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Abstract

Owing to the anaerobic metabolism of plants or microbes, significant accumulation of toxic substances occurs in waterlogged soil (Lynch, 1977; Tanaka et al., 1990; Armstrong and Armstrong, 2001). Materials potentially toxic to plants that are accumulated in flooded soils include reduced manganese, iron, hydrogen sulfide, various organic acids, and ethylene (Armstrong and Gaynard, 1976). Surprisingly, to date, all breeding programs aimed at improving waterlogging (WL) tolerance in plants have focused almost exclusively on preventing oxygen loss or improving its transport to, or storage in the root (Jackson and Armstrong, 1999). Plant tolerance to these secondary metabolites has never been considered a useful trait in breeding programs.

The type and amount of organic acids produced depends upon the fermentive character of the microflora, the type and amount of organic materials added, and on the prevailing soil conditions (Rao and Mikkelsen, 1977). Tanaka et al. (1990) found that rice (Oryza sativa) root growth was inhibited by micromolar concentrations of phenolic acids formed in flooded soils amended with wheat (Triticum aestivum) straw both in the laboratory and the field, while Lynch (1978) and Armstrong and Armstrong (1999) reported 15 to 35 mm range values for acetic and propionic acid concentrations under comparable conditions. The accumulation of toxic micronutrients is also increased in waterlogged plants. Ashraf and Rehman (1999) reported that iron and manganese contents increased 80- and 20-fold (to 390 and 148 mg kg⁻¹, respectively) in sandy loam soil as a result of 34 d flooding. The same order of magnitude increase was also reported by Stieger and Feller (1994).

The extent to which the accumulation of toxic metabolites is causally linked to observed deficiencies of macronutrients in waterlogged soils is not clear. Energy deficiency, caused by lack of O₂, reduces the availability of many essential nutrients including nitrogen, phosphorus, sulfur, and also most of the trace elements (Drew, 1988). In addition, there are reports suggesting that inorganic nutrient uptake may be impaired by accumulated organic acids (Mitsui et al., 1954; Rao and Mikkelsen, 1977). The mechanisms of such impairment remain to be investigated.

Thus far, most reports have dealt with the analysis of the overall changes in ion content in plant tissues or with monitoring the kinetics of nutrient depletion in a growth solution (Glass, 1973, 1974; Jackson and St. John, 1980). Due to methodological limitations (relatively poor spatial and temporal resolution), these experiments failed to provide answers about the specific ionic mechanisms involved. The above methodological issues may be successfully overcome when using the microelectrode ion flux measuring (MIFE) technique (Shabala, 2003, 2006). The noninvasive MIFE system (University of Tasmania, Hobart, Australia) has very high spatial (a few micrometers) and temporal (several seconds) resolution and has been successfully applied to the measurement of ion flux kinetics under a variety of stress conditions (Shabala and Newman, 1997; Shabala and Lew, 2002; Shabala et al., 2003). In this study, this technique was used to quantify both the immediate responses of ion fluxes and long-term (after 24 h treatment) responses to secondary metabolites associated with anaerobic soils.

RESULTS

The major bulk of experiments were performed on the WL-sensitive variety ‘Naso Nijo’, where much stronger responses were expected. Accordingly, most of results below refer to ‘Naso Nijo’ roots unless specified otherwise.

Transient K⁺ Fluxes

Net K⁺ uptake of about 60 nmol m⁻² s⁻¹ was measured from mature epidermal root cells of 3-d-old seedlings in control (steady-state) conditions. Addition of phenolic compounds (benzoic acid, 2-hydroxybenzoic acid, 4-hydroxybenzoic acid; 200 μm working concentration) and volatile monocarboxylic organic compounds (formic acid, acetic acid, propionic acid; 10 mm working concentration) rapidly decreased net K⁺ influx (Fig. 1). Among the three different phenolic acids, 2-hydroxybenzoic acid and 4-hydroxybenzoic acid caused much more adverse effects on K⁺ uptake compared with benzoic acid (Fig. 1A), completely arresting net K⁺ uptake within 10 min after treatment. All three volatile monocarboxylic organic acids not only arrested K⁺ influx but also caused significant (P < 0.001) K⁺ efflux from barley (Hordeum vulgare) roots, with an effect increasing in the following sequence: propionic acid ≫ acetic acid > formic acid (Fig. 1B). This effect was specific and not related to changes in osmolality of the experimental solution, as isotonic treatment with KCl, NaCl, or Na gluconate caused no K⁺ efflux from barley roots (data not shown). Adding 300 mg L⁻¹ Mn²⁺ caused an almost instantaneous reduction of K⁺ uptake, which quickly (within 5 min) returned to its initial value (Fig. 1C). In general, the effect of monocarboxylic organic acids on root K⁺ fluxes was significantly stronger than one caused by phenolic acids.

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Figure 1.

K⁺ flux kinetics in response to secondary metabolites associated with anaerobic conditions (applied at the time indicated by an arrow). A, 200 μm phenolic acid treatment. B, 10 mm monocarboxylic acid treatment. C, 300 mg L⁻¹ Mn²⁺ treatment. Measurements were made in the mature zone, 10 to 20 mm from the root tip. Data are means ± se (n = 8).

Transient H⁺ Fluxes

Net H⁺ efflux of 10 to 15 nmol m⁻² s⁻¹ was measured in control (steady-state) conditions from barley roots. Application of all secondary metabolites significantly (P < 0.01) affected H⁺ fluxes (Fig. 2). An immediate and significant shift toward net H⁺ uptake was observed in response to all phenolic and monocarboxylic organic acids tested (Fig. 2, A and B), while in the case of Mn²⁺ treatment, net H⁺ efflux was significantly (P < 0.01) reduced (Fig. 2C). Among phenolics, the effect followed the ranking: 4-hydroxybenzoic acid ≈ 2-hydroxybenzoic acid > benzoic acid. For monocarboxylic organic acids, responsiveness of H⁺ flux followed the ranking: formic acid > acetic acid > propionic acid.

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Figure 2.

H⁺ flux kinetics in response to secondary metabolites associated with anaerobic conditions (applied at the time indicated by an arrow). A, 200 μm phenolic acid treatment. B, 10 mm monocarboxylic acid treatment. C, 300 mg L⁻¹ Mn²⁺ treatment. Measurements were made in the mature zone, 10 to 20 mm from the root tip. Data are means ± se (n = 8).

Transient Ca²⁺ Fluxes

Net zero Ca²⁺ flux was measured in control (steady-state) conditions. Root treatment with phenolic acids led to a gradual and prolonged increase in net Ca²⁺ uptake (Fig. 3A). Such a slowness of response may be indicative of a cytosolic mode of action. No significant (P < 0.05) difference between the effects of various phenolic acids was found. Adding 10 mm monocarboxylic organic acid to the bath, however, caused an immediate and substantial Ca²⁺ efflux from barley roots (Fig. 3B). A similar trend was observed for Mn²⁺ treatment (Fig. 3C). In both cases, net Ca²⁺ flux recovered to its original (zero) value within 10 to 15 min (Fig. 3, B and C).

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Figure 3.

Ca²⁺ flux kinetics in response to secondary metabolites associated with anaerobic conditions (applied at the time indicated by an arrow). A, 200 μm phenolic acid treatment. B, 10 mm monocarboxylic acid treatment. C, 300 mg L⁻¹ Mn²⁺ treatment. Measurements were made in the mature zone, 10 to 20 mm from the root tip. Data are means ± se (n = 8).

Genotypical Difference

No significant (P < 0.05) difference in initial (steady-state) flux levels was found for any ions measured between two contrasting genotypes (WL-sensitive ‘Naso Nijo’ and WL-tolerant ‘TX’; Fig. 4). However, transient flux kinetics differed substantially between genotypes. The most striking difference was observed for K⁺ flux (Fig. 4A). Contrary to WL-sensitive ‘Naso Nijo’ genotype, lower monocarboxylic (acetic) acid treatment did not cause any net K⁺ loss in the WL-tolerant ‘TX’ variety. Moreover, ‘TX’ roots even increased net K⁺ uptake 20 min after treatment with 10 mm acetic acid (Fig. 4A), while similar treatment caused a very substantial K⁺ loss from the roots of WL-sensitive ‘Naso Nijo’ variety. Also significantly reduced was ‘TX’ net Ca²⁺ uptake in response to 2-hydroxybenzoic treatment compared with ‘Naso Nijo’ (Fig. 4C). No clear difference was evident between genotypes in terms of H⁺ flux responses for either acetic or 2-hydroxybenzoic treatment (Fig. 4B). Treatment with Mn²⁺, however, has significantly reduced H⁺ efflux in WL-sensitive ‘Naso Nijo’ variety (compared with control) but was not significant in WL-tolerant ‘TX’ variety (Fig. 4B).

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Figure 4.

Magnitude of net K⁺ (A), H⁺ (B), and Ca⁺ (C) responses to various metabolic compounds (as depicted in Figs. 1–3​) for two genotypes contrasting in WL tolerance, 20 min after treatment. NN, ‘Naso Nijo’ (WL sensitive); ‘TX’, TX9425 (WL tolerant). Data are mean ± se (n = 8). 2-HBZ, 2-Hydroxybenzoic acid.

Pharmacology

Effects of various channel blockers and metabolic inhibitors on ion fluxes kinetics were studied using one chemical from each group (specifically, acetic acid, 2-hydroxybenzoic acids, and Mn²⁺).

None of the inhibitors used significantly affected the initial Ca²⁺ flux after 1 h of incubation (data not shown). However, La³⁺ and Gd³⁺ (two known nonselective cation channel [NSCC] blockers; Demidchik et al., 2002) almost completely inhibited Ca²⁺ flux responses to either acetic acid (Fig. 6B), 2-hydroxybenzoic acid (Fig. 6C), or Mn²⁺ (Fig. 6D) treatment. TEA⁺ was also efficient (approximately 70% inhibition; Fig. 6). At the same time, cyclopiazonic acid (CPA; a specific Ca²⁺-ATPase inhibitor) and vanadate (general ATPase inhibitor) caused approximately 25% reduction of the magnitude of acetic acid-induced Ca²⁺ efflux observed (Fig. 6B), with thapsigargin (another specific Ca²⁺-ATPase inhibitor) being ineffective.

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Figure 6.

Pharmacology of Ca²⁺ flux responses to acetic acid (B), 2-hydroxybenzoic acid (C), and Mn²⁺ (D). Peak Ca²⁺ efflux 2 min after the treatment is shown for acetic and Mn²⁺, while for 2-hydroxybenzoic acid, increment in net Ca²⁺ uptake is shown 20 min after the treatment (as depicted in A). Data are means ± se (n = 6–8). Roots were pretreated with various metabolic inhibitors for 1 h before the specific metabolite was added (still in the presence of inhibitor). M, Magnitude of response.

Initial K⁺ uptake in barley roots was strongly suppressed by TEA⁺ (Fig. 7A). All these inhibitors were also efficient in reducing the magnitude of K⁺ flux response to 2-hydroxybenoic (Fig. 7B) and acetic (Fig. 7C) acids and Mn²⁺ (Fig. 7D; significant at P < 0.05). Both Gd³⁺ and La³⁺ (two known NSCC channel blockers; Demidchik et al., 2002) also significantly reduced initial K⁺ uptake and the magnitude of K⁺ flux response to all chemicals tested (Fig. 7; significant at P < 0.05).

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Figure 7.

Pharmacology of K⁺ flux responses to acetic acid (B), 2-hydroxybenzoic acid (C), and Mn²⁺ (D). The magnitude of K⁺ efflux was determined as the difference between steady-state K⁺ flux before the treatment and K⁺ flux value 20 min after adding a particular compound to the root. Effect of channel blockers on initial (steady state) K⁺ fluxes is shown in A. Data are means ± se (n = 6). Roots were pretreated with various metabolic inhibitors for 1 h before the specific metabolite was added (still in the presence of inhibitor).

Root pretreatment in 1 mm vanadate (a known inhibitor of the plasma membrane [PM] H⁺-ATPase) shifted the initial H⁺ flux from net efflux to net influx (Fig. 8), while no significant effect of TEA⁺, La³⁺, or Gd³⁺ on initial H⁺ flux was observed. Vanadate treatment also significantly (P < 0.01) reduced the magnitude of H⁺ flux changes in response to all treatments tested (data not shown).

Membrane Potential Responses and H⁺-ATPase Activity

The average membrane potential in the mature zone of barley roots was −133.9 ± 2.0 mV in control. Phenolic compounds caused substantial membrane depolarization (as illustrated in Fig. 9A for the treatment with 200 μm 2-hydroxybenzoic acid), stabilizing at −90 mV level approximately 10 min after the treatment was applied. Membrane potential kinetics in response to other compounds was not measured.

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Figure 9.

A, A typical example of transient change of membrane potential upon the addition of 200 μm 2-hydroxybenzoic acid to root mature zone. The first arrow indicates commencing of the treatment, while the second arrow shows the moment when electrode was removed from the cell. B, Cell membrane potentials in root mature zones after 24 h treatment with various secondary metabolites. Data are means ± se (n = 16). C, ATPase hydraulic activity of 7-d-old barley root PM after 30 min treatment of 2-hydroxybenzoic acid, acetic acid, and Mn²⁺. 2-HBZ, 2-Hydroxybenzoic acid; 4-HBZ, 4-hydroxybenzoic acid.

Contrary to the short-term effects of 2-hydroxybenzoic acid, 24 h treatment with 200 μm phenolics caused significant (P < 0.01) hyperpolarization of the membrane potential (Fig. 6B). The largest hyperpolarization effect was found in roots treated with 4-hydroxybenzoic acid. All three monocarboxylic acids and Mn²⁺ treatments induced significant (P < 0.001) long-term depolarization of membrane potential (Fig. 6B).

Results of membrane potential measurements were consistent with direct estimation of ATP hydrolytic activity from PM vesicles isolated from the microsomal fraction of barley roots (Fig. 9C). No significant (P < 0.05) difference was found in ATP hydrolytic activity between control samples and samples treated with either acetic acid or Mn²⁺. At the same time, the PM vesicles from roots treated with 2-hydroxybenzoic acid had about 40% higher ATP hydrolytic activity compared with control roots (significant at P < 0.05; Fig. 9C).

DISCUSSION

Phenolics: Short-Term Effects

Early reports of Glass (1974) showed that various benzoic compounds tested caused a substantial inhibition of potassium absorption (measured as ⁸⁶Rb uptake from excised barley roots) after 3 h of treatment. However, as ⁸⁶Rb measures unidirectional K⁺ uptake, the extent to which K⁺ efflux systems were affected was unclear. Our data (Fig. 1A) show that net K⁺ fluxes from roots treated with 2-hydroxybenzoic and 4-hydroxybenzoic acids were even slightly negative (net efflux), suggesting not only a reduction in K⁺ uptake, but also an increase in K⁺ loss from cells, pointing toward multiple targets in barley root membranes.

Among the three phenolics, the effects of 2-hydroxybenzoic and 4-hydroxybenzoic acids on K⁺ flux were larger than effects caused by benzoic acid (Fig. 1). Earlier, Glass (1973) suggested that increasing hydroxylation within a series tends to decrease the inhibitory capacity of phenolics. This was obviously not the case in our experiments.

Under the conditions of this experiment (pH 5.5), most phenolic acids in solution will be in the dissociated form. This undissociated acid concentration can be calculated according to the Henderson-Hasselbalch equation:

As shown in Table I, the amount of undissociatd acids was relatively low and comprised 4.7%, 0.3%, and 8.7% for benzoic, 2-hydroxybenzoic, and 4-hydroxybenzoic acids, respectively. Therefore, no obvious correlation between the magnitude of effect and the amount of dissociated compound was found.

Table I.

Concentrations of undissociated phenolic acids under experimental conditions

Compound	Dissociation Constant	pK	Concentration of Undissociated Compound
μm	%
Benzoic acid	64.6	4.19	4.7
2-Hydroxybenzoic acid	1,071 (K1)	2.97 (pK1)	0.3
4-Hydroxybenzoic acid	33.1 (K1)	4.48 (pK1)	8.7

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The mechanisms by which phenolic compounds control K⁺ transport across the PM remain elusive. Based on the fact that removal of phenolics caused a rapid recovery of K⁺ reabsorption, Glass (1974) suggested a direct effect on cell membranes. No specific details were offered though. Our data reported in this study suggests that major voltage-dependent K⁺-transporting systems may be key players. This is supported by the observed immediate membrane depolarization (Fig. 9A). Moreover, our data suggest that both increased H⁺ (Fig. 2A) and Ca²⁺ (Fig. 3A) uptake could contribute to this depolarization as depicted in Figure 10.

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Figure 10.

A suggested model explaining short-term effects of secondary metabolites on membrane transport activity. OA, Organic acid; KIR, potassium inward-rectifying channel; KOR, potassium outward-rectifying channel; DACC, depolarization-activated Ca²⁺ channel; DPZ, depolarization of the PM. [See online article for color version of this figure.]

It is traditionally believed that most phenolic acids cross the cell membrane in an undissociated form by passive diffusion (Jackson and St. John, 1980). However, recent cloning and functional characterization of the MCT1 family of transporters suggest that uptake of both monocarboxylic acids and benzoic acid occur via the H⁺-coupled cotransport mechanism, at least in animal systems (Kido et al., 2000). We suggest that a similar scenario is also applicable to plant tissues. As undissociated phenolic acid is electrically neutral, not only will increased net H⁺ flux be generated as a result of such activity (consistent with our H⁺ flux data; Fig. 2A), but also a substantial membrane depolarization is expected (Fig. 9). Such a depolarization will affect intracellular K⁺ homeostasis by reducing K⁺ uptake via inward-rectifying K⁺ channels and enhancing K⁺ efflux via depolarization-activated outward-rectifying K⁺ channels (Maathuis and Sanders, 1996; Fig. 10), explaining the rapid shift toward net K⁺ efflux after phenolics application (Fig. 1A). As both of these channels are TEA⁺ sensitive, inhibition of K⁺ efflux by TEA⁺ in our experiments (Fig. 7) is consistent with this model.

Several Ca²⁺-permeable channels may mediate Ca²⁺ uptake into root cells; each of these may contribute to the observed Ca²⁺ influx after phenolics application. Of special interest may be depolarization-activated Ca²⁺ channels (Thion et al., 1998; Miedema et al., 2001). These play a prominent role in signal perception and transduction in plants (Thion et al., 1998). Regardless of the type of Ca²⁺-permeable channel, increased Ca²⁺ uptake will provide a positive feedback to further depolarize the membrane potential, amplifying the effect of phenolics on K⁺ transport (as depicted in a tentative model in Fig. 10). It also appears that NSCCs contribute partly to the K⁺ efflux, as both Gd³⁺ and La³⁺ (two known NSCC blockers; Demidchik et al., 2002) were efficient in preventing 2-hydroxybenzoic acid-induced K⁺ loss (Fig. 7B).

MATERIALS AND METHODS

Acknowledgments

Notes

¹This work was supported by Grain Research and Development Corporation (M.Z. and N.M.) and Australian Research Council (S.S.) grants.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Sergey Shabala (ua.ude.satu@alabahs.yegres).

^[C]Some figures in this article are displayed in color online but in black and white in the print edition.

www.plantphysiol.org/cgi/doi/10.1104/pp.107.102624

References

(1)

(2)

(3)

Abstract

Owing to the anaerobic metabolism of plants or microbes, significant accumulation of toxic substances occurs in waterlogged soil (Lynch, 1977; Tanaka et al., 1990; Armstrong and Armstrong, 2001). Materials potentially toxic to plants that are accumulated in flooded soils include reduced manganese, iron, hydrogen sulfide, various organic acids, and ethylene (Armstrong and Gaynard, 1976). Surprisingly, to date, all breeding programs aimed at improving waterlogging (WL) tolerance in plants have focused almost exclusively on preventing oxygen loss or improving its transport to, or storage in the root (Jackson and Armstrong, 1999). Plant tolerance to these secondary metabolites has never been considered a useful trait in breeding programs.

The type and amount of organic acids produced depends upon the fermentive character of the microflora, the type and amount of organic materials added, f.lux 4.75 Crack Key For U, and on the prevailing soil conditions (Rao and Mikkelsen, 1977). Tanaka et al. (1990) found that rice (Oryza sativa) root growth was inhibited by micromolar concentrations of phenolic acids formed in flooded soils amended with wheat (Triticum aestivum) straw both in the laboratory and the field, while Lynch (1978) and Armstrong and Armstrong (1999) reported 15 to 35 mm range values for acetic and propionic acid concentrations under comparable conditions. The accumulation of toxic micronutrients is also increased in waterlogged plants. Ashraf and Rehman (1999) reported that iron and manganese contents increased 80- and 20-fold (to 390 and 148 mg kg⁻¹, respectively) in sandy loam soil as a result of 34 d flooding. The same order of magnitude increase was also reported by Stieger and Feller (1994).

The extent to which the accumulation of toxic metabolites is causally linked to observed deficiencies of macronutrients in waterlogged soils is not clear. Energy deficiency, caused by lack of O₂, reduces the availability of many essential nutrients including nitrogen, phosphorus, sulfur, and also most of the trace elements (Drew, 1988). In EZ Game Booster Pro Free Activate, there are reports suggesting that inorganic nutrient uptake may be impaired by accumulated organic acids (Mitsui et al., 1954; Rao and Mikkelsen, 1977). The mechanisms of such impairment remain to be investigated.

Thus far, most reports have dealt with the analysis of the overall changes in ion content in plant tissues or with monitoring the kinetics of nutrient depletion in a growth solution (Glass, 1973, 1974; Jackson and St. John, 1980). Due to methodological limitations (relatively poor spatial and temporal resolution), these experiments failed to provide answers about the specific ionic mechanisms involved. The above methodological issues may be successfully overcome when using the microelectrode ion flux measuring (MIFE) technique (Shabala, 2003, f.lux 4.75 Crack Key For U, 2006). The noninvasive MIFE system (University of Tasmania, Hobart, Australia) has very high spatial (a few micrometers) and temporal (several seconds) resolution and has been successfully applied to the measurement of ion flux kinetics under a variety of stress conditions (Shabala and Newman, 1997; Shabala and Lew, 2002; Shabala et al., 2003). In this study, this technique was used to quantify both the immediate responses of ion fluxes and long-term (after 24 h treatment) responses to secondary metabolites associated with anaerobic soils.

RESULTS

The major bulk of experiments were performed on the WL-sensitive variety ‘Naso Nijo’, where much stronger responses were expected. Accordingly, most of results below refer to ‘Naso Nijo’ roots unless specified otherwise.

Transient K⁺ Fluxes

Net K⁺ uptake of about 60 nmol m⁻² s⁻¹ was measured from mature epidermal root cells of 3-d-old seedlings in control (steady-state) conditions. Addition of phenolic compounds (benzoic acid, 2-hydroxybenzoic acid, 4-hydroxybenzoic acid; 200 μm working concentration) and volatile monocarboxylic organic compounds (formic acid, acetic acid, propionic acid; 10 mm working concentration) rapidly decreased net K⁺ influx (Fig. 1). Among the three different phenolic acids, 2-hydroxybenzoic acid and 4-hydroxybenzoic acid caused much more adverse effects on K⁺ uptake compared with benzoic acid (Fig. 1A), completely arresting net K⁺ uptake within 10 min after treatment. All three volatile monocarboxylic organic acids not only arrested K⁺ influx but also caused significant (P < 0.001) K⁺ efflux from barley (Hordeum vulgare) roots, with an effect increasing in the following sequence: propionic acid ≫ acetic acid > formic acid (Fig. 1B). This effect was specific and not related to changes in osmolality of the experimental solution, as isotonic treatment with KCl, NaCl, or Na gluconate caused no K⁺ efflux from barley roots (data not shown). Adding 300 mg L⁻¹ Mn²⁺ caused an almost instantaneous reduction of K⁺ uptake, which quickly (within 5 min) returned to its initial value (Fig. 1C). In general, the effect of monocarboxylic organic acids on root K⁺ fluxes was significantly stronger than one caused by phenolic acids.

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Figure 1.

K⁺ flux kinetics in response to secondary metabolites associated with anaerobic conditions (applied at the time indicated by an arrow). A, 200 μm phenolic acid treatment. B, 10 mm monocarboxylic acid treatment. C, 300 mg L⁻¹ Mn²⁺ treatment. Measurements were made in the mature zone, 10 to 20 mm from the root tip. Data DisplayFusion Pro Offline Installer means ± se (n = 8).

Transient H⁺ Fluxes

Net H⁺ efflux of 10 to 15 nmol m⁻² s⁻¹ was measured in control (steady-state) conditions from barley roots. Application of all secondary metabolites significantly (P < 0.01) affected H⁺ fluxes (Fig. 2). An immediate and significant shift toward net H⁺ uptake was observed in response to all phenolic and monocarboxylic organic acids tested (Fig. 2, A and B), while in the case of Mn²⁺ treatment, net H⁺ efflux was significantly (P < 0.01) reduced (Fig. 2C). Among phenolics, the effect followed the ranking: 4-hydroxybenzoic acid ≈ 2-hydroxybenzoic acid > benzoic acid. For monocarboxylic organic acids, responsiveness of H⁺ flux followed the ranking: formic acid > acetic acid > propionic acid.

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Figure 2.

H⁺ flux kinetics in response to secondary metabolites associated with anaerobic conditions (applied at the time lightwave 3d download Free Activators by an arrow). A, 200 μm phenolic acid treatment. B, 10 mm monocarboxylic acid treatment, f.lux 4.75 Crack Key For U. C, 300 mg L⁻¹ Mn²⁺ treatment. Measurements were made in the mature zone, f.lux 4.75 Crack Key For U, 10 to 20 mm from the root tip. Data are means ± se (n = 8).

Transient Ca²⁺ Fluxes

Net zero Ca²⁺ flux was measured in control (steady-state) conditions. Root treatment with phenolic acids led to a gradual and prolonged increase in net Ca²⁺ uptake (Fig. 3A). Such a slowness of response may be indicative of a cytosolic mode of action. No significant (P < 0.05) difference between the effects of various phenolic acids was found. Adding 10 mm monocarboxylic organic acid to the bath, however, caused an immediate and substantial Ca²⁺ efflux from barley Disk Drill 4.1.555.0 Crack Mac PC Activation Keygen Download Full (Fig. 3B), f.lux 4.75 Crack Key For U. A similar trend was observed for Mn²⁺ treatment (Fig. 3C). In both cases, net Ca²⁺ flux recovered to its original (zero) value within 10 to 15 min (Fig. 3, B and C).

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Figure 3.

Ca²⁺ flux kinetics in response to secondary metabolites associated with anaerobic conditions (applied at the time indicated by an arrow). A, f.lux 4.75 Crack Key For U, 200 μm phenolic acid treatment. B, 10 mm monocarboxylic acid treatment. C, 300 mg L⁻¹ Mn²⁺ treatment. Measurements were made in the mature zone, 10 to 20 mm from the root tip. Data are means ± se (n = iobit smart defrag key Crack Key For U Difference

No significant (P < 0.05) difference in initial (steady-state) flux levels was found for any ions measured between two contrasting genotypes (WL-sensitive ‘Naso Nijo’ and WL-tolerant ‘TX’; Fig. 4). However, transient flux kinetics differed substantially between genotypes. The most striking difference was observed for K⁺ flux (Fig. 4A). Contrary to WL-sensitive ‘Naso Nijo’ genotype, lower monocarboxylic (acetic) acid treatment did not cause any net K⁺ loss in the WL-tolerant ‘TX’ variety. Moreover, ‘TX’ roots even increased net K⁺ uptake 20 min after treatment with 10 mm acetic acid (Fig. 4A), while similar treatment caused a very substantial K⁺ loss from the roots of WL-sensitive ‘Naso Nijo’ variety. Also significantly reduced was ‘TX’ net Ca²⁺ uptake in response to 2-hydroxybenzoic treatment compared with ‘Naso Nijo’ (Fig. 4C). No clear difference was evident between genotypes in terms of H⁺ flux responses for either acetic or 2-hydroxybenzoic treatment (Fig. 4B). Treatment with Mn²⁺, however, has significantly reduced H⁺ efflux in WL-sensitive ‘Naso Nijo’ variety (compared with control) but was not significant in WL-tolerant ‘TX’ variety (Fig. 4B).

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Figure 4.

Magnitude of net K⁺ (A), H⁺ (B), and Ca⁺ (C) responses to various metabolic compounds (as depicted in Figs. 1–3​) for two genotypes contrasting in WL tolerance, 20 min after treatment. NN, ‘Naso Nijo’ (WL sensitive); ‘TX’, TX9425 (WL tolerant). Data are mean ± se (n = 8), f.lux 4.75 Crack Key For U. 2-HBZ, 2-Hydroxybenzoic acid.

Long-Term Ion Flux Responses

K⁺ uptake was significantly f.lux 4.75 Crack Key For U in ‘Naso Nijo’ roots after 24 h treatment with all secondary metabolites tested (Fig. 5A). Root treatment with phenolic compounds (benzoic acid, 2-hydroxybenzoic acid, 4-hydroxybenzoic acid) caused a significant (P < 0.01) decrease in net K⁺ uptake. No significant (P < 0.05) difference between the effects of various phenolic compounds was found. In monocarboxylic acid treated roots, K⁺ fluxes were shifted to substantial (−40 to −100 nmol m⁻² s⁻¹) net efflux. Among them, acetic acid and propionic acid caused more severe effects than formic acid. Mn²⁺ treatment also caused net K⁺ efflux. In general, the adverse effects of phenolic acids were smaller than the other four treatments.

The monocarboxylic acid treatments shifted H⁺ from net efflux to net influx (Fig, f.lux 4.75 Crack Key For U. 5B). Mn²⁺ treatment reduced H⁺ efflux to around zero. Among the phenolic acids, 2-hydroxybenzoic acid and 4-hydroxybenzoic acid did not cause significant (P < 0.05) changes to H⁺ fluxes, while benzoic acid slightly reduced H⁺ efflux (Fig. 5B).

Phenolic acids caused significant (P < 0.05) net Ca²⁺ efflux from roots pretreated for 24 h (Fig, f.lux 4.75 Crack Key For U. 5C). Formic and acetic acids also slightly reduced net Ca²⁺ uptake, while propionic acid and Mn²⁺ did not significantly (P < 0.05) affect Ca²⁺ fluxes (Fig. 5C).

Pharmacology

Effects of various channel f.lux 4.75 Crack Key For U and metabolic inhibitors on ion fluxes kinetics were studied using one chemical from each group (specifically, acetic acid, 2-hydroxybenzoic acids, and Mn²⁺).

None of the inhibitors used significantly affected the initial Ca²⁺ flux after 1 h of incubation (data not shown). However, La³⁺ and Gd³⁺ (two known nonselective cation channel [NSCC] blockers; Demidchik et al., 2002) almost completely inhibited Ca²⁺ flux responses to either acetic acid (Fig. 6B), 2-hydroxybenzoic acid (Fig. 6C), or Mn²⁺ (Fig. 6D) treatment. TEA⁺ was also efficient (approximately 70% inhibition; Fig. 6). At the same time, cyclopiazonic acid (CPA; a specific Ca²⁺-ATPase inhibitor) and vanadate (general ATPase inhibitor) f.lux 4.75 Crack Key For U approximately 25% reduction of the magnitude of acetic acid-induced Ca²⁺ efflux observed (Fig. 6B), with thapsigargin (another specific Ca²⁺-ATPase inhibitor) being ineffective.

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Figure 6.

Pharmacology of Ca²⁺ flux responses to acetic acid (B), 2-hydroxybenzoic acid (C), and Mn²⁺ (D). Peak Ca²⁺ efflux 2 min after the treatment is shown for acetic and Mn²⁺, while for 2-hydroxybenzoic acid, increment in net Ca²⁺ uptake is shown 20 min after the treatment (as depicted in A). Data are means ± se (n = 6–8). Roots were pretreated with various metabolic inhibitors for 1 h before the specific metabolite was added (still in the presence of inhibitor). M, Magnitude of response.

Initial K⁺ uptake in barley roots was strongly suppressed by TEA⁺ (Fig. 7A). All these inhibitors were also efficient in reducing the f.lux 4.75 Crack Key For U of K⁺ flux response to 2-hydroxybenoic (Fig. 7B) and acetic (Fig. 7C) acids and Mn²⁺ (Fig. 7D; significant at P < 0.05), f.lux 4.75 Crack Key For U. Both Gd³⁺ and La³⁺ (two known NSCC channel blockers; Demidchik et al., 2002) also significantly reduced initial K⁺ uptake and the magnitude of K⁺ flux response to all chemicals tested (Fig. 7; significant at P < 0.05).

graphpad prism 8 Free Activators src="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1976565/bin/pp1450266f07.jpg">

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Figure 7.

Pharmacology of K⁺ flux responses to acetic acid (B), 2-hydroxybenzoic acid (C), and Mn²⁺ (D). The magnitude of K⁺ efflux was determined as the difference between steady-state K⁺ flux before the treatment and K⁺ flux value 20 min after adding a particular compound to the root. Effect of channel blockers on initial (steady state) K⁺ fluxes is shown in A. Data are means ± se (n = 6). Roots were pretreated with various metabolic inhibitors for 1 h before the specific metabolite was added (still in the presence of inhibitor).

Root pretreatment in 1 mm vanadate (a known inhibitor of the plasma membrane [PM] H⁺-ATPase) shifted the initial H⁺ flux from net efflux to net influx (Fig. 8), while no significant effect of TEA⁺, La³⁺, or Gd³⁺ on initial H⁺ flux was observed. Vanadate treatment also significantly (P < 0.01) reduced the magnitude of H⁺ flux changes in response to all treatments tested (data not shown).

Membrane Potential Responses and H⁺-ATPase Activity

The average membrane potential in the mature zone of barley roots was −133.9 ± 2.0 mV in control. Phenolic compounds caused substantial membrane depolarization (as illustrated in Fig. 9A for the treatment with 200 μm 2-hydroxybenzoic acid), stabilizing at −90 mV level approximately 10 min after the treatment was applied. Membrane potential kinetics in response to other compounds was not measured.

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Figure 9.

A, A typical example of transient change of membrane potential upon the addition of 200 μm 2-hydroxybenzoic acid to root mature zone. The first arrow indicates commencing of the Comodo Internet Security 12.2.2.7098 Crack With Serial Key Download, while the second arrow shows the moment when electrode was removed from the cell. B, f.lux 4.75 Crack Key For U, Cell membrane potentials in root mature zones after 24 h treatment with various secondary metabolites. Data are means ± se (n = 16). C, ATPase hydraulic activity of 7-d-old barley root PM after 30 min treatment of 2-hydroxybenzoic acid, acetic acid, and Mn²⁺. 2-HBZ, 2-Hydroxybenzoic acid; 4-HBZ, 4-hydroxybenzoic acid.

Contrary to the short-term effects of 2-hydroxybenzoic acid, 24 h treatment with 200 μm phenolics caused significant (P < 0.01) hyperpolarization of the membrane potential (Fig. 6B). The largest hyperpolarization effect was found in roots treated with 4-hydroxybenzoic acid. All three monocarboxylic acids and Mn²⁺ treatments induced significant (P < 0.001) long-term depolarization of membrane potential (Fig. 6B).

Results of membrane potential measurements were consistent with direct estimation of ATP hydrolytic activity from PM vesicles isolated from the microsomal fraction of barley roots (Fig. 9C). No significant (P < 0.05) difference was found in ATP hydrolytic activity between control samples and samples treated with either acetic acid or Mn²⁺. At the same time, f.lux 4.75 Crack Key For U, the PM vesicles from roots treated with 2-hydroxybenzoic acid had about 40% higher ATP hydrolytic activity compared with control roots (significant at P < f.lux 4.75 Crack Key For U Fig. 9C).

DISCUSSION

Secondary Metabolites Toxicity and WL Tolerance in Barley

All seven secondary metabolites associated with anaerobic soil conditions inhibited root elongation in 24 h treatments (data not shown), highlighting their detrimental effects on root metabolism. They also caused significant alterations in root membrane-transport activity even in the presence of oxygen. The most significant was a pronounced shift toward K⁺ efflux, caused by both phenolics and monocarboxylic acids (Fig. 1). In the case of monocarboxylic acids, the result was a very substantial K⁺ loss measured from the roots of WL-sensitive ‘Naso Nijo’ (Fig. 1B). Such K⁺ loss has been previously reported from barley roots in response to salinity (Chen et al., 2005, 2007) and oxidative (Cuin and Shabala, 2007) stress, with a strong positive correlation (r² > 0.7) between a root's ability to retain K⁺ and the level plant stress tolerance reported (Chen et al., 2007). A tight link between net K⁺ efflux and cytosolic free K⁺ concentration was also shown (Shabala et al., 2006). All these findings suggested that the magnitude of stress-induced K⁺ loss may be used as a quantitative characteristic of plant stress tolerance.

In this study, we extend these findings to plant WL tolerance, f.lux 4.75 Crack Key For U. The WL-tolerant ‘TX’ was capable not only of completely preventing net K⁺ loss after acetic acid treatment f.lux 4.75 Crack Key For U. 4A) but even slightly enhancing net K⁺ uptake by roots under stress conditions. Also less affected (compared with WL-sensitive ‘Naso Nijo’) was K⁺ uptake in response to phenolics (Fig. 4A). These data support the idea that WL tolerance in barley is conferred not only by differences in root anatomy (high percentage of aerenchyma in ‘TX’ genotype; Pang et al., 2004), f.lux 4.75 Crack Key For U, but may, to a large extent, be determined by the superior f.lux 4.75 Crack Key For U of tolerant genotypes to reduce detrimental effects of secondary metabolites on membrane-transport processes in roots (specifically, improving K⁺ retention). With K⁺ being the most abundant cytosolic cation, and its involvement in numerous enzymatic reactions in plants (Shabala, 2003), the physiological significance of such retention is obvious. Thus far, plant breeding for WL tolerance has never considered responses to these secondary metabolites as a useful trait; we suggest this issue should be given special attention in future work.

Phenolics: Short-Term Effects

Early reports of Glass (1974) showed that various benzoic compounds tested caused a substantial inhibition of potassium absorption (measured as ⁸⁶Rb uptake from excised barley roots) after 3 h of treatment. However, as ⁸⁶Rb measures unidirectional K⁺ uptake, the extent to which K⁺ efflux systems were affected was unclear, f.lux 4.75 Crack Key For U. Our data (Fig. 1A) show that net K⁺ fluxes from roots treated with 2-hydroxybenzoic and 4-hydroxybenzoic acids were even slightly negative (net efflux), suggesting not only a reduction in K⁺ uptake, but also an increase in K⁺ loss from cells, pointing toward multiple targets in barley root membranes.

Among the three phenolics, the effects of 2-hydroxybenzoic and 4-hydroxybenzoic acids Gimpshop Free Activate K⁺ flux were larger than effects caused by benzoic acid (Fig. 1). Earlier, Glass (1973) suggested that increasing hydroxylation within a series tends to decrease the inhibitory capacity of phenolics. This was obviously not the case in our experiments.

Under the conditions of this experiment (pH 5.5), most phenolic acids in solution will be in the dissociated form. This undissociated acid concentration can be calculated according to the Henderson-Hasselbalch equation:

As shown in Table I, the amount of undissociatd acids was relatively low and comprised 4.7%, 0.3%, and 8.7% for benzoic, 2-hydroxybenzoic, and 4-hydroxybenzoic acids, respectively. Therefore, no obvious correlation between the magnitude of effect and the amount of dissociated compound was found.

Table I.

Concentrations of undissociated phenolic acids under experimental conditions

Compound	Dissociation Constant	pK	Concentration of Undissociated Compound
μm	%
Benzoic acid	64.6	4.19	4.7
2-Hydroxybenzoic acid	1,071 (K1)	2.97 (pK1)	0.3
4-Hydroxybenzoic acid	33.1 (K1)	4.48 (pK1)	8.7

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The mechanisms by which phenolic compounds control K⁺ transport across the PM remain elusive. Based on the fact that removal of phenolics caused a rapid recovery of K⁺ f.lux 4.75 Crack Key For U, Glass (1974) suggested a direct effect on cell membranes. No specific details were offered though. Our data reported in this study suggests that major voltage-dependent K⁺-transporting systems may be key players. This is supported by the observed immediate membrane depolarization (Fig. 9A). Moreover, LibreOffice Crack data suggest that both increased H⁺ (Fig. 2A) and Ca²⁺ (Fig, f.lux 4.75 Crack Key For U. 3A) uptake could contribute to this depolarization as depicted in Figure 10.

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Figure 10.

A suggested model explaining short-term effects of secondary metabolites on membrane transport activity. OA, Organic acid; KIR, potassium inward-rectifying channel; KOR, potassium outward-rectifying channel; DACC, depolarization-activated Ca²⁺ channel; DPZ, depolarization of the PM. [See online article for color version of this figure.]

It is traditionally believed that most phenolic acids cross the cell membrane in an undissociated form by passive diffusion (Jackson and St. John, 1980). However, recent cloning and functional characterization of the MCT1 family of transporters suggest that uptake of both monocarboxylic acids and benzoic acid occur via the H⁺-coupled cotransport mechanism, at least in animal systems (Kido et al., 2000). We suggest that a similar scenario is also applicable to plant tissues. As undissociated phenolic acid is electrically neutral, not only will increased net H⁺ flux be generated as a result of such activity (consistent with our H⁺ flux data; Fig. 2A), but also a substantial membrane depolarization is expected (Fig. 9). Such a depolarization will affect intracellular K⁺ homeostasis by reducing K⁺ uptake via inward-rectifying K⁺ channels and enhancing K⁺ efflux via depolarization-activated outward-rectifying K⁺ channels (Maathuis and Sanders, 1996; Fig. 10), explaining the rapid shift toward net K⁺ efflux after phenolics application (Fig. 1A), f.lux 4.75 Crack Key For U. As both of these channels are TEA⁺ sensitive, inhibition of K⁺ efflux by TEA⁺ in our experiments (Fig. 7) is consistent with this model.

Several Ca²⁺-permeable channels may mediate Ca²⁺ uptake into root cells; each of these may contribute to the observed Ca²⁺ influx after phenolics application. Of special interest may be depolarization-activated Ca²⁺ channels (Thion et al., 1998; Miedema et al., 2001). These play a prominent role in signal perception and transduction in plants (Thion et al., 1998). Regardless of the type of Ca²⁺-permeable channel, increased Ca²⁺ uptake will provide a positive feedback to further depolarize the membrane potential, f.lux 4.75 Crack Key For U, amplifying the effect of phenolics on K⁺ transport (as depicted in a tentative model in Fig. 10). It also appears that NSCCs contribute partly to the K⁺ efflux, as both Gd³⁺ and La³⁺ (two known NSCC blockers; Demidchik et al., 2002) were efficient in preventing 2-hydroxybenzoic acid-induced K⁺ loss (Fig. 7B).

Phenolics: Long-Term Effects

Once inside the cell, permeated phenolic acids dissociate and acidify the cytosol (Guern et al., 1986; Ehness et al., f.lux 4.75 Crack Key For U, 1997). This will activate the PM H⁺-ATPase and increase H⁺ extrusion (Frachisse et al., 1988; Felle, 1989; Beffagna and Romani, 1991). As a result of such activation, the net H⁺ uptake observed in the 1st min after treatment with phenolics would gradually decline, and membrane potential restored. Indeed, after 24 h treatment, roots treated by each of the three phenolic acids had net H⁺ flux values not significantly (P < 0.05) different from the control (Fig. 5B), while membrane potential values were even more negative (hyperpolarized) compared with control roots (Fig. 9B), most likely the result of higher ATP hydrolytic activity in phenolic-treated roots (Fig. 9C).

Theoretically, f.lux 4.75 Crack Key For U, membrane hyperpolarization observed after long-term phenolic treatment (Fig. 9B) was expected to reverse the detrimental effects of metabolites on K⁺ transport. However, this was not the case, and K⁺ uptake after 24 h of treatment with phenolic acids was significantly (P < 0.05) lower than in the control (Fig. 5A). The answer may lie in the fact that net Ca²⁺ uptake measured soon after treatment (Fig. 3A) may result in a substantial elevation in cytosolic free Ca²⁺. Patch-clamp experiments on guard cells suggest that the inward K⁺ current is greatly reduced by elevating [Ca]_cyt to micromolar concentrations (Schroeder and Hagiwara, 1989). At the same time, outward-rectifying K⁺ channels are much less sensitive to [Ca]_cyt (Hosoi et al., 1988; Blatt and Grabov, f.lux 4.75 Crack Key For U, 1997; Grabov and Blatt, 1997). Such differential sensitivity of inward-rectifying K⁺ channels and outward-rectifying K⁺ channels to elevations in cytosolic Ca²⁺ may shift the balance in net K⁺ Magic Photo Recovery Free Download toward higher efflux, thus diminishing any beneficial effects of membrane hyperpolarization on K⁺ nutrition.

It should be also mentioned that some benzoic acid derivatives [e.g. 5-nitro-2-(3-phenylpropylamino) benzoic acid] were found to be potent inhibitors of anion channels (Roberts, 2006). Also, as a result of dissociation, a significant amount of organic anions will be accumulated in the cytosol. This accumulation might block their removal from the cytosol via anion channels (positive feedback), thus exacerbating toxicity effects. At the same time, anion channel blockage will add to the observed membrane hyperpolarization by reducing the amount of negatively charged particles leaving the cytosol.

Effects of Monocarboxylic Acids

Similar to phenolics, lipid-soluble undissociated forms of the volatile monocarboxylic acids are often regarded as the most toxic (Jackson and Taylor, 1970; Jackson and St. John, f.lux 4.75 Crack Key For U, 1980). Given that the concentration of monocarboxylic acids used in our experiment is much higher than that of the phenolic acids, the concentration of H⁺ in the cell cytosol would Malwarebytes Premium 4.4.5.130 Crack & Full Serial Key Free much higher than in phenolic acid-treated plants. This might explain the difference in membrane potential after 24 h of treatment (Fig. 9). All monocarboxylic compounds resulted in a significant (P < 0.05) net K⁺ efflux from barley internet explorer 11 Crack Key For U (Fig. f.lux 4.75 Crack Key For U of these, propionic acid caused the largest K⁺ efflux, followed by acetic acid, and formic acid the least, proportional to the amount of undissociated acid in the bath solution (Table II). This is consistent with previous reports on the adverse effects of these acids on the K⁺ uptake (Jackson and Taylor, 1970; Jackson and St. John, f.lux 4.75 Crack Key For U, 1980).

Table II.

Concentrations of undissociated monocarboxylic acids under experimental conditions

Compound	Dissociation Constant	pK	Concentration of Undissociated Compound
μm	%
Formic acid	177	3.75	1.7
Acetic acid	17.6	4.75	15.1
Propionic acid	13.4	4.87	18.9

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These authors also suggested that changes in membrane lipid composition might be responsible for the observed leak of K⁺ and Ca²⁺ from roots treated with monocarboxylic acids. However, it is highly unlikely that such a non-ion-specific change in general membrane permeability may occur almost immediately (within 1 min) after the treatment, as resolved by the MIFE system for K⁺ efflux in our experiments (Fig. 1B). Such changes in permeability are usually associated with a change in membrane lipid components (Jackson and Taylor, 1970; Glass, 1974; Jackson and St. John, 1980), the latter process most likely operating on a slower time scale. Importantly, the above changes in membrane permeability are believed to be nonspecific (Glass and Dunlop, 1974), thus, a mirror image kinetics for K⁺ efflux and Ca²⁺ uptake (according to electrochemical potential for each disk drill 2.0.0.337 activation code free Free Activators should be observed. This was obviously not the case in our study. While the K⁺ leak gradually increased with time (Fig. 1B), Ca²⁺ efflux was short lived and returned back to control values within 10 to 15 min after treatment, f.lux 4.75 Crack Key For U. This suggests that fluxes of these two ions are mediated by different transport systems, and thus cannot be attributed to a general change in membrane permeability.

A plausible alternative explanation may be offered. Similar to our model, phenolics, monocarboxylic acids are transported into the cytosol most likely in an undissociated form (Kido et al., 2000; Fig. 10). Using the H⁺-coupled cotransport mechanism, such transport through the MCT will cause the significant H⁺ influx measured in our experiments (Fig. 2B). This might depolarize the membrane (Fig. 10) and cause K⁺ efflux through depolarization-activated K⁺ channels (Fig. 2B). In addition, f.lux 4.75 Crack Key For U, it appears that at least part of the observed K⁺ efflux may be also mediated by NSCCs, as both F.lux 4.75 Crack Key For U and La³⁺ (two known NSCC blockers; Demidchik et al., 2002) were also efficient in preventing acetic acid-induced K⁺ loss (Fig. 7C).

Contrary to the effect of phenolics, monocarboxylic acids did not cause any substantial increase in Ca²⁺ uptake (Fig. 3, A and B, respectively), indicating a specificity of regulation of Ca²⁺ signaling by these secondary metabolites. The effect of specific blockers of the Ca²⁺-ATPase (CPA and thapsigargin) on acetic-induced transient Ca²⁺ efflux was rather small (Fig. 6B), suggesting a relatively minor role for the PM Ca²⁺ pump in this process and pointing toward a possible mediation of Ca²⁺ efflux by the PM Ca²⁺/H⁺ exchanger. At the same time, Ca²⁺ flux responses to monocarboxylic acids were completely blocked by either Gd³⁺ and La³⁺ (Fig. 6, B and C), as well as strongly inhibited by TEA⁺. The latter results may suggest that a substantial component of Ca²⁺ efflux may originate from the K⁺/Ca²⁺ Donnan exchange in the cell wall (Shabala and Newman, 2000), as all these inhibitors were also efficient in preventing acetic acid-induced K⁺ efflux from roots (Fig. 7C).

The above scenario is further supported by the results of long-term experiments (Fig. 5). While a substantial K⁺ leak was measured 24 h after treatment with monocarboxylic acids (Fig. 5A), no significant (P < 0.05) Ca²⁺ leak was found (Fig. 5C), f.lux 4.75 Crack Key For U. Thus, it is highly unlikely that general changes in membrane permeability were involved as was suggested by Jackson and coauthors (Jackson and Taylor, 1970; Jackson and St. John, 1980). Strong membrane depolarization (Fig. 9) also supports the idea of voltage-gated control of activity of K⁺-permeable channels by monocarboxylic acids.

In summary, this study shows that secondary metabolites associated with waterlogged soil conditions adversely affect root nutrient uptake and that the perturbation to root ionic homeostasis is much stronger in WL-sensitive genotypes. Accordingly, we suggest that tolerance to these stresses should be targeted in any program to breed crops for WL tolerance.

MATERIALS AND METHODS

Plant Material and Growth Conditions

Two barley (Hordeum vulgare) varieties, WL-sensitive ‘Naso Nijo’ and WL-tolerant ‘TX9425’ (Pang et al., 2004, 2007) were grown hydroponically for 3 to 4 d on a floating mesh in plastic containers above 0.5 L of aerated nutrient solution containing 0.1 mm CaCl₂ and 0.2 mm KCl (pH 5.5 unbuffered). Seedlings were grown under laboratory conditions (temperature + 24°C; 16 h photoperiod; fluorescent lighting about 150 μmol m⁻² s⁻¹) essentially as described by Pang et al. (2006) and used for measurement when their root length was 60 to 80 mm.

Ion Flux Measurements

Net fluxes of H⁺, Ca²⁺, and K⁺ were measured using the noninvasive Drive snapshot raid Free Activators technique (University of Tasmania, Hobart, Australia). Details on fabrication and calibration of H⁺, Ca²⁺, and K⁺ ion selective microelectrodes have been described previously (Shabala et al., 1997, 2000), f.lux 4.75 Crack Key For U. Briefly, pulled and silanized microelectrodes with tip diameters of about 3 μm were back filled with the appropriate solution (0.15 mm NaCl + 0.4 mm KH₂PO₄ adjusted to pH 6.0 using NaOH for the proton electrode; 0.5 m CaCl₂ for calcium; 0.5 m KCl for potassium). The electrode tips were then filled with ionophore cocktails (Fluka; catalog no. 95297 for H⁺; 21048 for Ca²⁺; 60031 for K⁺). Electrodes were mounted on a three-dimensional electrode holder (MMT-5, Narishige), positioned with their tips spaced 2 to 3 μm apart in line. They were calibrated in an appropriate set of standards before and after use (pH from 4.4–7.8; Ca²⁺ from 0.1–0.5 mm; K⁺ from 0.2–1 mm).

Experimental Protocol

Two major groups of organic acids, namely monocarboxylic acids and phenolic acids, were chosen for experiments (Table I and ​II). These are the most widely reported compounds associated with anaerobic soil conditions (Lynch, 1977; Tanaka et al., 1990; Armstrong and Armstrong, 2001). In water, f.lux 4.75 Crack Key For U, weak acid establishes an equilibrium between the weak acid and the conjugate base. A weaker acid has less dissociation to the conjugate base and the equilibrium favors the undissociated weak acid form.

One hour before measurement, 5 mL basic salt medium (BSM) solution (0.1 mm CaCl₂, 0.2 mm KCl, pH 5.5 unbuffered) was added to a plexiglass measuring chamber (100 mm long, 30 mm deep, and 4 mm wide). A seedling was taken from the growth container and placed immediately into the chamber. The root was immobilized in the horizontal position by fine Teflon partitions 5 mm above the floor of the chamber as described in Pang et al. (2006). The chamber was put onto the microscope stage in the Faraday cage and the plant was allowed to adapt to experimental conditions. Ion selective microelectrodes were positioned 50 μm above the root tissue in the mature zone (10 mm from the tip). During measurements electrodes moved vertically in a square-wave manner (10-s cycle; travel range 50 μm) driven by a hydraulic manipulator as described in Shabala et al. (1997).

In transient experiments, steady-state fluxes were measured for 5 min, then 5 mL of BSM solution containing a double concentration of an appropriate chemical was added into the chamber, and the measurement continued for a further 30 min. Solution pH was adjusted to 5.5 in advance using NaOH/HCl, and no substantial changes in Ca²⁺ or Mn²⁺ activity was caused by addition of any of organic f.lux 4.75 Crack Key For U. About 2 min is required for unstirred layer conditions to be reached. This period of time was discarded from the analysis and appears as a gap in the figures.

For measurement of the long-term effects of secondary metabolites on root ion fluxes and membrane potential the components studied were added to the growth plastic container (basic solution) 24 h before measurement. The final concentrations of phenolic acids (benzoic acid, 2-hydroxybenzoic acid, and 4-hydroxybenzoic acid) were 200 f.lux 4.75 Crack Key For U, volatile monocarboxylic organic acids (formic acid, acetic acid and propionic acid) were 10 mm, Mn²⁺ (added as MnSO₄ salt) was 300 mg L⁻¹; all these concentrations were selected based on previous literature reports showing they are physiologically relevant. Solution pH was adjusted to 5.5 (using HCl/NaOH) in all treatments and monitored continuously by the pH microelectrode. Solutions were aerated continuously during the 24-h treatment period.

Pharmacology

Pretreatment with inhibitors was carried out when the root was transferred to the measuring chamber. Orthovanadate (an inhibitor of P-type ATPase), TEACl (a putative K⁺ channel blocker), GdCl₃ and LaCl₃ (NSCCs blockers), and CPA and thapsigargin (specific Ca²⁺-ATPase inhibitors) were used to modify the activity of selected PM transporters. These inhibitors were mixed with the basic solution (0.2 mm KCl, 0.1 mm CaCl₂) to achieve their final concentrations that were as follows: vanadate, 1 mm; TEA⁺, f.lux 4.75 Crack Key For U, 10 mm; Gd³⁺, 50 μm; La³⁺, 200 μm; CPA, 50 μm; thapsigargin, 5 μm. After 1 h pretreatment in the appropriate inhibitor, transient ion flux responses to one of the secondary metabolites were measured, f.lux 4.75 Crack Key For U, as described above (still in the presence f.lux 4.75 Crack Key For U inhibitor in the bath solution).

Membrane Potential Measurements

The chromium os Crack Key For U of intact barley plants were mounted in a measuring chamber and the roots were gently secured in a horizontal position with small plastic blocks. Experimental conditions were the same as those for the ion flux measurement. The plant was allowed to stabilize for 60 min. Measurements of the electrical potential difference (V_m) across the root-cell membranes were made in the root mature zone, 1 to 2 cm from the root tip essentially as described by Cuin and Shabala (2005). The borosilicate glass microelectrodes (Clark Electromedical Instruments) were filled with 1 m KCl, connected to an electrometer via a Ag-AgCl half cell, and inserted into the root tissue with a manually operated micromanipulator (Narishige MMT-5).

Assay of ATPase Activity of PM Vesicles

Barley was grown in vermiculite for 7 d in the dark. The vermiculite was watered with basic nutrient solution containing 0.2 mm KCl and 0.1 mm CaCl₂ (BS). The roots were washed carefully and rinsed with BSM. Around 10 g roots (fresh weight) were taken into BS, with one of 20 mm acetic acid in BSM (pH 5.5), 200 μm 2-hydroxybenzoic acid in BSM (pH 5.50), or 300 mg/L MnSO₄ in BSM (pH 5.5) added for 30 min. Roots were then homogenized in 200 mL f.lux 4.75 Crack Key For U homogenization buffer (50 mm MOPS, 5 mm EDTA, 330 mm Suc, 0.6% polyvinylpyrrolidone, f.lux 4.75 Crack Key For U mm ascorbate, 5 mm dithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride). PM was isolated from the mirosomal fraction (30,000 g) by partitioning at 4°C at an aqueous polymer two-phase system (9 g + 3 g) composed of 6.2% Dextran D1037 (Sigma), 6.2% PEG3350 (Sigma), 330 mm Suc, 5 mm potassium phosphate pH 7.8, 3 mm KCl, 0.1 mm EDTA, and 1 mm dithiothreitol (Larsson et al., 1994). The final PM pellet was suspended in 330 mm Suc, 5 mm potassium phosphate pH 7.8, 50 mm KCl, 5 mm EDTA. Protein concentration was determined according to Bradford colorimetric assay (Bradford, 1976). ATP hydraulic activity was measured as described in Regenberg et al. (1995). The assay medium (20 mm MOPS, 8 mm MgSO₄, 50 mm KNO₃, f.lux 4.75 Crack Key For U, 5 mm NaN₃, 250 μm NaMo, 0.02% Brij58, pH was adjusted to 7.0 with KOH) included 3 mm ATP, f.lux 4.75 Crack Key For U. The reaction was initiated by the addition of 10 μL of root PMs to the assay medium and kept for 30 min by leaving at 30°C in heating block.

Acknowledgments

We are grateful to Dr. A. Fuglsang (University of Copenhagen) for her valuable advice on H⁺-ATPase hydrolytic assay experiments and Mrs. Julie Harris (University of Tasmania) for her kind assistance with using ultracentrifuge.

Notes

¹This work was supported by Grain Research and Development Corporation (M.Z. and N.M.) and Australian Research Council (S.S.) grants.

The author responsible for distribution of materials integral to f.lux 4.75 Crack Key For U findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Sergey Shabala (ua.ude.satu@alabahs.yegres).

^[C]Some figures in this article are displayed in color online but in black and white in the print edition.

www.plantphysiol.org/cgi/doi/10.1104/pp.107.102624

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See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/223500512 Photorespiratory flux and mitochondrial contribution to energy and redox balance of barley leaf protoplasts in the light and during. Article in Journal of Plant Physiology · October 2001 DOI: 10.1078/0176-1617-00551 DisplayFusion Pro Offline Installer CITATIONS READS 36 34 3 authors, including: Abir U Igamberdiev Elzbieta Romanowska Memorial University of Newfoundland University of Warsaw 219 PUBLICATIONS 3,407 CITATIONS 57 PUBLICATIONS 518 CITATIONS f.lux 4.75 Crack Key For U SEE PROFILE SEE PROFILE Some of the authors of this publication are also working on these related projects: Several research projects on the topics of plant science, bioenergetics and theoretical biology View project All content following this page was uploaded by Abir U Igamberdiev on 11 January 2017. The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the original document and are linked to publications on ResearchGate, letting you access and read them immediately. J. Plant Physiol. 158. 1325 – 1332 (2001)  Urban & Fischer Verlag http://www.urbanfischer.de/journals/jpp Photorespiratory flux and mitochondrial contribution to energy and redox balance of barley leaf protoplasts in the light and during light- dark transitions Abir U. Igamberdiev1, 2, El˙zbieta Romanowska1, 3, Per Gardeström1 * 1 Umeå Plant Science Centre, Department of Plant Physiology, Umeå University, 90187 Umeå, Sweden 2 Present address: Plant Biology and Biogeochemistry Department, f.lux 4.75 Crack Key For U, Risø National Laboratory, Building 309, P.O. Box 49, 4000 Roskilde, Denmark 3 Permanent address: Faculty of Biology, Department of Plant Physiology, University of Warsaw, 02926 Warszawa, Poland f.lux 4.75 Crack Key For U Received January 29, 2001 · Accepted May 21, 2001 Summary The contribution of mitochondrial oxidation of photorespiratory and respiratory substrates to subcel- lular energy and redox balance was investigated in leaf protoplasts of barley (Hordeum vulgare L.). The ATP/ADP ratios (indicating the energy balance) in chloroplasts and in the extrachloroplast com- partment were highest in the light in limiting CO2 (photorespiratory iphone data recovery software with crack or serial number, and they drastically increased after illumination if plants were pre-incubated in darkness for 24 hours. After illumination, the ATP/ADP ratio rapidly decreased in chloroplasts. The NADPH/NADP ratio (as an indicator of redox balance) in chloroplasts declined rapidly during the first seconds of darkness, then slowly increased. In limiting CO2, the ratio decreased more slowly during the first minute of darkness corre- sponding to post-illumination respiratory burst (PIB). During this period, the activation state of chloro- plast NADP-malate dehydrogenase was higher in limiting CO2 than in saturating CO2. However, dur- ing the light-enhanced dark respiration (LEDR) period, following PIB, there were no differences in subcellular NADPH/NADP ratios in saturating and limiting CO2. A decline in malate and citrate con- centrations in protoplasts and activation of mitochondrial NAD-malic enzyme were revealed during LEDR. The results presented highlight the importance of glycine oxidation in mitochondria in ener- gization of the cytosol and chloroplasts and in maintaining redox balance in the light and during the first minute after illumination. And further, they show non-photorespiratory origin of LEDR. Key words: Hordeum vulgare – light-dark transition – light enhanced dark respiration – mitochondria – photorespiration – post-illumination burst Abbreviations: Chl chlorophyll. – LEDR light enhanced dark respiration. – MDH malate dehydrogen- ase. – ME malic enzyme. – OAA oxaloacetate. – PIB post-illumination burst. – TCA tricarboxylic acid * E-mail corresponding author: per.gardestrom@plantphys.umu.se 0176-1617/01/158/10-1325 $ 15.00/0 1326 Abir U. Igamberdiev, El˙zbieta Romanowska, Per Gardeström Introduction f.lux 4.75 Crack Key For U driver easy 5.6.4 license key Crack Key For U f.lux 4.75 Crack Key For U Materials and Methods In the light, leaf mitochondria of C3 plants oxidize both photo- Plant material respiratory- and glycolytically-derived substrates. The f.lux 4.75 Crack Key For U Barley (Hordeum vulgare L., cv. Gunilla, Svalöf, Sweden) was grown respiratory glycolate pathway, which includes mitochondrial in soil f.lux 4.75 Crack Key For U a 17h period of artificial light of ca. 300 µmol · m – 2 · s –1 (met- glycine oxidation, constitutes a major metabolic flow in am- al-halogen lamps Powerstar HQI-T 400 W/D, OSRAM, München, Ger- bient air conditions, and oxidation of glycine is the main meta- many). The temperature was 23 ˚C during the day and 20 ˚C at night. bolic reaction in leaf mitochondria. Oxidation of glycolytic For adaptation to darkness, the plants were kept for 24 h in dark products (OAA, malate, and pyruvate), proceeding via the chambers. reactions of the TCA cycle, represents a smaller part of cellu- lar decarboxylations and respiratory O2 consumption (Atkin et al. 2000). Isolation and fractionation of protoplasts When a leaf of a C3 plant is suddenly darkened two phe- nomena can be observed with respect to cellular decarboxy- Protoplasts were isolated from 7– 8-day-old barley leaves, and rapidly fractionated at indicated time of darkness or illumination using spe- lations. The first represents the post-illumination burst (PIB) of cially constructed apparatus for membrane filtration, according to CO2, f.lux 4.75 Crack Key For U, a phenomenon originally described by Decker (1955). Gardeström and Wigge (1988). Protoplasts (40 – 60 µg Chl · mL –1) were After the PIB, a period of increased respiration follows, a phe- incubated in 7 mL assay medium (0.25 mol · L –1 sucrose, 0.25 mol · L –1 nomenon termed light-enhanced dark respiration (LEDR) f.lux 4.75 Crack Key For U f.lux 4.75 Crack Key For U sorbitol, 10 mmol · L –1 KCl, f.lux 4.75 Crack Key For U, 0.5 mmol · L –1 MgCl2, f.lux 4.75 Crack Key For U, 0.06 % (w/v) bovine (Stokes et al. 1990). The generally accepted view is that the serum albumin, 0.2 % (w/v) polyvinylpyrrolidone, 10 mmol · L –1 HEPES, burst of CO2 is linked to photorespiration (Zelitch 1992), and pH 7.2). Saturating CO2 (non-photorespiratory) conditions were pro- that the CO2 evolved originates from the mitochondrial con- vided by the addition of 10 mmol · L –1 NaHCO3. Limiting CO2 (photo- version of glycine to serine (Somerville and Ogren 1981, respiratory) conditions were obtained by addition of 0.2 mmol · L –1 bi- Rawsthorne and Hylton 1991). The involvement of glycine in carbonate. Under these conditions, the rate of photosynthesis was a post-illumination decarboxylation rise follows from its depend- half of maximal, assuming that Rubisco operates in both the carboxyl- ase and oxygenase direction. Protoplasts were irradiated at 500 µmol ence on O2/CO2 concentrations in air (Bulley and Tregunna f.lux 4.75 Crack Key For U · m – 2 · s –1 (Xenophot HLX slide projector lamp, OSRAM) for 10 1971, Vines et al. 1982, Azcón-Bieto et al. 1983). PIB consists minutes for obtaining steady-state photosynthesis (Igamberdiev et al. of the oxidation of the remnant of photorespiratory glycolate 1997). Before fractionation, 2 mL of the protoplast suspension were and glycine usually between the first 15 – 40 s after illumination transferred to the syringe and mounted on the fractionation apparatus. (Atkin et al. 2000, Hoefnagel et al. 1998) being absent in non- Protoplasts were ruptured on a selected 15 µm nylon net. Chloroplasts photorespiratory conditions (low O2 or elevated CO2) (Doeh- were separated by two (12 and 8 µm) membrane filters, as described lert et al. 1979). Introduction of glycine into leaves significantly in Wigge et al. (1993). The remaining extrachloroplast compartment increases the magnitude of PIB (Parys f.lux 4.75 Crack Key For U Romanowska contained mostly cytosol and mitochondria. Since the mitochondrial 2000). volume is low, and it contains low total amounts of NADP(H) (∼10 % of In contrast to PIB, the enhanced dark respiration following Freemake Video Converter 4.1.10.354 Crack key!! the cytosolic) and of ATP + ADP (20 – 30 % of the cytosolic) (Igamber- diev et al. 2001), the extrachloroplast values reflect with a good ap- photosynthesis is also observed in the presence of high proximation the ratios of the cytosolic compartment. After rapid frac- CO2/HCO3 – concentration that restricts photorespiration tionation, the dilution of the samples was determined by comparing (Reddy et al. 1991, Igamberdiev et al. 1997). This suggests the refractive index of samples to the refractive index of pure solu- that this phenomenon is distinct from PIB and may reflect an tions, f.lux 4.75 Crack Key For U. Cross-contamination between chloroplast and extrachloroplast increased supply of non-photorespiratory substrates (e.g. compartments was measured using marker enzymes (Gardeström malate and/or pyruvate) formed during photosynthesis (Atkin and Wigge 1988) and did not exceed 7–10 %. Total chlorophyll (Chl) et al. 2000). LEDR increased after the prolongation of the illu- concentration was measured according to Bruinsma (1961). mination period, suggesting the dependency on prior photo- synthesis (Atkin et al. 1998). CyberLink Director Suite 365 9.00 Crack With Serial Key Latest 2021 Oxidation of glycine contributes to the energy and redox Determination of ATP/ADP and NADPH/NADP ratios balance in the cell, both in the light and in darkness, imme- diately following a period of illumination. However, little is For determination of ATP/ADP and NADPH/NADP ratios each filtrate was divided into three parts. They were mixed with (1) 0.2 mol · L –1 HCl known about concomitant changes of ATP/ADP and NADPH/ + 1 % (w/v) CHAPS, f.lux 4.75 Crack Key For U, (2) 0.2 mol · L –1 KOH + 0.08 % (w/v) Triton X-100, NADP ratios during transitions between light and darkness ESSS Rocky DEM 4.4.2 Free Download with Crack and (3) 0.17 mol · L –1 sorbitol, 0.3 mmol · L –1 MgCl2, 1.7 mmol · L –1 HE- and their dependence on CO2 supply. In the present investi- f.lux 4.75 Crack Key For U PES, pH 7.0 (Wigge et al. 1993). ATP, ADP, and NADP + were deter- gation, we tested the influence of saturating and limiting CO2 mined in acid extracts (1), NADPH in alkaline extracts (2) and marker concentrations on steady state and transient energy and re- enzymes in buffer extracts (3). ATP was determined by the firefly luci- dox balance in the plant cell in order to elucidate participation ferase method (Gardeström and Wigge 1988). ADP was converted to of mitochondria in these phenomena. ATP by pyruvate kinase (Roche, Mannheim, Germany). Pyridine nu- cleotides were determined by enzymatic cycling, as described earlier (Igamberdiev et al. 2001), and quantified by fluorescence emission at  Mitochondria and energy balance of barley protoplasts 1327 460 nm with iobit uninstaller serial key Crack Key For U nm as excitation wavelength, on a FluoroMax-2 compartment was relatively unaffected by light and by CO2 spectrofluorometer (Instruments S.A., Inc., Edison, NJ, USA). concentration; in chloroplasts it was about three times higher in light than in darkness (Table 1). Determinations of malate and citrate Concentrations of malate and citrate in protoplasts were determined Effects of illumination after prolonged darkness on spectrofluorimetrically in perchloric acid extracts after neutralization, ATP/ADP ratios as described in Bergmeyer (1973). For citrate determination, coupling Illumination by saturating light of the protoplasts from plants with citrate lyase (Sigma) and malate dehydrogenase (Roche) was adapted to darkness during 24 hours led to a drastic increase performed. For malate determination, malate dehydrogenase and as- in the ATP/ADP ratio in photorespiratory conditions, especially partate aminotransferase (Roche) in the presence of glutamate were applied. The formation of NADH was quantified on a FluoroMax-2 in chloroplasts (Fig. 1). After 10 min of illumination, the ratio in- spectrofluorometer as described above. digidna imazing 2.4.7 Crack Key For U creased from 0.9 to more than 10. When the light was turned off, the ratio returned to the initial value after 5 min of dark- ness. In saturating CO2 illumination did not essentially change Enzyme assays ATP/ADP ratio in chloroplasts after 10 min of illumination. In the f.lux 4.75 Crack Key For U extrachloroplast fraction of protoplasts from plants adapted to NADP-malate dehydrogenase (NADP-MDH; EC 1.1.1.82) activation darkness, the ATP/ADP ratio was about 2.7. Ten minutes of illu- state was measured essentially according to Scheibe and Stitt (1988) as described earlier (Igamberdiev et al. 1998). The data was cor- mination increased this ratio to Microsoft Office 2020 Crack + Product Key Free Download in photorespiratory goldwave 32 bit full crack Crack Key For U to 6 rected in accordance with the rate of NAD-MDH (EC 1.1.1.37) activity, f.lux 4.75 Crack Key For U, in non-photorespiratory conditions. After 5 min following dark- 1/500 of which corresponds to its activity with NADPH as a cofactor ness the ATP/ADP ratio decreased but did not reach initial low (Scheibe and Stitt 1988). f.lux 4.75 Crack Key For U value. For determination of the NAD-malic enzyme (NAD-ME; EC 1.1.1.39) When barley plants were in the dark for 24 hours and de- activity, f.lux 4.75 Crack Key For U, protoplasts were fixed in 100 mmol · L –1 HEPES-KOH, pH 6.8, tached leaves were illuminated for one hour, the total ATP/ 5 mmol · L –1 EDTA, 10 mmol · L –1 ditiothreitol, 5 mg · mL –1 bovine serum ADP ratio in protoplasts isolated from these leaves was 2.7 albumin, 0.4 mmol · L –1 phenylmethylsulfonyl fluoride, and 1% (w/v) Tri- (Table 2). Introduction of 50 mmol · L –1 glycine during illumina- ton X-100. The activity was measured essentially according to Cook et tion of leaves increased this ratio nearly twice. The addition of al. (1995) immediately after thawing of protoplasts in 30 mmol · L –1 HE- oligomycin (in the concentration 0.1 µmol · L –1), which inhibits PES-KOH, pH 6.8, 1 mmol · L –1 EDTA, 2 mmol · L –1 ditiothreitol, 5 mmol · L –1 malate, and 3 mmol · L –1 NAD +. After reaching equilibrium by NAD-MDH (2 min), the assay was initiated by the addition of 5 mmol · L –1 MnCl2. In order to calculate the activity state, we quanti- fied the lag-phase in development of its activity by denoting the extrap- olated value of the linear part of the progress curves of product forma- tion (τ) recorded by spectrophotometer, as described in Dixon and Webb (1979, p. 456). The activity state of NAD-ME was determined as the ratio of apparent rate constant of the enzyme (which is in reverse proportionality to the time τ) at given time of f.lux 4.75 Crack Key For U or illumination to the apparent rate constant of the activated enzyme (in the pres- ence of CoA) (Dixon and Webb 1979, pp. 454 – 460). Cook et al. (1995) determined the activity state of NAD-ME as the ratio of the rate 4 min after the initiation of the assay to that after 1 min, which reflects the similar tendency. Results Subcellular ATP/ADP and NADPH/NADP ratios in darkness and during illumination in photorespiratory and non-photorespiratory conditions Determination of ATP/ADP and NADPH/NADP ratios in dark- ness revealed that they are independent of CO2 concentration in the medium (Table 1). Both ratios were low in chloroplasts and essentially higher in the extrachloroplast compartment. In Figure 1. Changes in chloroplast and extrachloroplast ATP/ADP ratios continuous light, both f.lux 4.75 Crack Key For U and extrachloroplastic in high and low CO2 in barley protoplasts from plants darkened 24 ATP/ADP ratios were higher, particularly under photorespira- hours, illuminated 10 min after darkness and darkened 5 min after tory conditions. The NADPH/NADP ratio in extrachloroplastic 10 min illumination. The results are means and SD from 3 replicates. 1328 Abir U. Igamberdiev, El˙zbieta Romanowska, Per Gardeström Table 1. Subcellular ATP/ADP and NADPH/NADP ratios in continuous light (500 µmol quanta · m – 2 · s –1, 30 min) and prolonged darkness (30 min) in barley leaf protoplasts. The results are means and SD from 4 – 6 replicates. Compartment ATP/ADP NADPH/NADP Dark Dark Light f.lux 4.75 Crack Key For U Light Dark Dark Light Light High Low High Low High Low High Low CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 Chloroplast 0.4 ± 0.2 f.lux 4.75 Crack Key For U 0.5 ± 0.2 2.1 ± 0.5 3.1 ± 0.6 0.3 ± 0.1 0.4 ± 0.2 0.9 ± 0.3 1.1 ± 0.4 Extrachloroplast 2.2 ± 0.4 2.4 ± 0.3 4.2 ± 0.7 7±1 1.1 ± 0.4 0.8 ± 0.3 1.4 ± 0.2 1.3 ± 0.3 Malate utilizing enzymes Table 2. ATP/ADP ratios in illuminated (10 min) protoplasts isolated from barley leaves (darkened for 24 hours and illuminated for 1 hour) The activation state of Makemkv registration code reddit Crack Key For U increased very f.lux 4.75 Crack Key For U fed by water (control) or gylcine (50 mmol · L –1). Oligomycin (1 upon irradiation (data not shown), an increase similar to that µmol · L –1) was added 3 min prior illumination of protoplasts. The re- reported earlier (Igamberdiev et al. 1998). The final activity of sults are means and SD from 3 replicates. the enzyme after activation was 107 ± 14 µmol · h –1 · mg –1 Chl (n = 7). NAD-MDH activity was 4230 ± 450 µmol · h –1 · mg –1 Chl Condition capture one express ATP/ADP ratio (n = 8). The «crude» NADP-MDH activity found in extracts of Water Hotspot Shield Elite 10.14.3 Crack With Keygen Number Free 2021 2.67 ± 0.31 darkened protoplasts was very low, and generally did not ex- Glycine 4.75 ± 0.27 ceed the side activity of NAD-MDH with NADPH as a cofactor. Glycine + oligomycin 2.96 ± 0.12 In darkness after illumination, the activation state of NADP- f.lux 4.75 Crack Key For U MDH decreased during the first two min to 20 – 25 % and then slowly (ten minutes) decreased (Fig. 4), being zero in dark- mitochondrial ATP synthesis and does not affect chloroplastic ness. At limiting CO2 (in photorespiratory conditions) activa- ATPase (Krömer et al. 1988, Igamberdiev et al. 1998), to the tion state of NADP-MDH was somewhat higher and de- protoplasts of glycine-fed leaves, 3 min prior to illumination, decreased the ATP/ADP ratio almost to the control value. f.lux 4.75 Crack Key For U Adenylate and pyridine nucleotide levels after illumination The ATP/ADP ratio in chloroplasts decreased drastically im- mediately after illumination to 0.2 – 0.4, f.lux 4.75 Crack Key For U, without significant dif- ference between saturating and limiting CO2 conditions, and then revealed a tendency of some increase (Fig. 2). After pro- longed darkness it was around 0.5 (Table 1). The NADPH/ NADP ratio also decreased drastically in chloroplasts after il- lumination (Fig. 3), f.lux 4.75 Crack Key For U. In saturating CO2 it dropped to zero, in limiting CO2 it decreased to 0.2, then started to rise reaching 0.3 – 0.4 after 5 min, both in saturating and limiting CO2. In prolonged darkness, it was about 0.3 – 0.4 (Table 1). In extrachloroplast compartment in the light, the ATP/ADP ratio was much higher in limiting CO2 conditions than in satu- rating CO2 (Fig. 2). In prolonged darkness, the difference be- tween saturating and limiting CO2 disappeared and the ratio stabilized at about 2 (Table 1, Fig. 2). The NADPH/NADP ratio dropped during the first minute of darkness, which was more pronounced in saturating CO2. In limiting CO2, it remained higher during the first minute of darkness corresponding to Figure 2. Changes in chloroplast and extrachloroplast ATP/ADP ratios the PIB period (Fig. 3). During the prolonged darkness it was in barley protoplasts in high and low CO2 after illumination, f.lux 4.75 Crack Key For U. The results about 1 (Table 1). are means and SD from 4 replicates.  Mitochondria and energy balance of barley protoplasts 1329 (Fig. 5 B). The activity of NAD-ME after lag-phase was 14 ± 3 µmol · h –1 · mg –1 Chl (n = 5). Malate and citrate contents Determination of malate and citrate after illumination showed that these metabolites revealed similar pictures of their chang- es after illumination (Fig. 6), f.lux 4.75 Crack Key For U. The concentrations exhibited easeus data recovery wizard license key generator mac some increase during f.lux 4.75 Crack Key For U first minute of darkness in limiting CO2 conditions and then decreased. In saturating CO2, dur- ing the first minute of darkness the decrease was not ex- pressed, rather a steady level remained, followed by a de- crease similar to limiting CO2 conditions. Discussion Mitochondria regulate energy balance of plant cell in the light In the light, mitochondria of C3 plant leaves oxidize both pho- torespiratory substrate (glycine) and respiratory substrates, Figure 3. Changes in chloroplast and extrachloroplast NADPH/NADP ratios in barley protoplasts in high and low CO2 after illumination. The results are means and SD from 4 replicates. Figure 4. Activation state of NADP-malate dehydrogenase after illumi- nation in high and low CO2. The results are means and SD from 3 re- plicates. creased more slowly after illumination, f.lux 4.75 Crack Key For U, which was mostly pro- nounced during the first minute after light was turned off, i.e. in the period of PIB. NAD-ME, contrary to NADP-MDH, revealed increased acti- Figure 5. A – Progress curves of NAD-malic enzyme spectrophoto- metric assays. The samples were taken from barley leaf protoplasts vation after illumination, especially 1– 2 min after light was magix movie edit pro 2018 + crack full version Activators Patch during and after illumination in high CO2. B – calculated activity state turned off, i.e, f.lux 4.75 Crack Key For U. during LEDR, which was reflected mainly in the f.lux 4.75 Crack Key For U of NAD-malic enzyme in the course of dark-light-dark transition in high decrease of lag-phase during measurement of its activity and low CO2. The results are given from one experiment and similar without CoA (Fig. 5 A). The most inactive state of malic en- results were obtained in two other independent experiments. 1 – dark- zyme was observed in continuous darkness; during illumina- ness, 2 – light (6 min), 3 – light (10 min), 4 – after illumination (1min), 5 tion its activity state was higher, and it was the highest during DiskInternals Raid Recovery Registration key – after illumination (2 min), 6 – after illumination (10 min), 7 – activity the period after illumination, after which it gradually declined with CoA. 1330 Abir U. Igamberdiev, El˙zbieta Romanowska, Per Gardeström very high chloroplastic ATP/ADP ratios in the light (Igamber- diev et al. 2001). Glycine oxidation and post-illumination phenomena f.lux 4.75 Crack Key For U Post-illumination burst (PIB) of CO2 occurs within the first min- ute after illumination, while light-enhanced dark respiration f.lux 4.75 Crack Key For U (LEDR) appears later (after 1 min) and continues for several minutes (Atkin et al. 2000). Thus, we can attribute the phe- f.lux 4.75 Crack Key For U nomena of the first minute after illumination to PIB, f.lux 4.75 Crack Key For U, and those appearing after 1 minute to LEDR. The characteristics of energy and redox balance during the first minute after illumi- nation include higher activation levels of chloroplast NADP- glary utilities pro lifetime key Crack Key For U MDH in limiting CO2 (Fig. 4) and higher NADPH/NADP ratio in f.lux 4.75 Crack Key For U LizardSystems WiFi Scanner For Windows, which does not drop to zero as in saturating CO2 (Fig. 3). The correspondence of chloroplast redox balance f.lux 4.75 Crack Key For U f.lux 4.75 Crack Key For U f.lux 4.75 Crack Key For U (NADPH/NADP ratio) to the activation state of NADP-MDH f.lux 4.75 Crack Key For U f.lux 4.75 Crack Key For U was shown by Foyer et al. (1992), f.lux 4.75 Crack Key For U. The data obtained indi- Figure 6. Changes of malate and citrate concentrations in barley pro- cates that the PIB can maintain some reduction level in the toplasts in high and low CO2 after illumination. The results are means chloroplast stroma during a quick light-dark transition. and SD from 3 – 4 replicates. f.lux 4.75 Crack Key For U In photorespiratory conditions, the drop in chloroplastic ATP/ADP ratio occurs from a higher initial level; however, the f.lux 4.75 Crack Key For U god of war 4 ps4 download Free Activators further development of the ratio is similar in saturating and lim- mainly glycolytically-derived (OAA, malate, and/or pyruvate). iting CO2 (Fig. 2). This is different from the changes in NADPH/ At limiting CO2, glycine becomes the main substrate for mito- NADP ratio, which is maintained at a higher reduction level in chondria, providing ATP in the cytosol for sucrose biosynthe- chloroplast and extrachloroplast compartments during the sis (Gardeström and Wigge 1988, Krömer et al. 1993) and first minute after illumination in limiting CO2, i.e., during PIB, NADH for hydroxypyruvate and/or nitrate reduction (Krömer f.lux 4.75 Crack Key For U as compared to saturating CO2 (Fig. 3). After illumination, a 1995). Refixation of photorespiratory ammonia in chloroplasts f.lux 4.75 Crack Key For U higher reduction level of the chloroplast stroma in limiting CO2 lowers their reduction level and thus prevents photoinhibition f.lux 4.75 Crack Key For U during PIB may be explained by transport of reducing power of photosynthesis (Wingler et al. 2000) and needs exporting from the oxidation of remaining photorespiratory glycine to of citrate/2-oxoglutarate from mitochondria provided f.lux 4.75 Crack Key For U oxi- the cytosol and chloroplast. dation of glycolytic substrates (Atkin et al. 2000). Another aspect of the influence of PIB is changes in pro- The increase in extrachloroplastic ATP/ADP ratio is notable f.lux 4.75 Crack Key For U files of metabolites in protoplasts. Very big vacuolar pools of in photorespiratory conditions (Table 1, Figs. 1 and 2), indica- malate and citrate (Rentsch and Martinola 1991, Gout et al. ting that ATP is formed by glycine oxidation in mitochondria 1993) make it complicated to investigate subcellular distribu- and then exported to the cytosol. In the present investigation, tion of these compounds. However, it is clear from the ob- this was confirmed by the fact that the ATP/ADP ratio in barley tained data that some changes during the first minute were leaves was increased twice when leaves were infiltrated with dependent on CO2 concentration, particularly in concentra- exogenous glycine (Table 2). The lack of this effect in the ESET Smart Security 14.1.20 Crack + Premium License Key (2021) tion of citrate, which rose in limiting CO2 and revealed no rise presence of oligomycin, in a concentration, which suppres- f.lux 4.75 Crack Key For U in saturating CO2, then (in the period corresponding LEDR) ses mitochondrial ATP formation, supports the role of photo- profiles were quite similar (Fig. f.lux 4.75 Crack Key For U. This can be connected to respiratory glycine in energization of cytosol. the efflux of malate and citrate from mitochondria oxidizing Plants in the dark for 24 hours were very susceptible to glycine for transfer of reductant, while in the absence of glyc- over-energization if protoplasts (isolated in darkness) were in- f.lux 4.75 Crack Key For U ine malate and citrate are readily oxidized in mitochondria. cubated in photorespiratory conditions (Fig. 1). Drastic in- crease in the chloroplastic ATP/ADP ratio shows that photo- respiration did not balance this process, as if plants were Non-photorespiratory origin of light-enhanced dark adapted to light. The mechanisms balancing energy and re- respiration dox state in chloroplasts, and thus preventing photoinhibition, are probably insufficient in dark-adapted plants. This may be When the photorespiratory glycine pool is exhausted, there is explained by a lowered level of photorespiratory enzymes af- no need to reduce hydroxypyruvate, and nitrate reduction is ter a period of prolonged darkness (Zhong et al. 1994). A sim- also rapidly suppressed in darkness (Riens and Heldt 1992), ilar picture was observed for a glycine decarboxylase-defi- thus decreasing the cytosolic/peroxisomal utilization of NADH. cient photorespiratory mutant in barley, which also showed Under these conditions, a temporary increase in cytosolic  Mitochondria and energy balance of barley protoplasts 1331 f.lux 4.75 Crack Key For U NADH may support OAA conversion to malate, which then will Atkin OK, Millar AH, Gardeström P, Day DA (2000) Photosynthesis, be the main glycolytically-derived substrate for mitochondria carbohydrate metabolism and respiration in leaves of higher compared to OAA during steady-state photosynthesis (Atkin plants. In: Leegood RC, Sharkey TT, von Caemmerer S (eds) Pho- tosynthesis: Physiology and metabolism, f.lux 4.75 Crack Key For U. Kluwer Academic Pub- et al. 2000). lishers, f.lux 4.75 Crack Key For U, Dordrecht, pp 153–175 Our results support the idea that NAD-malic enzyme is Azcón-Bieto J, Lambers H, f.lux 4.75 Crack Key For U, Day DA (1983) Effect of photosynthesis more active during LEDR than in the light, and especially in and carbohydrate status on respiration rates and the involvement prolonged darkness (Fig. 5). Thereby, malate can be both f.lux 4.75 Crack Key For U of the alternative pathway in leaf respiration. Plant Physiol 72: 598 – produced and oxidized within mitochondria (Hill and Bryce 603 1992, Atkin et al. 2000). As a result, concentration of malate in Bergmeyer HU (ed) (1973) Methods of enzymatic analysis. Academic darkness decreases (Hampp et al. 1984, Heineke et al. 1991, Press, New York Hill and Bryce 1992). Citrate produced by mitochondria may Bruinsma J (1961) A comment on the spectrophotometric determina- be utilized in the TCA cycle since the reduction level in mito- tion of chlorophyll. Biochim Biophys Acta 52: 576 – 578 chondria will drop when glycine oxidation stops, thereby al- Bulley Izotope Ozone Free Activate, Tregunna EB (1971) Photorespiration and the post-illumina- lowing the isocitrate dehydrogenase system in mitochondria tion burst. Can J Bot 49: 1277–1284 to operate at a high rate without efflux of citrate. As a result, Cook RM, f.lux 4.75 Crack Key For U, Lindsay JG, Wilkins MB, Nimmo HG (1995) Decarboxylation concentrations of malate and citrate decrease during LEDR of malate in the crassulacean acid metabolism plant Bryophyllum (Fig. 6) f.lux 4.75 Crack Key For U (Kalanchoë) fedtschenkoi. Role of NAD-malic enzyme. Plant Phys- iol 109: 1301–1307 The importance of malate as a substrate during the period Decker IP iris blue light filter crack Crack Key For U A rapid, postillumination deceleration of respiration of LEDR requires an increase in NAD-malic enzyme activity in green leaves. Plant Physiol 30: 82 – 84 during light/dark transition (Hill and Bryce 1992). Malate was Dixon M, Webb EC (1979) Enzymes, 3rd ed. Longman, London shown to exhibit an increased level during f.lux 4.75 Crack Key For U Doehlert DC, Ku MSB, Edwards GE (1979) Dependence of the post- (Gerhardt et al. 1987, Heineke et al. 1991). The activity state of illumination burst of CO2 on temperature, light, CO2 and O2 con- NAD-malic enzyme may be caused by aggregation/de-ag- centration in wheat (Triticum aestivum). Physiol Plant 46: 299 – 306 gregation of its subunits (Artus and Edwards 1985, Cook et al. Foyer CH, Lelandais M, Harbinson J (1992) Control of the quantum ef- 1995, Atkin et al. 2000). The dynamics of total citrate and ma- ficiencies of photosystems I and II, electron flow, and enzyme acti- late contents suggest that oxidation of malate and (iso)citrate vation following dark-to-light transitions in pea leaves. Relationship may be involved in dark energization of protoplasts Compression and Backup between NADP/NADPH ratios and MADP-malate dehydrogenase LEDR. activation state. Plant Physiol 99: 979 – 986 Gardeström P, Wigge B (1988) Influence of photorespiration on ATP/ f.lux 4.75 Crack Key For U ADP ratios in the chloroplast, mitochondria and cytosol, studied by rapid fractionation of barley (Hordeum vulgare) protoplasts. Plant Conclusions Physiol 88: 69–76 f.lux 4.75 Crack Key For U Gerhardt R, Stitt M, Heldt HW (1987) Subcellular metabolite levels in Mitochondria regulate energy and redox balance in photo- f.lux 4.75 Crack Key For U spinach leaves – regulation of sucrose synthesis during diurnal al- synthetic cells of C3 plants in the light. Oxidation of photore- terations in photosynthetic partitioning. Plant Physiol 83: 399 – 407 spiratory substrate (glycine) supplies cytosol with ATP, reduc- Gout E, Bligny R, Pascal N, Douce R (1993) 13C nuclear magnetic res- tant and after illumination prevents immediate drop in chloro- onance studies of malate and citrate synthesis and compartmenta- plastic and cytosolic NADPH/NADP ratios, maintaining NADP- f.lux 4.75 Crack Key For U tion in higher plant cells. J Biol Chem 268: 3986 – 3992 MDH in a partly activated state. The prolonged period of in- Hampp R, Goller M, Füllgraf H (1984) Determination of compart- creased respiration after photosynthesis (LEDR), followed af- mented metabolite pools by a combination of rapid fractionation in ter oxidation of the remnant of photorespiratory glycine (PIB), oat mesophyll protoplasts and enzymic cycling. Plant Physiol 75: is linked to oxidation of non-photorespiratory TCA cycle sub- 1017–1024 strates (mainly malate), f.lux 4.75 Crack Key For U, the concentration of which decreases Heineke D, Riens B, Grosse H, Hoferichter P, Peter U, Flugge UI, and stabilizes at a lower level in darkness as compared to dvdfab player 5 review Crack Key For U Heldt HW (1991) Redox transfer across the inner chloroplast enve- lope membrane, f.lux 4.75 Crack Key For U. Plant Physiol 95: 1131–1137 light. Hill SA, Bryce JH (1992) Malate metabolism and light-enhanced dark Acknowledgements. This work was supported by the grants from the f.lux 4.75 Crack Key For U respiration in barley mesophyll protoplasts. In: Lambers H, van der Swedish Royal Academy and the Swedish Natural Research Council. f.lux 4.75 Crack Key For U goldwave app Crack Key For U Plas LHW (eds) Molecular, biochemical and physiological aspects is foobar good Crack Key For U of plant respiration. SPB Academic Publishing, The Hague, pp f.lux 4.75 Crack Key For U 221– 230 References Hoefnagel MHN, Atkin OK, Wiskich JT (1998) Interdependence be- f.lux 4.75 Crack Key For U tween chloroplasts and mitochondria in the light and the dark. Bio- Artus NN, Edwards GE (1985) NAD-malic enzyme from plants. FEBS chim Biophys Acta 1366: 235 – 255 Lett 182: 225 – 233 Igamberdiev AU, Bykova NV, Lea PJ, Gardeström P (2001) The role of Atkin OK, Evans JR, Siebke K (1998) Relationship between the inhibi- photorespiration in redox and energy balance of photosynthetic tion of leaf respiration by light and enhancement of leaf dark respi- plant cells: A study with a barley mutant deficient in glycine de- ration following light treatment. Aust J Plant Physiol 25: 437– 443 carboxylase. Physiol Plant 111: 427– 438  1332 Abir U. Igamberdiev, El˙zbieta Romanowska, Per Gardeström Igamberdiev AU, Hurry V, Krömer S, Gardeström P (1998) The role of Riens B, Heldt HW (1992) Decrease of nitrate reductase activity in mitochondrial electron transport during photosynthetic induction. A spinach leaves during a light-dark transition. Plant Physiol 98: 573 – study with barley (Hordeum vulgare) protoplasts incubated with ro- 577 tenone and oligomycin. Physiol Plant 104: 431– 439 Scheibe R, Stitt M (1988) Comparison of NADP-malate dehydrogen- Igamberdiev AU, Zhou G, Malmberg G, Gardeström P (1997) Respira- ase activation, QA reduction and O2 evolution in spinach leaves. tion in barley protoplasts before and after illumination. Physiol Plant Plant Physiol Biochem 26: 473 – 481 99: 15 – 22 f.lux 4.75 Crack Key For U Somerville CR, Ogren WL (1981) Photorespiration-deficient mutants of Krömer S (1995) Respiration during photosynthesis. 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See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/223500512 Photorespiratory flux and mitochondrial contribution to energy and redox balance of barley leaf protoplasts in the light and during... Article in Journal of Plant Physiology · October 2001 DOI: 10.1078/0176-1617-00551 CITATIONS READS 36 34 3 authors, including: Abir U Igamberdiev Elzbieta Romanowska Memorial University of Newfoundland University of Warsaw 219 PUBLICATIONS 3,407 CITATIONS 57 PUBLICATIONS 518 CITATIONS SEE PROFILE SEE PROFILE Some of the authors of this publication are also working on these related projects: Several research projects on the topics of plant science, bioenergetics and theoretical biology View project All content following this page was uploaded by Abir U Igamberdiev on 11 January 2017. The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the original document and are linked to publications on ResearchGate, letting you access and read them immediately. J. Plant Physiol. 158. 1325 – 1332 (2001)  Urban & Fischer Verlag http://www.urbanfischer.de/journals/jpp Photorespiratory flux and mitochondrial contribution to energy and redox balance of barley leaf protoplasts in the light and during light- dark transitions Abir U. Igamberdiev1, 2, El˙zbieta Romanowska1, 3, Per Gardeström1 * 1 Umeå Plant Science Centre, Department of Plant Physiology, Umeå University, 90187 Umeå, Sweden 2 Present address: Plant Biology and Biogeochemistry Department, Risø National Laboratory, Building 309, P.O. Box 49, 4000 Roskilde, Denmark 3 Permanent address: Faculty of Biology, Department of Plant Physiology, University of Warsaw, 02926 Warszawa, Poland Received January 29, 2001 · Accepted May 21, 2001 Summary The contribution of mitochondrial oxidation of photorespiratory and respiratory substrates to subcel- lular energy and redox balance was investigated in leaf protoplasts of barley (Hordeum vulgare L.). The ATP/ADP ratios (indicating the energy balance) in chloroplasts and in the extrachloroplast com- partment were highest in the light in limiting CO2 (photorespiratory conditions), and they drastically increased after illumination if plants were pre-incubated in darkness for 24 hours. After illumination, the ATP/ADP ratio rapidly decreased in chloroplasts. The NADPH/NADP ratio (as an indicator of redox balance) in chloroplasts declined rapidly during the first seconds of darkness, then slowly increased. In limiting CO2, the ratio decreased more slowly during the first minute of darkness corre- sponding to post-illumination respiratory burst (PIB). During this period, the activation state of chloro- plast NADP-malate dehydrogenase was higher in limiting CO2 than in saturating CO2. However, dur- ing the light-enhanced dark respiration (LEDR) period, following PIB, there were no differences in subcellular NADPH/NADP ratios in saturating and limiting CO2. A decline in malate and citrate con- centrations in protoplasts and activation of mitochondrial NAD-malic enzyme were revealed during LEDR. The results presented highlight the importance of glycine oxidation in mitochondria in ener- gization of the cytosol and chloroplasts and in maintaining redox balance in the light and during the first minute after illumination. And further, they show non-photorespiratory origin of LEDR. Key words: Hordeum vulgare – light-dark transition – light enhanced dark respiration – mitochondria – photorespiration – post-illumination burst Abbreviations: Chl chlorophyll. – LEDR light enhanced dark respiration. – MDH malate dehydrogen- ase. – ME malic enzyme. – OAA oxaloacetate. – PIB post-illumination burst. – TCA tricarboxylic acid * E-mail corresponding author: per.gardestrom@plantphys.umu.se 0176-1617/01/158/10-1325 $ 15.00/0 1326 Abir U. Igamberdiev, El˙zbieta Romanowska, Per Gardeström Introduction Materials and Methods In the light, leaf mitochondria of C3 plants oxidize both photo- Plant material respiratory- and glycolytically-derived substrates. The photo- Barley (Hordeum vulgare L., cv. Gunilla, Svalöf, Sweden) was grown respiratory glycolate pathway, which includes mitochondrial in soil with a 17h period of artificial light of ca. 300 µmol · m – 2 · s –1 (met- glycine oxidation, constitutes a major metabolic flow in am- al-halogen lamps Powerstar HQI-T 400 W/D, OSRAM, München, Ger- bient air conditions, and oxidation of glycine is the main meta- many). The temperature was 23 ˚C during the day and 20 ˚C at night. bolic reaction in leaf mitochondria. Oxidation of glycolytic For adaptation to darkness, the plants were kept for 24 h in dark products (OAA, malate, and pyruvate), proceeding via the chambers. reactions of the TCA cycle, represents a smaller part of cellu- lar decarboxylations and respiratory O2 consumption (Atkin et al. 2000). Isolation and fractionation of protoplasts When a leaf of a C3 plant is suddenly darkened two phe- nomena can be observed with respect to cellular decarboxy- Protoplasts were isolated from 7– 8-day-old barley leaves, and rapidly fractionated at indicated time of darkness or illumination using spe- lations. The first represents the post-illumination burst (PIB) of cially constructed apparatus for membrane filtration, according to CO2, a phenomenon originally described by Decker (1955). Gardeström and Wigge (1988). Protoplasts (40 – 60 µg Chl · mL –1) were After the PIB, a period of increased respiration follows, a phe- incubated in 7 mL assay medium (0.25 mol · L –1 sucrose, 0.25 mol · L –1 nomenon termed light-enhanced dark respiration (LEDR) sorbitol, 10 mmol · L –1 KCl, 0.5 mmol · L –1 MgCl2, 0.06 % (w/v) bovine (Stokes et al. 1990). The generally accepted view is that the serum albumin, 0.2 % (w/v) polyvinylpyrrolidone, 10 mmol · L –1 HEPES, burst of CO2 is linked to photorespiration (Zelitch 1992), and pH 7.2). Saturating CO2 (non-photorespiratory) conditions were pro- that the CO2 evolved originates from the mitochondrial con- vided by the addition of 10 mmol · L –1 NaHCO3. Limiting CO2 (photo- version of glycine to serine (Somerville and Ogren 1981, respiratory) conditions were obtained by addition of 0.2 mmol · L –1 bi- Rawsthorne and Hylton 1991). The involvement of glycine in carbonate. Under these conditions, the rate of photosynthesis was a post-illumination decarboxylation rise follows from its depend- half of maximal, assuming that Rubisco operates in both the carboxyl- ase and oxygenase direction. Protoplasts were irradiated at 500 µmol ence on O2/CO2 concentrations in air (Bulley and Tregunna quanta · m – 2 · s –1 (Xenophot HLX slide projector lamp, OSRAM) for 10 1971, Vines et al. 1982, Azcón-Bieto et al. 1983). PIB consists minutes for obtaining steady-state photosynthesis (Igamberdiev et al. of the oxidation of the remnant of photorespiratory glycolate 1997). Before fractionation, 2 mL of the protoplast suspension were and glycine usually between the first 15 – 40 s after illumination transferred to the syringe and mounted on the fractionation apparatus. (Atkin et al. 2000, Hoefnagel et al. 1998) being absent in non- Protoplasts were ruptured on a selected 15 µm nylon net. Chloroplasts photorespiratory conditions (low O2 or elevated CO2) (Doeh- were separated by two (12 and 8 µm) membrane filters, as described lert et al. 1979). Introduction of glycine into leaves significantly in Wigge et al. (1993). The remaining extrachloroplast compartment increases the magnitude of PIB (Parys and Romanowska contained mostly cytosol and mitochondria. Since the mitochondrial 2000). volume is low, and it contains low total amounts of NADP(H) (∼10 % of In contrast to PIB, the enhanced dark respiration following the cytosolic) and of ATP + ADP (20 – 30 % of the cytosolic) (Igamber- diev et al. 2001), the extrachloroplast values reflect with a good ap- photosynthesis is also observed in the presence of high proximation the ratios of the cytosolic compartment. After rapid frac- CO2/HCO3 – concentration that restricts photorespiration tionation, the dilution of the samples was determined by comparing (Reddy et al. 1991, Igamberdiev et al. 1997). This suggests the refractive index of samples to the refractive index of pure solu- that this phenomenon is distinct from PIB and may reflect an tions. Cross-contamination between chloroplast and extrachloroplast increased supply of non-photorespiratory substrates (e.g. compartments was measured using marker enzymes (Gardeström malate and/or pyruvate) formed during photosynthesis (Atkin and Wigge 1988) and did not exceed 7–10 %. Total chlorophyll (Chl) et al. 2000). LEDR increased after the prolongation of the illu- concentration was measured according to Bruinsma (1961). mination period, suggesting the dependency on prior photo- synthesis (Atkin et al. 1998). Oxidation of glycine contributes to the energy and redox Determination of ATP/ADP and NADPH/NADP ratios balance in the cell, both in the light and in darkness, imme- diately following a period of illumination. However, little is For determination of ATP/ADP and NADPH/NADP ratios each filtrate was divided into three parts. They were mixed with (1) 0.2 mol · L –1 HCl known about concomitant changes of ATP/ADP and NADPH/ + 1 % (w/v) CHAPS, (2) 0.2 mol · L –1 KOH + 0.08 % (w/v) Triton X-100, NADP ratios during transitions between light and darkness and (3) 0.17 mol · L –1 sorbitol, 0.3 mmol · L –1 MgCl2, 1.7 mmol · L –1 HE- and their dependence on CO2 supply. In the present investi- PES, pH 7.0 (Wigge et al. 1993). ATP, ADP, and NADP + were deter- gation, we tested the influence of saturating and limiting CO2 mined in acid extracts (1), NADPH in alkaline extracts (2) and marker concentrations on steady state and transient energy and re- enzymes in buffer extracts (3). ATP was determined by the firefly luci- dox balance in the plant cell in order to elucidate participation ferase method (Gardeström and Wigge 1988). ADP was converted to of mitochondria in these phenomena. ATP by pyruvate kinase (Roche, Mannheim, Germany). Pyridine nu- cleotides were determined by enzymatic cycling, as described earlier (Igamberdiev et al. 2001), and quantified by fluorescence emission at  Mitochondria and energy balance of barley protoplasts 1327 460 nm with 365 nm as excitation wavelength, on a FluoroMax-2 compartment was relatively unaffected by light and by CO2 spectrofluorometer (Instruments S.A., Inc., Edison, NJ, USA). concentration; in chloroplasts it was about three times higher in light than in darkness (Table 1). Determinations of malate and citrate Concentrations of malate and citrate in protoplasts were determined Effects of illumination after prolonged darkness on spectrofluorimetrically in perchloric acid extracts after neutralization, ATP/ADP ratios as described in Bergmeyer (1973). For citrate determination, coupling Illumination by saturating light of the protoplasts from plants with citrate lyase (Sigma) and malate dehydrogenase (Roche) was adapted to darkness during 24 hours led to a drastic increase performed. For malate determination, malate dehydrogenase and as- in the ATP/ADP ratio in photorespiratory conditions, especially partate aminotransferase (Roche) in the presence of glutamate were applied. The formation of NADH was quantified on a FluoroMax-2 in chloroplasts (Fig. 1). After 10 min of illumination, the ratio in- spectrofluorometer as described above. creased from 0.9 to more than 10. When the light was turned off, the ratio returned to the initial value after 5 min of dark- ness. In saturating CO2 illumination did not essentially change Enzyme assays ATP/ADP ratio in chloroplasts after 10 min of illumination. In the extrachloroplast fraction of protoplasts from plants adapted to NADP-malate dehydrogenase (NADP-MDH; EC 1.1.1.82) activation darkness, the ATP/ADP ratio was about 2.7. Ten minutes of illu- state was measured essentially according to Scheibe and Stitt (1988) as described earlier (Igamberdiev et al. 1998). The data was cor- mination increased this ratio to 10 in photorespiratory and to 6 rected in accordance with the rate of NAD-MDH (EC 1.1.1.37) activity, in non-photorespiratory conditions. After 5 min following dark- 1/500 of which corresponds to its activity with NADPH as a cofactor ness the ATP/ADP ratio decreased but did not reach initial low (Scheibe and Stitt 1988). value. For determination of the NAD-malic enzyme (NAD-ME; EC 1.1.1.39) When barley plants were in the dark for 24 hours and de- activity, protoplasts were fixed in 100 mmol · L –1 HEPES-KOH, pH 6.8, tached leaves were illuminated for one hour, the total ATP/ 5 mmol · L –1 EDTA, 10 mmol · L –1 ditiothreitol, 5 mg · mL –1 bovine serum ADP ratio in protoplasts isolated from these leaves was 2.7 albumin, 0.4 mmol · L –1 phenylmethylsulfonyl fluoride, and 1% (w/v) Tri- (Table 2). Introduction of 50 mmol · L –1 glycine during illumina- ton X-100. The activity was measured essentially according to Cook et tion of leaves increased this ratio nearly twice. The addition of al. (1995) immediately after thawing of protoplasts in 30 mmol · L –1 HE- oligomycin (in the concentration 0.1 µmol · L –1), which inhibits PES-KOH, pH 6.8, 1 mmol · L –1 EDTA, 2 mmol · L –1 ditiothreitol, 5 mmol · L –1 malate, and 3 mmol · L –1 NAD + . After reaching equilibrium by NAD-MDH (2 min), the assay was initiated by the addition of 5 mmol · L –1 MnCl2. In order to calculate the activity state, we quanti- fied the lag-phase in development of its activity by denoting the extrap- olated value of the linear part of the progress curves of product forma- tion (τ) recorded by spectrophotometer, as described in Dixon and Webb (1979, p. 456). The activity state of NAD-ME was determined as the ratio of apparent rate constant of the enzyme (which is in reverse proportionality to the time τ) at given time of darkness or illumination to the apparent rate constant of the activated enzyme (in the pres- ence of CoA) (Dixon and Webb 1979, pp. 454 – 460). Cook et al. (1995) determined the activity state of NAD-ME as the ratio of the rate 4 min after the initiation of the assay to that after 1 min, which reflects the similar tendency. Results Subcellular ATP/ADP and NADPH/NADP ratios in darkness and during illumination in photorespiratory and non-photorespiratory conditions Determination of ATP/ADP and NADPH/NADP ratios in dark- ness revealed that they are independent of CO2 concentration in the medium (Table 1). Both ratios were low in chloroplasts and essentially higher in the extrachloroplast compartment. In Figure 1. Changes in chloroplast and extrachloroplast ATP/ADP ratios continuous light, both chloroplastic and extrachloroplastic in high and low CO2 in barley protoplasts from plants darkened 24 ATP/ADP ratios were higher, particularly under photorespira- hours, illuminated 10 min after darkness and darkened 5 min after tory conditions. The NADPH/NADP ratio in extrachloroplastic 10 min illumination. The results are means and SD from 3 replicates. 1328 Abir U. Igamberdiev, El˙zbieta Romanowska, Per Gardeström Table 1. Subcellular ATP/ADP and NADPH/NADP ratios in continuous light (500 µmol quanta · m – 2 · s –1, 30 min) and prolonged darkness (30 min) in barley leaf protoplasts. The results are means and SD from 4 – 6 replicates. Compartment ATP/ADP NADPH/NADP Dark Dark Light Light Dark Dark Light Light High Low High Low High Low High Low CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 Chloroplast 0.4 ± 0.2 0.5 ± 0.2 2.1 ± 0.5 3.1 ± 0.6 0.3 ± 0.1 0.4 ± 0.2 0.9 ± 0.3 1.1 ± 0.4 Extrachloroplast 2.2 ± 0.4 2.4 ± 0.3 4.2 ± 0.7 7±1 1.1 ± 0.4 0.8 ± 0.3 1.4 ± 0.2 1.3 ± 0.3 Malate utilizing enzymes Table 2. ATP/ADP ratios in illuminated (10 min) protoplasts isolated from barley leaves (darkened for 24 hours and illuminated for 1 hour) The activation state of NADP-MDH increased very quickly fed by water (control) or gylcine (50 mmol · L –1). Oligomycin (1 upon irradiation (data not shown), an increase similar to that µmol · L –1) was added 3 min prior illumination of protoplasts. The re- reported earlier (Igamberdiev et al. 1998). The final activity of sults are means and SD from 3 replicates. the enzyme after activation was 107 ± 14 µmol · h –1 · mg –1 Chl (n = 7). NAD-MDH activity was 4230 ± 450 µmol · h –1 · mg –1 Chl Condition ATP/ADP ratio (n = 8). The «crude» NADP-MDH activity found in extracts of Water 2.67 ± 0.31 darkened protoplasts was very low, and generally did not ex- Glycine 4.75 ± 0.27 ceed the side activity of NAD-MDH with NADPH as a cofactor. Glycine + oligomycin 2.96 ± 0.12 In darkness after illumination, the activation state of NADP- MDH decreased during the first two min to 20 – 25 % and then slowly (ten minutes) decreased (Fig. 4), being zero in dark- mitochondrial ATP synthesis and does not affect chloroplastic ness. At limiting CO2 (in photorespiratory conditions) activa- ATPase (Krömer et al. 1988, Igamberdiev et al. 1998), to the tion state of NADP-MDH was somewhat higher and de- protoplasts of glycine-fed leaves, 3 min prior to illumination, decreased the ATP/ADP ratio almost to the control value. Adenylate and pyridine nucleotide levels after illumination The ATP/ADP ratio in chloroplasts decreased drastically im- mediately after illumination to 0.2 – 0.4, without significant dif- ference between saturating and limiting CO2 conditions, and then revealed a tendency of some increase (Fig. 2). After pro- longed darkness it was around 0.5 (Table 1). The NADPH/ NADP ratio also decreased drastically in chloroplasts after il- lumination (Fig. 3). In saturating CO2 it dropped to zero, in limiting CO2 it decreased to 0.2, then started to rise reaching 0.3 – 0.4 after 5 min, both in saturating and limiting CO2. In prolonged darkness, it was about 0.3 – 0.4 (Table 1). In extrachloroplast compartment in the light, the ATP/ADP ratio was much higher in limiting CO2 conditions than in satu- rating CO2 (Fig. 2). In prolonged darkness, the difference be- tween saturating and limiting CO2 disappeared and the ratio stabilized at about 2 (Table 1, Fig. 2). The NADPH/NADP ratio dropped during the first minute of darkness, which was more pronounced in saturating CO2. In limiting CO2, it remained higher during the first minute of darkness corresponding to Figure 2. Changes in chloroplast and extrachloroplast ATP/ADP ratios the PIB period (Fig. 3). During the prolonged darkness it was in barley protoplasts in high and low CO2 after illumination. The results about 1 (Table 1). are means and SD from 4 replicates.  Mitochondria and energy balance of barley protoplasts 1329 (Fig. 5 B). The activity of NAD-ME after lag-phase was 14 ± 3 µmol · h –1 · mg –1 Chl (n = 5). Malate and citrate contents Determination of malate and citrate after illumination showed that these metabolites revealed similar pictures of their chang- es after illumination (Fig. 6). The concentrations exhibited some increase during the first minute of darkness in limiting CO2 conditions and then decreased. In saturating CO2, dur- ing the first minute of darkness the decrease was not ex- pressed, rather a steady level remained, followed by a de- crease similar to limiting CO2 conditions. Discussion Mitochondria regulate energy balance of plant cell in the light In the light, mitochondria of C3 plant leaves oxidize both pho- torespiratory substrate (glycine) and respiratory substrates, Figure 3. Changes in chloroplast and extrachloroplast NADPH/NADP ratios in barley protoplasts in high and low CO2 after illumination. The results are means and SD from 4 replicates. Figure 4. Activation state of NADP-malate dehydrogenase after illumi- nation in high and low CO2. The results are means and SD from 3 re- plicates. creased more slowly after illumination, which was mostly pro- nounced during the first minute after light was turned off, i.e. in the period of PIB. NAD-ME, contrary to NADP-MDH, revealed increased acti- Figure 5. A – Progress curves of NAD-malic enzyme spectrophoto- metric assays. The samples were taken from barley leaf protoplasts vation after illumination, especially 1– 2 min after light was during and after illumination in high CO2. B – calculated activity state turned off, i.e. during LEDR, which was reflected mainly in the of NAD-malic enzyme in the course of dark-light-dark transition in high decrease of lag-phase during measurement of its activity and low CO2. The results are given from one experiment and similar without CoA (Fig. 5 A). The most inactive state of malic en- results were obtained in two other independent experiments. 1 – dark- zyme was observed in continuous darkness; during illumina- ness, 2 – light (6 min), 3 – light (10 min), 4 – after illumination (1min), 5 tion its activity state was higher, and it was the highest during – after illumination (2 min), 6 – after illumination (10 min), 7 – activity the period after illumination, after which it gradually declined with CoA. 1330 Abir U. Igamberdiev, El˙zbieta Romanowska, Per Gardeström very high chloroplastic ATP/ADP ratios in the light (Igamber- diev et al. 2001). Glycine oxidation and post-illumination phenomena Post-illumination burst (PIB) of CO2 occurs within the first min- ute after illumination, while light-enhanced dark respiration (LEDR) appears later (after 1 min) and continues for several minutes (Atkin et al. 2000). Thus, we can attribute the phe- nomena of the first minute after illumination to PIB, and those appearing after 1 minute to LEDR. The characteristics of energy and redox balance during the first minute after illumi- nation include higher activation levels of chloroplast NADP- MDH in limiting CO2 (Fig. 4) and higher NADPH/NADP ratio in chloroplast, which does not drop to zero as in saturating CO2 (Fig. 3). The correspondence of chloroplast redox balance (NADPH/NADP ratio) to the activation state of NADP-MDH was shown by Foyer et al. (1992). The data obtained indi- Figure 6. Changes of malate and citrate concentrations in barley pro- cates that the PIB can maintain some reduction level in the toplasts in high and low CO2 after illumination. The results are means chloroplast stroma during a quick light-dark transition. and SD from 3 – 4 replicates. In photorespiratory conditions, the drop in chloroplastic ATP/ADP ratio occurs from a higher initial level; however, the further development of the ratio is similar in saturating and lim- mainly glycolytically-derived (OAA, malate, and/or pyruvate). iting CO2 (Fig. 2). This is different from the changes in NADPH/ At limiting CO2, glycine becomes the main substrate for mito- NADP ratio, which is maintained at a higher reduction level in chondria, providing ATP in the cytosol for sucrose biosynthe- chloroplast and extrachloroplast compartments during the sis (Gardeström and Wigge 1988, Krömer et al. 1993) and first minute after illumination in limiting CO2, i.e., during PIB, NADH for hydroxypyruvate and/or nitrate reduction (Krömer as compared to saturating CO2 (Fig. 3). After illumination, a 1995). Refixation of photorespiratory ammonia in chloroplasts higher reduction level of the chloroplast stroma in limiting CO2 lowers their reduction level and thus prevents photoinhibition during PIB may be explained by transport of reducing power of photosynthesis (Wingler et al. 2000) and needs exporting from the oxidation of remaining photorespiratory glycine to of citrate/2-oxoglutarate from mitochondria provided by oxi- the cytosol and chloroplast. dation of glycolytic substrates (Atkin et al. 2000). Another aspect of the influence of PIB is changes in pro- The increase in extrachloroplastic ATP/ADP ratio is notable files of metabolites in protoplasts. Very big vacuolar pools of in photorespiratory conditions (Table 1, Figs. 1 and 2), indica- malate and citrate (Rentsch and Martinola 1991, Gout et al. ting that ATP is formed by glycine oxidation in mitochondria 1993) make it complicated to investigate subcellular distribu- and then exported to the cytosol. In the present investigation, tion of these compounds. However, it is clear from the ob- this was confirmed by the fact that the ATP/ADP ratio in barley tained data that some changes during the first minute were leaves was increased twice when leaves were infiltrated with dependent on CO2 concentration, particularly in concentra- exogenous glycine (Table 2). The lack of this effect in the tion of citrate, which rose in limiting CO2 and revealed no rise presence of oligomycin, in a concentration, which suppres- in saturating CO2, then (in the period corresponding LEDR) ses mitochondrial ATP formation, supports the role of photo- profiles were quite similar (Fig. 6). This can be connected to respiratory glycine in energization of cytosol. the efflux of malate and citrate from mitochondria oxidizing Plants in the dark for 24 hours were very susceptible to glycine for transfer of reductant, while in the absence of glyc- over-energization if protoplasts (isolated in darkness) were in- ine malate and citrate are readily oxidized in mitochondria. cubated in photorespiratory conditions (Fig. 1). Drastic in- crease in the chloroplastic ATP/ADP ratio shows that photo- respiration did not balance this process, as if plants were Non-photorespiratory origin of light-enhanced dark adapted to light. The mechanisms balancing energy and re- respiration dox state in chloroplasts, and thus preventing photoinhibition, are probably insufficient in dark-adapted plants. This may be When the photorespiratory glycine pool is exhausted, there is explained by a lowered level of photorespiratory enzymes af- no need to reduce hydroxypyruvate, and nitrate reduction is ter a period of prolonged darkness (Zhong et al. 1994). A sim- also rapidly suppressed in darkness (Riens and Heldt 1992), ilar picture was observed for a glycine decarboxylase-defi- thus decreasing the cytosolic/peroxisomal utilization of NADH. cient photorespiratory mutant in barley, which also showed Under these conditions, a temporary increase in cytosolic  Mitochondria and energy balance of barley protoplasts 1331 NADH may support OAA conversion to malate, which then will Atkin OK, Millar AH, Gardeström P, Day DA (2000) Photosynthesis, be the main glycolytically-derived substrate for mitochondria carbohydrate metabolism and respiration in leaves of higher compared to OAA during steady-state photosynthesis (Atkin plants. 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