Cytochrome and Alternative Pathway Respiration in Green ... - NCBI

Plant Physiol. (1990) 93, 356-360

Received for publication November 16, 1989 and in revised form February 12, 1990

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Cytochrome and Alternative Pathway Respiration in Green Algae1 Measurements Using Inhibitors and 1802 Discrimination Harold G. Weger, Robert D. Guy, and David H. Turpin* Department of Biology, Queen's University, Kingston, ON, Canada K7L 3N6 (H.G.W., D.H.T.), and Department of Forest Sciences, University of British Columbia, Vancouver, BC, Canada V6T 1W5 (R.D.G.) in the absence versus the presence of a Cyt pathway inhibitor at various alternative pathway inhibitor concentrations (1), or from the SHAM-induced decline in respiration in the absence of Cyt pathway inhibitors. Potential problems with these methods are numerous, and results are often

ABSTRACT Inhibitor titration curves and discrimination against 1802 by mitochondrial respiration in three strains of green algae (Selenastrum minutum [Naeg.] Collins, and two strains of Chlamydomonas reinhardtii Dangeard) with differing respiratory capabilities were determined. Discrimination for cytochrome pathway respiration ranged from 19.89 to 20.43%. Discrimination for altemative pathway respiration by wild-type C. reinhardtii (measured in the presence of KCN) was 25.46%, while discrimination values for a cytochrome oxidase deficient mutant of C. reinhardtii ranged from 24.24 to 24.96%. In the absence of KCN, the alternative pathway was not engaged in wild-type C. reinhardtii, the only algal strain that possessed both cytochrome and altemative pathway capacities.

ambiguous (13). Some of the problems in assessing the contribution of the alternative pathway can be overcome by the use of isotopic discrimination techniques (8). The discrimination against 1802 by the alternative pathway is greater than by the cytochrome pathway (23.5-25.5%o and 17.1-19.4%o, respectively). By measuring 1802 discrimination in the presence of inhibitors of the Cyt or alternative pathways, it is possible to establish 1802 discrimination (D) values associated with the operation of each pathway for a plant tissue (i.e. the 'end members'). 1802 discrimination by tissue in the absence of inhibitor can then be used to calculate alternative pathway contribution to total respiratory 02 consumption. While measuring alternative pathway respiration via the 1802 discrimination method is much more laborious and time consuming than traditional inhibitor-based methods, it provides a useful check when inhibitor-based estimates are in doubt (8). In this report, we extend the work of our previous study (8) to mitochondrial respiration by green algae. While the potential for cyanide-resistant respiration has been demonstrated in a few green algae (17, 18, 22, 24), even fewer studies have attempted to quantify the engagement of the alternative pathway. Grant and Hommersand (7) detected no effect of 1 mM m-CLAM on respiration in the absence of KCN in Chlorella protothecoides, and Peltier and Thibault (15) obtained similar results for 5 mM SHAM in Chlamydomonas reinhardtii. In a recent study, Goyal and Tolbert (6) found that alternative pathway capacity in C. reinhardtii changed as a function of growth conditions, but were unable to quantify the engagement of the alternative pathway using inhibitors. In this study, we report 1802 discrimination factors for Cyt and alternative pathways in green algae, and estimates of alternative pathway capability and engagement based on inhibitors and isotopic discrimination. The three strains of green algae used in the study all have different respiratory characteristics. C. reinhardtii R34 ('wild type') possesses the capability for both Cyt

Mitochondrial electron transport in higher plants and microorganisms may be mediated via the phosphorylating Cyt pathway or the nonphosphorylating alternative pathway. The branch-point between these pathways is the mobile ubiquinone pool. The function of the apparently wasteful alternative pathway has long been debated (see ref. 19 for a recent review). The 'energy overflow' hypothesis suggests that the alternative pathway becomes engaged only when the Cyt pathway is working at full capacity, or is restricted (e.g. by adenylates; ref. 10). Recently, it has been demonstrated that the degree of engagement of the alternative pathway is dependent upon the reduction state of the ubiquinone pool. The alternative pathway is only engaged at a high ubiquinone reduction state, possibly due to a high activation energy required for the first step in that pathway (4). The capacity and engagement of the alternative pathway is usually determined by the use ofsubstituted hydroxamic acids (especially SHAM2), which inhibit alternative pathway activity. Engagement can be calculated from 'p plots' of respiration Supported by the Natural Sciences and Engineering Research Council of Canada. H. G. W. acknowledges an Ontario Graduate Scholarship. 2 Abbreviations: SHAM, salicylhydroxamic acid; m-CLAM, mchlorobenzhydroxamic acid; D, discrimination against 1802. 356

MITOCHONDRIAL RESPIRATION IN GREEN ALGAE

and alternative pathway activity, C. reinhardtii R4 ('Cyt ox-') has greatly reduced Cyt pathway activity, and Selenastrum minutum displays no detectable alternative pathway capacity (25).

MATERIALS AND METHODS Algal Strains

Selenastrum minutum (UTEX 2459) is an original isolate from Lake Ontario. The two strains of Chlamydomonas reinhardtii were the products of crosses between C. reinhardtii CC-1039 (wild type) and CC-314 (Cyt ox-, nitrate reductase deficient). The parental strains (CC-314, CC-1039) were obtained from the Chlamydomonas Genetics Center at Duke University (Durham, NC). Chlamydomonas CC-314 (dk97) has been reported to exhibit 3 to 11 % of wild-type Cyt oxidase activity (9, 28). For the crosses, gametogenesis was induced by suspending cells of each strain in N-free 'high-salt' medium (21) overnight in the light. Cells were subsequently mixed, left in the light for 4 h, vortexed, pipetted onto maturation plates (-N medium; ref. 23), and incubated in the light for 4 d. Zygotes were then transferred to germination plates (2.5% agar, 1 mM NaNO3 as the sole N source); germination occurred after 2 d. Germination plates were then flooded with sterile medium, and cell suspensions were plated onto another set of germination plates. The colonies thus produced all displayed nitrate reductase activity. These were transferred to two sets of replicate plates, one set supplemented with acetate. The plates without acetate were incubated in the light, while the acetatesupplemented plates were kept in darkness. Cyt ox- strains were identified by the inability to grow heterotrophically on acetate. The presence or absence of Cyt oxidase activity was confirmed for all strains by measurement of mitochondrial 02 consumption in the dark using an 02 electrode (Hansatech, King's Lynn, England), in the presence and absence of SHAM and KCN. Two strains (R4, Cyt ox-; R34, Cyt ox') were selected for further experiments. The ability to grow on NO3in the Cyt ox- mutant was necessary for further investigations on nitrogen metabolism in this species, not reported in this paper. Culture Methods S. minutum was grown in

NO3--limited chemostats as previously described (5). C. reinhardtii R4 was grown in NH4+-limited chemostats at 30°C using a modification of Surzycki's medium (26) containing 1 mM NH4Cl, at a growth rate of 0.3 d-'. C. reinhardtii R34 was grown in batch culture in the same medium containing 5 mM NH4Cl. Cultures were bubbled with air enriched with 2.5% CO2.

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suspended in 11.6 mm potassium phosphate (pH 7.2) for C. reinhardtii or 25 mm NaHepes-HCl (pH 7.6) for S. minutum, and placed in a collapsible reaction vessel. Cell suspensions were bubbled with 100% 02 until 02 saturation was reached; 02 concentration was monitored with an 02 electrode (Hansatech) fitted into the base of the reaction vessel. Inhibitors were added just prior to the experiment, through a septum in the top of the vessel. KCN was added to a concentration of 1 mm and SHAM to 5 mM. At discrete time intervals, aliquots of known volume (17-80 mL) were withdrawn from the reaction vessel into evacuated glass bulbs containing phosphoric acid as a killing agent (final concentration approximately 4%). After the experiment, samples were processed within 10 h using a vacuum line as described by Guy et al. (8). The stability of the acidified samples over this time period was verified in preliminary experiments. 02 was stripped from the sample by bubbling with He, separated from N2 and Ar via chromatography, combusted to CO2 by reaction with hot graphite, and the CO2 was collected in a sample tube attached to a port on the vacuum line. Yields were determined manometrically. These samples were later analyzed for 180/160 ratios via a VG Isotech Prism triple-collecting mass spectrometer (Middlewich, England). Internal precision was better than ±0.02%. These ratios represent the isotopic composition of the 02 remaining in the reaction vessel at the time of sampling, i.e. the nonrespired 02. Discrimination Calculations Oxygen isotope discrimination was calculated as described previously (8). Discrimination (D) was calculated as the slope of a linear regression through the origin of (ln R/Ro)* 1000 versus -lnf('Rayleigh distillation plots'), where R is the 1802/ 1602 composition of the dissolved 02 in the sample, R. is the initial composition, and f is the fraction of 02 remaining in the reaction vessel at the time of sampling. Since the data are normalized, results from replicate experiments were pooled. To calculate R/Ro values, every sample isotope ratio (180/ 160) was compared to every other isotope ratio within an experiment. If the number of comparisons (minus 1) is used as the denominator in the calculation of the residual mean square, both residual mean square and the standard error would be underestimated. To avoid this problem, n (sample size) was calculated as the total number of samples (not ratios) minus the number of experiments (i.e. the sum of [sample number minus 1] for each experiment). Standard error of the regression was then calculated in the usual manner for a regression through the origin (using n-1 as the denominator in the calculation of residual mean square). RESULTS AND DISCUSSION

Inhibitor Titrations

Mitochondrial 02 consumption was measured polarographically. KCN was added to a final concentration of 1 mm, and SHAM was added from a 1 M stock in methoxyethanol.

02 Extraction and Analysis The general procedure was as previously described (8). Cells were harvested and concentrated by centrifugation, and re-

Inhibitor titration curves for the three algal strains indicate that all three display different respiratory capabilities (Fig. 1, A-C). In Chlamydomonas reinhardtii R34 (wild type), evidence for the capacity for both Cyt and alternative pathway activity was found (Fig. IA). In the absence of SHAM, respiration was entirely cyanide resistant; in fact, there was a KCN stimulation of respiration. The absence of KCN inhibition suggests that electron flow was diverted to the alternative

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[SHAM] (mM) Figure 1. SHAM titration curves in the presence (0) and absence (0) of 1 mm KCN. A, C. reinhardtii R34 (wild type); B, C. reinhardtii R4 (Cyt ox-); and C, S. minutum. Note the different scale for (C). Control respiration rates averaged 135, 175, and 98 Amol O2-mg-' Chl * h-1, respectively.

pathway when the Cyt pathway was blocked, and implies that the capacity of the alternative pathway is at least as great as the engagement of the Cyt pathway in the absence of KCN (approximately 135 ,umol 02 mg-' Chl h-'). The addition of low concentrations (I1-2 mM) of SHAM alone had no inhibi02 consumption by C. reinhardtii R34 (Fig. IA), indicating that the alternative pathway was probably not engaged under control conditions. The 802 discrimination data are consistent with the inhibitor results for C. reinhardtii R34; Rayleigh distillation plots are shown in Figure 2A. In the absence of inhibitor, D was found to be 19.89%oo. In the presence of SHAM, D was unchanged (20.02%o). The similar values of D in the presence

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Figure 2. Rayleigh distillation plots for A, C. reinhardtui R34 (wild type); B, C. reinhardtii R4 (Cyt ox-); and C, S. minutum. Discrimination (D) values for respiration were calculated as outlined in 'Materials and Methods," and values are %o ± SE (n).

and absence of alternative pathway inhibitor are consistent with the lack of engagement of the alternative pathway under control conditions, indicated by the SHAM titration curve (Fig. IA). Blocking Cyt pathway activity with KCN, resulting in engagement of the alternative pathway, increased D to 25.5%o (Fig. 2A). There was no observable KCN inhibition of respiration by the Cyt oxidase-deficient mutant C. reinhardtii R4, in the presence or absence of SHAM (Fig. 1 B). Respiration was entirely SHAM sensitive, and no evidence for in vivo Cyt pathway activity was found. In the absence of KCN, respiration by Cyt ox- cells resulted in a D of 24.96%o (Fig. 2B). In

MITOCHONDRIAL RESPIRATION IN GREEN ALGAE

the presence of KCN, D was calculated to be 24.24%o, not significantly different from control. These results confirm the inhibitor-based conclusion that C. reinhardtii R4 has no detectable Cyt pathway activity. Oxygen consumption by S. minutum was approximately 80% inhibited by 1 mM KCN in the absence of SHAM (Fig. 1C). Addition of higher KCN concentrations (up to 20 mM) resulted in an inhibition of up to 94% of 02 consumption (data not shown). There was only a minor inhibitory effect of SHAM in the presence of KCN, suggesting that S. minutum has little or no capacity for alternative pathway activity. Addition of 5 mM SHAM to S. minutum resulted in a slight decline in D from 20.43 to 18.69%o (P < 0.001). This unexpected decrease in D may be associated with the large SHAMstimulation of 02 consumption seen in the titration curve (Fig. 1C). The nature ofthis stimulation is unknown; however, other workers have suggested the presence of a SHAM-stimulated plasma membrane NADH oxidase for some higher plant roots ( 12, 20). Estimates of D for alternative pathway activity in these algae ranged from 24.24 to 25.46%o (Fig. 2, A and B), comparable to values of 23.5 to 25.5%o found by Guy et al. (8) for higher plants. Estimates of discrimination by the Cyt pathway ranged from 19.89 to 20.43%o, slightly higher than those previously reported for higher plant material and yeast (8). Nonetheless, these results demonstrate the clear dichotomy in D values for Cyt and alternative pathway activity in these green algae. The residual respiration rate was determined at the end of every isotopic discrimination experiment, and was always similar to that determined from the titration curves (Fig. 1). Attempts to assign a D value to residual 02 consumption were unsuccessful, as this 02 consumption did not persist for a sufficient length oftime. Discrimination associated with residual respiration by alfalfa sprouts (Medicago sativa L.) (in the presence of both KCN and SHAM) was previously determined to be 19.4%o (8). There are many potential problems associated with the use of SHAM in measuring alternative pathway activity. Stimulation of 02 consumption by low SHAM concentrations and inhibition at higher concentrations have been reported, and may be associated with peroxidases or plasma membrane NADH oxidases (3, 20). High SHAM concentrations have also been reported to inhibit Cyt pathway activity (2, 11). Other potential complications arising from the use of SHAM are inhibition of lipoxygenase or other 02-consuming enzymes (14, 16), as well as the recent report that use of alternative pathway inhibitors may result in a small increase in electron flow through the cytochrome pathway (27). More specific inhibitors of the alternative pathway have been found, but they are not suitable for working with intact tissues (13). Some of the problems associated with interpreting inhibitor results are apparent from Figure 1. In both C. reinhardtii R34 (wild type) and in S. minutum, intermediate SHAM concentrations (in the absence of KCN) had a stimulatory effect on 02 consumption (Fig. 1, A and C). In the latter organism, 02 consumption in the presence of 5 mm SHAM reached 220% of control. In contrast, Cyt ox- (R4) cells exhibited no detectable SHAM stimulation of 02 consumption (Fig. 1 B), sug-

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gesting that at least part of the SHAM stimulation observed in wild-type cells (R34) may be due to effects of SHAM on the Cyt pathway. High SHAM concentrations (10-20 mM) caused an inhibition of Cyt pathway activity in both C. reinhardtii R34 and S. minutum (Fig. 1, A and C). The various effects of SHAM described above can make interpretation difficult, especially when using 'p plots.' Even when using isotopic discrimination techniques, the use of inhibitors is still required to verify the end members for Cyt and alternative pathway discrimination for a plant tissue or cells. A previous study indicated that the value of D for Cyt pathway respiration was somewhat lower for isolated mitochondria and yeast cells than for intact plant tissues (15.8-17.7%oo; ref. 8), and from the present study it is evident that the presence of SHAM may affect the value obtained (e.g. for S. minutum). However, any errors associated with determining end members appear to be small. Discrimination values for alternative pathway respiration are quite consistent between plant sources, and while some small differences in D for Cyt pathway respiration have been detected, intact higher plant tissues and green algae have produced similar results. The results of this study represent the first report of alternative pathway 1802 discrimination by an organism that lacks apparent Cyt pathway capacity. The calculated discrimination was consistent with that calculated for higher plant material from various sources (8), and that calculated from wild-type cells in the presence of KCN (Fig. 2). Values of D for Cyt pathway respiration for these green algae were marginally higher than those reported for intact higher plant tissues (8), but still much lower than the alternative pathway values. Thus, once the end members have been established, use of 1802 discrimination techniques allow for the measurement of partitioning of respiratory electron flow between the Cyt and alternative pathways without the complications produced by alternative pathway inhibitors. ACKNOWLEDGMENTS We wish to thank Dr. E. H. Harris and Ms. H. E. Hemmingsen for advice on performing genetic crosses, Dr. P. F. Pedersen for use of his mass spectrometer, Ms. B. Nielsen for processing the isotope samples, and Ms. A. K. Horsey for aid in identifying mutant strains.

LITERATURE CITED 1. Bahr JT, Bonner WD Jr (1973) Cyanide-insensitive respiration. I. The steady states of skunk cabbage spadix and bean hypocotyl mitochondria. J Biol Chem 248: 3441-3445 2. Bingham IJ, Farrar JF (1987) Respiration of barley roots: Assessment of activity of the alternative path using SHAM. Physiol Plant 70: 491-498 3. de Visser R, Blacquiere T (1984) Inhibition and stimulation of root respiration in Pisurm and Plantago by hydroxamate. Plant Physiol 75: 813-817 4. Dry IB, Moore AL, Day DA, Wiskich JT (1989) Regulation of

alternative pathway activity in plant mitochondria: nonlinear relationship between electron flux and the redox poise of the quinone pool. Arch Biochem Biophys 273: 148-157 5. Elrifi IR, Turpin DH (1986) Nitrate and ammonium induced photosynthetic suppression in N-limited Selenastrum minutum. Plant Physiol 81: 273-279 6. Goyal A, Tolbert NE (1989) Variations in the alternative oxidase in Chlamvdomonas grown in air or high CO2. Plant Physiol 89: 958-962

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7. Grant NG, Hommersand MH (1974) The respiratory chain of

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Chliorella proiothecoides. I. Inhibitor responses and cytochrome components of whole cells. Plant Physiol 54: 50-56 Guy RD, Berry JA, Fogel ML, Hoering TC (1989) Differential fractionation of oxygen isotopes by cyanide-resistant and cyanide-sensitive respiration in plants. Planta 177: 483-491 Husic DW, Tolbert NE (1987) Inhibition of glycolate and Dlactate metabolism in a Chlamydomonas reinhardtii mutant deficient in mitochondrial respiration. Proc Natl Acad Sci USA 84: 1555-1559 Lambers H (1982) Cyanide-resistant respiration: A non-phosphorylating electron pathway acting as an energy overflow. Physiol Plant 55: 478-485 Lambers H, Day DA, Azcon-Bieto J (1983) Cyanide-resistant respiration in roots and leaves. Measurements with intact tissues and isolated mitochondria. Physiol Plant 58: 148-154 Moller IM, Berczi A (1986) Salicylhydroxamic acid-stimulated NADH oxidation by purified plasmalemma vesicles from wheat roots. Physiol Plant 68: 67-74

pyrenoidosa. Plant Physiol 49: 775-778 19. Siedow JN, Berthold DA (1986) The alternative oxidase: A cyanide-resistant respiratory pathway in higher plants. Physiol Plant 66: 569-573 20. Spreen Brouwer K, van Valen T, Day DA, Lambers H (1986) Hydroxamate-stimulated 02 uptake in roots of Pisum sativum and Zea mays, mediated by a peroxidase. Plant Physiol 82: 236-240 21. Sueoka N (1960) Mitotic replication of deoxyribonucleic acid in Chiamydomonas reinhardtii. Proc Natl Acd Sci USA 46: 8391 22. Syrett PJ (1951) The effect of cyanide on the respiration and the oxidative assimilation of glucose by Chlorella vulgaris. Ann Bot 15: 473-492 23. van Winkle-Swift KP (1977) Maturation of algal zygotes: Alternate experimental approaches for Chlamydomonas reinhardtii (Chlorophyceae). J Phycol 13: 225-231 24. Webster DA, Hackett DP (1965) Respiratory chain of colorless algae. I. Chlorophyta and Euglenopyta. Plant Physiol 41: 10911100 25. Weger HG, Birch DG, Elrifi IR, Turpin DH (1988) Ammonium assimilation requires mitochondrial respiration in the light. A study with the green alga Selenastrum minutum. Plant Physiol 86: 688-692 26. Williams TG, Turpin DH (1987) The role of external carbonic anhydrase in inorganic carbon acquisition by Chlamydomonas reinhardtii at alkaline pH. Plant Physiol 83: 92-96 27. Wilson SB (1988) The switching of electron flux from the cyanide-insensitive oxidase to the cytochrome pathway in mung bean (Phaseolus aureus L.) mitochondria. Biochem J 249: 301-303 28. Wiseman A, Gillham NW, Boynton JE (1977) Nuclear mutations affecting mitochondrial structure and function in Chiamydomonas. J Cell Biol 73: 56-77

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Corrections Vol. 92: 41-47, 1990

Vol. 92: 543-546, 1990

Dieter Strack and Wiltrud Gross. Properties and Activity Changes of Chlorogenic Acid:Glucaric Acid Caffeoyltransferase from Tomato (Lycopersicon esculentum). Page 44, left column, change "Mr of about 513,000 (line 10) and "153 kD" (line 11) to "Mr of about 51,000" and "51 kD," respectively.

Keerti S. Rathore, Kevin B. Hotary, and Kenneth R. Robinson. A Two-Dimensional Vibrating Probe Study of Currents around Lateral Roots of Raphanus sativus Developing in Culture. Page 543, right column, second line from bottom, change ". . . silicone cone grease" to "silicone grease." Page 544, Table I, lower right straddle heading, change "Current density (uA -cm-')" to "Current density (,uA-cm-A)."