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Keywords:

  • oats;
  • victorin;
  • apoptosis;
  • programmed cell death;
  • mitochondria;
  • permeability transition

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Conclusions
  7. Experimental procedures
  8. Supplementary Material
  9. References
  10. Supporting Information

In this study, we determined the timing of events associated with cell death induced by the host-selective toxin, victorin. We show that the victorin-induced collapse in mitochondrial transmembrane potential (Δψm), indicative of a mitochondrial permeability transition (MPT), on a per cell basis, did not occur simultaneously in the entire mitochondrial population. The loss of Δψm in a predominant population of mitochondria preceded cell shrinkage by 20–35 min. Rubisco cleavage, DNA laddering, and victorin binding to the P protein occurred concomitantly with cell shrinkage. During and following cell shrinkage, tonoplast rupture did not occur, and membranes, including the plasma membrane and tonoplast, retained integrity. Ethylene signaling was implicated upstream of a victorin-induced loss in mitochondrial motility and the collapse in Δψm. Results suggest that the victorin-induced collapse in Δψm is a consequence of an MPT and that the timing of the victorin-induced MPT is poised to influence the cell death response. The retention of plasma membrane and tonoplast integrity during cell shrinkage supports the interpretation that victorin induces an apoptotic-like cell death response.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Conclusions
  7. Experimental procedures
  8. Supplementary Material
  9. References
  10. Supporting Information

Victorin is a host-selective toxin produced by Cochliobolus victoriae (Macko et al., 1985; Wolpert et al., 1985, 1986), the causal agent of victoria blight of oats (Meehan and Murphy, 1946). Victorin is required for pathogenesis, and both oat sensitivity to victorin and susceptibility to C. victoriae are conditioned by the dominant Vb allele (Meehan and Murphy, 1947; Scheffer et al., 1967). Genetically, the Vb allele is inseparable from Pc2 (Mayama et al., 1995; Rines and Luke, 1985), an oat gene that conditions resistance to specific races of Puccinia coronata f. sp. avenea (Welsh et al., 1954), the causal agent of crown rust. Although victorin is causal to host susceptibility to C. victoriae, the response induced by victorin shares similarities with an elicitor-induced defense response, including rapid cell death (Wolpert et al., 2002). Localized, rapid cell death in the context of resistance is referred to as the hypersensitive response (HR) and is a form of programmed cell death (PCD; Heath, 2000; Lam et al., 2001). Cell death induced by victorin is also a form of PCD (Curtis and Wolpert, 2002; Navarre and Wolpert, 1999; Wolpert et al., 2002; Yao et al., 2002). The defense-like response induced by victorin and the genetic relationship between Vb and Pc2, a race-cultivar-specific resistance (R) gene, indicate that the host actively responds to victorin ultimately initiating PCD.

In animals, PCD pathways culminate in ‘organized’ cell disassembly, which can be facilitated by a family of proteases called caspases (Thornberry and Lazebnik, 1998). Recent studies demonstrate that cell shrinkage, a characteristic feature of PCD, is necessary for, and precedes, a portion of the caspase-dependent cell disassembly (Maeno et al., 2000; Okada et al., 2001; Yu et al., 2001). Cell shrinkage is mediated by cellular efflux of K+ and Cl via specific Ca2+- and ATP-regulated ion channels (Okada et al., 2001). In contrast, necrotic cell death arises from a rapid loss of volume regulation, and subsequently cellular swelling and lysis. The loss of volume regulation arises from ion dysregulation, cytosolic Ca2+ overloading, ATP depletion and generation of reactive oxygen species (ROS; Okada et al., 2001). Ca2+ (Yu et al., 2001) and ROS (Christophe et al., 2002; Curtin et al., 2002), depending on their concentration and temporal and spatial distribution, are recognized as regulators of both PCD and necrosis. In contrast, ATP depletion drives necrotic cell death while PCD requires cellular energy (Crompton, 1999; Dussmann et al., 2002; Kroemer et al., 1998; Leist et al., 1999).

A collapse in mitochondrial transmembrane potential (Δψm), which is considered to be an early, rate-limiting, committal step to cell death (Kroemer et al., 1998), precedes cell shrinkage during animal PCD (Kroemer et al., 1998; Zamzami et al., 1995). The collapse in Δψm is indicative of a mitochondrial permeability transition (MPT) that is likely mediated by the irreversible opening of the permeability transition pore (PTP; Zoratti and Szabo, 1995). Opening of the PTP is promoted by cell stress (for example, decreases in adenine nucleotides, oxidative stress, and overloading of mitochondrial matrix Ca2+; Crompton, 1999; Lemasters et al., 1998) and ‘death’ signals, such as pro-apoptotic bcl-2 family members and caspases (Guo et al., 2002; Marzo et al., 1998; Pastorino et al., 1999). An MPT results in the collapse of Δψm, generation of ROS, a cessation in ATP synthesis, and a release of matrix solutes, including Ca2+ (Kroemer et al., 1998). Opening of the PTP is also associated with the release of apoptogenic proteins that are necessary for, or amplify, caspase-dependent and -independent PCD events (Susin et al., 1996; Thress et al., 1999; Waterhouse and Green, 1999). In contrast, depletion of ATP, cytosolic Ca2+ overload, and ROS can promote necrotic cell death (Crompton, 1999; Kroemer et al., 1998; Okada et al., 2001). The relative rates of the necrotic promoting events versus activation of downstream PCD events by the apoptogenic proteins are thought to determine whether cell death will be necrotic or PCD (Kroemer et al., 1998).

Similar to PCD in animals, changes in Ca2+ (Levine et al., 1996; Navarre and Wolpert, 1999; Xu and Heath, 1998), ROS (Jabs, 1999; Neil et al., 2002; Vranova et al., 2002), and mitochondrial functions (Curtis and Wolpert, 2002; Lam et al., 2001; Tiwari et al., 2002; Yu et al., 2002), and activation of caspase-like enzymes (Lam and del Pozo, 2000; Woltering et al., 2002) may participate in plant PCD. Unique to plants is a possible role for vacuoles during PCD (Kuriyama and Fukuda, 2002). Differentiation of tracheary elements (TEs) into empty cell corpses (mature TE) provides such an example. A significant event during TE death is the rupture of the tonoplast and collapse of the vacuole. Rupture of the tonoplast results in the release of hydrolytic enzymes that are thought to degrade the cytosolic constituents, a process referred to as autolysis (Groover et al., 1997; Kuriyama and Fukuda, 2002; Obara et al., 2001). During autolysis, organelles become disorganized and swollen (Groover et al., 1997), a typical feature of necrotic cell death in animals. Although the rupture of the tonoplast drives cell death by autolysis, rupture of the tonoplast is apparently regulated by a PCD pathway sharing conserved features with animal PCD such as a collapse in Δψm (Yu et al., 2002).

Tonoplast rupture is apparently not involved in cell collapse during the HR of cowpea epidermal cells to the cowpea rust fungus (Heath et al., 1997). However, the HR in potato epidermal cells involves instantaneous cell collapse that, Freytag et al. (1994) suggests, involves a breakdown of the ‘entire membrane system’. The mode of disassembly during the HR has been suggested to be different for different host–microbe pairs (Heath, 2000). Considering the dramatic change in cell compartmentation that occurs with tonoplast rupture, the timing of changes in the integrity of the tonoplast during an HR likely influences the form of cell death.

Alterations in mitochondrial function, similar to animal PCD, are also apparent during plant PCD. The generation of ROS has been proposed as one mitochondrial contribution to plant PCD (Jones, 2000; Lam et al., 2001). Several studies also suggest that a change in mitochondrial permeability, similar to the MPT in animals, occurs during plant PCD (Curtis and Wolpert, 2002; Lacomme and Santa Cruz, 1999; Tiwari et al., 2002). Recently, we demonstrated that isolated oat mitochondria can undergo an MPT, and that the MPT is sufficient to permit victorin access to the mitochondrial matrix. In addition, victorin induces an in vivo collapse in Δψm that is indicative of an MPT (Curtis and Wolpert, 2002). In vivo, genotype-specific binding of victorin to the mitochondrial matrix-localized P protein (Wolpert and Macko, 1989; Wolpert et al., 1994) supports the notion that a change in mitochondrial permeability is induced during victorin-induced PCD. As discussed above, the timing of changes in mitochondrial permeability in animals can influence the form of cell death.

In this study, we sought to determine the timing of victorin-induced PCD events, including the collapse of Δψm, cell shrinkage, DNA laddering, cleavage of the chloroplast-localized Rubisco large subunit, and victorin binding to the P protein. A fluorescent conjugate of victorin was utilized in this study to allow visualization of the in vivo distribution of victorin and for quantification of victorin binding to the P protein. The distribution of labeled victorin and two additional dyes were used to infer changes in the permeability and integrity of the plasma membrane and the tonoplast. Ethylene antagonists were also used to address the role of ethylene signaling in victorin-induced PCD events.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Conclusions
  7. Experimental procedures
  8. Supplementary Material
  9. References
  10. Supporting Information

Temporal relationship of the victorin-induced Δψm collapse with other events associated with cell death

In this paper, we used a fluorescein conjugate of victorin (VicFluor) to detect victorin distribution in vivo and victorin binding to P protein. VicFluor was used at a concentration of 0.35 µg ml−1, which has an activity equivalent to 5–7.5 ng ml−1 victorin (based on the timing of induced Rubisco cleavage and DNA laddering; data not shown) as reported for similar derivatives (Wolpert and Macko, 1988). Oat sensitivity to VicFluor displayed the same characteristics and genotype specificity as sensitivity to victorin. The toxin-insensitive cultivar (X424) displayed no detectable response to VicFluor (as shown in this paper), and thus all reference to an effect of VicFluor refers exclusively to toxin-sensitive tissue.

As with native victorin, VicFluor induces the collapse of Δψm in sensitive-oat mesophyll cells. As shown in Figure 1, the cellular distribution of tetramethylrhodamine methyl ester (TMRM), a potentiometric dye that accumulates in mitochondria maintaining Δψm, changes in response to VicFluor. Initially, TMRM predominantly accumulates in the mitochondria and, to a lesser degree, in the cytosol (Figure 1a). Prior to 95 min, cytosolic TMRM staining is more intense relative to the vacuole in the majority of cells (Figure 1a). Chloroplasts appear to exclude TMRM (Figure 1a–f). This staining pattern persists in control tissue (Figure 1e), as well as VicFluor-treated insensitive tissue (Figure 1f), for at least 360 min. However, by 95–125 min in VicFluor-treated sensitive tissue, the majority of cells display vacuolar TMRM staining in addition to cytoplasmic staining (Figure 1b,c). By 125 min (Figure 1c), VicFluor-treated sensitive cells have fewer TMRM-stained mitochondria, suggesting that within a cell individual mitochondria loses Δψm while other mitochondria retain Δψm. As shown in Figure 1(d), by 175 min, there are many cells that lack any TMRM-stained mitochondria. In addition, by 175 min, portions of the cell population are shrunken. In shrunken cells, TMRM is retained in the cytosol (Figure 1d) and vacuoles lose TMRM. The compartmentation of TMRM in shrunken cells suggests that the plasma membrane and the tonoplast retain integrity.

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Figure 1. Image collection for counting mitochondria stained with the potentiometric dye, TMRM, in live tissue.

(a) Sensitive-oat tissue floated on VicFluor and viewed at 45 min.

(b) Sensitive-oat tissue floated on VicFluor and viewed at 95 min.

(c) Sensitive-oat tissue floated on VicFluor and viewed at 125 min.

(d) Sensitive-oat tissue floated on VicFluor and viewed at 175 min.

(e) Sensitive-oat tissue untreated and viewed at 360 min.

(f) Insensitve-oat tissue floated on VicFluor and viewed at 360 min.

Peeled oat leaf slices were floated exposed mesophyll side down on a solution of 0.35 µg ml−1 VicFluor, or without VicFluor, and 50 nm TMRM. At selected time points, the abaxial mesophyll layer was viewed by confocal laser scanning microscopy with excitation at 568 nm and emission collected with a BP-TRITC filter to visualize TMRM. The images shown are representative of four experiments. Bars, 10 µm.

Examples of mitochondria (m), upper focal plane mitochondria (ufm), cytosol with TMRM (cy), vacuole with TMRM (dv), vacuole excluding TMRM (ev), shrunken cell (sc), chloroplast (chl), and apoplast (ap) are indicated.

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The timing of the victorin-induced Δψm collapse was determined by counting the number of observable (TMRM stained) mitochondria per cell over time (four experiments with a total of six repetitions of controls and VicFluor-treated tissue). The number of mitochondria observed per cell was organized into four categories including cells with 0, 1–5, 6–10, and 11+ mitochondria. The data for a representative experiment are indicated in Figure 2. As shrunken cells were never observed to contain TMRM-stained mitochondria, all shrunken cells were grouped into the category of ‘zero observable mitochondria per cell’. Consequently, the data presented in Figure 2(a) represent the percentage of all cells with the indicated number of mitochondria and include cells that had shrunken. To demonstrate more clearly changes in the number of observable mitochondria per cell prior to shrinkage, data from only non-shrunken VicFluor-treated cells are indicated in Figure 2(b).

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Figure 2. Timing of VicFluor-induced collapse in mitochondrial transmembrane potential (Δψm) in victorin-sensitive tissue.

(a) The percentage of cells (of the total number of cells imaged) with a given number of mitochondria in control tissue at 80, 120, 250, and 355 min (black bars) or VicFluor-treated tissue at 95, 130, 175, and 220 min (gray bars).

(b) The number of non-shrunken cells with a given number of mitochondria in VicFluor-treated tissue at 95, 130, 175, and 220 min.

TMRM-stained mitochondria were counted from images collected as described in Figure 1. For each time point, mitochondria were counted from 60 non-shrunken cells, and the total number of shrunken and non-shrunken cells were counted, as described in the Experimental procedures. Cells were grouped into categories of 0, 1–5, 6–10, or >11 observable mitochondria per cell. Shrunken cells had zero observable mitochondria. These data are representative of four experiments.

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In control tissue, the percentage of cells in each category remained relatively constant, and the majority of cells typically had >11 observable mitochondria even at the latest time point checked (355 min; Figure 2a). In contrast, by 95 min following treatment with VicFluor, the majority of cells contained <11 observable mitochondria. By 175 min, there was a gradual increase in the percentage of cells with <5 observed mitochondria over time. By 220 min, the majority of cells treated with VicFluor had no observable mitochondria (Figure 2a). These data indicate that VicFluor induces a progressive loss of mitochondria maintaining Δψm, and all mitochondria do not lose their potential simultaneously. Significantly, the data of only non-shrunken cells clearly demonstrate that there is an enrichment in the population of cells that contain one to five observed mitochondria (Figure 2b) prior to an accumulation of cells with zero observable mitochondria. This trend was consistent among all four experiments.

The victorin-induced Δψm collapse was given a quantitative value by calculating the percentage of total cells with zero to five mitochondria (including shrunken cells) as a function of time, as shown for representative data in Figure 3(a). The percentage of shrunken cells over time was also plotted in Figure 3(a). The Δψm collapse typically preceded cell shrinkage by 20–35 min. Δψm collapse preceded cell shrinkage by 20–35 min in four repetitions and 55–90 min in two repetitions. Among the six repetitions, 100% of the cells were shrunken by 160 (n = 1), 175 (n = 1), 210 (n = 1), 220 (n = 1), and 250 (n = 2) min. Although there was leaf-to-leaf variability in the absolute timing of the Δψm collapse, the order of events, as described for Figure 1, was consistent among all experiments and repetitions.

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Figure 3. Temporal relationship of the VicFluor-induced mitochondrial transmembrane potential (Δψm) collapse to biochemical markers of cell death in victorin-sensitive tissue.

(a) Rate of Δψm collapse and cell shrinkage in the abaxial mesophyll cell layer. The rate of Δψm collapse is represented by the percentage of total cells with zero to five mitochondria. The rate of cell shrinkage is the percentage of total cells with a shrunken morphology. The data presented are representative of four experiments.

(b) Rate of Rubisco large subunit cleavage and VicFluor binding to the P protein. Cleavage of the Rubisco large subunit was detected by Coomassie staining and quantified by densitometry after protein separation on a 12% SDS–PAGE gel. P protein labeled with VicFluor was detected and quantified by scanning fluorimetry after protein separation on an 8% SDS–PAGE gel.

(c) Time course of VicFluor-induced DNA laddering. DNA was analyzed by ethidium bromide staining on a 1.5% agarose gel. Lanes 1–3, 5, 6, and 8, treated with VicFluor; lanes 4 and 7, untreated controls.

Peeled oat leaf slices were floated exposed mesophyll side down on a solution of 0.35 µg ml−1 VicFluor, or without VicFluor for 1–6 h prior to extraction of (b) protein and (c) DNA. Data for the collapse of Δψm and cell shrinkage were adapted from the experiments described in Figure 2.

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The timing of the victorin-induced Δψm collapse was compared to victorin-induced Rubisco cleavage, DNA laddering, and victorin binding to the P protein. In Figure 3(b), the average (n = 4) rates of both in vivo VicFluor binding to the P protein and in vivo Rubisco cleavage are presented. Also, a representative DNA gel demonstrating the timing of DNA laddering is presented in Figure 3(c). All three of these biochemical events represent all mesophyll cells, in contrast to the data obtained by confocal microscopy, which only visualizes the abaxial mesophyll cell layer. However, in cross-sections prepared from VicFluor-treated tissue, the majority of mesophyll cells are shrunken by 4 h in all cell layers (data not shown). Thus, the rate of cellular shrinkage that occurs at the abaxial mesophyll cell layer closely represents the rate of cellular shrinkage through all the mesophyll cell layers. Comparing Figure 3(a) to Figure 3(b,c), it is clear that Δψm collapse precedes cell shrinkage, victorin binding to the P protein, Rubisco cleavage, and DNA laddering. In addition, cell shrinkage appears to occur around the same time as victorin binding to the P protein, Rubisco cleavage, and DNA laddering.

Observations of cell shrinkage

Cell shrinkage is a definitive characteristic of PCD, in particular, apoptotic cell death, and so we were interested in observing cell shrinkage in real time. Groups of mesophyll cells, lacking or with only a few observable mitochondria, proximal to cells that had already shrunk were selected for time series image collection. A time series of images (12 sec per image) were collected for a maximum of 5 min. As the cells of the abaxial mesophyll do not respond with complete synchrony, selected groups of cells, sometimes, were not observed to shrink within the 5-min interval. Image collection of these cells was not continued to avoid artifacts generated by photobleaching or light-induced damage to the cell.

Figure 4(a) (as well as Supplementary Material; Fig. S1) demonstrates victorin-induced cell shrinkage. Shrinkage was observed on four occasions (in three separate experiments), and each time it occurred as demonstrated in Figure 4(a). Interestingly, cells typically shrunk in pairs, and the pairs were within the same cell file. Just prior to shrinkage (84 sec), and during shrinkage (94 sec), there is a re-distribution of TMRM such that TMRM becomes excluded from the vacuole. By the end of cell shrinkage, the chloroplasts are rounded up and are no longer pressed against the periphery of the cell, and regions of the plasma membrane and adjacent cytosol have moved away from the cell wall (120 sec). Other regions of the plasma membrane and adjacent cytosol remain positioned next to the cell wall. In addition, cytosolic TMRM staining persists in shrunken cells, indicating that both the plasma membrane and the tonoplast retain integrity.

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Figure 4. VicFluor-induced cell shrinkage.

(a) VicFluor-treated tissue undergoing cell shrinkage stained with TMRM. The initial image was designated as time 0 sec, with subsequent images shown for 84, 96, and 120 sec. The images are representative of four replicates.

(b) VicFluor-treated tissue stained with neutral red. (i) Sensitive-oat tissue viewed at 65 min prior to re-distribution of neutral red. (ii) Insensitive-oat tissue viewed at 330 min.

(c) Sensitive-oat tissue undergoing cell shrinkage. The initial image was designated as time 0 sec, with subsequent images shown for 12 and 24 sec. The images are representative of six replicates.

(d) Oat tissue floated on 300 mm sorbitol in the presence of 6 μm neutral red.

(e) Shrunken cells stained with VicFluor. VicFluor was excited with the 488 laser line and emission collected with an BP-FITC filter.

(f) Shrunken cells stained with VicFluor after a 10-min incubation in a solution of 3.4 kDa PEG (>1 Osmol).

Peeled oat leaf slices were floated exposed mesophyll side down on a solution of 0.35 µg ml−1 VicFluor except in (d), and (a) 50 nm TMRM or (b–d) 6 µm neutral red. TMRM and neutral red were excited with the 568 laser line and emission collected with a BP-TRITC filter. For (a,c), unshrunken cells were selected as described in the text and a series of images collected at 12-sec intervals by confocal scanning laser microscopy. Bars, 10 µm.

Examples of mitochondria (m), cytosol with TMRM or neutral red (cy), vacuole with TMRM or neutral red (dv), vacuole excluding TMRM or neutral red (ev), shrunken cell (sc), chloroplast (chl), apoplast (ap), invagination (inv), neutral red (nr), and VicFluor (VicF) are indicated.

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The apparent lack of tonoplast rupture during cell shrinkage was also evident after staining cells with neutral red, a dye that accumulates in the acidic, vacuolar compartment. In untreated tissue, and VicFluor-treated insensitive tissue (Figure 4bii) and early time points of VicFluor-treated tissue (Figure 4bi), neutral red accumulates and remains only in the vacuole. However, as shown in Figure 4(c) (as well as Supplementary Material; Fig. S2), VicFluor treatment results in neutral red staining of both the vacuole and cytosol before cell shrinkage. This is obvious by the negative staining of chloroplasts (Figure 4c, 0 sec) and is similar to what is observed during TMRM staining (Figure 4a) prior to cell shrinkage. During shrinkage, neutral red is re-distributed such that it is excluded from the vacuole while being retained in the cytosol (Figure 4c, 12 and 24 sec). In addition, neutral red was observed re-distributing to adjacent, shrunken mesophyll cells. In the newly shrunken cell and adjacent cells, neutral red re-distributes only into the cytosolic compartment, suggesting that compartmentation, and plasma membrane and tonoplast integrity, persists after cell shrinkage. This same sequence of events, including re-distribution to adjacent, shrunken cells, was observed six times in three separate experiments.

Plasmolysis is a physiological form of cell shrinkage that occurs in response to a hyperosmotic solution. Oat mesophyll cells stained with neutral red and floated on a solution of 0.3 m sorbitol, to induce a degree of plasmolysis, are shown in Figure 4(d). In mesophyll cells undergoing plasmolysis, invaginations are observed that cross the diameter of the cell and reflect regions where the plasma membrane has pulled away from the cell wall. These invaginations are not observed in control cells floated on 20 mm MOPS (3-[N-morpholino]propane-sulfonic acid). Similar invaginations are observed in VicFluor-induced shrunken cells (Figure 4e) that are visualized by VicFluor staining of the cell. Significantly, incubation of VicFluor-induced shrunken cells in a solution of 3.4 kDa polyethylene glycol (PEG) with an osmolality >1 Osmol results in further shrinkage (Figure 4f). These results further support that membranes retain integrity after shrinkage.

VicFluor accumulates in mitochondria after cell shrinkage

Prior to cell shrinkage in toxin-sensitive tissue (Figure 5ai), VicFluor is predominantly visible in the apoplast and the majority of VicFluor is apparently excluded from the cell. In toxin-insensitive tissue (Figure 5b), VicFluor remained in the apoplast for at least 360 min (latest time point checked). As shown in Figure 5(aii,iii), after cell shrinkage in victorin-sensitive tissue, VicFluor enters cells, staining both the cytosol and vacuole. In addition, invaginations are observed (as discussed above), and VicFluor accumulates most intensely in mitochondria (also Figure 4e). Accumulation of VicFluor in mitochondria was confirmed by immunolocalization of the mitochondrial matrix-localized P protein, as shown in Figure 5(c). In tissue fixed 4 h after treatment with VicFluor, immunolocalization of P protein (Figure 5ci) and VicFluor fluorescence (Figure 5cii) generate an almost identical punctate pattern. When these images are merged (Figure 5ciii), it is clear that VicFluor labels the mitochondria detected by immunolocalization of P protein.

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Figure 5. Distribution of VicFluor in shrunken cells.

(a) Sensitive-oat tissue floated on VicFluor and viewed at (i) 110 and (ii,iii) 230 min.

(b) Insensitive-oat tissue floated on VicFluor and viewed at 360 min.

(c) Sensitive-oat tissue fixed 4 h after treatment with VicFluor and probed with anti-P protein antibody and an Alexa 350 secondary antibody. Fixed mesophyll cells viewed for anti-P protein by excitation with a UV laser and emission collected with a 440 BP filter (i) and VicFluor (ii). (iii) Merged image from (i,ii).

(d) Sensitive-oat tissue floated on 7.5 ng ml−1 of unlabeled victorin and viewed at (i) 120 min and (ii,iii) 240 min.

(e) Sensitive-oat tissue floated on VicFluor and viewed at 260 min. (ii) Digitally magnified image of the circled area in (i). (iii) Chloroplast autofluorescence from same cells as in (ii) collected with excitation at 488 nm and emission collected with a LP-665 filter.

Peeled oat leaf slices were floated exposed mesophyll side down on a solution of 0.35 µg ml−1 VicFluor, or 7.5 ng ml−1 victorin (d). At selected time points, the abaxial mesophyll layer was viewed by confocal laser scanning microscopy with excitation at 488 nm and emission collected with an BP-FITC filter (except diii and eiii) to visualize VicFluor. The images shown here are representative of at least three experiments. Bars, 10 µm.

Examples of shrunken cell (sc), non-shrunken cell (nc), chloroplast (chl), mitochondria (m), apoplast (ap), autofluorescence (af), and VicF (VicFluor) are indicated.

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In the BP (band pass)-FITC filter, with excitation at 488 nm, there is weak chloroplast autofluorescence. In the absence of VicFluor (either in control tissue or tissue treated with 7.5 ng ml−1 unlabeled victorin, 5 days), the predominant signal present is because of chloroplast autofluorescence. Some shrunken cells do contain autofluorescent vesicles as shown in Figure 5(diii). Typically, this autofluorescence appears as speckles, but occasionally, vesicles appear that have an appearance similar to that of a VicFluor-labeled mitochondria. However, vesicular autofluorescence in fixed tissue treated with unlabeled victorin did not co-localize with anti-P protein antibody (data not shown). This result confirms that the intensely stained punctate bodies in VicFluor-stained shrunken cells are mitochondria that have accumulated VicFluor. Also, in VicFluor-stained cells, the chloroplasts appear negatively stained (Figure 5aii,iii), suggesting VicFluor is excluded from the chloroplasts.

In the majority of shrunken cells, VicFluor accumulated in the cell, and in the same cells, chloroplasts, as visualized by autofluorescence, maintained a distinct shape. In contrast, a minor population of cells (<1% of the total cells), as shown in Figure 5(e), did not accumulate VicFluor. These cells typically contained autofluorescent vesicles that were larger than mitochondria. In addition, chloroplast autofluorescence (detected by excitation at 488 nm and emission collection with an LP (long pass)-665 filter) was weaker than that found in shrunken cells, and the chloroplasts in this minor population of cells did not have a distinct shape (Figure 5eiii). These cells appear to have lost compartmentation and may represent cell death by necrosis, which is likely caused while peeling off the abaxial epidermal cell layer.

Inhibition of VicFluor-induced events by ethylene antagonists

Previously, Navarre and Wolpert (1999) demonstrated that the ethylene antagonists, aminooxyacetic acid (AOA) and silver thiosulfate (STS), delayed symptom development in a whole-leaf assay and inhibited victorin-induced Rubisco cleavage. As shown in Figure 6(a), both AOA and STS were effective inhibitors of VicFluor-induced Rubsico cleavage and victorin binding to the P protein. Both antagonists also inhibited DNA laddering (data not shown). STS and AOA were also effective at inhibiting the VicFluor-induced collapse of mitochondrial Δψm, as shown in Figure 6(b). By 230 min, oat mesophyll cells treated with VicFluor alone are shrunken, and TMRM is only present in the cytosol (Figure 6bii). In contrast, in the presence of 1 mm STS (Figure 6biv) or 2 mm AOA (Figure 6bvi), VicFluor-treated mesophyll cells retain Δψm. STS (1 mm; Figure 6biii) or AOA (2 mm; Figure 6bv) alone have no obvious effect on mesophyll cells. By 300 min, in tissue treated with VicFluor and 2 mm AOA, TMRM does stain the vacuole and mitochondria lose motility, as shown in Figure 7.

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Figure 6. Ethylene antagonists inhibit VicFluor-induced cell death events.

(a) VicFluor binding to the P protein in tissue treated in the presence of (i) STS or (iii) AOA. Cleavage of the Rubisco large subunit in tissue treated with VicFluor in the presence of (ii) STS or (iv) AOA. The data presented are the average of three experiments.

(b) Images collected by confocal laser scanning microscopy. (i) Control tissue at 300 min. (ii) VicFluor-treated tissue at 230 min. (iii) 1 mm STS alone at 280 min. (iv) VicFluor-treated tissue with 1 mm STS at 255 min. (v) 2 mm AOA alone at 310 min. (vi) VicFluor-treated tissue with 2 mm AOA at 300 min. The data presented is representative of three experiments. Bars, 10 µm.

(c) Oat tissue treated with VicFluor in the presence of 5 mm AOA viewed at 185 min. Bar, 10 µm.

(d) VicFluor binding to the P protein and Rubisco detected by scanning fluorimetry after protein separation on an 8% SDS–PAGE gel.

Peeled oat leaf slices were floated exposed mesophyll side down on a solution of 0.35 µg ml−1 VicFluor, or without VicFluor, and with or without STS or AOA. At given time points, the tissue was (a,d) extracted for protein, or (b,c) viewed by confocal laser scanning microscopy. For microscopy, oat tissue was floated on 50 nm TMRM (except (c)) and viewed (b) with excitation at 568 nm with emission collected with a BP-TRITC filter, or (c) with excitation at 488 nm with emission collected with an BP-FITC filter.

Examples of mitochondria (m), cytosolic TMRM (cy), vacuole with TMRM (dv), vacuole excluding TMRM (ev), shrunken cell (sc), chloroplast (chl), and chloroplast-associated punctate staining (c) are indicated.

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Figure 7. Ethylene antagonists inhibit VicFluor-induced lose of mitochondrial motility.

(a) Sensitive-oat tissue floated on VicFluor viewed at 100 min.

(b) Untreated tissue viewed at 360 min.

(c) Insensitive-oat tissue floated on VicFluor and viewed at 360 min.

(d) Sensitive-oat tissue floated on VicFluor and 1 mm STS, and viewed at 300 min.

(e) Sensitive-oat tissue floated on VicFluor and 2 mm AOA, and viewed at 155 min.

(f) Sensitive-oat tissue floated on VicFluor and 2 mm AOA, and viewed at 300 min.

Peeled oat leaf slices were floated exposed mesophyll side down on a solution of 0.35 µg ml−1 VicFluor, or without VicFluor, and 50 nm TMRM. Images were collected with excitation at 568 nm and emission collected with a BP-TRITC filter. Mitochondrial motility was assessed by collecting a time series of four images, each 12 sec apart. The first image was false-colored white and the last image false-colored green, and then the images were merged. Mitochondria that are not moving appear purple. Bars, 10 µm.

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Staining of mitochondria with TMRM, in addition to permitting the visualization of mitochondria with Δψm, allows the observation of mitochondrial motility. As shown in Figure 7(a), VicFluor induces the loss of mitochondrial motility in toxin-sensitive tissue. The majority of cells lose mitochondrial motility between 90 and 120 min after VicFluor treatment. In untreated (Figure 7b) and VicFluor-treated insensitive (Figure 7c) tissues, mitochondria retain motility at least for 360 min. The timing of the VicFluor-induced loss of mitochondrial motility indicates that motility is lost prior to the collapse of Δψm in a predominant population of a cell's mitochondria.

As shown in Figure 7(d,e), 1 mm STS and 2 mm AOA inhibited the VicFluor-induced loss of mitochondrial motility. Inhibition of the loss of mitochondrial motility by 1 mm STS was effective even after 300 min (Figure 7d). AOA (2 mm) inhibited the loss of mitochondrial motility at 155 min (Figure 7e) after VicFluor treatment; however, by 300 min, mitochondria lost motility (Figure 7f). Inhibition or delay (in the case of 2 mm AOA) of VicFluor-induced cell death events by these ethylene antagonists suggest ethylene has a significant role in promoting victorin-induced PCD, and the inhibition of the loss of mitochondrial motility implicates ethylene in early signaling.

In contrast to treatment with 2 mm AOA, 5 mm AOA was detrimental to oat mesophyll cells. AOA (5 mm) alone caused a loss of mitochondrial motility (typically between 50 and 90 min), a reduction in the number of observable mitochondria per cell (typically between 90 and 150 min) and cell shrinkage (typically between 165 and 220 min) in four experiments (data not shown). Thus, 5 mm AOA apparently induces cell death that, similar to victorin, involves a loss of mitochondrial motility and Δψm, as well as cell shrinkage. These three events induced by 5 mm AOA occurred in the same order at approximately the same rate as in tissue treated with VicFluor alone. However, cells that shrink in tissue treated with 5 mm AOA and VicFluor versus tissue treated with only VicFluor have distinct differences in the permeability of the vacuole, chloroplasts, and mitochondria.

After cell shrinkage in the presence of 5 mm AOA in combination with VicFluor, VicFluor distributes into the cytosol and is visible as punctate staining associated with the chloroplasts, but does not permeate the vacuole or appear to accumulate in mitochondria (Figure 6c). In contrast, after cell shrinkage in the presence of only VicFluor, VicFluor permeates the vacuole, staining the cell diffusely, and accumulates in mitochondria, but is apparently excluded from the chloroplasts (Figures 4e and 5a). The permeability of chloroplasts, but not mitochondria, to victorin in tissue treated with 5 mm AOA is also reflected by VicFluor binding to the Rubisco large subunit, but not the P protein as shown in Figure 6(d, lanes 3 and 4). In contrast, in tissue treated with only VicFluor, victorin binds to the P protein but not the Rubisco large subunit (Figure 6d, lane 2). In addition to these differences in the permeability of organelle membranes, 5 mm AOA alone did not induce DNA laddering or Rubisco cleavage, and 5 mm AOA inhibited DNA laddering and Rubisco cleavage induced by VicFluor.

Although 5 mm AOA alone induces a loss of mitochondrial motility and Δψm, the lack of both victorin binding to the P protein and accumulation in mitochondria suggest that 5 mm AOA induces cell death independent of an MPT. This is consistent with the apparent role of ethylene in promoting the VicFluor-induced MPT, as indicated by STS and 2 mm AOA inhibition of VicFluor-induced Δψm collapse. Furthermore, the distinct differences in the permeability of the vacuole, chloroplasts, and mitochondria after cell shrinkage induced in tissue treated with 5 mm AOA versus tissue treated with VicFluor alone indicates that the process of cell shrinkage itself is not sufficient to permeabilize mitochondria. Therefore, the binding of victorin to the P protein in tissue treated with VicFluor alone indicates that the VicFluor-induced collapse in Δψm is indicative of an MPT.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Conclusions
  7. Experimental procedures
  8. Supplementary Material
  9. References
  10. Supporting Information

Victorin-induced collapse of mitochondrial transmembrane potential (Δψm) precedes cell shrinkage and biochemical markers of PCD

In animals, an MPT mediated by the PTP is typically considered to be a rate-limiting and committal step during PCD (Kroemer et al., 1998). An MPT results in the collapse of Δψm. Recently, we demonstrated that oat mitochondria in vitro can undergo an MPT and that victorin induces an in vivo collapse of Δψm indicative of an MPT in sensitive oat tissue (Curtis and Wolpert, 2002). The MPT in vitro is sufficient to permit victorin access to the mitochondrial matrix-localized P protein, and we suggested that an MPT in vivo accounts for genotype-specific binding of victorin to the P protein. In this work, we present further evidence that victorin binding to the P protein depends on an MPT and that loss of Δψm, indicative of an MPT, precedes cell shrinkage, Rubisco cleavage, DNA laddering, and detectable victorin binding to the mitochondrial-localized P protein.

The victorin-induced collapse of Δψm preceded cell shrinkage by at least 20–35 min. Rubisco cleavage, DNA laddering, and victorin binding to the P protein occurred concomitantly with cell shrinkage (Figure 3). The lag phase after the collapse in Δψm is likely sufficient for changes in mitochondrial function to influence the coordinated pathways that regulate Rubisco cleavage, DNA laddering, and cell shrinkage. In animals, an MPT is associated with the release of apoptogenic proteins including procaspases, cytochrome c, Smac/Diablo, Endo G, HtrAL/Omni, and apoptosis-inducing factor (AIF; Parone et al., 2002; Ravagna et al., 2002). Procaspases, cytochrome c, and Smac/Diablo influence the activity of caspases and subsequent proteolytic cascades, where as AIF, Endo G, and HtrAL/Omni influence caspase-independent PCD events. Preliminary evidence indicates that victorin-induced Rubisco cleavage is the end result of a proteolytic cascade that involves caspase-like activity (Coffeen and Wolpert, 2004). Also, induction of an MPT in isolated plant mitochondria does result in the release of mitochondrial proteins including cytochrome c (Arpagaus et al., 2002; Curtis and Wolpert, 2002). Recently, Balk et al. (2003) identified a Mg2+-dependent nuclease activity in the mitochondrial innermembrane space that cleaved nuclear DNA into 30-kbp fragments and induced chromatin condensation. A second mitochondrial-derived activity, which was dependent on the addition of cytosolic extract, was also identified that induced DNA cleavage into oligonucleosome fragments. Thus, similar to animal mitochondria, plant mitochondria contain releasable proteins that have the potential to influence PCD.

In addition to releasing apoptogenic proteins, the MPT in animals also results in the release of mitochondrial matrix solutes, including Ca2+, the generation of ROS, and a cessation in ATP synthesis. Yao et al. (2002) recently suggested that victorin induces a mitochondrial oxidative burst that precedes a collapse in Δψm. Generation of intracellular ROS was detected with reduced dichlorofluorescein, which fluoresces after oxidation by ROS. The mitochondrial potentiometric dye, Mitotracker Red, was used to detect Δψm. However, the Mitotracker series of potentiometric fluorescent probes were developed for retention after fixation by covalently cross-linking to proteins (Haugland, 2002), and therefore are not necessarily released from mitochondria after a collapse of Δψm. Therefore, it is not clear whether the generation of ROS precedes or follows the victorin induced Δψm collapse. Nonetheless, victorin apparently does induce mitochondrial generation of ROS (Yao et al., 2002). Chloroplasts also may provide a source of ROS in response to victorin. Navarre and Wolpert (1999) detected light-dependent lipid peroxidation in victorin-treated leaf slices. In the present study, however, all treatments were carried out in the dark, so the chloropolast contribution to ROS generation was likely minimal if any. Mitochondrial generation of ROS is thought to promote PCD in plants, although the mechanism by which ROS induces cell death is not known (Neil et al., 2002).

An MPT also results in depletion of ATP. Depletion of ATP promotes necrosis, while PCD is an active process requiring energy (Crompton, 1999; Dussmann et al., 2002; Kroemer et al., 1998; Leist et al., 1999). In animals, pro-apoptotic bcl-2 family members may promote the release of apoptogenic proteins prior to irreversible opening of the PTP and collapse of the Δψm, thereby promoting PCD prior to the depletion of ATP (Kroemer et al., 1998; Waterhouse and Green, 1999). In addition, within a single animal cell, a fraction of the mitochondria can undergo an MPT, while the remaining mitochondria retain Δψm (Lemasters et al., 1998). As shown in Figure 2, victorin induces a collapse in Δψm in a subfraction of the mitochondria within a cell before all mitochondria lose Δψm prior to cell shrinkage. Thus, a subpopulation of mitochondria likely respond to ‘death’ signals or cellular stress, induced by victorin, by undergoing an MPT to promote the cell death process, while a different subpopulation of mitochondria continue to generate energy to keep necrotic events in check.

The victorin-induced collapse of Δψm also precedes detectable victorin binding to the P protein (Figure 3a,b). As observed by confocal microscopy, detectable amounts of victorin entered oat mesophyll cells only after cell shrinkage, and subsequently accumulated in the mitochondrial matrix (Figures 4e and 5aiii). Although victorin binding to the P protein occurs after cell shrinkage, cell shrinkage alone is not sufficient to permeabilize mitochondria. As discussed below, victorin binding to the P protein does not accompany cell shrinkage induced by toxic quantities of 5 mm AOA, an ethylene synthesis inhibitor, although victorin is clearly present in the cytosol (Figure 6c). Thus, these results are consistent with the hypothesis that the Δψm collapse is indicative of an MPT that permits victorin access to the mitochondrial matrix and binding to the P protein (Curtis and Wolpert, 2002).

Cell shrinkage occurs without loss of membrane integrity

Apoptosis is a form of PCD that has distinct morphological features including cell shrinkage. In plants, cell volume is dominated by a large central vacuole that typically contains hydrolytic enzymes. The lytic vacuole is considered to be a key player during the terminal differentiation of TE. Execution of cell death in TEs occurs as the tonoplast loses integrity and the vacuole collapses (Groover et al., 1997; Kuriyama and Fukuda, 2002; Obara et al., 2001). Rupture of the tonoplast results in the release of hydrolytic enzymes that degrade the cellular contents. This form of PCD is referred to as autolysis and results in a cellular morphology distinct from that of apoptosis.

Similar to differentiating TEs, oat mesophyll cells have a large central vacuole that is directly visualized by the accumulation of neutral red in the vacuole as shown in Figure 4(b). TMRM also stains the vacuole 90–120 min after victorin treatment (Figure 1b–d). In contrast to TEs, PCD induced by victorin occurs without rupture of the tonoplast. This is evident by the re-distribution, during cell shrinkage, of TMRM or neutral red out of the vacuole while TMRM or neutral red persists in the cytosol as shown in Figure 4(a,c). This compartmentalized dye staining demonstrates that neither the plasma membrane nor the tonoplast ruptures.

Interestingly, neutral red that re-distributed to the cytoplasm also flowed into adjacent shrunken cells, indicating that plasmodesmata were not closed. In the adjacent cell, neutral red did not stain the entire cell, but only the compartment surrounding chloroplasts indicative of the cytosol, demonstrating that cells retain compartmentation after cell shrinkage. Open plasmodesmata during the initiation and effector phase of PCD could provide a mechanism by which a cytosolic signal promoting PCD could be propagated cell to cell.

The distribution of VicFluor after cell shrinkage also supports the notion that cells retain membrane integrity. VicFluor, in addition to accumulating in mitochondria, was present in both the cytosol and the vacuole, which is distinct from the exclusion of either TMRM or neutral red from the vacuole. The distinct distribution of these molecules indicates that the tonoplast has selective permeability properties, suggesting that the tonoplast has retained integrity. In addition, victorin-shrunken cells had a morphology similar to that of cells undergoing plasmolysis, a physiological form of cell shrinkage, as shown in Figure 4(d,e). The victorin-shrunken cells also retained the ability to respond osmotically because floating the victorin treated tissue on 3.4 kDa PEG (>1 Osmol) resulted in a further shrinking of the cell such that victorin was observed only at the periphery of the cell cavity (Figure 4f), demonstrating that victorin is contained within the extremely shrunken cell. Thus, apparently most membranes retain integrity. Furthermore, these results support the inference that victorin induces an apoptotic-like response and that execution of cell disassembly, presumably involving the activation of lytic enzymes, is distinct from autolysis mediated by tonoplast rupture.

PCD induced by victorin depends on ethylene

Ethylene evolution has been shown to occur in response to victorin (Shain and Wheeler, 1975), and AOA, an ethylene synthesis inhibitor, and STS, an ethylene mode of action inhibitor, have previously been shown to inhibit victorin-induced symptom development and Rubisco cleavage (Navarre and Wolpert, 1999). Both AOA (2 mm) and STS (1 mm) were effective at inhibiting all of the victorin-induced responses including the loss of mitochondrial motility (Figure 7d,e) and collapse in Δψm, as shown in Figure 6(biv,vi). Inhibition by 2 mm AOA appeared to be more of a delay, instead of complete inhibition, because by 300 min mitochondria had lost motility (Figure 7f). Thus, ethylene appears to be involved in early signaling events because the inhibitors prevented or delayed all responses including the earliest detectable event, the victorin-induced loss of mitochondrial motility. These results are consistent with previous reports that ethylene promotes PCD (Wang et al., 2002).

In contrast to 2 mm AOA, 5 mm AOA alone was toxic to oat cells and caused a loss of mitochondrial motility and Δψm as well as cell shrinkage. Treatment of tissue with 5 mm AOA in the presence of VicFluor did not result in victorin binding to the P protein even though VicFluor was present in the cytosol after cell shrinkage (Figure 6c,d). This result demonstrates that victorin accessibility to the mitochondrial matrix is dependent on an MPT, and cell shrinkage alone is not sufficient to permit victorin access to the matrix. Thus, this provides further evidence that victorin binding to the P protein and the victorin-induced collapse in Δψm are distinct and likely the consequence of an MPT.

Conclusions

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Conclusions
  7. Experimental procedures
  8. Supplementary Material
  9. References
  10. Supporting Information

In this paper, we have examined the timing of events associated with victorin-induced PCD, and changes in the permeability and integrity of cellular membranes. We show that an early event induced by victorin is a loss of mitochondrial motility and a collapse in Δψm indicative of an MPT. Ethylene synthesis and action was implicated upstream of the victorin-induced loss of mitochondrial motility and the MPT. On a per cell basis, the victorin-induced collapse in Δψm did not occur simultaneously in the entire mitochondrial population. The initial fraction of mitochondria that lose Δψm could act as signals for PCD while the fraction of mitochondria that retain Δψm could provide cellular ATP to prevent necrotic cell death. The collapse of Δψm in a predominant population of mitochondria in a cell preceded cell shrinkage by at least 20–35 min. Thus, the timing of the victorin-induced MPT is poised to influence the form of PCD.

We also show that detectable amounts of victorin are extracellular prior to cell shrinkage. While it is possible that biologically significant quantities of labeled victorin are undetectable under these conditions, the results suggest that victorin induces a response in sensitive oats by interacting with an extracellular or plasma membrane-localized host protein, which may be the product of the dominant Vb allele. After cell shrinkage, the majority of victorin enters the cell and accumulates in mitochondria. In addition, detectable victorin binding to the P protein occurs concomitantly with cell shrinkage. Thus, victorin binding to the P protein appears to be a consequence of the response induced by victorin, and not the cause.

We also show that cell shrinkage, as induced by toxic amounts of AOA, is not sufficient to permit victorin access to the mitochondria even though victorin is present in the cytosol. This result further demonstrates that an MPT must occur to permit victorin access to the P protein, and supports the hypothesis that the victorin-induced collapse in Δψm is a consequence of an MPT.

In animals, the timing of the MPT can influence the form of cell death, whether PCD or necrosis. In plants, in addition to mitochondrial functions influencing cell death, the rupture of the tonoplast can drive the process of cell death as it does during TE differentiation. Under those circumstances, rupture of the tonoplast results in degradation of organelles similar to what occurs during necrosis in animals. In this work, we demonstrate that the tonoplast and the plasma membrane retain integrity during and after cell shrinkage. Thus, the disassembly of oat cells in response to victorin appears to occur by an apoptotic-like process as opposed to autolysis.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Conclusions
  7. Experimental procedures
  8. Supplementary Material
  9. References
  10. Supporting Information

Plant material and growth conditions

Oat (Avena sativa) seedlings were grown in a growth chamber with a 16-h photoperiod at 24°C for 5–6 days with daily watering. The susceptible (toxin-sensitive) line X469 and resistant (toxin-insensitive) line X424 were used.

Preparation of fluorescein-conjugated victorin (VicFluor)

Victorin (1.0 mg) was incubated for 4 h at 25°C in 1 ml of 0.1 m MES (2[N-morpholino]ethane-sulfonic acid) (pH 6.5; KOH), 10% DMSO, and 1.0 mg of 5-carboxyfluorescein, succinimidyl ester (NHS (N-hydroxysuccinimide)-Fluorescein, Pierce, Rockford, IL, USA). The fluorescein–victorin conjugate was purified essentially as described for biotinylated victorin (Wolpert and Macko, 1988).

Confocal microscopy

A Leica DM IRBETCS confocal microscope equipped with an Omnichrome Ar/Kr laser (Leica, Wetzler, Germany) with emission lines 488 and 568 and an INNOVA Enterprise Ion Laser (Coherent, Santa Clara, CA, USA) with UV output of 351–364 nm for excitation were used for microscopy. Images were collected with a 40×/1.0–0.5 NA (numerical aperature) Oil PL Fluotar objective. Scanning and imaging were controlled by a computer equipped with scanware 5.0 software. The potentiometric dye TMRM was used for visualizing Δψm in abaxial mesophyll cells. TMRM, as well as neutral red, was visualized with excitation at 568 nm and emission collected using a BP-TRITC filter. Chloroplast autofluorescence was visualized with excitation at 488 or 568 nm and emission collected using a LP-665 filter. VicFluor was visualized with excitation at 488 nm and emission collected using a BP-FITC filter.

The epidermis was peeled off of the abaxial surface of 5–6-day-old oat seedling leaves. For each treatment, six 0.25 cm × 1 cm leaf slices were cut from a single peeled leaf and floated in a well (of a 12-well microtiter dish) with the exposed mesophyll side in contact with the 20 mm MOPS (pH 6.5; KOH), 400 ml incubation solution. After peeling the last sample for an experiment, all the samples were placed in the dark for 15 min. After 15 min, the incubation solution was replaced with the same MOPS buffer amended with 50 nm TMRM and placed in the dark for 10 min. VicFluor (0.35 µg ml−1) was then added to the solution (except for the untreated controls and native victorin, 7.5 ng ml−1, treatments), and the samples were placed into a water bath (25°C) in the dark. For inhibitor studies, AOA or STS was added at the concentration indicated and at the same time as VicFluor.

After adding treatments to the oat leaf slices, images were collected at varying times by confocal microscopy. A single leaf slice from each treatment was used for each time point. Typically, for each time point, confocal microscopy images of VicFluor, TMRM, and chloroplast fluorescence were collected from four focal planes (each 4 mm deep), starting with the upper cell focal plane, at four regions of the leaf slice. Each region included 75–100 mesophyll cells. In addition, a time series of four images (12 sec per image) of a single focal plane was collected at two regions of the leaf slice for assessment of mitochondrial motility. Motility was evaluated by animating the series of four images. For time course experiments of Δψm collapse, images were evaluated by counting the number of mitochondria with Δψm per cell in 15 non-shrunken cells per region (a total of 60 cells per time point per experiment). Observable mitochondria were counted from three of the four focal planes such that the upper focal plane of each cell was included. The total number of shrunken and non-shrunken cells was also counted for each region imaged. For confirming the accumulation of VicFluor in mitochondria, untreated, victorin-treated and VicFluor-treated oat tissue were fixed and probed with the anti-P protein antibody as previously described by Curtis and Wolpert (2002).

The dynamics of cell shrinkage was observed in VicFluor-treated tissue by collecting a time series (12 sec per image) of images of a single focal plane for up to 5 min. If the selected cells did not shrink during the 5 min, another group of cells was selected at a different region of the leaf slice. Cell shrinkage was observed in leaf slices that were incubated with TMRM, as described above. In separate experiments, VicFluor-treated leaf slices were incubated in the absence of TMRM, and at selected time points, a single leaf slice was transferred to an incubation solution containing 6 µm neutral red. After 15 min, the neutral red-loaded leaf slice was briefly dipped in incubation solution without neutral red and then visualized by confocal microscopy. Collection of a time series of cell shrinkage in the presence of neutral red was performed, as described for TMRM-loaded cells.

For comparing plasmolysis to victorin-induced cell shrinkage, untreated oat leaf slices loaded with neutral red and incubated for 15 min on a solution of 0.3 m sucrose were visualized by confocal microscopy. To evaluate if VicFluor-shrunken cells were osmotically responsive, VicFluor-treated tissue, after collapse of the abaxial mesophyll cell layer, was incubated for 15 min on a solution of 3.4 kDa PEG (>1 Osmol) and then visualized by confocal microscopy.

Cross sectioning of oat tissue

Oat leaf slices with the epidermis peeled off were prepared and floated on the MOPS buffer, as described for confocal microscopy. Oat tissue treated with 0.35 µg ml−1 VicFluor or untreated controls were fixed, 4 h after adding VicFluor, in 4% paraformaldehyde (0.1 m sodium phosphate buffer, pH 7.2) at 4°C. The fixative was vacuum-infiltrated for 30 min followed by 3.5-h fixation at atmospheric pressure. After fixation, the tissue was washed three times with 0.1 m sodium phosphate buffer with the last wash extending overnight. The tissue was dehydrated by an acetone series (70%, 1 h; 95%, 1 h; 100% 1.5 h) followed by a 5-h incubation in a 1 : 1 solution of Technovit 7100 (Kulzer Histo-Technik, Germany) under vacuum. The tissue was then embedded with Technovit 7100 following the manufacturer's directions. Buffer washes, acetone dehydration, and plastic embedding were performed at 4°C. Embedded oat tissue was then cut into 4-mm thick slices with a microtome and counterstained with toludine blue. Cell morphology was assessed by light microscopy.

Biochemical markers of victorin-induced PCD

The epidermis was peeled off of the abaxial surface of 5–6-day-old oat seedling leaves. A single peeled leaf was used for each time point in the time course studies, and a single peeled leaf was used for each treatment in the inhibitor studies. For each treatment or time point, six 0.25 cm × 1 cm leaf slices were cut from a single leaf and floated in a well (of a 12-well microtiter dish) with the exposed mesophyll side in contact with the 20 mm MOPS (pH 6.5; KOH), 400 ml incubation solution. After peeling the last sample for an experiment, all the samples were placed in the dark for 15 min. VicFluor (0.35 µg ml−1) was then added to the solution (except for untreated controls), and the samples were placed into a water bath (25°C) in the dark. For inhibitor studies, AOA or STS was added at the concentration indicated and at the same time as VicFluor.

Protein and DNA were extracted and re-suspended as previously described by Curtis and Wolpert (2002). DNA was separated on a 1.5% agarose gel and visualized by ethidium bromide staining. For Rubisco cleavage, 40 µg protein was loaded per lane on a 12% SDS–PAGE gel. The cleaved and uncleaved bands of the Rubisco large subunit were then quantified after gels were stained with Coomassie Brilliant Blue G, de-stained, and scanned with a Molecular Dynamics Personal Densitometer SI (Sunnyvale, CA, USA). For P protein binding, 160 µg protein was loaded per lane on an 8% SDS–PAGE gel. Fluorescence from VicFluor-labeled P protein was quantified using a Hitachi FMBIO II (Oakland, CA, USA) with a 505 nm LP filter, and Hitachi software for volume calculations.

Supplementary Material

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Conclusions
  7. Experimental procedures
  8. Supplementary Material
  9. References
  10. Supporting Information

Fig. S1. VicFluor-treated tissue stained with TMRM and undergoing cell shrinkage. An image series collected at 12-sec intervals by confocal scanning laser microscopy was compiled into a movie.

Fig. S2. VicFluor-treated tissue stained with neutral red and undergoing cell shrinkage. An image series collected at 12-sec intervals by confocal scanning laser microscopy was compiled into a movie.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Conclusions
  7. Experimental procedures
  8. Supplementary Material
  9. References
  10. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Conclusions
  7. Experimental procedures
  8. Supplementary Material
  9. References
  10. Supporting Information

Figure S1.  VicFluor-treated tissue stained with TMRM and under-going cell shrinkage. An image series collected at 12-sec intervals by confocal scanning laser microscopy was compiled into a movie.

Figure S2.  VicFluor-treated tissue stained with red and under-going cell shrinkage. An image series collected at 12-sec intervals by confocal scanning laser microscopy was compiled into a movie.

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TPJ_2040_sm_FigureS2.avi2053KSupporting info item

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.