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IN THIS ISSUE

Programmed Cell Death in Plants: A Role for Mitochondrial-Associated Hexokinases

neckardt{at}aspb.org

"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 = from, and ptosis = 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 mitochondrial 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. Azoulay-Zohar 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 (Azoulay-Zohar 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 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 mitochondrial membrane potential, declined in Hxk1-silenced 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 Hxk1-silenced plants, indicating that mitochondrial release of cytochrome c accompanied PCD in the Hxk1-silenced plants.

View larger version (62K): [in this window] [in a new window]   Phenotype of Hxk1-Silenced N. benthamiana. 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.

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 dose-dependent 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 dose-dependent manner and was also dependent on the presence of the Hxk1 N-terminal membrane anchor, as recombinant protein lacking the N-terminal fragment 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.

	Footnotes

 
www.plantcell.org/cgi/doi/10.1105/tpc.106.046623

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Mitochondria-Associated Hexokinases Play a Role in the Control of Programmed Cell Death in Nicotiana benthamiana

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