequence of events and the resulting
cellular morphological changes may differ depending on
the particular inductive death signal [2,3,6–9]. This may be
a consequence of the role that cell death may have in different situations. In the case of developmental cell death
such as senescence and TE differentiation, the recycling of
the dead cell’s content may be an important ‘purpose’. In
the case of cell death related to pathogen containment, cell
Die and let live — programmed cell death in plants Lam, Pontier and del Pozo
death may be used to rapidly ‘isolate’ the infected cells
and efficient transport of the dead cell’s content may be
secondary [10]. In most cases of plant PCD, vacuolization
of the cytoplasm and disruption of the tonoplast are common events that appear to precede mass disruption of the
mitochondria and nucleus. On the other hand, recent flow
cytometry studies with tobacco protoplasts suggest that
nuclear DNA condensation could be activated before the
first irreversible step of PCD [11•]. Evidence for disruption of the cytoskeleton early on in plant PCD has also
been suggested by the finding that disruption of microfilaments by cytochalasin E could suppress cell death induced
by fungal pathogens in cowpea [12] and that cytoplasmic
streaming is arrested during TE differentiation [4••].
What’s new in PCD signaling in plants?
The signaling processes that activate PCD in plants
promise to be as diverse as those that have been found in
animals. One of the most well-characterized systems to
date is the differentiation of Zinnia TEs in vitro. Earlier
work with the Zinnia TE system has established that
wounding and hormonal balance (auxin and cytokinin
treatments) are critical signaling components for initiating
the transformation of mesophyll cells to TE [6]. More
recently, the involvement of calcium, heterotrimeric
G-proteins and extracellular proteases has been
suggested [4••].
In contrast to other plant PCD systems, a large body of
literature has accumulated on the study of cell death during plant pathogen interactions [2,3,7,13]. Two major
types of cell death can result when plants are inoculated
with a pathogen: the rapid hypersensitive response (HR),
which is typically associated with resistance, and a slower
form of death that is the result of disease. Previous work
has implicated the involvement of protein phosphorylation, calcium channel functions and reactive oxygen
species (ROS) in HR cell death. Studies of disease-related cell death are less developed as it is not obvious that
they resulted from a suicide process. However, recent
work with the fungal toxin victorin has implicated an
active participation of the dying cells and more particularly, the requirement of ethylene in disease-related cell
death induction [14•]. Consistent with these observations, ethylene insensitivity in tomato was found to
suppress cell death due to infection with biotrophic
pathogens [15]. These observations raised the possibility
that ethylene action may be involved in the slower cell
death that is manifested during plant diseases. In contrast, resistance response does not appear to require
ethylene action [16]. Interestingly, ethylene may also be
involved in developmental cell death such as endosperm
degeneration in wheat and during maize aerenchyma formation induced by hypoxia [9,17]. Taking into account its
classic role in tissue senescence, it appears that ethylene
may be an important signal for slower forms of cell death
during which efficient recycling of the dead cell’s content
might be important for the plant. This interpretation is
503
consistent with our recent observation that a senescencespecific marker gene, SAG12, is activated in cells
neighboring HR lesions, where chlorotic symptoms of
cell death are often observed [10].
Salicylic acid (SA) has emerged in the past several years as
a positive feedback regulator of cell death during the HR
[2,3]. This was first suggested in the study of disease lesion
mimics in Arabidopsis [18], where spontaneous cell death
in the mutants lsd6 and lsd7 was repressed by transgenic
expression of a gene encoding a bacterial salicylate hydroxylase (NahG), which converts SA to catechol. Two new
disease lesion mimic mutants, ssi1 and acd6, appeared to
show the same dependence on SA [19•,20••]. Interestingly,
ozone induced cell death in Arabidopsis has also been
shown to be potentiated by SA [21]. A cell wall associated
protein kinase has recently been suggested to be involved
in suppressing cell death signaling by SA [22•].
The specific requirement of calcium signaling in HR cell
death had been suggested through studies using calcium
channel blockers [2,3,7]. The recent work of Heo et al.
[23••] suggested that this dependence on calcium may
involve specific isoforms of calmodulin. HR-like cell
death, pathogenesis-related (PR) gene induction and
broad spectrum disease resistance are activated by transgenic expression of two soybean calmodulin-encoding
genes. These phenotypes are not SA dependent and the
level of SA is not altered by the transgenes. Thus, distinct
pathways can give rise to phenotypes that are morphologically very similar. As these soybean genes are induced
during the defense response, they may participate in the
long term signaling for resistance rather than the initial signaling process to activate the HR pathway.
Another likely signaling molecule for the activation of HR
cell death is ROS (reactive oxygen species) [24,25•]. Two
phases of ROS generation by plants are known to occur
upon incompatible interactions with pathogens [24]. We
have shown that ambient oxygen pressure is required for
HR cell death while PR gene induction remains little
affected by low oxygen pressure [26]. HR-like cell death
and defense gene activation can also be induced ectopically by either transgenic expression of a gene encoding
glucose oxidase [27] or suppression of endogenous catalase
[28•]. The action of ROS may be augmented by nitric
oxide (NO) to potentiate and propagate the cell death signals [25•]. It has been speculated for a number of years that
ROS involved in activating the HR may be generated by
an NADPH oxidase similar to the well-characterized ROS
signaling system in neutrophils [24,25•]. Recent identification of six Arabidopsis genes homologous to the large
subunit of the human NADPH oxidase should facilitate
testing of this hypothesis [29,30]. In support of the
involvement of NADPH oxidase as an activator of ROS for
HR induction, recent work by Kawasaki et al. [31••]
showed that the small GTP-binding protein Rac may
mediate the HR response of rice in an ROS-dependent
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Cell biology
manner. Rac is known to regulate mammalian NADPH
oxidase as well.
How does ROS lead to the activation of cell death and how
is it managed in the cell? Reichheld et al. [32] suggest that
alteration of the cell cycle may be one aspect of death
induction by ROS. Interestingly, the role of SA in cell proliferation as well as cell death was postulated to explain
some of the abnormal leaf morphologies observed in the
acd6 mutant [20••]. Thus, SA and ROS may cooperate to
arrest the cell cycle and tip the balance toward cell death.
As plants are likely to be exposed to a variety of agents or
environmental conditions that can activate ROS production, a buffering system that can deal with normal
fluctuations in ROS level should be in place to prevent
chance activation of PCD. The work of Tamagnone et al.
[33•] showed that transgenic expression of a transcription
regulator leads to plant cells that are hyper-responsive to
pathogen challenge, abnormal cell expansion and PCD in
the leaf palisade cells, and increase in lipid peroxidation.
Addition of phenolic precursors can rescue cell cultures
derived from these transgenic plants from cell death and
abnormal morphologies, thus showing nicely that these
phenotypes are reversible. This work provided evidence
that phenolic products could play an important role in ROS
homeostasis during normal plant development.
inhibited by the serine protease inhibitor AEBSF (4-[2aminoethyl]-benzenesulfonyl-flonyl-fluoride), but not by
caspase inhibitors [40]. Activation of cell death by victorin
in oat also appears to be mediated by cysteine proteases
sensitive to E-64 and calpeptin [14•]. These studies suggest that proteases with different specificities may be
used to regulate cell death induced by different agents in
different species. Involvement of intracellular cysteine
proteases [6] as well as an extracellular serine protease
[4••] have also been proposed to regulate cell death activation during TE differentiation.
In recent years, the role of mitochondria in controlling cell
death activation has been recognized in the animal field.
Cytochrome c leakage from this organelle appears to
mediate many signaling pathways for PCD. Once in the
cytosol, cytochrome c can activate caspases through interaction with Ced-4/APAF1 in conjunction with dATP
[35••]. In addition, disruption of electron flow through the
electron transport chain of the mitochondria is likely to
lead to accumulation of reducing equivalents that can
result in ROS. Bcl-2 related proteins can either activate or
repress the leakage of cytochrome c through the voltage
dependent anion channel (VDAC) located in the membrane of the mitochondria [41]. They can also suppress or
activate caspases through interaction with the Ced4/APAF1 regulator.
The nuts and bolts of the death engine
The identities of the key executioners of plant cells
remain elusive, whereas in animal systems a large number
of caspases and their regulators have been defined in the
past five years [34•,35••]. Caspase-like proteolytic activity
has been observed to be transiently activated in plants
synchronized to undergo the HR [36••]. Peptide inhibitors
of caspases can abolish HR cell death induced by avirulent bacteria without affecting the induction of defense
genes significantly. Furthermore, transgenic expression of
the gene encoding a broad range caspase inhibitor from
Baculovirus, p35, also leads to the delay of HR cell death
in tobacco (O del Pozo, E Lam, unpublished data). More
recently, Mitsuhara et al. [37•] reported that in tobacco
expression of transgenes encoding the pro-survival cell
death regulators Bcl-xL and Ced-9 can delay HR cell
death as well as death induced by UVB and paraquat,
whereas expression of the transgene encoding the proapoptotic Bax is found to activate HR-like cell death
[38••]. These observations suggest the possibility that caspase-like proteases and a Ced-4/APAF1 like regulatory
switch may operate in plants to control cell death [35••].
However, alternative explanations for these results are
possible (see below).
In addition to caspases, other cysteine and serine proteases may also be involved in HR cell death. Solomon et al.
[39•] found that expression of the cysteine protease
inhibitor cystatin can suppress ROS and bacteria-induced
cell death in soybean cell cultures, while activation of cell
death by the xylanase from Trichoderma viride can be
In plants, the fungal toxin victorin appears to induce cell
death via inhibition of the mitochondrial enzyme glycine
decarboxylase [14•]. More recently, the mitochondrial connection to PCD in plants was implicated by the work of
Lacomme and Santa Cruz [38••]. Bax was found to activate
HR-like cell death and its localization to plant mitochondria was required. Bax expression was also known to
induce cell death in yeast with similar dependence on its
targeting to the mitochondria [42]. Thus, it appears that
this organelle may be a conserved site where death signals
can be generated in eukaryotes. This may act via leakage
of cytochrome c, which has not been demonstrated in plant
PCD or in yeast. It may also be mediated by other cell
death factors that bypass the caspase switch and act directly at the nuclear level [43••]. This type of cell death
activation may be responsible for caspase-independent
PCD pathways that can be modulated by Bcl-2 [44].
Interestingly, using the phenotype of Bax expressing yeast,
a novel gene called Bax inhibitor (BI)-1 was cloned from
human cells [42]. BI-1 appears to be well conserved in animals and genes with weak sequence homology to BI-1
have been identified in C. elegans and Arabidopsis sequence
databases. Although its function is unclear at present, BI-1
may be an ancient regulator of cell death activation
through the mitochondria.
The mitochondrial connection in HR cell death has also
been made at the level of the Alternative oxidase (AOX).
AOX is not found in animals and its function as a heat generator in floral tissues such as the Voodoo Lily has been
Die and let live — programmed cell death in plants Lam, Pontier and del Pozo
established. However, plants suppressed in AOX expression remain largely normal until stressed by inhibitors of
the mitochondrial electron transport chain such as
antimycin A (AA). The recent paper by Maxwell et al. [45••]
showed that AOX suppressed plants produce rapid and dramatic amounts of ROS in their mitochondria upon
treatment with AA. Moreover, they showed that AOX-deficient plants constitutively expressed defense gene markers
whereas overexpressors of AOX repressed the basal expression levels of these genes. Thus, ROS generated in the
mitochondria can lead to the induction of PR gene expression. Furthermore, these observations suggest that
interruption of normal electron transport functions leads to
rapid generation of ROS and the role of AOX may be to act
as a safety valve to ‘siphon’ off the reducing intermediates
from the clogged electron transport chains. Consistent with
this idea is the observation that treatments with inhibitors
of oxidative electron transport such as AA and cyanide activate AOX expression [46••]. In addition, mutations or
transgenes that lead to an impairment of heme biosynthesis result in spontaneous lesion formation [13,47,48]. The
disruption of heme biosynthesis may lead to a loss of functional cytochromes and excess ROS generation from the
mitochondria. Together with expected defects in ROS protectants that require heme as their prosthetic group such as
catalases and peroxidases, it is plausible that in these plants
ROS can easily reach a threshold sufficient to activate cell
death spontaneously. Induction of AOX has been observed
under various stress conditions and a recent study in
Arabidopsis showed that rapid localized AOX induction by
avirulent bacterial pathogens requires SA [49••]. On the
other hand, ethylene is absolutely required for AOX induction by pathogens. This work suggests that localized
induction of AOX may act to protect the cells at or near the
infection sites against ROS damage. This work complemented the studies of Chivasa and Carr [46••] who showed
that cyanide treatment, which activates AOX synthesis, can
reverse the spreading cell death phenotype observed in
NahG expressing tobacco plants. The effects of cyanide
can be reversed by salicylhydroxamic acid (SHAM), an
inhibitor of AOX. Obviously, the results with chemical
inhibitors have to be interpreted cautiously since SHAM
can also inhibit other enzymes such as peroxidases that may
function to protect against ROS. Nevertheless, it is tempting to speculate that AOX may be involved in cell death
regulation in the development of spreading lesions such as
those observed during tobacco mosaic virus (TMV) infection. Future manipulation of AOX levels should lead to a
better understanding of the role for this protein in determining lesion size and disease resistance.
Conclusions
The mitochondrion has emerged recently as a conserved
site where cell death signals in the form of ROS and protein
factors can be generated. Our understanding of its role in
plant PCD will be aided by comparative analyses with
results from animal and yeast fields, in addition to the
wealth of genomic information that is rapidly accumulating.
505
Ultimately, forward or reverse genetic definition of candidates for cell death regulation will be required to sort out
the complex interplay between the varieties of regulators
that are likely to control this important process.
Acknowledgements
Research on cell death in our laboratory is funded in part by the New Jersey
Commission on Science and Technology, and a competitive research grant
from the United States Department of Agriculture. Sharing of preprints and
unpublished information by Jean Greenberg, Ko Shimamoto and Yuko
Ohashi is gratefully acknowledged.
References and recommended reading
Papers of particular interest, published within the annual period of review,
have been highlighted as:
• of special interest
•• of outstanding interest
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found to accelerate hormone-dependent programmed cell death. Vacuole
collapse and cessation of cytoplasmic streaming preceded nuclear DNA
fragmentation, suggestive of an autolytic process of programmed cell
death. Interestingly, these authors found that addition of 1% (w/v) trypsin
activated vacuole collapse and nuclear DNA fragmentation in a calciumdependent manner whereas exogenous soybean trypsin inhibitor has the
opposite effect of inhibiting TE differentiation. A 40 kDa secreted protease
that can be inhibited with soybean trypsin inhibitor was found to correlate
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Bethke PC, Lonsdale JE, Fath A, Jones RL: Hormonally regulated
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•
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Using flow cytometry, these authors reported large changes in chromatin
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•
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respond with an HR upon challenge with an avirulent bacteria and phytoalexin synthesis is not activated. Interestingly, the phenotype of acd6 is SA
dependent, as it can be reversed by NahG expression, but is partially NPR1
independent. A novel phenotype of the acd6 mutant in comparison to other
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and fungal elicitors. Overexpression of these genes in transgenic tobacco
resulted in the appearance of hypersensitive-response-like phenotypes
including spontaneous cell death in older mature leaves, pathogen-related
gene expression and broad spectrum resistance to various types of
pathogens. Surprisingly, salicylic acid (SA) levels are not affected by these
transgenes suggesting that they act via SA-independent pathways to
induce lesions and resistance. This work provides evidence that intracellular calcium levels can play important roles in the control of cell death activation in plants via specific calmodulin isoforms that are transcriptionally
regulated. Moreover, it clearly demonstrates the existence of SA-independent cell death and resistance pathways.
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of the temporal behavior of various markers of cell death and defense, as
well as a working model integrating recent observations of the various
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inducing pathogenesis-related gene expression and resistance without concomitant cell death.
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The role of the small GTP-binding protein OsRac1 was examined by expression of its constitutively active or dominant-negative mutations in transgenic
rice cell cultures and plants. Expression of the transgene encoding the constitutively active mutant of OsRac1 resulted in ROS (reactive oxygen
species) production and programmed cell death which correlated with
TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick endlabeling) staining. Involvement of the NADPH oxidase is suggested by inhibition of these phenotypes by diphenylene iodonium. Conversely, transgenic
expression of a dominant-negative variant of OsRac1 in a rice lesion mimic
mutant resulted in suppression of ROS generation and cell death induction
by a rice blast fungus and calyculin A, a protein phosphatase I inhibitor. This
work provides the first clear evidence for the involvement of Rac in the regulation of plant NADPH oxidase in ROS generation and cell death induction.
It complements nicely the identification of multiple plant genes encoding the
large subunit of the plant NADPH oxidase and suggests that analogous to
the neutrophil NADPH oxidase, the plant enzyme is also regulated by Rac
and phosphorylation.
32. Reichheld JP, Vernoux T, Lardon F, Van Montagu M, Inze D: Specific
checkpoints regulate plant cell cycle progression in response to
oxidative stress. Plant J 1999, 17:647-656.
33. Tamagnone L, Merida A, Stacey N, Plaskitt K, Parr A, Chang CF,
•
Lynn D, Dow MJ, Roberts K, Martin C: Inhibition of phenolic acid
metabolism results in precocious cell death and altered cell
morphology in leaves of transgenic tobacco plants. Plant Cell
1998, 10:1801-1816.
An extensive study characterizing the phenotypic defects resulting from
downregulation of phenolic acid metabolism in transgenic tobacco through
the transgenic expression of the gene encoding the transcription factor
AmMYB308. Concomitant with the decrease of phenolic compounds,
Die and let live — programmed cell death in plants Lam, Pontier and del Pozo
abnormal development of the leaf palisade layer is correlated with premature
programmed cell death as detected by TUNEL staining. This ‘early senescence’ phenotype could be rescued in cell cultures by supplying exogenous
phenolic precursors. Interestingly, the hypersensitive response is activated
much faster in these transgenic plants and this response correlated with a
heightened level of lipid peroxidation. These and earlier results suggest that
phenolic compounds act as important buffers against the effects of ROS
(reactive oxygen species) in plants.
34. Aravind L, Dixit VM, Koonin EV: The domains of death: evolution of
•
the apoptosis machinery. Trends Biochem Sci 1999, 24:47-53.
A thorough and provocative review of the various types of cell death regulators that have been defined in animal systems. Interesting comparisons
between molecules that are or may be involved in cell death regulation
from plants, animals and prokaryotes are presented and the evolutionary
implications summarized.
35. Wolf BB, Green DR: Suicidal tendencies: apoptotic cell death by
•• caspase family proteinases. J Biol Chem 1999, 274:20049-20052.
A well-written and comprehensive summary of the state-of-the-art knowledge on the structure, function and regulation of caspases, the growing family of cysteine proteases that specializes in controlling cell death activation.
36. del Pozo O, Lam E: Caspases and programmed cell death in the
•• hypersensitive response of plants to pathogens. Curr Biol 1998,
8:1129-1132.
This work showed that synthetic peptide inhibitors of animal caspases
can suppress hypersensitive response (HR) cell death induced by avirulent bacteria when co-infiltrated into tobacco leaves. The induction of two
HR cell death gene markers was inhibited whereas pathogenesis-related
gene induction was not affected by these inhibitors, thus showing that
plant–pathogen signaling remains intact and that defense gene activation
can be uncoupled from cell death. Transient induction of caspase-like
proteolytic activity was detected in extracts from leaf tissues during
tobacco mosaic virus (TMV)-induced HR that was synchronized by temperature shift. This work constitutes the first report of caspase-like protease activities in plants and provided evidence for their participation in
HR cell death activation.
37.
•
Mitsuhara I, Malik KA, Miura M, Ohashi Y: Animal cell-death
suppressors Bcl-xL and Ced-9 inhibit cell death in tobacco plants.
Curr Biol 1999, 9:775-778.
This work showed that expression in transgenic tobacco of the genes
encoding the pro-survival cell death regulators Bcl-x L and Ced-9 from animal systems can delay cell death induced by ultraviolet light, paraquat
and pathogen challenge. Although the mechanism responsible for these
observations is unclear at present, the results suggest that these regulators may suppress one or more evolutionarily conserved cell death
switches in plants.
38. Lacomme C, Santa Cruz S: Bax-induced cell death in tobacco is
•• similar to the hypersensitive response. Proc Natl Acad Sci USA
1999, 96:7956-7961.
The gene encoding the pro-apoptotic regulator Bax from mammalian systems is expressed in tobacco by a tobacco mosaic virus (TMV) viral vector
and found to induce hypersensitive-response-like phenotypes such as cell
death and pathogenesis-related gene expression. Mutational analyses show
that the likely target site of Bax in plants is the mitochondria and that
domains required for Bax dimerization are required for optimal cell death
induction. This work provides evidence that plant mitochondria can be the
site of origin for signals leading to hypersensitive-response-like phenomena.
39. Solomon M, Belenghi B, Delledonne M, Menachem E, Levine A: The
•
involvement of cysteine proteases and protease inhibitor genes in
the regulation of programmed cell death in plants. Plant Cell
1999, 11:431-444.
Cell death induction in soybean cells by oxidative stress or avirulent bacterial pathogen was shown to depend on protein synthesis and multiple proteases are induced. Ectopic expression of the soybean cysteine protease
inhibitor cystatin resulted in suppression of programmed cell death. This
work showed that a cysteine protease or proteases that can interact with
cystatin may be good candidates for activities that are required to execute
the death signal upon oxidative stress and during the HR in this system.
40. Yano A, Suzuki K, Shinshi H: A signaling pathway, independent of
the oxidative burst, that leads to hypersensitive cell death in
cultured tobacco cells includes a serine protease. Plant J 1999,
18:105-109.
41. Shimizu S, Narita M, Tsujimoto Y: Bcl-2 family proteins regulate the
release of apoptogenic cytochrome c by the mitochondrial
channel VDAC. Nature 1999, 399:483-487.
507
42. Shaham S, Shuman MA, Herskowitz I: Death-defying yeast identify
novel apoptosis genes. Cell 1998, 92:425-427.
43. Susin SA, Lorenzo HK, Zamzami N, Marzo I, Snow BE, Brothers GM,
•• Mangion J, Jacotot E, Costantini P, Loeffler M et al.: Molecular
characterization of mitochondrial apoptosis-inducing factor.
Nature 1999, 397:441-445.
A flavoprotein that has homology to bacterial oxidoreductases was purified
from mouse liver mitochondria. It is a mitochondrial protein that is released
by the transition pore which can be regulated by Bcl-2. Purified apoptosis
inducing factor (AIF) can induce apoptotic phenotypes in isolated nuclei as
well as activate cytochrome c release from mitochondria in whole cells with
subsequent caspase activation. AIF thus may function directly in inducing
nuclear events as well as serving to amplify cell death signals. It may be
involved in caspase-independent cell death signaling as AIF activity in
microinjected cells is insensitive to the broad range inhibitor Z–VAD–fmk.
(Z—Val—Ala—Asp—fluoromethylketone).
44. Okuno S, Shimizu S, Ito T, Nomura M, Hamada E, Tsujimoto Y,
Matsuda H: Bcl–2 prevents caspase-independent cell death.
J Biol Chem 1998, 273:34272-34277.
45. Maxwell DP, Wang Y, McIntosh L: The alternative oxidase lowers
•• mitochondrial reactive oxygen production in plant cells.
Proc Natl Acad Sci USA 1999, 96:8271-8276.
Using a transgenic strategy, the level of the alternative oxidase (AOX)
enzyme was increased or decreased in tobacco plants using sense or antisense transcript expression, respectively. In the absence of stress, plants
deficient in AOX show detectable levels of ROS (reactive oxygen species)
in their mitochondria and expression of PR-1. These plants are also hypersensitive to treatment with antimycin A (AA) — a specific inhibitor of Complex
III of the oxidative electron transport chain — and rapid cell death is activated concomitant with production of dramatic levels of ROS in the mitochondria. Overexpression of AOX suppresses induction of ROS by AA treatment
as well as suppressing basal levels of PR-1 expression. This work provides
the first evidence that interference with electron transport in plant mitochondria can lead to significant ROS generation and this process is regulated by
AOX. Furthermore, ROS generated from plant mitochondria can activate cell
death and defense gene markers coordinately, analogous to that observed
during the hypersensitive response.
46. Chivasa S, Carr JP: Cyanide restores N gene-mediated resistance
•• to tobacco mosaic virus in transgenic tobacco expressing salicylic
acid hydroxylase. Plant Cell 1998, 10:1489-1498.
Antimycin A and cyanide treatments were found to induce expression of
AOX to the same level as salicylic acid (SA) and its analog 2,2-dichloroisonicotinic acid (INA) in tobacco. Cyanide treatment can reverse the
effects of NahG expression by inhibiting tobacco mosaic virus (TMV) cellto-cell movement and lesion proliferation, as well as virus replication.
Interestingly, the effect of cyanide can be reversed by treatment with SHAM
(salicylhydroxamic acid), an inhibitor of AOX. Treatment with SHAM alone
can block SA-dependent resistance to TMV, induce PR-1 in an SA-dependent manner, but does not affect resistance to bacterial or fungal
pathogens. This work provided evidence that SA-mediated cell death activation that is required for optimal virus restriction probably involves AOX.
However, the effects of SHAM and cyanide on resistance may be more
complex to interpret at present.
47.
Hu G, Yalpani N, Briggs SP, Johal GS: A porphyrin pathway
impairment is responsible for the phenotype of a dominant
disease lesion mimic mutant of maize. Plant Cell 1998,
10:1095-1105.
48. Molina A, Volrath S, Guyer D, Maleck K, Ryals J, Ward E: Inhibition of
protoporphyrinogen oxidase expression in Arabidopsis causes a
lesion-mimic phenotype that induces systemic acquired
resistance. Plant J 1999, 17:667-678.
49. Simons BH, Millenaar FF, Mulder L, Van Loon LC, Lambers H:
•• Enhanced expression and activation of the alternative oxidase
during infection of Arabidopsis with Pseudomonas syringae pv
tomato. Plant Physiol 1999, 120:529-538.
AOX (alternative oxidase) expression was found to be induced rapidly in the
infected leaves by challenge with avirulent, but more slowly with virulent,
bacterial pathogens in Arabidopsis. In addition, the rapid but not the slower
rise in AOX is dependent on salicylic acid but independent of NPR1.
Furthermore, ethylene sensing through ETR1 is absolutely required for AOX
induction. This work suggests that the local induction of AOX involves ethylene production during the hypersensitive response HR and that AOX could
serve as a protectant for ROS (reactive oxygen species) damage to the cells
at or near the infection site.
Die and let live — programmed cell death in plants
Eric Lam*, Dominique Pontier and Olga del Pozo
Cysteine and serine proteases are prominent players in the
control of developmental and pathogen-activated cell deaths in
plants. Ethylene, salicylic acid, the small G-protein Rac,
calcium and reactive oxygen species are recurring mediators of
death signaling. Lastly, the mitochondrion has emerged in both
plant and animal systems as a ‘central depot’ that interprets
multiple signals and in some instances determines the fate of
the cell.
Addresses
Biotech Center, Foran Hall, Cook College, 59 Dudley Road, Rutgers
University, New Brunswick, NJ 08903, USA
*e-mail: [email protected]
Current Opinion in Plant Biology 1999, 2:502–507
1369-5266/99/$ — see front matter © 1999 Elsevier Science Ltd.
All rights reserved.
Abbreviations
AA
antimycin A
AEBSF
4-(2-aminoethyl)-benzenesulfonyl-flonyl-fluoride
AOX
Alternative oxidase
BI-1
Bax inhibitor-1
HR
hypersensitive response
NO
nitric oxide
PCD
programmed cell death
PR
pathogenesis-related
ROS
reactive oxygen species
SA
salicylic acid
SHAM
salicylhydroxamic acid
TE
tracheary element
VDAC
voltage dependent anion channel
Introduction
The study of programmed cell death (PCD) has seen a
remarkable boom in the past 10 years since the early recognition in the 1970’s by Andrew Wyllie and his coworkers
that a variety of animal cells die with similar morphological
features which appeared to be the result of an active suicidal process (for review see [1]). This renaissance of PCD
research was brought about by the convergence of work on
PCD with studies of animal genes involved in oncogenesis
such as Bcl-2, and genetic research on cell death mutants
by Bob Horvitz and his coworkers in the nematode
Caenorhabditis elegans (reviewed in [1]). The realization that
molecules with similar biochemical functions and structural domains are conserved in diverse organisms to regulate
PCD convinced most skeptics that this process can indeed
be studied at the molecular level with defined players.
Remarkably, although many signaling pathways and regulators of animal PCD have been defined in the past
10 years, they revolved around only a small number of central players and their modes of action appeared to be
variations on a few themes. The central enzymatic activity
that has been most well-characterized is a growing family
of cysteine proteases called caspases. They are synthesized
as zymogens that require specific cleavage and subsequent
oligomerization in order to reveal their enzymatic activities. Once activated via proteolysis, they appear to activate
a cascade of proteases in a serial fashion that is reminescent
of the classic blood clotting factor cascade.
PCD research in plants has also begun to blossom in the
past five years [2,3]. There are three major reasons for our
interest in this process in plants. First, it is a way of life for
plants, just as it is for animals. Essential processes such as
xylem differentiation and tapetal cell degeneration all
involve PCD. Second, comparison of the mechanisms and
molecules that are involved in the two kingdoms should
enlighten us about the evolution of PCD and may help to
determine the indispensible players in the chain of events
that lead to cell death in eukaryotes. Third, from an
applied perspective, the ability to regulate cell death in
plants may have important applications in agriculture and
post-harvest industries. For example, suppression of PCD
induced by biotrophic pathogens could minimize disease
symptoms and inhibition of cell death during senescence
may prolong the shelf-life of crops and vegetables. In this
review, we discuss work published in the past year that has
begun to reveal some of the players that are involved in
controling PCD in plant cells.
The demise of a cell — the phenomenon
What actually occurs when a plant cell commits suicide? In
contrast to apoptosis in animals, the presence of a rigid wall
in plant cells poses a distinct problem: because the dying
cell cannot be packaged into small apoptotic bodies and
then engulfed by its neighbors, as in the case for their animal counterparts, plants must deal with suicidal cells and
their contents in fundamentally different ways. A distinguishing feature of plant cells is the prominant vacuole,
which contains many catabolic enzymes such as proteases
and nucleases. It is thus not surprising that this organelle
appears to play an important role in tracheary element
(TE) differentiation [4••] as well as degeneration of the
aleurone in barley [5•]. The disruption of the vacuole during TE development appears to catalyze the autolysis of
the cell’s content in a systematic fashion after the synthesis of the reticulated secondary cell wall [4••,6].
The self-ingestion approach to the dismantling of cells
destined for PCD is likely to be a common strategy for
plants. However, the sequence of events and the resulting
cellular morphological changes may differ depending on
the particular inductive death signal [2,3,6–9]. This may be
a consequence of the role that cell death may have in different situations. In the case of developmental cell death
such as senescence and TE differentiation, the recycling of
the dead cell’s content may be an important ‘purpose’. In
the case of cell death related to pathogen containment, cell
Die and let live — programmed cell death in plants Lam, Pontier and del Pozo
death may be used to rapidly ‘isolate’ the infected cells
and efficient transport of the dead cell’s content may be
secondary [10]. In most cases of plant PCD, vacuolization
of the cytoplasm and disruption of the tonoplast are common events that appear to precede mass disruption of the
mitochondria and nucleus. On the other hand, recent flow
cytometry studies with tobacco protoplasts suggest that
nuclear DNA condensation could be activated before the
first irreversible step of PCD [11•]. Evidence for disruption of the cytoskeleton early on in plant PCD has also
been suggested by the finding that disruption of microfilaments by cytochalasin E could suppress cell death induced
by fungal pathogens in cowpea [12] and that cytoplasmic
streaming is arrested during TE differentiation [4••].
What’s new in PCD signaling in plants?
The signaling processes that activate PCD in plants
promise to be as diverse as those that have been found in
animals. One of the most well-characterized systems to
date is the differentiation of Zinnia TEs in vitro. Earlier
work with the Zinnia TE system has established that
wounding and hormonal balance (auxin and cytokinin
treatments) are critical signaling components for initiating
the transformation of mesophyll cells to TE [6]. More
recently, the involvement of calcium, heterotrimeric
G-proteins and extracellular proteases has been
suggested [4••].
In contrast to other plant PCD systems, a large body of
literature has accumulated on the study of cell death during plant pathogen interactions [2,3,7,13]. Two major
types of cell death can result when plants are inoculated
with a pathogen: the rapid hypersensitive response (HR),
which is typically associated with resistance, and a slower
form of death that is the result of disease. Previous work
has implicated the involvement of protein phosphorylation, calcium channel functions and reactive oxygen
species (ROS) in HR cell death. Studies of disease-related cell death are less developed as it is not obvious that
they resulted from a suicide process. However, recent
work with the fungal toxin victorin has implicated an
active participation of the dying cells and more particularly, the requirement of ethylene in disease-related cell
death induction [14•]. Consistent with these observations, ethylene insensitivity in tomato was found to
suppress cell death due to infection with biotrophic
pathogens [15]. These observations raised the possibility
that ethylene action may be involved in the slower cell
death that is manifested during plant diseases. In contrast, resistance response does not appear to require
ethylene action [16]. Interestingly, ethylene may also be
involved in developmental cell death such as endosperm
degeneration in wheat and during maize aerenchyma formation induced by hypoxia [9,17]. Taking into account its
classic role in tissue senescence, it appears that ethylene
may be an important signal for slower forms of cell death
during which efficient recycling of the dead cell’s content
might be important for the plant. This interpretation is
503
consistent with our recent observation that a senescencespecific marker gene, SAG12, is activated in cells
neighboring HR lesions, where chlorotic symptoms of
cell death are often observed [10].
Salicylic acid (SA) has emerged in the past several years as
a positive feedback regulator of cell death during the HR
[2,3]. This was first suggested in the study of disease lesion
mimics in Arabidopsis [18], where spontaneous cell death
in the mutants lsd6 and lsd7 was repressed by transgenic
expression of a gene encoding a bacterial salicylate hydroxylase (NahG), which converts SA to catechol. Two new
disease lesion mimic mutants, ssi1 and acd6, appeared to
show the same dependence on SA [19•,20••]. Interestingly,
ozone induced cell death in Arabidopsis has also been
shown to be potentiated by SA [21]. A cell wall associated
protein kinase has recently been suggested to be involved
in suppressing cell death signaling by SA [22•].
The specific requirement of calcium signaling in HR cell
death had been suggested through studies using calcium
channel blockers [2,3,7]. The recent work of Heo et al.
[23••] suggested that this dependence on calcium may
involve specific isoforms of calmodulin. HR-like cell
death, pathogenesis-related (PR) gene induction and
broad spectrum disease resistance are activated by transgenic expression of two soybean calmodulin-encoding
genes. These phenotypes are not SA dependent and the
level of SA is not altered by the transgenes. Thus, distinct
pathways can give rise to phenotypes that are morphologically very similar. As these soybean genes are induced
during the defense response, they may participate in the
long term signaling for resistance rather than the initial signaling process to activate the HR pathway.
Another likely signaling molecule for the activation of HR
cell death is ROS (reactive oxygen species) [24,25•]. Two
phases of ROS generation by plants are known to occur
upon incompatible interactions with pathogens [24]. We
have shown that ambient oxygen pressure is required for
HR cell death while PR gene induction remains little
affected by low oxygen pressure [26]. HR-like cell death
and defense gene activation can also be induced ectopically by either transgenic expression of a gene encoding
glucose oxidase [27] or suppression of endogenous catalase
[28•]. The action of ROS may be augmented by nitric
oxide (NO) to potentiate and propagate the cell death signals [25•]. It has been speculated for a number of years that
ROS involved in activating the HR may be generated by
an NADPH oxidase similar to the well-characterized ROS
signaling system in neutrophils [24,25•]. Recent identification of six Arabidopsis genes homologous to the large
subunit of the human NADPH oxidase should facilitate
testing of this hypothesis [29,30]. In support of the
involvement of NADPH oxidase as an activator of ROS for
HR induction, recent work by Kawasaki et al. [31••]
showed that the small GTP-binding protein Rac may
mediate the HR response of rice in an ROS-dependent
504
Cell biology
manner. Rac is known to regulate mammalian NADPH
oxidase as well.
How does ROS lead to the activation of cell death and how
is it managed in the cell? Reichheld et al. [32] suggest that
alteration of the cell cycle may be one aspect of death
induction by ROS. Interestingly, the role of SA in cell proliferation as well as cell death was postulated to explain
some of the abnormal leaf morphologies observed in the
acd6 mutant [20••]. Thus, SA and ROS may cooperate to
arrest the cell cycle and tip the balance toward cell death.
As plants are likely to be exposed to a variety of agents or
environmental conditions that can activate ROS production, a buffering system that can deal with normal
fluctuations in ROS level should be in place to prevent
chance activation of PCD. The work of Tamagnone et al.
[33•] showed that transgenic expression of a transcription
regulator leads to plant cells that are hyper-responsive to
pathogen challenge, abnormal cell expansion and PCD in
the leaf palisade cells, and increase in lipid peroxidation.
Addition of phenolic precursors can rescue cell cultures
derived from these transgenic plants from cell death and
abnormal morphologies, thus showing nicely that these
phenotypes are reversible. This work provided evidence
that phenolic products could play an important role in ROS
homeostasis during normal plant development.
inhibited by the serine protease inhibitor AEBSF (4-[2aminoethyl]-benzenesulfonyl-flonyl-fluoride), but not by
caspase inhibitors [40]. Activation of cell death by victorin
in oat also appears to be mediated by cysteine proteases
sensitive to E-64 and calpeptin [14•]. These studies suggest that proteases with different specificities may be
used to regulate cell death induced by different agents in
different species. Involvement of intracellular cysteine
proteases [6] as well as an extracellular serine protease
[4••] have also been proposed to regulate cell death activation during TE differentiation.
In recent years, the role of mitochondria in controlling cell
death activation has been recognized in the animal field.
Cytochrome c leakage from this organelle appears to
mediate many signaling pathways for PCD. Once in the
cytosol, cytochrome c can activate caspases through interaction with Ced-4/APAF1 in conjunction with dATP
[35••]. In addition, disruption of electron flow through the
electron transport chain of the mitochondria is likely to
lead to accumulation of reducing equivalents that can
result in ROS. Bcl-2 related proteins can either activate or
repress the leakage of cytochrome c through the voltage
dependent anion channel (VDAC) located in the membrane of the mitochondria [41]. They can also suppress or
activate caspases through interaction with the Ced4/APAF1 regulator.
The nuts and bolts of the death engine
The identities of the key executioners of plant cells
remain elusive, whereas in animal systems a large number
of caspases and their regulators have been defined in the
past five years [34•,35••]. Caspase-like proteolytic activity
has been observed to be transiently activated in plants
synchronized to undergo the HR [36••]. Peptide inhibitors
of caspases can abolish HR cell death induced by avirulent bacteria without affecting the induction of defense
genes significantly. Furthermore, transgenic expression of
the gene encoding a broad range caspase inhibitor from
Baculovirus, p35, also leads to the delay of HR cell death
in tobacco (O del Pozo, E Lam, unpublished data). More
recently, Mitsuhara et al. [37•] reported that in tobacco
expression of transgenes encoding the pro-survival cell
death regulators Bcl-xL and Ced-9 can delay HR cell
death as well as death induced by UVB and paraquat,
whereas expression of the transgene encoding the proapoptotic Bax is found to activate HR-like cell death
[38••]. These observations suggest the possibility that caspase-like proteases and a Ced-4/APAF1 like regulatory
switch may operate in plants to control cell death [35••].
However, alternative explanations for these results are
possible (see below).
In addition to caspases, other cysteine and serine proteases may also be involved in HR cell death. Solomon et al.
[39•] found that expression of the cysteine protease
inhibitor cystatin can suppress ROS and bacteria-induced
cell death in soybean cell cultures, while activation of cell
death by the xylanase from Trichoderma viride can be
In plants, the fungal toxin victorin appears to induce cell
death via inhibition of the mitochondrial enzyme glycine
decarboxylase [14•]. More recently, the mitochondrial connection to PCD in plants was implicated by the work of
Lacomme and Santa Cruz [38••]. Bax was found to activate
HR-like cell death and its localization to plant mitochondria was required. Bax expression was also known to
induce cell death in yeast with similar dependence on its
targeting to the mitochondria [42]. Thus, it appears that
this organelle may be a conserved site where death signals
can be generated in eukaryotes. This may act via leakage
of cytochrome c, which has not been demonstrated in plant
PCD or in yeast. It may also be mediated by other cell
death factors that bypass the caspase switch and act directly at the nuclear level [43••]. This type of cell death
activation may be responsible for caspase-independent
PCD pathways that can be modulated by Bcl-2 [44].
Interestingly, using the phenotype of Bax expressing yeast,
a novel gene called Bax inhibitor (BI)-1 was cloned from
human cells [42]. BI-1 appears to be well conserved in animals and genes with weak sequence homology to BI-1
have been identified in C. elegans and Arabidopsis sequence
databases. Although its function is unclear at present, BI-1
may be an ancient regulator of cell death activation
through the mitochondria.
The mitochondrial connection in HR cell death has also
been made at the level of the Alternative oxidase (AOX).
AOX is not found in animals and its function as a heat generator in floral tissues such as the Voodoo Lily has been
Die and let live — programmed cell death in plants Lam, Pontier and del Pozo
established. However, plants suppressed in AOX expression remain largely normal until stressed by inhibitors of
the mitochondrial electron transport chain such as
antimycin A (AA). The recent paper by Maxwell et al. [45••]
showed that AOX suppressed plants produce rapid and dramatic amounts of ROS in their mitochondria upon
treatment with AA. Moreover, they showed that AOX-deficient plants constitutively expressed defense gene markers
whereas overexpressors of AOX repressed the basal expression levels of these genes. Thus, ROS generated in the
mitochondria can lead to the induction of PR gene expression. Furthermore, these observations suggest that
interruption of normal electron transport functions leads to
rapid generation of ROS and the role of AOX may be to act
as a safety valve to ‘siphon’ off the reducing intermediates
from the clogged electron transport chains. Consistent with
this idea is the observation that treatments with inhibitors
of oxidative electron transport such as AA and cyanide activate AOX expression [46••]. In addition, mutations or
transgenes that lead to an impairment of heme biosynthesis result in spontaneous lesion formation [13,47,48]. The
disruption of heme biosynthesis may lead to a loss of functional cytochromes and excess ROS generation from the
mitochondria. Together with expected defects in ROS protectants that require heme as their prosthetic group such as
catalases and peroxidases, it is plausible that in these plants
ROS can easily reach a threshold sufficient to activate cell
death spontaneously. Induction of AOX has been observed
under various stress conditions and a recent study in
Arabidopsis showed that rapid localized AOX induction by
avirulent bacterial pathogens requires SA [49••]. On the
other hand, ethylene is absolutely required for AOX induction by pathogens. This work suggests that localized
induction of AOX may act to protect the cells at or near the
infection sites against ROS damage. This work complemented the studies of Chivasa and Carr [46••] who showed
that cyanide treatment, which activates AOX synthesis, can
reverse the spreading cell death phenotype observed in
NahG expressing tobacco plants. The effects of cyanide
can be reversed by salicylhydroxamic acid (SHAM), an
inhibitor of AOX. Obviously, the results with chemical
inhibitors have to be interpreted cautiously since SHAM
can also inhibit other enzymes such as peroxidases that may
function to protect against ROS. Nevertheless, it is tempting to speculate that AOX may be involved in cell death
regulation in the development of spreading lesions such as
those observed during tobacco mosaic virus (TMV) infection. Future manipulation of AOX levels should lead to a
better understanding of the role for this protein in determining lesion size and disease resistance.
Conclusions
The mitochondrion has emerged recently as a conserved
site where cell death signals in the form of ROS and protein
factors can be generated. Our understanding of its role in
plant PCD will be aided by comparative analyses with
results from animal and yeast fields, in addition to the
wealth of genomic information that is rapidly accumulating.
505
Ultimately, forward or reverse genetic definition of candidates for cell death regulation will be required to sort out
the complex interplay between the varieties of regulators
that are likely to control this important process.
Acknowledgements
Research on cell death in our laboratory is funded in part by the New Jersey
Commission on Science and Technology, and a competitive research grant
from the United States Department of Agriculture. Sharing of preprints and
unpublished information by Jean Greenberg, Ko Shimamoto and Yuko
Ohashi is gratefully acknowledged.
References and recommended reading
Papers of particular interest, published within the annual period of review,
have been highlighted as:
• of special interest
•• of outstanding interest
1.
Raff M: Cell suicide for beginners. Nature 1998, 396:119-122.
2.
Pennell RI, Lamb C: Programmed cell death in plants. Plant Cell
1997, 9:1157-1168.
3.
Richberg MH, Aviv DH, Dangl JL: Dead cells do tell tales.
Curr Opin Plant Biol 1998, 1:480-485.
4.
••
Groover A, Jones AM: Tracheary element differentiation uses a
novel mechanism coordinating programmed cell death and
secondary cell wall synthesis. Plant Physiol 1999, 119:375-384.
In vitro differentiation of tracheary element (TE) was studied. The heterotrimeric G-protein activator mastoparan and a calcium ionophore were
found to accelerate hormone-dependent programmed cell death. Vacuole
collapse and cessation of cytoplasmic streaming preceded nuclear DNA
fragmentation, suggestive of an autolytic process of programmed cell
death. Interestingly, these authors found that addition of 1% (w/v) trypsin
activated vacuole collapse and nuclear DNA fragmentation in a calciumdependent manner whereas exogenous soybean trypsin inhibitor has the
opposite effect of inhibiting TE differentiation. A 40 kDa secreted protease
that can be inhibited with soybean trypsin inhibitor was found to correlate
with TE differentiation.
5.
•
Bethke PC, Lonsdale JE, Fath A, Jones RL: Hormonally regulated
programmed cell death in barley aleurone cells. Plant Cell 1999,
11:1033-1045.
This paper describes the careful microscopic analysis of gibberellic acid
induced aleurone cell death with isolated protoplasts. Cell death is preceded by an abrupt loss of membrane integrity and vacuolization of cytoplasmic
vesicles. Signaling by cGMP is suggested by the effects of LY83583, a
guanylyl cyclase inhibitor, on DNA degradation and programmed cell death
of aleurone cells. The authors concluded that gibberellic acid activated aleurone cell death is likely to occur via autolysis rather than apoptosis.
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Fukuda H: Xylogenesis: initiation, progression, and cell death.
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Young TE, Gallie DR: Analysis of programmed cell death in wheat
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hypersensitive response and senescence show distinct patterns
of expression. Plant Mol Biol 1999, 39:1243-1255.
11. O’Brian IEW, Baguley BC, Murray BG, Morris BAM, Ferguson IB:
•
Early stages of the apoptotic pathway in plant cells are reversible.
Plant J 1998, 13:803-814.
Using flow cytometry, these authors reported large changes in chromatin
condensation during cell death induction of tobacco protoplasts by chemical treatments. Annexin V binding, indicative of phosphatidylserine exposure,
as well as chromatin condensation appeared to be reversible at the early
stage of the cell death process in this assay system.
12. Skalamera D, Heath MC: Changes in the cytoskeleton
accompanying infection-induced nuclear movements and the
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506
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mechanisms in maize. Trends Plant Sci 1998, 3:218-223.
14. Navarre DA, Wolpert TJ: Victorin induction of an
•
apoptotic/senescence-like response in oats. Plant J 1999,
11:237-249.
Cell death induction by the host-selective fungal toxin victorin in susceptible
oat leaves resulted in chromosomal laddering, similar to those previously
reported for Alternaria alternate toxin and tomato. Victorin interacts with
subunits of the photorespiratory enzyme glycine decarboxylase (GDC) and
effectively inhibits this mitochondrial enzyme in vivo. Parallels between victorin induced cell death and senescence are drawn. An interesting phenotype is the cleavage of the 14 amino-terminal amino acid residues from the
large subunit of ribulose 1,5-bisphosphate carboxylase (LSU) and its inhibition by a variety of treatments, including cysteine protease inhibitors such as
E-64 and calpeptin. This work suggests the plant mitochondria may be the
site where an apoptotic signal can be activated by victorin inhibition of photorespiration through the GDC. Ethylene also appears to be required for victorin induced cell death and LSU cleavage.
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19. Shah J, Kachroo P, Klessig DF: The Arabidopsis ssi1 mutation
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restores pathogenesis-related gene expression in npr1 plants
and renders defensin gene expression salicylic acid dependent.
Plant Cell 1999, 11:191-206.
The authors describe a semidominant mutant of Arabidopsis that constitutively expresses PR genes in an NPR1 independent manner. Spontaneous
HR-like cell death lesions are observed and the phenotypes are SA dependent as revealed through NahG expression. This work suggests that SA is
required, although not sufficient, to induce HR cell death.
20. Rate DN, Cuenca JV, Bowman GR, Guttman DS, Greenberg JT:
•• A gain-of-function Arabidopsis acd6 mutant reveals novel
regulation and function of the salicylic acid signaling pathway in
controlling cell death, defenses and cell growth. Plant Cell 1999,
11:191-206.
This report documents the characterization of a new lesion mimic mutant in
Arabidopsis, acd6. It is caused by a dominant mutation that activates cell
death and resistance to bacterial pathogen; however, the plant is unable to
respond with an HR upon challenge with an avirulent bacteria and phytoalexin synthesis is not activated. Interestingly, the phenotype of acd6 is SA
dependent, as it can be reversed by NahG expression, but is partially NPR1
independent. A novel phenotype of the acd6 mutant in comparison to other
previously described lesion mimics is the observation of abnormal cell
growth and enlargement that is SA dependent. A model is presented whereby SA potentiates both cell death and cell growth and/or proliferation and
the acd6 mutation effectively decreases the threshold of response for SA
signaling to these processes.
21. Rao M, Davis KR: Ozone-induced cell death occurs via two distinct
mechanisms in Arabidopsis: the role of salicylic acid. Plant J
1999, 17:603-614.
22. He ZH, He D, Kohorn BD: Requirement for the induced expression
•
of a cell wall associated receptor kinase for survival during the
pathogen response. Plant J 1998, 14:55-63.
Suppression of a cell wall associated receptor kinase by antisense technology and a dominant-negative mutant resulted in heightened sensitivity
to 2,2-dichloroisonicotinic acid (INA) whereas overexpression of this
kinase resulted in higher tolerance for SA treatment.
23. Heo WD, Lee SH, Kim MC, Kim JC, Chung WS, Chun HJ, Lee KJ,
•• Park CY, Park HC, Choi JY, Cho MJ: Involvement of specific
calmodulin isoforms in salicylic acid-independent activation of
plant disease resistance responses. Proc Natl Acad Sci USA
1999, 96:766-771.
The expression of two isoforms encoded by SCaM4 and SCaM5 of the
calmodulin gene family from soybean was found to respond to pathogen
and fungal elicitors. Overexpression of these genes in transgenic tobacco
resulted in the appearance of hypersensitive-response-like phenotypes
including spontaneous cell death in older mature leaves, pathogen-related
gene expression and broad spectrum resistance to various types of
pathogens. Surprisingly, salicylic acid (SA) levels are not affected by these
transgenes suggesting that they act via SA-independent pathways to
induce lesions and resistance. This work provides evidence that intracellular calcium levels can play important roles in the control of cell death activation in plants via specific calmodulin isoforms that are transcriptionally
regulated. Moreover, it clearly demonstrates the existence of SA-independent cell death and resistance pathways.
24. Lamb C, Dixon RA: The oxidative burst in plant disease resistance.
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25. Van Camp W, Van Montagu M, Inze D: H2O2 and NO: redox signals
•
in disease resistance. Trends in Plant Sci 1998, 3:330-334.
A succinct review on the role of ROS and various plant signaling components such as nitric oxide, ethylene and salicylic acid on the activation of
cell death and disease resistance. The authors provide a good summary
of the temporal behavior of various markers of cell death and defense, as
well as a working model integrating recent observations of the various
signaling components.
26. Mittler R, Shulaev V, Seskar M, Lam E: Inhibition of programmed
cell death in tobacco plants during a pathogen-induced
hypersensitive response at low oxygen pressure. Plant Cell 1996,
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Kazan K, Murray FR, Goulter KC, Llewellyn DJ, Manners JM: Induction
of cell death in transgenic plants expressing a fungal glucose
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28. Chamnongpol S, Willekens H, Moeder W, Langebartels C,
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Sandermann H, Van Montagu M, Inze D, Van Camp W: Defense
activation and enhanced pathogen tolerance induced by H2O2 in
transgenic tobacco. Proc Natl Acad Sci USA 1998, 95:5818-5823.
Expression of catalase in tobacco plants was suppressed by transgenic
expression of antisense transcripts. High light treatment of these plants
results in activation of hypersensitive-response-like cell death as well as
defense gene markers and disease resistance, presumably due to the production of H2O2. These responses were shown to be systemic and the
induction of cell death can be uncoupled from disease resistance and
defense markers, with sublethal levels of ROS (reactive oxygen species)
inducing pathogenesis-related gene expression and resistance without concomitant cell death.
29. Torres MA, Onouchi H, Hamada S, Machida C, Hammond-Kosack KE,
Jones JJ: Six Arabidopsis thaliana homologues of the human
respiratory burst oxidase (gp91phox). Plant J 1998, 14:365-370.
30. Keller T, Damude HG, Werner D, Doerner P, Dixon RA, Lamb C: A
plant homolog of the neutrophil NADPH oxidase gp91phox subunit
gene encodes a plasma membrane protein with Ca2+ binding
motifs. Plant Cell 1998, 10:255-266.
31. Kawasaki T, Henmi K, Ono E, Hatakeyama S, Iwano M, Satoh H,
•• Shimamoto K: The small GTP-binding protein Rac is a regulator of
cell death in plants. Proc Natl Acad Sci USA 1999,
96:10922-10926.
The role of the small GTP-binding protein OsRac1 was examined by expression of its constitutively active or dominant-negative mutations in transgenic
rice cell cultures and plants. Expression of the transgene encoding the constitutively active mutant of OsRac1 resulted in ROS (reactive oxygen
species) production and programmed cell death which correlated with
TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick endlabeling) staining. Involvement of the NADPH oxidase is suggested by inhibition of these phenotypes by diphenylene iodonium. Conversely, transgenic
expression of a dominant-negative variant of OsRac1 in a rice lesion mimic
mutant resulted in suppression of ROS generation and cell death induction
by a rice blast fungus and calyculin A, a protein phosphatase I inhibitor. This
work provides the first clear evidence for the involvement of Rac in the regulation of plant NADPH oxidase in ROS generation and cell death induction.
It complements nicely the identification of multiple plant genes encoding the
large subunit of the plant NADPH oxidase and suggests that analogous to
the neutrophil NADPH oxidase, the plant enzyme is also regulated by Rac
and phosphorylation.
32. Reichheld JP, Vernoux T, Lardon F, Van Montagu M, Inze D: Specific
checkpoints regulate plant cell cycle progression in response to
oxidative stress. Plant J 1999, 17:647-656.
33. Tamagnone L, Merida A, Stacey N, Plaskitt K, Parr A, Chang CF,
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Lynn D, Dow MJ, Roberts K, Martin C: Inhibition of phenolic acid
metabolism results in precocious cell death and altered cell
morphology in leaves of transgenic tobacco plants. Plant Cell
1998, 10:1801-1816.
An extensive study characterizing the phenotypic defects resulting from
downregulation of phenolic acid metabolism in transgenic tobacco through
the transgenic expression of the gene encoding the transcription factor
AmMYB308. Concomitant with the decrease of phenolic compounds,
Die and let live — programmed cell death in plants Lam, Pontier and del Pozo
abnormal development of the leaf palisade layer is correlated with premature
programmed cell death as detected by TUNEL staining. This ‘early senescence’ phenotype could be rescued in cell cultures by supplying exogenous
phenolic precursors. Interestingly, the hypersensitive response is activated
much faster in these transgenic plants and this response correlated with a
heightened level of lipid peroxidation. These and earlier results suggest that
phenolic compounds act as important buffers against the effects of ROS
(reactive oxygen species) in plants.
34. Aravind L, Dixit VM, Koonin EV: The domains of death: evolution of
•
the apoptosis machinery. Trends Biochem Sci 1999, 24:47-53.
A thorough and provocative review of the various types of cell death regulators that have been defined in animal systems. Interesting comparisons
between molecules that are or may be involved in cell death regulation
from plants, animals and prokaryotes are presented and the evolutionary
implications summarized.
35. Wolf BB, Green DR: Suicidal tendencies: apoptotic cell death by
•• caspase family proteinases. J Biol Chem 1999, 274:20049-20052.
A well-written and comprehensive summary of the state-of-the-art knowledge on the structure, function and regulation of caspases, the growing family of cysteine proteases that specializes in controlling cell death activation.
36. del Pozo O, Lam E: Caspases and programmed cell death in the
•• hypersensitive response of plants to pathogens. Curr Biol 1998,
8:1129-1132.
This work showed that synthetic peptide inhibitors of animal caspases
can suppress hypersensitive response (HR) cell death induced by avirulent bacteria when co-infiltrated into tobacco leaves. The induction of two
HR cell death gene markers was inhibited whereas pathogenesis-related
gene induction was not affected by these inhibitors, thus showing that
plant–pathogen signaling remains intact and that defense gene activation
can be uncoupled from cell death. Transient induction of caspase-like
proteolytic activity was detected in extracts from leaf tissues during
tobacco mosaic virus (TMV)-induced HR that was synchronized by temperature shift. This work constitutes the first report of caspase-like protease activities in plants and provided evidence for their participation in
HR cell death activation.
37.
•
Mitsuhara I, Malik KA, Miura M, Ohashi Y: Animal cell-death
suppressors Bcl-xL and Ced-9 inhibit cell death in tobacco plants.
Curr Biol 1999, 9:775-778.
This work showed that expression in transgenic tobacco of the genes
encoding the pro-survival cell death regulators Bcl-x L and Ced-9 from animal systems can delay cell death induced by ultraviolet light, paraquat
and pathogen challenge. Although the mechanism responsible for these
observations is unclear at present, the results suggest that these regulators may suppress one or more evolutionarily conserved cell death
switches in plants.
38. Lacomme C, Santa Cruz S: Bax-induced cell death in tobacco is
•• similar to the hypersensitive response. Proc Natl Acad Sci USA
1999, 96:7956-7961.
The gene encoding the pro-apoptotic regulator Bax from mammalian systems is expressed in tobacco by a tobacco mosaic virus (TMV) viral vector
and found to induce hypersensitive-response-like phenotypes such as cell
death and pathogenesis-related gene expression. Mutational analyses show
that the likely target site of Bax in plants is the mitochondria and that
domains required for Bax dimerization are required for optimal cell death
induction. This work provides evidence that plant mitochondria can be the
site of origin for signals leading to hypersensitive-response-like phenomena.
39. Solomon M, Belenghi B, Delledonne M, Menachem E, Levine A: The
•
involvement of cysteine proteases and protease inhibitor genes in
the regulation of programmed cell death in plants. Plant Cell
1999, 11:431-444.
Cell death induction in soybean cells by oxidative stress or avirulent bacterial pathogen was shown to depend on protein synthesis and multiple proteases are induced. Ectopic expression of the soybean cysteine protease
inhibitor cystatin resulted in suppression of programmed cell death. This
work showed that a cysteine protease or proteases that can interact with
cystatin may be good candidates for activities that are required to execute
the death signal upon oxidative stress and during the HR in this system.
40. Yano A, Suzuki K, Shinshi H: A signaling pathway, independent of
the oxidative burst, that leads to hypersensitive cell death in
cultured tobacco cells includes a serine protease. Plant J 1999,
18:105-109.
41. Shimizu S, Narita M, Tsujimoto Y: Bcl-2 family proteins regulate the
release of apoptogenic cytochrome c by the mitochondrial
channel VDAC. Nature 1999, 399:483-487.
507
42. Shaham S, Shuman MA, Herskowitz I: Death-defying yeast identify
novel apoptosis genes. Cell 1998, 92:425-427.
43. Susin SA, Lorenzo HK, Zamzami N, Marzo I, Snow BE, Brothers GM,
•• Mangion J, Jacotot E, Costantini P, Loeffler M et al.: Molecular
characterization of mitochondrial apoptosis-inducing factor.
Nature 1999, 397:441-445.
A flavoprotein that has homology to bacterial oxidoreductases was purified
from mouse liver mitochondria. It is a mitochondrial protein that is released
by the transition pore which can be regulated by Bcl-2. Purified apoptosis
inducing factor (AIF) can induce apoptotic phenotypes in isolated nuclei as
well as activate cytochrome c release from mitochondria in whole cells with
subsequent caspase activation. AIF thus may function directly in inducing
nuclear events as well as serving to amplify cell death signals. It may be
involved in caspase-independent cell death signaling as AIF activity in
microinjected cells is insensitive to the broad range inhibitor Z–VAD–fmk.
(Z—Val—Ala—Asp—fluoromethylketone).
44. Okuno S, Shimizu S, Ito T, Nomura M, Hamada E, Tsujimoto Y,
Matsuda H: Bcl–2 prevents caspase-independent cell death.
J Biol Chem 1998, 273:34272-34277.
45. Maxwell DP, Wang Y, McIntosh L: The alternative oxidase lowers
•• mitochondrial reactive oxygen production in plant cells.
Proc Natl Acad Sci USA 1999, 96:8271-8276.
Using a transgenic strategy, the level of the alternative oxidase (AOX)
enzyme was increased or decreased in tobacco plants using sense or antisense transcript expression, respectively. In the absence of stress, plants
deficient in AOX show detectable levels of ROS (reactive oxygen species)
in their mitochondria and expression of PR-1. These plants are also hypersensitive to treatment with antimycin A (AA) — a specific inhibitor of Complex
III of the oxidative electron transport chain — and rapid cell death is activated concomitant with production of dramatic levels of ROS in the mitochondria. Overexpression of AOX suppresses induction of ROS by AA treatment
as well as suppressing basal levels of PR-1 expression. This work provides
the first evidence that interference with electron transport in plant mitochondria can lead to significant ROS generation and this process is regulated by
AOX. Furthermore, ROS generated from plant mitochondria can activate cell
death and defense gene markers coordinately, analogous to that observed
during the hypersensitive response.
46. Chivasa S, Carr JP: Cyanide restores N gene-mediated resistance
•• to tobacco mosaic virus in transgenic tobacco expressing salicylic
acid hydroxylase. Plant Cell 1998, 10:1489-1498.
Antimycin A and cyanide treatments were found to induce expression of
AOX to the same level as salicylic acid (SA) and its analog 2,2-dichloroisonicotinic acid (INA) in tobacco. Cyanide treatment can reverse the
effects of NahG expression by inhibiting tobacco mosaic virus (TMV) cellto-cell movement and lesion proliferation, as well as virus replication.
Interestingly, the effect of cyanide can be reversed by treatment with SHAM
(salicylhydroxamic acid), an inhibitor of AOX. Treatment with SHAM alone
can block SA-dependent resistance to TMV, induce PR-1 in an SA-dependent manner, but does not affect resistance to bacterial or fungal
pathogens. This work provided evidence that SA-mediated cell death activation that is required for optimal virus restriction probably involves AOX.
However, the effects of SHAM and cyanide on resistance may be more
complex to interpret at present.
47.
Hu G, Yalpani N, Briggs SP, Johal GS: A porphyrin pathway
impairment is responsible for the phenotype of a dominant
disease lesion mimic mutant of maize. Plant Cell 1998,
10:1095-1105.
48. Molina A, Volrath S, Guyer D, Maleck K, Ryals J, Ward E: Inhibition of
protoporphyrinogen oxidase expression in Arabidopsis causes a
lesion-mimic phenotype that induces systemic acquired
resistance. Plant J 1999, 17:667-678.
49. Simons BH, Millenaar FF, Mulder L, Van Loon LC, Lambers H:
•• Enhanced expression and activation of the alternative oxidase
during infection of Arabidopsis with Pseudomonas syringae pv
tomato. Plant Physiol 1999, 120:529-538.
AOX (alternative oxidase) expression was found to be induced rapidly in the
infected leaves by challenge with avirulent, but more slowly with virulent,
bacterial pathogens in Arabidopsis. In addition, the rapid but not the slower
rise in AOX is dependent on salicylic acid but independent of NPR1.
Furthermore, ethylene sensing through ETR1 is absolutely required for AOX
induction. This work suggests that the local induction of AOX involves ethylene production during the hypersensitive response HR and that AOX could
serve as a protectant for ROS (reactive oxygen species) damage to the cells
at or near the infection site.