The results of the present study are described in more detail in original publications. Here we present data, which reflect the main tendencies in the plant response to anoxia and reoxygenation, and are of crucial importance for discussion and conclusions. Data on control samples (before anoxic treatment) and aerated controls (during the treatment) are given when necessary. The experiments on mitochondrial characteristics under anoxia and identification of the PTP in plant mitochondria have not been published yet and, therefore, are described here in details.
Anoxic treatment brought about both H 2 O 2 accumulation and changes in the cell ultrastructure. The ability to maintain cell intactness correlated positively with anoxia tolerance. I. pseudacorus rhizome tissue showed undisturbed structures after 15 days and even after 45 days of anoxia ( IV ) with thin layers of cytoplasm along the cell walls and amorphous fructan stores in the vacuoles ( IV) . In the roots of the anoxia intolerant wheat plasmolysis could be observed already after 1 day of oxygen deprivation.
In rice continued stress led to swelling of mitochondria and to plasmolysis and finally to the disintegration of the plasma membrane after 7 days of anoxia ( IV ).
In the cereals imposition of anoxia resulted in the accumulation of H 2 O 2 associated with the plasma membrane and the cell wall ( IV ) . Both the enhancement of stress and the restoration of normoxic conditions caused rapid membrane degradation and an increase in H 2 O 2 formation. Reoxygenation injury caused by a 2 h reaeration period resulted in deteriorated membrane structures, and formation of H 2 O 2 not only on the plasma membrane but inside the protoplasts of both wheat and rice. In rhizomatous species the difference in anoxia tolerance, estimated as H 2 O 2 production, was more pronounced in comparison to that of the cereals. In the cells of the anoxia intolerant I. germanica intensive accumulation of H 2 O 2 was detected on the plasma membrane and less intensive in the cell wall after 8 days of oxygen deprivation. Anoxia tolerant I. pseudacorus showed no H 2 O 2 formation on the plasma membrane after up to 45 days of anoxia. Reoxygenation after 15 days of treatment did not cause any changes neither in the cell ultrastructure nor in H 2 O 2 accumulation in the rhizomes of I. pseudacorus ( IV ) .
The degree of membrane peroxidation was measured as conjugated diene (CD) and conjugated triene (CT) formation ( II ). This process (i.e. free radical attack on the double bonds of polyunsaturated fatty acids) is characteristic of the propagation phase of LP (Recknagel and Glende 1984). The initial content of CD in the roots of cereals was several times lower than that of Iris rhizomes, and this difference was preserved during the anoxic treatment. In general, varying lengths of anoxia, which preceeded the 2 h reaeration period, resulted in enchanced LP in both cereal roots and Iris rhizomes. The intensity of the peroxidative reaction was different between species and correlated with anoxia tolerance. In anoxia tolerant species the content of both dienes and trienes was lower after long-term exposure as compared with intolerant species. The highest CD and CT content and degree of increase were detected in the rhizomes of the intolerant I. germanica . The increase was 1300% of the initial CD value after 12 days of anoxia. In I. pseudacorus the same parameter was only 144% of the initial value after 15 days of treatment. In wheat and oat roots the accumulation of CD and CT had a similar pattern as in Iris spp. However, the difference between the absolute levels of dienes in the tolerant oat and intolerant wheat was not so pronounced as in the Iris spp.
Figure 7. Simple UV (A) and second derivative (SD) spectra (B) of lipid extracts from anoxically treated (1 day) wheat roots. Minima in SD spectra correspond with: 1, conjugated trienes; 2, cis-trans conjugated dienes; 3, trans-trans conjugated dienes .
Second derivative (SD) spectrophotometry of lipid extracts from oxygen-deprived plants confirmed the formation of conjugated double bonds with specific absorption minima, and hence the presence of peroxidized lipids. The minima in SD spectra corresponded with the maxima in the original UV spectra of lipid extracts and allowed the quantification of conjugated bonds via the peak heights. Two characteristic minima in SD spectra could be detected and ascribed to two stereoisomers of CD: trans-trans with a minimum at a shorter wavelength region (228-230 nm) and cis-trans shifted to longer wavelengths (237-240 nm) (Fig 7). There were no significant differences between the peak heights of trans-trans and cis-trans dienes, a fact which suggests an equal probability for stereoisomer formation. The additional minima in the SD spectrum, clearly seen at about 280 nm, probably corresponded with conjugated triene stereoisomers. Two peaks resolved in this region were at about 274 and 283 nm. In general, anoxia was shown to result in an increase in the peaks in the SD spectra corresponding with CD and CT stereoisomers especially in anoxia-intolerant plants. However, in the anoxia-tolerant I. pseudacorus no CD signal was observed in the SD spectra even after 45 days of anoxia. A strong signal was detected in this species at about 280 nm with two minima (271 and 282 nm), which probably corresponded with conjugated triene isomers.
Formation of substances, which react with thiobarbituric acid (TBARS), is characteristic of the terminal stage of LP and indicates the breakdown of peroxidised lipids. The anoxia-intolerant wheat contained initially (before the treatment) a higher amount of TBARS as compared with the tolerant rice. Monitoring of the degradative stage of LP in connection with anoxia indicated a general increase in TBARS with the exception of the anoxia-tolerant I. pseudacorus ( I , II ). The dynamics of this process in the roots of cereals and I. germanica rhizomes correlated positively with that of CD and CT formation. Determination of LP level in mitochondria, isolated from the roots of wheat and rice ( I ), revealed the same tendency, i.e. accumulation of peroxidative products in response to anoxic stress and higher intensity of this process in anoxia intolerant species.
Figure 8 . Determination of lipid peroxidation products (TBARS) under normoxia and hypoxia in wheat and rice roots after anoxic treatment. Circles = wheat, triangles = rice; open labels = normoxia, closed labels = hypoxia. Bars represent standard errors n = 5-7.
Extremely high levels of TBARS were found in the untreated rhizomes of I. pseudacorus . These levels dropped significantly during anoxic treatment, while in the aerated controls only a slight decrease was observed. According to the TBA test, no lipid peroxidation had occurred in the rhizomes of anoxia tolerant I. pseudacorus . (This is discussed further in II : Probably LP was terminated at the earlier stages or polyphenols interfered with the assay.)
In order to evaluate the impact of re-admission of oxygen into the samples during the assay procedure and the role of low oxygen concentration on LP, an experimental series on TBARS accumulation in cereal roots was performed in an air-tight chamber under hypoxic conditions (ca. 2% of O 2 ) (Fig. 8). In the controls (anoxically treated plants, in which TBARS content was determined under normal aeration) the same, as described above, increase in LP products was detected. Under the hypoxic TBARS assay, only in the anoxia intolerant wheat a significant increase was observed, while in rice roots there were no detectable changes in LP.
In the present study the activity of SOD was determined without a reoxygenation period, immediately after termination of the anoxic treatment in the roots of cereals with different tolerance to anoxia. To elucidate the stress response early under anoxia, the experiments were performed after 5, 14, 24, 72, 168 hours ( I ). In the roots of the control wheat seedlings SOD activity was two times higher than in rice. In the course of the experiment the activity decreased in wheat under both aeration and anoxia, however, in the anoxic samples this decline was slower. As a result, after 3 days of anoxia the activity was by 65% higher than in the control roots. In the more anoxia tolerant rice, anoxia did not affect SOD activity. The determination of SOD activity in the rhizomes of Iris spp. has been done earlier (Monk et al. 1989).
Cereals and Iris spp. were different from each other both in absolute levels of ascorbate and in its reduction state. Oxidised dehydroascorbic (DHA) acid was the main form in wheat and rice roots in the initial control plants and under anoxia and aeration (58.4% of total ascorbate for wheat and 78% for rice, with AA/DHA ratio of 0.7 and 0.3 for wheat and rice, respectively). On the contrary, the rhizomes of Iris spp. were characterised by elevated levels of reduced ascorbic acid (AA), which was the predominant form in the ascorbate pool. In the rhizomes of the anoxia tolerant I. pseudacorus the initial level of total ascorbate and the degree of ascorbate reduction were higher than in the intolerant I. germanica . AA/DHA ratio was 4.8 for I. pseudacorus and 2.7 for I. germanica . The imposition of anoxia and subsequent reoxygenation caused a decrease both in the content of ascorbate and in its reduction state. However, no consequent changes in DHA content were detected. Prolongation of the anoxic treatment led to a decline in the antioxidant level, both reduced and oxidised forms, in all plants tested. A decrease in AA/DHA ratio indicated a shift in the reduction state of the ascorbate pool under oxygen deprivation.
In the roots of wheat and rice the initial level of glutathione (under aeration, before treatment) was approximately the same, while the reduction state (measured as GSH/GSSG ratio) appeared to be higher in the roots of the anoxia tolerant rice (III). The highest content of glutathione in the control plants was detected in the anoxia intolerant rhizomes of I. germanica , while in I. pseudacorus the amount of glutathione was three times lower. A remarkable drop in the level of reduced glutathione was detected after anoxia and reoxygenation in all plant species studied. The most rapid decrease was observed after short periods (considering anoxia tolerance of the species) of oxygen deprivation. The GSH content in the plant tissues after short term oxygen deprivation was only 13% and 55% of the initial level in wheat and rice roots, while in the rhizomes it was 36% and 11% for I. germanica and I. pseudacorus , respectively. These changes were not associated with a corresponding increase in GSSG, as the same tendency for decline was observed for the changes in GSSG content.
Tocopherol levels in the cereals and in the Iris spp. were measured by two different methods: TLC and HPLC, respectively ( I, III ). Both methods gave similar results considering the order of tocopherol content in the species studied. The HPLC-method allowed the detection of alterations in both a - and b -tocopherols under anoxia.
Tocopherol content ( I ) in the wheat control plants at the beginning of the experiment was similar to that of rice (0.5 - 0.8 m gg -1 FW). Short-term anoxic treatment revealed a different response of the tocopherol system in anoxia tolerant and intolerant plants: A significant increase in wheat and preservation of the initial tocopherol level in rice roots were recorded. Continuation of anoxia up to 3 days led to the decline of the tocopherol content in wheat roots back to the control level, while in rice this parameter remained unaltered. In the aerated control plants of both wheat and rice, the tocopherol level was not affected.
HPLC of tocopherols in rhizomes of Iris spp. confirmed the presence of both a - and b -tocopherols ( III ). b -Tocopherol appeared to be the predominant compound in rhizomatous tissue. In I. pseudacorus however, the difference in a - and b -tocopherol levels was less pronounced. I. germanica rhizomes were characterised by a very high content of b -tocopherol (about 7 m gg -1 FW, in comparison to 1.5 m gg -1 FW in I. pseudacorus ). To our knowledge, there are no previous reports of as high a b -tocopherol content in plant tissues. Under experimental conditions (anoxia and aeration) the same pattern of changes was shown in Iris spp. for both a - and b -tocopherols, i.e. the preservation of the tocopherol content during the first days of anoxia. Prolonged anoxia (45 days for the tolerant I. pseudacorus and 12 days for the intolerant I. germanica ) caused a decrease in a - and b -tocopherols in the rhizomes of both species. At the end of the anoxic treatment 43% of the initial a -tocopherol content in the rhizomes of I. pseudacorus remained, while for I. germanica this value was 62%.
Although the retention times of the HPLC-peaks suggested the presence of a few tocopherol isomers, the existence of only a -tocopherol could be confirmed by a mass spectrometry (MS) analysis. Another tocopherol isomer with a molecular mass of 414 was found and this was most probably b -tocopherol (MW= 416.7).
The main aims of the experiments were to characterise any alterations in particular mitochondrial functions caused by oxygen deprivation, and to identify the permeability transition pore in the inner membrane of plant mitochondria. The choice of the parameters measured was, therefore, determined by the factors important for PTP induction in animal mitochondria: high amplitude swelling, Ca 2+ overload and membrane depolarisation. Oxygen consumption by isolated mitochondria was used as an integral parameter for evaluation of mitochondrial viability and the degree of coupling.
Since a high matrix Ca 2+ -concentration has been reported as a crucial condition for PTP induction in animal mitochondria, it was necessary to show the ability of plant mitochondria (and particular mitochondrial preparation) to take up external Ca 2+ . The choice of incubation medium was determined by the facts that Ca 2+ uptake is facilitated in the presence of inorganic phosphorus (Pi) and is dependent on membrane potential.
Figure 9. Ca 2+ uptake by isolated plant mitochondria. Incubation medium: 0.25 M sucrose in 0.010 M TRIS-HEPES pH 7.4, 30 m M EGTA, Arsenazo III, 10 m M Pi, 5 mM; rotenone, 1 M;mitochondria 0.4 mg protein ml -1 . Additions: 100 nM Ca 2+ was added each time, Suc = 5 mM succinate, 1 mM ADP, 1 mM NADH.
Addition of 100 nmol of Ca 2+ to the incubation medium and subsequent additions of a respiratory substrate (5mM succinate or 1 mM NADH) and 5 mM Pi induced Ca 2+ uptake by isolated plant mitochondria (Fig. 9).
It was observed that NADH was more effective than succinate in the induction of Ca 2+ uptake. It should be taken into consideration, that only qualitative evaluation of Ca 2+ uptake is given, since plant mitochondria can, as it seems, slowly metabolise the Ca 2+ -specific dye Arsenazo III used. This results in gradual fading of the indicator colour and hence introduces an error, which is difficult to take in consideration in the calculation of Ca 2+ uptake. Nevertheless, high sensitivity of Arsenazo III to Ca 2+ allows the use of the indicator in the detection of changes in Ca 2+ -concentrations below 100 m M. However, we were not able to detect any Ca 2+ -induced Ca 2+ -release from plant mitochondria - a feature which is characteristic of permeability transition and the resulting diffusion of solutes according to the concentration gradient.
In our experiment high-amplitude mitochondrial swelling was observed under physiological conditions. The effects of different PTP inducers and inhibitors were examined. Employment of cyclosporin A (CsA), a specific inhibitor of PT in mammalian mitochondria, permits the distinction between PT and other processes. In all the cases studied here swelling was observed in actively respiring mitochondria after the addition of a respiratory substrate (succinate, NADH). In contrast to facts known about animal mitochondria, succinate-driven swelling of mitochondria was inhibited by 100 m M Ca 2+ . Addition of Pi and KCl prior to succinate greatly enhanced the amplitude of swelling (Fig. 10). Application of CsA did not affect plant mitochondria, however in a few experiments some CsA sensitivity was observed (data not shown). According to our results, the high-amplitude swelling of mitochondria observed, was not caused by PTP operation in the inner mitochondrial membrane, and can be attributed to the 'high energy' swelling, as defined by Bernardi (1999). The next step was to characterise this type of swelling by other parameters (i.e. membrane potential measurements) and to monitor changes in mitochondrial volume under anoxia.
Figure 10. Dependence of the amplitude of the mitochondrial swelling on Ca 2+ , Pi and respiratory substrates. I ncubation medium: 0.25 M sucrose in 0.010 M TRIS-HEPES pH 7.4, 30 M EGTA, mitochondria 0.4 mg protein ml -1 . Additions: R= 1 m M rotenone, 100 m M Ca 2+ , 5 mM Pi, Suc = 5mM Succinate, 1 mM NADH. Trace A: effect of Ca 2+ ions and respiratory substrates on the amplitude of mitochondrial swelling; trace B: effect of the respiratory substrates; trace C: effect of Pi
Among many factors and compounds that increase PTP opening probability, anoxia takes an important place. Anoxia itself is an integral factor, which induces several PTP-triggering conditions: alterations in membrane potential, pH shift, GSH depletion, cytosolic Ca 2+ increase and release of free fatty acids (have been shown to act as uncouplers). Monitoring of anoxia induced changes in mitochondrial swelling revealed that upon the depletion of oxygen from the medium due to respiration, swelling stopped immediately and the opposite process - shrinkage of mitochondria took place. These changes were detected as an increase in light scattering at 540 nm, indicating increased matrix density (Fig.11). Restoration of the oxygen supply reversed the response. This fact indicates an active physiological state of mitochondria and that the changes observed cannot be considered degradative. Oxygen consumption measurement under the same conditions confirmed mitochondrial viability and the timing of anoxic conditions in the spectrophotometer cuvette: All oxygen was consumed after 8-13 min from the addition of 0.4 mg mitochondrial protein ml -1 into 1 ml of incubation medium at +25 ° C. This time was dependent on the quality of the mitochondrial preparation and number of additions to the cuvette (since each time some oxygen is introduced).
The magnitude of the inner membrane potential is an important parameter for PTP regulation and under oxygen deprivation. Moreover, it was necessary to characterise basic electrophysiology of plant mitochondria and in our particular mitochondrial preparation, since such experiments have not been executed with plant mitochondria earlier.
Figure 11. Time-dependent decrease in the inner mitochondrial membrane potential of NEM-treated mitochondria. Mitochondria were loaded with 2mM Pi, then treated with 0.8 mM NEM (an inhibitor of Pi uniporter) and washed. Addition of 5 mM succinate caused the generation of D Y , which slowly decreased because of the inhibition of Succinate/Pi exchange. Incubation medium: 0.25 M sucrose in 0.010 M TRIS-HEPES pH 7.4, 30 m M EGTA, 10 nmol ml -1 safranine O, 0.4 mg mitochondrial protein ml -1 , Suc= 5mM succinate.
Qualitative estimation of membrane potential with safranine O revealed the absolute necessity of Pi (when succinate, but not when NADH was used as a substrate) for the generation of a membrane potential. Together with the observation that Pi enhanced succinate-induced mitochondrial swelling, and the fact that Complex II succinate dehydrogenase active sites face mitochondrial matrix, these observations suggest the existence of a Pi/succinate exchanger on the inner mitochondrial membrane. To check this hypothesis mitochondria were loaded with 2mM Pi, then treated with N-ethylmaleimide (an inhibitor of the Pi uniporter), washed and treated with 5mM succinate. As predicted, an initial generation of membrane potential was followed by a linear decrease, because of Pi exhaustion inside mitochondria and, therefore, inhibition of succinate uptake by mitochondria (Fig 11). This finding is of particular importance for PTP investigation, since Pi also facilitates Ca 2+ uptake, which requires a high membrane potential.
Figure 12. Changes in mitochondrial volume and inner membrane potential induced by anoxia. Incubation medium: 0.25 M sucrose in 0.010 M TRIS-HEPES pH 7.4, 30 m M EGTA, 0.4 mg mitochondrial protein ml -1 . Suc= 5mM succinate, 1 mM ADP, Trace A, inner membrane potential; trace B, swelling of mitochondria. Both parameters were recorded simultaneously.
Changes in the membrane potential brought about by anoxia were monitored simultaneously with swelling. The results show a clear negative correlation between the magnitude of the inner membrane potential and the degree of mitochondrial shrinkage (Fig.12). The same tendency was observed in the experiments on PTP identification: As soon as oxygen was consumed by mitochondrial respiration, an increase in light scattering at 540 nm (swelling) was detected. The amplitude of the increase was lower, probably because of the different additions of chemicals and a different incubation system.