Crop Science 42:423-427 (2002)
© 2002 Crop Science Society of America
CROP PHYSIOLOGY & METABOLISM
Influence of Trinexapac-Ethyl on Respiration of Isolated Wheat Mitochondria
Neil L. Heckmana,
Thomas E. Elthonb,
Garald L. Horst*,a and
Roch E. Gaussoina
a Dep. of Agronomy and Horticulture, Univ. of Nebraska, Lincoln, NE 68583-0724
b School of Biological Sciences and Center for Biotechnology, Univ. of Nebraska, Lincoln, NE 68588-0666
* Corresponding author (ghorst{at}unlserve.unl.edu)
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ABSTRACT
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The plant growth regulators (PGRs) 2,4-D [(2,4-dichlorophenoxy) acetic acid] and malic hydrazide (1,2-dyhydro-3,6-pyridazinedione) have been shown to reduce plant respiration. The effect of other PGRs such as trinexapac-ethyl [(4-cyclopropyl-
-hydroxy-methylene)-3,5-dioxocyclohexanecarboxylic acid methyl ester] on respiration is unknown. Experiments were conducted to evaluate the effects of trinexapac-ethyl and 2-oxoglutarate on the respiration of mitochondria isolated from wheat (Triticum aestivum ‘Arapahoe’) seedlings. Trinexapac-ethyl applied at increasing concentrations inhibited nicotinamide adenine dinucleotide (NADH)-dependent O uptake, while 2-oxoglutarate had no significant effect. This is different than other reports that trinexapac-ethyl may compete with 2-oxoglutarate for binding sites. Different regions of the mitochondria were tested to determine the site of inhibition caused by trinexapac-ethyl. Nicotinamide adenine dinucleotide dehydrogenase activity, duroquinol-dependent O uptake, and cytochrome bc1 activity were all reduced by 
30% in the presence of 10 mM trinexapac-ethyl. Succinate-dependent O uptake, alternative oxidase, and cytochrome oxidase were not reduced by any trinexapac-ethyl concentration. This revealed possible interference of trinexapac-ethyl with ubiquinone binding sites. The reduced form of trinexapac-ethyl was observed to inhibit the electron transport chain greater than the oxidized form. Reduction in respiration from trinexapac-ethyl may result in greater stress tolerance in treated plants.
Abbreviations: cyt c, cytochrome c • GA, gibberellic acid • NADH, nicotinamide adenine dinucleotide • PGR, plant growth regulator
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INTRODUCTION
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THE USE OF PGRs in the turfgrass industry has increased in recent years. Trinexapac-ethyl is a foliar-applied PGR that is rapidly absorbed (Fagerness and Penner, 1998). Trinexapac-ethyl, a cyclohexanedione, reduces cell elongation in vegetative tissue by blocking gibberellic acid (GA) biosynthesis. Cyclohexanediones inhibit 3ß-hydroxylase from converting GA20 to GA1 (Nakayama et al., 1990a,b; Adams et al., 1992; Rademacher et al., 1992). Griggs et al. (1991) proposed that cyclohexanediones mimic 2-oxoglutarate and could act as competitive inhibitors. The cyclohexanedione PGR, LAB 198 999, has a structure similar to trinexapac-ethyl and has been shown to compete with the cofactor 2-oxoglutarate for the binding site on the 3ß-hydroxylase enzyme (Sponsel, 1995).
Trinexapac-ethyl has recently been shown to decrease Kentucky bluegrass (Poa pratensis L.) sod heating during storage (Heckman et al., 2001). The authors speculated that trinexapac-ethyl may reduce respiration, which would, in turn, decrease the amount of heat generated during sod storage. Levitt (1980) estimated that heat generated by plant respiration can be as high as 69.8 W m-2 (10-5 kcal cm-2 leaf min-1). Other PGRs have also been shown to inhibit respiration. 2,4-D inhibited respiration of oat (Avena sativa L.) coleoptiles and pea (Pisum sativum L.) stems at concentrations >450 µM (Kelly and Avery, 1949). French and Beevers (1953) showed a 40% decrease in respiration of corn (Zea mays L.) mitochondria by 2,4-D at 400 µM concentrations. Switzer (1957) observed nearly a complete inhibition of respiration in soybean [Glycine max (L.) Merr.] mitochondria by 2,4-D at concentrations of 200 µM. Another PGR, malic hydrazide, has also been shown to inhibit respiration of isolated soybean mitochondria by 25% at 20 mM levels (Switzer, 1957). In addition, dinitroaniline herbicides also have been shown to inhibit mitochondrial O uptake. For example, Trifluralin [2,6-dinitro-N, N-dipropyl-4-(trifluoromethyl)benzenamine] inhibited respiration of mitochondria isolated from sorghum [Sorghum bicolor (L.) Moench], corn, and soybean by >23% at 400 µM concentrations (Negi et al., 1967). Similar results were found upon exposing isolated mung bean (Vigna radiata L.) mitochondria to other substituted 2,6-dinitroaniline herbicides (Moreland et al., 1972; Moreland and Huber, 1979).
Little is known about nontarget biochemical effects on plants from use of turfgrass PGRs. Our objective was to determine the effect of 2-oxoglutarate and trinexapac-ethyl on isolated mitochondria respiration. Wheat is used as a cool-season grass model system to efficiently test this hypothesis. If inhibition is detected, specific site of action would be identified.
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MATERIALS AND METHODS
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Isolated wheat mitochondria were used to determine the effects of 2-oxogluarate and trinexapac-ethyl on respiration. Eight biochemical experiments were conducted in this study. Nicotinamide adenine dinucleotide-dependent O uptake was first examined to evaluate the entire electron transport chain. Subsequent tests of succinate-dependent O uptake, alternative oxidase activity, cytochrome oxidase, duroquinol-dependant O uptake, and cytochrome bc1 were used to measure individual segments of the electron transport chain and identify possible inhibition sites. A final comparison of a reduced form of trinexapac-ethyl compared with an unreduced form was performed to support the results.
Mitochondrial Isolation
Wheat seedlings were grown in the dark at 30 ± 2 °C in moist vermiculite. Mitochondria were isolated from shoots of 2.5- to 3-d-old seedlings using a modification of the Day and Hanson (1977) procedure. Seventy-five grams of fresh shoots were ground using a mortar and pestle and 250 mL of grinding media consisting of 350 mM mannitol, 30 mM 3-[N-morpholino]propane sulfonic acid (MOPS), and 1 mM ethylenediaminetetraacetic acid (EDTA), pH 7.6. Prior to crushing the shoots, 1.5 g of polyvinylpolypyrrolidone and 0.32 g L-cystine were added to the grinding media. The pulp was squeezed through muslin and centrifuged for 2 min at 5110 g. The pellet containing cellular debris was discarded, and the supernatant was centrifuged at 19150 g for 5 min to yield a crude mitochondrial pellet. The pellet was washed with media containing 300 mM mannitol, 20 mM MOPS, and 1 mM EDTA at pH 7.2 and resuspended. The suspension was centrifuged at 5110 g for 2 min, and the pellet was discarded. The supernatant was underlain with 8 mL of 0.6 M sucrose solution and spun at 10350 g for 20 min. The pellet contained washed mitochondria. The mitochondria were resuspended in 600 µL of suspension media composed of 250 mM sucrose and 30 mM MOPS at pH 7.2 and stored at -80 °C until assays were performed. Mitochondrial protein was estimated using the method of Lowry et al. (1951) as modified by Larson et al. (1977) with bovine serum albumin (BSA) (fraction V) as the standard.
Nicotinamide Adenine Dinucleotide-Dependent Oxygen Uptake
Isolated mitochondria were sonicated for 1 min to disrupt membranes. Oxygen consumption was measured polarographically using a Rank Brothers O electrode (Rank Brothers, Ltd., Cambridge, UK) at 25 °C in 0.5 mL of reaction medium containing 30 mM MOPS adjusted to pH 7.2. The O content of air-saturated water was estimated according to Estabrook (1967). The reaction was initiated with 1 mM NADH. Mitochondria were activated using two State 3-State 4 cycles [200 nmol adenosine diphosphate] prior to the addition of trinexapac-ethyl dissolved in 80% ethanol or 2-oxoglutarate dissolved in deionzed distilled water during State 4. Trinexapac-ethyl and 2-oxoglutarate treatments were 0, 2, 4, 6, 8, and 10 mM. Controls for ethanol addition were performed.
Succinate-Dependent Oxygen Uptake
Oxygen consumption was measured using sonicated mitochondria at 25 °C in 0.5 mL of reaction medium containing 30 mM MOPS, 250 mM sucrose, 5 mM KH2PO4, 5 mM MgCl2, and 0.1% (w/v) BSA adjusted to pH 7.2. The assay was initiated with 1 mM adenosine triphosphate followed by 10 mM succinate. Treatments of 10 mM trinexapac-ethyl were added to respective samples prior to the assay initiation to observe complete inhibition. Controls for ethanol addition were performed.
Alternative Oxidase Activity
Alternative oxidase activity was measured at 25 °C in 0.5 mL of reaction medium containing 30 mM MOPS and 1 mM KCN at pH 7.2. Sonicated mitochondria were used, and the reaction was initiated with 1 mM duroquinol. Alternative oxidase activity was evaluated as duroquinol-dependent O uptake that was sensitive to 1 mM salicylhydroxamic acid (SHAM). A no-mitochondrial protein control for autooxidation of duroquinol was performed. Alternative oxidase activity was also measured using mitochondria treated with dithiothreitol as described by Umbach and Siedow (1993) and in the presence of 1 mM pyruvate (Millar et al., 1993). These treatments result in the alternative oxidase being fully activated. Trinexapac-ethyl treatments were the same as for the succinate-dependent O uptake assays.
Cytochrome Oxidase
Oxygen uptake of sonicated mitochondria was measured at 25 °C in 0.5 mL of reaction medium containing 30 mM MOPS, 1 mM EDTA, and 50 µM cytochrome c (cyt c) adjusted to pH 7.2. The assay was initiated with 250 µM N, N, N, N-tetramethyl-p-phenylenediamine and 1 mM ascorbate. The difference between the initial rate and the rate after adding 1 mM KCN was used to determine cytochrome oxidase activity. Treatments were the same as for the succinate-dependent O uptake assays and the alternative pathway assays.
Nicotinamide Adenine Dinucleotide Dehydrogenase
NADH dehydrogenase activity was measured with a spectrophotometer (Lambda 3B uv/vis, Perkin-Elmer Corporation, Norwalk, CT) at 25 °C. Total NADH dehydrogenase activity was measured on sonicated mitochondria in a 1-mL reaction medium containing 35 µM 2,6-dichloroindophenol (DCIP), 30 mM MOPS, 1 mM SHAM, 1 mM KCN, and 1 µM antimycin A and was initiated with 1 mM NADH. Rotenone-sensitive NADH dehydrogenase activity was measured through addition of 1 mM rotenone. An extinction coefficient (600 nm) of 21 mM -1 cm-1 was used for DCIP (Douce et al., 1973). Trinexapac-ethyl treatments were 0, 2, 4, 6, 8, and 10 mM.
Reduction of Duroquinone
Duroquinone was reduced as described by Rich (1978). One-half millimole of duroquinone was dissolved in 50 mL diethyl ether and placed in a separating funnel. Sodium dithionite (1 g in 50 mL water) was added, and the solution was vigorously shaken. After the quinone had been reduced to its colorless form, the aqueous layer was discarded. The ethereal layer was washed twice with 25 mL saturated sodium chloride solution. The washed ethereal layer was filtered through anhydrous sodium sulfate and the ether was dried off by vacuum desiccation under a N flow. The duroquinol was dissolved in 5 mL of 95% ethanol and stored under N to avoid autoxidation.
Duroquinol-Dependent Oxygen Uptake
Oxygen uptake was measured at 25 °C in 0.5 mL of reaction medium containing 30 mM MOPS at pH 7.2. Sonicated mitochondria were used, and the reaction was initiated with 1 mM duroquinol. Trinexapac-ethyl treatments were 0, 2, 4, 6, 8, and 10 mM. A no-mitochondrial protein control was included to estimate duroquinol autoxidation.
Cytochrome bc1
Cytochrome bc1 activity was measured spectrophotometrically at 25 °C in 1 mL of reaction medium containing 30 mM MOPS, 50 µM cyt c, 1 mM SHAM, 1 mM KCN, and was initiated with 1 mM reduced duroquinone. An extinction coefficient (550 nm) of 21 mM -1 cm-1 was used for cyt c (Douce et al., 1973). Trinexapac-ethyl treatments were 0, 2, 4, 6, 8, and 10 mM.
Nicotinamide Adenine Dinucleotide-Dependent Oxygen Uptake in the Presence of Reduced Trinexapac-Ethyl
Trinexapac-ethyl was reduced as described for duroquinone reduction. Nicotinamide adenine dinucleotide-dependent O uptake was measured in the same manner as the other NADH-dependent O uptake assays using both reduced and oxidized trinexapac-ethyl at the six previously mentioned concentrations.
Data Analysis
All O consumption rates are expressed as nmol min-1 O2 mg-1 protein. Spectrophotometrically, data were expressed as amount of DCIP reduced or cyt c reduced min-1 mg-1 protein. Experimental design for all experiments was a randomized complete block with three replications and all experiments were repeated. Nicotinamide adenine dinucleotide-dependent O uptake, NADH dehydrogenase activity, quinol-dependent O uptake, and cyt bc1 activity were analyzed with linear regression and curvilinear regression and separated with standard errors (P = 0.05). Student's paired t test was used for the analysis of succinate-dependent O uptake, alternative oxidase, and cytochrome oxidase activities.
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RESULTS AND DISCUSSION
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Nicotinamide Adenine Dinucleotide-Dependent Oxygen Uptake
Nicotinamide adenine dinucleotide-dependent O uptake rates at 0 mM trinexapac-ethyl and 0 mM 2-oxoglutarate were 135 nmol min-1 O2 mg-1 protein (Fig. 1)
. Trinexapac-ethyl decreased O consumption; respiratory capacity was reduced 35% at 10 mM trinexapac-ethyl. In contrast, O uptake rates were not reduced significantly at the tested concentrations of 2-oxoglutarate. Rademacher (2000) indicated that cyclohexanediones mimic 2-oxoglutarate and could act as a competitive inhibitor during some metabolic reactions. Our results show that this is not the case in the mitochondrial electron transport chain. In contrast to 2-oxoglutarate, trinexapac-ethyl did partially inhibit NADH-dependent O uptake. Therefore, it was our second objective to identify the specific site of inhibition by trinexapac-ethyl, through analysis of different activities and regions of the electron transport chain.
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Fig. 1. Nicotinamide adenine dinucleotide (NADH)-dependent O uptake of wheat mitochondria in the presence of trinexapac-ethyl and 2-oxogluarate. Data points represent means of two experiments (n = 6) ± SE.
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Succinate-Dependent Oxygen Uptake
Rates of O uptake with succinate as substrate were similar between ethanol controls (18.6 nmol min-1 O2 mg-1 protein) and assays containing 10 mM trinexapac-ethyl (Table 1). Variability was high and there was no statistical difference between the two treatments. The control rates are much lower than the NADH-dependent O uptake rates, showing that little of the electron transport chain capacity was in use. Because the electron transport chain was operating at a lower capacity than the NADH-dependent O uptake, we cannot draw any conclusions about its inhibition by trinexapac-ethyl.
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Table 1. Activities of succinate-dependent oxygen uptake, alternative oxidase, and cytochrome oxidase of wheat mitochondria in the presence of 10 mM trinexapac-ethyl and ethanol controls. Data represent means of two experiments.
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Alternative Oxidase
Duroquinol-dependent O consumption rates in the presence of 1 mM KCN were 7.38 and 8.70 nmol min-1 O2 mg-1 protein for controls and assays containing 10 mM trinexapac-ethyl, respectively (Table 1). No statistical difference was detected between the treatments. These rates are within the range of 4.9 to 24.1 nmol min-1 O2 mg-1 protein for alternative oxidase activities found in other varieties of wheat (McCaig and Hill, 1977). Fully activated alternative oxidase was also tested and results indicated no response to trinexapac-ethyl (Table 1). Therefore, other regions of the mitochondrial electron transport chain were tested.
Cytochrome Oxidase
Oxygen uptake rates for cytochrome oxidase were 898 and 872 nmol min-1 O2 mg-1 protein for controls and assays containing 10 mM trinexapac-ethyl, respectively (Table 1). These rates were approximately six times greater than the rates of NADH-dependent O uptake, suggesting that this is not the limiting step of the overall electron transport chain. Treatment means were not statistically different, thus inhibition of the electron transport chain by trinexapac-ethyl must occur between the NADH dehydrogenases and the cyt bc1 complex, or within the cyt bc1 complex.
Nicotinamide Adenine Dinucleotide Dehydrogenase Activities
NADH-dependent DCIP reduction activities in sonicated mitochondria were reduced by trinexapac-ethyl (Fig. 2)
. This observed inhibition is very similar in magnitude to that observed for NADH-dependent O uptake, suggesting that NADH dehydrogenases may be one site of trinexapac-ethyl inhibition. In sonicated mitochondria, the NADH-dependent reduction of DCIP is due to a combination of all of the NADH dehydrogenases present in plant mitochondria. Of the NADH dehydrogenases present, complex I activity is believed to be rotenone sensitive. However, no statistical difference was detected between the rotenone sensitive and insensitive NADH dehydrogenase activities. Rotenone is not a very effective inhibitor in plant mitochondria, thus the rotenone results are inconclusive. These results do indicate that one or more of the NADH dehydrogenases present in plant mitochondria is inhibited by trinexapac-ethyl.
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Fig. 2. Nicotinamide adenine dinucleotide (NADH) dehydrogenase activities of wheat mitochondria in the presence of trinexapac-ethyl. Data points represent means of two experiments (n = 6) ± SE.
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Duroquinol-Dependent Oxygen Uptake
Duroquinol-dependent O uptake declined linearly with trinexapac-ethyl concentration (Fig. 3)
. Approximately 40% reduction of activity was shown in the presence of 10 mM trinexapac-ethyl. This decrease is similar to that observed for NADH-dependent O uptake and NADH dehydrogenase activity.
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Fig. 3. Duroquinol-dependent O uptake of wheat mitochondria in the presence of trinexapac-ethyl. Data points represent means of two experiments (n = 6) ± SE.
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Cytochrome bc1
Cytochrome bc1 activity was linearly reduced by trinexapac-ethyl (Fig. 4)
. Activity decreased by 30% in the presence of 10 mM trinexapac-ethyl. This percentage reduction in activity was also comparable with that observed for NADH-dependent O uptake, NADH dehydrogenase activities, and duroquinol-dependent O uptake.
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Fig. 4. Cytochrome bc1 activity of wheat mitochondria in the presence of trinexapac-ethyl. Data points represent means of two experiments (n = 6) ± SE.
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The common component of all the previous assays showing inhibition from trinexapac-ethyl is ubiquinone and ubiquinol binding sites. Ubiquinone is a substituted 1,4-benzoquinone, which contains two double bonded oxygens to a hexane ring (Lambers, 1995). Trinexapac-ethyl also contains two double bonded oxygens on a hexane ring (Fagerness and Penner, 1998). This similarity in structure may allow trinexapac-ethyl to accept electrons in a similar manner as ubiquinione. However, the difference in the other functional groups may not allow trinexapac-ethyl to pass electrons to other components of the electron transfer chain. Therefore, trinexapac-ethyl could act as an electron sink. Alternatively, trinexapac-ethyl could fit into quinone or quinol binding sites and competitively inhibit these activities.
Nicotinamide Adenine Dinucleotide-Dependent Oxygen Uptake in the Presence of Reduced Trinexapac-Ethyl
If trinexapac-ethyl accepts electrons from the mitochondrial NADH dehydrogenases, then it is possible that the oxidized and reduced forms of trinexapac-ethyl may differ in their ability to inhibit electron transport. The commercial form of trinexapac-ethyl is in the oxidized form, and the reduced form was prepared using the same procedure used to reduce duroquinone. Nicotinamide adenine dinucleotide-dependent O uptake was partially inhibited by both the oxidized and reduced forms of trinexapac-ethyl (Fig. 5) . A 50% decrease in O uptake was shown with 10 mM reduced trinexapac-ethyl compared with controls. Oxygen uptake was decreased by 40% with 10 mM oxidized trinexapac-ethyl. The increased inhibition due to the reduced from of trinexapac-ethyl was statistically significant (P = 0.05). If trinexapac-ethyl accepts electrons from the mitochondrial NADH dehydrogenases and becomes reduced, its inhibitory properties would be increased somewhat. However from our experiments, it is not possible to establish if trinexapac-ethyl accepts electrons from the mitochondrial NADH dehydrogenases, or whether it simply acts as an inhibitor at quinone and quinol binding sites.
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Fig. 5. Nicotinamide adenine dinucleotide-dependent O uptake of wheat mitochondria in the presence of oxidized or reduced trinexapac-ethyl. Ethanol (95%) was used as the solvent for all treatments and controls. Data points represent means of two experiments (n = 6) ± SE.
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Trinexapac-ethyl has been suggested to act as a competitive inhibitor during GA biosynthesis (Griggs et al., 1991; Rademacher, 2000). However, we have shown that trinexapac-ethyl partially inhibits mitochondrial electron transport, indicating another potential site of action for trinexapac-ethyl. Our results further show that trinexapac-ethyl inhibition occurs at sites of quinol and quinone binding within the mitochondrial electron transport chain. This may be one reason for the observed decrease in sod heating during storage of trinexapac-ethyl treated sod (Heckman et al., 2001). Other stress tolerances resulting from trinexapac-ethyl application may also be partially a result of less respiration.
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ACKNOWLEDGMENTS
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The authors would like to thank Beth Whitaker for her support and assistance in this research, and Syngenta for trinexapac-ethyl technical information.
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NOTES
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University of Nebraska-Lincoln, Agricultural Research Division journal series no. 13223.
Received for publication May 15, 2001.
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ABSTRACT
INTRODUCTION
