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The Plant Cell, Vol. 18, 2097–2099, September 2006, www.plantcell.org ª 2006 American Society of Plant Biologists
IN THIS ISSUE
Programmed Cell Death in Plants: A Role for
Mitochondrial-Associated Hexokinases
‘‘By the time I was born, more of me
had died than survived. It was no wonder I
cannot remember; during that time I went
through brain after brain for nine months,
finally contriving the one model that could
be human, equipped for language.’’ This
quote from Lewis Thomas (1992) speaks to
the importance of programmed cell death
(PCD) in human development. PCD is a
fundamental process in both plants and animals (reviewed in Greenberg, 1996; Gilbert,
2001). A major pathway of PCD in animals is
known as apoptosis, a highly regulated process of cell death that differs from autophagy (lysosomal degradation of organelles
and certain proteins) and from necrosis
(which results from acute tissue injury and
provokes an inflammatory response in animals). Kerr et al. (1972) coined the term
apoptosis from Greek words (apo ¼ from,
and ptosis ¼ falling) used to describe plant
leaf abscission, and they correctly hypothesized that it plays a broad role in normal cell
metabolism and that abnormalities in this
process contribute to a variety of diseases,
including cancer. PCD plays a number of
important roles in plant developmental
pathways, including xylogenesis, formation
of woody tissues in trees and perennials,
leaf abscission, self-incompatibility, and
defense responses to a wide variety of
pathogens and environmental stresses.
Researchers have noted a number of
similarities between apoptosis in animals
and PCD in plants (Greenberg, 1996), and
comparing the functions of key components involved in these processes between
kingdoms may serve fruitful for understanding the regulation of PCD in both.
For example, cytochrome c release from
mitochondria is a key event in apoptosis in
animal cells and has also been shown to be
an early event in PCD in plants (Balk et al.,
1999; Balk and Leaver, 2001). Interestingly,
it has been shown that preventing mito-
chondrial cytochrome c release inhibits
apoptosis in animal cells, in a manner
that is intimately involved with the activity
of mitochondrial hexokinase activity. In human tumor cells, elevated levels of mitochondria-bound hexokinases HK-I and
HK-II prevent apoptosis and allow the cells
to continue proliferating. Majewski et al.
(2004) provided evidence that the protein
kinase Akt functions in the suppression
of apoptosis by eliciting translocation of
hexokinase to the mitochondria. AzoulayZohar et al. (2004) showed that HK-I binds
directly to the voltage-dependent anion
channel (VDAC), an integral protein of the
mitochondrial outer membrane that forms
a large channel that transports ions, adenine nucleotides, and other metabolites into
and out of the mitochondria. Binding of
HK-1 to the VDAC induces closure of this
channel, thereby preventing cytochrome
c release and inhibiting apoptosis (AzoulayZohar et al., 2004). It also has been shown
that excessive VDAC closure leads to mitochondrial swelling and apoptosis (Majewski
et al., 2004; Rostovtseva et al., 2005).
Because it is such a major factor in the
life of a cell, perhaps it should come as no
surprise that glucose metabolism (and hexokinase activity) also plays a key role in PCD.
Some of the major routes of glucose
metabolism are glycolysis (the principal
pathway for cellular energy production in
the form of ATP), the pentose phosphate
pathway (which generates NADPH and precursors for a variety of anabolic pathways),
and biosynthesis of structural and storage
polysaccharides (e.g., starch, cellulose, and
glycogen). The first step in glucose metabolism, ATP-dependent phosphorylation to
yield glucose-6-phosphate, is catalyzed by
hexokinase. Plants and animals contain
a number of isozymes of hexokinase, which
differ in subcellular localization and in their
catalytic and regulatory properties, and
selective expression of the various hexokinases is thought to be a major factor in the
regulation of glucose metabolism (Wilson,
2003). Thus, hexokinase is well-positioned
to be a major player in controlling life and
death processes within the cell.
In this issue of The Plant Cell, Kim et al.
(pages 2341–2355) show that mitochondrial-associated hexokinase functions in
the control of PCD in plants. The authors
present evidence that virus-induced gene
silencing (VIGS) of Hxk1, which encodes
a mitochondrial hexokinase, caused PCD
in Nicotiana benthamiana. In addition, overexpression of mitochondrial hexokinases in
Arabidopsis led to enhanced survival of
cells exposed to PCD-inducing conditions,
such as treatment with H2O2.
First, the authors examined expression
of an Hxk1:GFP fusion construct and found
that Hxk1:GFP was localized primarily in
Phenotype of Hxk1-Silenced N. benthamiana.
www.plantcell.org/cgi/doi/10.1105/tpc.106.046623
TRV:Hxk1-silenced plant (right) shows necrotic lesions, abnormal leaf development, and reduced plant
height relative to control plant (left) infected with a TRV empty vector construct.
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mitochondria, dependent on the presence
of an N-terminal putative membrane anchor sequence. Expression analysis in
wild-type plants showed that Hxk1 mRNA
was most abundant in flowers, flower buds,
and young leaves and was detected at
lower levels in mature leaves, stems, and
roots. Furthermore, Hxk1 expression was
found to be induced in leaves following
H2O2, heat treatment, or treatment with the
chemical thapsigargin, which is known to
induce apoptosis in animal cells.
The authors next employed VIGS, using
the tobacco rattle virus (TRV) vector described by Ratcliff et al. (2001), to examine
the effect of suppression of Hxk1 expression in N. benthaniana. VIGS is based on
the principle that virus vectors carrying
host-plant-derived sequence inserts induce silencing of the corresponding genes
in infected plants. The TRV vector designed
by Ratcliff et al. (2001) mediates VIGS of
endogenous plant genes in Nicotiana species without causing virus-induced symptoms and is able to target mRNAs in plant
meristems. VIGS provides a means of rapidly assessing gene function through silencing without the need of creating transgenic
plants; construction of the virus vector, including the target gene sequence, and monitoring symptoms on infected plants can be
completed in a matter of weeks instead of
months (Ratcliff et al., 2001).
The TRV:Hxk1-silenced plants created
by Kim et al. were found to have reduced
hexokinase activity and exhibited necrotic
lesions on leaves, abnormal leaf development, and reduced plant height, relative to
control plants infected with a TRV empty
vector (see figure on previous page). The
areas surrounding necrotic lesions showed
some of the hallmarks of PCD, such as
DNA laddering and increased reactive oxygen species production, indicating that suppression of Hxk1 activity activates a PCD
pathway in plants. Enhanced cell death in
the Hxk1-silenced plants was also found to
be accompanied by disruption of mitochondrial membrane potential and release
of cytochrome c—two other hallmarks of
PCD. Mitochondrial membrane potential,
measured using a fluorescent lipophilic
cationic dye (TMRM) that accumulates in
mitochondria in proportion to the mitochon-
drial membrane potential, declined in Hxk1silenced plants to 12.5% of that measured
in controls (plants infected with the empty
VIGS vector). Cytochrome c, measured by
immunoblot analysis, was found to be associated mainly with the mitochondrial protein fraction in leaves of control plants but
mainly with the cyotsolic fraction in Hxk1silenced plants, indicating that mitochondrial release of cytochrome c accompanied
PCD in the Hxk1-silenced plants.
Kim et al. also analyzed transgenic Arabidopsis plants that overexpressed the Arabidopsis hexokinase genes HXK1 or HXK2,
which have N-terminal membrane anchor
sequences and are localized primarily to
the mitochondria. Isolated protoplasts from
these plants were treated with H2O2 and
a-picolinic acid (in separate experiments)
to induce cell death, stained with propidium
iodide, and assessed using flow cytometry
(cells with disrupted membrane potential
allow propidium iodide to enter the cell
and fluoresce red). In each case, the
percentage of live cells remaining following
treatment was significantly higher in the
HXK-overexpressing cells than in wild-type
controls, suggesting that elevated hexokinase activity conferred partial protection
from either H2O2- or a-picolinic acid–
induced PCD.
Finally, experiments were performed to
examine the possibility that PCD in the
Hxk1-silenced plants was not due to a direct effect of Hxk1 but occurred as an indirect result of perturbed carbon metabolism.
Using a mitochondria-enriched fraction isolated from leaves, the authors showed that
exogenous addition of Hxk1 blocked cytochrome c release induced by clotrimazole
and H2O2. Clotrimazole is an antifungal azole
derivative that acts to dissociate hexokinases from mitochondria in a dosedependent manner. The authors first showed
that clotrimazole had no effect on cytochrome c release alone, but it enhanced or
potentiated H2O2-induced cytochrome c
release when the two agents were combined. The addition of Hxk1 blocked this
induction of cytochrome c release in a dosedependent manner and was also dependent on the presence of the Hxk1
N-terminal membrane anchor, as recombinant protein lacking the N-terminal frag-
ment had no effect. Other experiments
showed that Hxk1 did not bind directly to
cytochrome c, but rather, Hxk1 prevented
disruption of mitochondrial membrane potential. These results strongly suggest that
Hxk1 association with mitochondria inhibits cytochrome c release associated with
PCD by acting to maintain mitochondrial
membrane potential.
The authors speculate that, as in animal
cells, mitochondrial-associated hexokinase
influences mitochondrial membrane potential and PCD by binding to the VDAC protein, which has been shown to play a role in
both plant and animal PCD (Godbole et al.,
2003). This intriguing possibility will need to
be addressed in future experiments.
Nancy A. Eckardt
News and Reviews Editor
[email protected]
REFERENCES
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September 2006
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Kim, M., Lim, J.-H., Ahn, C.S., Park, K., Kim,
G.T., Kim, W.T., and Pai, H.-S. (2006).
Mitochondria-associated hexokinases play
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Programmed Cell Death in Plants: A Role for Mitochondrial-Associated Hexokinases
Nancy A. Eckardt
Plant Cell 2006;18;2097-2099
DOI 10.1105/tpc.106.046623
This information is current as of June 15, 2017
References
This article cites 10 articles, 3 of which can be accessed free at:
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