Department of Biology, Queen's University, Kingston, Ontario, Canada K7L 3N6 (H.G. W., ... and 1802 discrimination experiments indicated that respiratory.
Received for publication May 11, 1990 Accepted June 28, 1990
Plant Physiol. (1990) 94, 1131-1136 0032-0889/90/94/1131/06/$01 .00/0
Cytochrome and Alternative Pathway Respiration during Transient Ammonium Assimilation by N-Limited Chiamydomonas reinhardtii' Harold G. Weger, Antony R. Chadderton, Min Lin, Robert D. Guy, and David H. Turpin* Department of Biology, Queen's University, Kingston, Ontario, Canada K7L 3N6 (H.G. W., A.R.C., M.L., D.H. T.); and Department of Forest Sciences, University of British Columbia, Vancouver, British Columbia, Canada V6T 1W5 (R.D.G.) TCA cycle reductant (NADH, FADH2). One possible mechanism for this oxidation is via the mitochondrial electron transport chain (17, 18), utilizing either the phosphorylating Cyt pathway or the nonphosphorylating alternative pathway. While the capacity for alternative pathway respiration is widespread among plants and algae, the in vivo role is poorly understood. One hypothesis is that operation of the alternative pathway allows oxidation of TCA cycle reductant during adenylate restriction of Cyt pathway activity, thus maintaining TCA cycle carbon flow for provision of biosynthetic intermediates (2, 12, 14). This may be especially important in the light, when photophosphorylation may meet cellular ATP demands. In the present study, we examine the role of the Cyt and alternative electron transport chains in support of respiratory carbon flow to ammonium assimilation by N-limited cells of a green alga, Chlamydomonas reinhardtii. This organism displays substantial capacities for both Cyt and alternative pathway activity (19). Use of inhibitors of the alternative and Cyt pathways, as well as examination of the discrimination against 802, permits the conclusion that enhanced rates of respiratory carbon flow during amino acid synthesis are supported by increased activity of the Cyt pathway. Under conditions where Cyt pathway activity is absent (i.e. in a Cyt oxidase-deficient mutant, or in the presence of cyanide), the alternative pathway may support biosynthetic carbon flow.
ABSTRACT Mass spectrometric analysis of gas exchange in light and dark by N-limited cells of Chiamydomonas reinhardtii indicated that ammonium assimilation was accompanied by an increase in respiratory carbon flow to provide carbon skeletons for amino acid synthesis. Tricarboxylic acid (TCA) cycle carbon flow was maintained by the oxidation of TCA cycle reductant via the mitochondrial electron transport chain. In wild-type cells, inhibitor studies and 1802 discrimination experiments indicated that respiratory electron flow was mediated entirely via the cytochrome pathway in both the light and dark, despite a large capacity for the alternative pathway. In a cytochrome oxidase deficient mutant, or in wild-type cells in the presence of cyanide, the altemative pathway could support the increase in TCA cycle carbon flow. These different mechanisms of oxidation of TCA cycle reductant were reflected by the much greater SHAM sensitivity of ammonium assimilation by cytochrome oxidase-deficient cells as compared to wild type.
Ammonium assimilation by higher plants and microalgae predominately via the glutamine synthetase/glutamine-2-oxo-glutarate amino-transferase pathway (1, 9). Operation of this pathway for net glutamate production requires the provision of aKG2, while the subsequent biosynthesis of other amino acids by transamination requires TCA cycle and glycolytic intermediates (9). Nitrogen assimilation also requires ATP and reducing power. In darkness, the requirements for carbon skeletons, ATP, and reducing power, can be met by mitochondrial respiration, whereas in the light ATP and reducing power may also be supplied by the light reactions of photosynthesis. Ammonium assimilation results in increased TCA cycle carbon flow to provide the carbon skeletons necessary for amino acid synthesis (10, 16). Maintenance of TCA cycle carbon flow is dependent upon the continued oxidation of occurs
MATERIALS AND METHODS Algal Strains and Culture Methods
Wild type Chlamydomonas reinhardtii R34 and Cyt ox- C. reinhardtii R4 were obtained via crosses using existing strains (19). Respiratory capabilities of both strains have been assessed using both inhibitor titrations and discrimination against 1802 (19). C. reinhardtii R34 was shown to possess a substantial capacity for both Cyt and alternative pathway respiration, while in C. reinhardtii R4 Cyt pathway capacity was undectable ( 19). Cells were grown in N-limited chemostats (4), using a modification of Surzycki's medium (20). Cultures were bubbled with air enriched with 2.5% CO2. Dissolved inorganic carbon in the culture was approximately 2.5 mm. Nitrogen was supplied as 1 mm NH4Cl, at a dilution rate of 0.3 d-'. Temperature was maintained at 30°C.
'
Supported by the Natural Sciences and Engineering Research Council of Canada. H.G.W. acknowledges an Ontario Graduate Scholarship. 2 Abbreviations: aKG, a-ketoglutarate; TCA, tricarboxylic acid; SHAM, salicylhydroxamic acid; Cyt ox-, Cyt oxidase deficient; FADH2, reduced flavin adenine dinucleotide; DIC, dissolved inorganic carbon. 1131
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Use of Isotope Discrimination
Discrimination against 1802 by mitochondrial respiration calculated from changes in the stable oxygen isotope composition of the medium with time (7, 19). Samples were analyzed for stable oxygen isotope ratios via a VG Isotech (Middlewich, England) Prism triple-collecting mass spectrometer. Due to the production of 02 in the light from water photolysis, the isotopic discrimination method of measuring respiratory electron partitioning is not appropriate under photosynthetic conditions. Consequently, this technique was only used to evaluate partitioning of respiratory electron flow between Cyt and alternative pathways in the dark. was
Inhibitor Effects on Gas Exchange
Respiratory electron flow through the alternative pathway (in both light and dark) was also evaluated by the SHAM inhibition of 02 consumption. Concomitant TCA cycle CO2 release was also recorded. On-line gas exchange was measured using a membrane inlet-equipped VG Gas Analysis (Middlewich, England) MM 14-80SC mass spectrometer (17). Inorganic carbon was added as 99% ['3C]Na2CO3 to a concentration of 2 mm, sufficient to inhibit photorespiration. 1802 was added as a bubble to the algal suspension; the bubble was removed prior to the experiment. Total [02] (1602 + 1802) was never allowed to reach more than 1 10% air saturation. Illumination, when provided, was at 250 ,E m-2 s-' from a 300 W projector lamp (General Electric). Gas exchange rates were calculated as previously described (16, 18). Isotopes were obtained from Merck, Sharpe & Dohme (Canada). One potential problem with the use of SHAM in assessing alternative pathway engagement in the light is that high SHAM concentrations may result in a partial inhibition of photosynthesis, possibly due to effects on CO2 transport (6). Thus, SHAM concentrations used with each strain were optimized to yield maximal inhibition of alternative pathway activity (measured in the presence of KCN) with a minimal effect on gross 02 evolution (measured by mass spectrometry in the absence of KCN). Based on these preliminary experiments SHAM was added to a concentration of 1 mm to Cyt ox- cells, and 2 mm to wild-type cells. The SHAM solution was prepared fresh daily as a M stock in methoxyethanol. KCN was added to a concentration of mm, which was found to be saturating for the inhibition of Cyt pathway activity (19). Since KCN is a potent inhibitor of photosynthesis, its use in the light was restricted to the end of experiments, allowing assessment of residual respiration. Other Measurements
Ammonium assimilation was measured as disappearance from a cell suspension in a water-jacketted cuvette (18). Unfiltered samples (2.5 mL) were combined with 25 ,uL of 10 N NaOH and mixed vigorously to convert NH4+ to NH3. The total NH3 concentration was then measured with an NH3 electrode. As this treatment releases internal NH4+, the rate of NH4+ disappearance represents the rate of NH4' assimilation. Chl was measured spectrophotometrically following extraction in 90% acetone (8).
Plant Physiol. Vol. 94, 1990
RESULTS Oxygen Isotope Discrimination Previous work has established values for the 1802 discrimination associated with Cyt and alternative pathway-mediated 02 consumption in wild type Chlamydomonas reinhardtii cells (Table I). These values were determined by measuring the 1802 discrimination in the presence of KCN for alternative pathway activity, and in the presence of SHAM for Cyt pathway activity (19). Under control conditions in the dark (i.e. absence of inhibitors or ammonium) the 1802 discrimination associated with respiratory 02 consumption was completely consistent with operation of only the Cyt pathway in wild-type cells, and only the alternative pathway in Cyt oxcells (19). These results agreed with the inhibitor data presented in the same study. In the present study we measured the 1802 discrimination associated with respiratory 02 consumption during dark ammonium assimilation by N-limited wild-type cells (Table I), which was accompanied by a marked stimulation of mitochondrial 02 consumption (Fig. 1). The '"02 discrimination associated with this increased respiratory 02 consumption was the same as that previously reported for the Cyt pathway (Table I), indicating that 02 consumption was mediated entirely via that pathway during dark ammonium assimilation. Effects of Inhibitors on Mitochondrial Respiration and Photosynthesis SHAM concentrations used for both algal strains were optimized to produce maximal inhibition of alternative pathway activity while minimizing inhibition of photosynthetic 02 evolution. Mitochondrial 02 consumption by wild-type cells was only slightly affected by 2 mM SHAM in the absence of KCN in light or dark (Table II). This SHAM concentration resulted in approximately a 20% decrease in gross photosynthetic 02 evolution, and a similar decrease in CO2 fixation (Table III). Subsequent addition of KCN resulted in a 70% decrease in mitochondrial 02 consumption (Table II). Residual respiration (02 consumption) under these conditions was 30% of control. This could be reduced to less than 10% by using higher SHAM concentrations (up to 40 mM; see ref. 19); however, gross photosynthetic 02 evolution was also greatly reduced. Addition of KCN alone resulted in a 30% stimulation of 02 consumption in the dark (Table II).
Table I. Discrimination Against 1802 by Dark Respiration in WildType Cells Discrimination in the presence of KCN is due to 02 consumption by the alternative pathway, whereas discrimination in the presence of SHAM is due to discrimination by the Cyt pathway. Treatment
Wild type (R34) +KCNa +SHAMa
+NH4+ a
From Weger et al. (19).
Discrimination %o + SE (n)
25.46 ± 0.18 (6) 20.02 ± 0.24 (7) 20.70 ± 0.54 (7)
RESPIRATION AND AMMONIUM ASSIMILATION
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Table II. Cyt and Alternative Pathway Respiration by Wild-Type and Cyt ox- Cells as a Percentage of Control KCN was added to a concentration of 1 mm. SHAM was added to 2 mm in wild-type cells and to 1 mm in Cyt ox cells. Control 02 consumption in the dark was 139 and 175 ,qmol2-* mg-1 Chl * h-1 for wild-type and Cyt ox- cells, respectively, and 138 ,mol 2* mg-1 Chl h-1 for both strains in the light. 02 consumption was measured via mass spectrometry. Percent Control 02 Consumption Treatment Wild type (R34) Cyt ox- (R4) % ± SE (n)
Dark Control +SHAM +SHAM +KCN +KCN Light Control +SHAM +SHAM +KCN
0
2
4
6
8
10 12 14 16 18 20 22
TIME (min) Figure 1. Mitochondrial 02 consumption (0) and TCA cycle C02 release (0) in the dark by (A) C. reinhardtii R34 (wild-type) and (B) C. reinhardtii R4 (Cyt ox-) cells. The data have been corrected for the effects of changing isotopic composition and mass spectrometer consumption of gases (16). Rates are expressed as Mmol. mg-1 Chl. h-1. Scale bars represent 100 gM 02 or C02. Representative experiments are shown.
Addition of 1 mm SHAM (in the absence of KCN) to Cyt ox- (R4) cells resulted in a 70% inhibition of mitochondrial 02 consumption (Table II). This SHAM concentration resulted in a 22% decrease in gross photosynthetic 02 evolution and a slightly larger decrease in photosynthetic CO2 fixation (Table II). A very small additional inhibition of mitochondrial 02 consumption was observed upon subsequent addition of KCN. Residual respiration under these conditions was approximately 30% of control. This could be reduced to 5% at 40 mM SHAM (19), but gross photosynthetic 02 evolution also decreased substantially. Addition of KCN alone did not affect 02 consumption by Cyt ox- cells, but resulted in approximately a 75% decrease in gross 02 evolution by both strains when added in the light (Table III).
Effects of Ammonium Assimilation Respiration
on
Mitochondrial
Addition of 500 ,uM NH4Cl to both wild-type and Cyt oxcells resulted in a large stimulation of mitochondrial respiration in the dark, measured both as increased respiratory 02 consumption and increased TCA cycle CO2 release (Fig. 1, A and B). However, the SHAM sensitivity of this increased respiration differed between the two strains. Addition of SHAM to wild-type cells had little effect on gas exchange (Fig.
100 109 ± 1 (6) 30 ± 2 (6) 131 ± 4 (6)
100 39 ± 3 (6) 31 ± 3 (6) 100 ± 3 (6)
100 96 ± 2 (3) 21 ± 5 (3)
100 30 ± 2 (3) 29 ± 4 (3)
IA), while in Cyt ox- cells SHAM addition resulted in a large decrease in both 02 consumption and CO2 release (Fig. 1B). Subsequent addition of KCN resulted in a large decrease in mitochondrial respiration by wild type cells, but had little effect on Cyt ox- cells. The addition of 1 mM KCN to wild-type cells in the dark stimulated 02 consumption (Table I; Fig. 2). Dark 02 consumption in the presence of KCN (i.e. via the alternative pathway) could be further increased by the addition of ammonium, and inhibited by the subsequent addition of SHAM (Fig. 2). Patterns of TCA cycle CO2 release were consistent with the effects on 02 consumption (Fig. 2). Results similar to those in the dark were observed during photosynthesis (Fig. 3, A and B). Increased mitochondrial respiration resulting from NH4' assimilation was largely SHAM insensitive in wild-type cells and SHAM sensitive in Cyt ox- cells. Conversely, the wild type exhibited substantial KCN-sensitivity, while Cyt ox- cells did not. It should be noted that rates of CO2 release observed during photosynthesis probably underestimate actual TCA cycle activity due to photosynthetic refixation of respired CO2 (18). On the other hand, measurement of respiratory 02 consumption as deter-
Table Ill. Effect of NH4+ and Inhibitors of Mitochondrial Respiration on Photosynthetic Gas Exchange by C. reinhardtii Rates are expressed as percentage of control. Gas exchange was measured via mass spectrometry. Treatment 02 Evolution CO2 Fixation % ± SE (n)
Wild type (R34) +SHAM (2 mM) +KCN Cyt ox- (R4) +SHAM (1 mM) +KCN
81 ± 2 (7) 16 ± 1 (3)
72 ± 9 (7) 18 ± 18 (3)
78 ± 3 (5) 22 ± 4 (5)
83 ± 3 (3) 7 ± 5 (3)
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of biosynthesis when mitochondrial electron transport chain activity is restricted by ADP supply (2, 12, 14). Maintenance of TCA Cycle Carbon Flow during Dark Ammonium Assimilation
TIME (min) Figure 2. Dark mitochondrial 02 consumption (0) and TCA cycle C02 release (0) by C. reinhardtii R34 (wild-type) cells; stimulation of
alternative pathway activity. The data have been corrected for the effects of changing isotopic composition and mass spectrometer consumption of gases. Rates are expressed as Amol mg-' Chl * h-'. Scale bars represent 100 MM 02 or C02. A representative experiment is shown. -
mined from 1802 disappearance tends not to be biased by preferential local consumption of photosynthetically-produced 1602 (5). The 02 consumption observed in the light was due almost exclusively to mitochondrial respiration. High-DIC grown green algae assayed in high DIC exhibit very low rates of photorespiration (21). Furthermore, addition of DCMU to photosynthesizing cells had no short-term effects on gross 02 consumption (data not shown), precluding significant rates of 02 consumption by either the Mehler reaction (02 photore-
The onset of dark ammonium assimilation resulted in a large increase in respiratory CO2 release in wild-type cells, consistent with an increase in glycolytic and TCA cycle carbon flow necessary for the provision ofcarbon skeletons for amino acid biosynthesis (Figs. 1 and 2; cJf ref. 16). Concomitant with the increase in respiratory CO2 release was an increase in mitochondrial 02 consumption. During dark ammonium assimilation by wild-type cells, the addition of SHAM had little effect on respiratory CO2 release or 02 consumption (Fig. lA), and only a 30% inhibition of NH4' assimilation (Table IV). In the Cyt ox- strain, the ammonium assimilation-induced stimulation of respiratory CO2 release and 02 consumption (Fig. lB) was much less than observed in the wild type. This was consistent with the lower rates ofammonium assimilation exhibited by Cyt ox- cells in the dark (Table IV). Both 02 consumption and TCA cycle CO2 release were SHAM-sensitive in Cyt ox- cells. Furthermore, the addition of SHAM completely inhibited dark ammonium assimilation (Table
duction) or photorespiration. Ammonium Assimilation
The ammonium assimilation rate was maximal in the light for both strains (Table IV); however, assimilation by Cyt oxcells was much more sensitive to the presence of SHAM. Darkness only slightly decreased the rate of ammonium assimilation by wild type, but resulted in a 75% decrease in Cyt ox- cells. Addition of SHAM in the dark resulted in approximately a 30% decrease in ammonium assimilation by wild type, and completely inhibited assimilation by the Cyt oxstrain. DISCUSSION The oxidation of mitochondrial reductant (NADH, FADH2) necessary for the maintenance of respiratory carbon flow may occur, in part, by either the Cyt or alternative electron transport chains. Cyt pathway activity is coupled to the synthesis of 3 ATP per electron pair passed to 02, while operation of the alternative pathway yields a maximum of 1 ATP per electron pair (generated via site I [ 11]). There has been considerable discussion as to the role of the alternative pathway in plant metabolism, but a satisfactory picture has yet to emerge (15). One hypothesis is that the alternative pathway serves to maintain TCA cycle carbon flow in support
TIME (min) Figure 3. Mitochondrial 02 consumption (0) and TCA cycle C02 release (0) in the light by (A) C. reinhardtii R34 (wild-type) and (B) C. reinhardtii R4 (Cyt ox-) cells. The data have been corrected for the effects of changing isotopic composition and mass spectrometer consumption of gases. Rates are expressed as ,umol * mg-' Chl h-'. Scale bars represent 75 AM 02 or C02. Representative experiments are shown.
RESPIRATION AND AMMONIUM ASSIMILATION
Table IV. Ammonium Assimilation Rates by Wild-Type and Cyt oxCells under Various Conditions NH4+ Assimilation Rate
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pathway. The addition of SHAM also resulted in approximately a 50% decline in the rate of ammonium assmilation (Table IV).
Treatment Wild type (R34)
Light Light + SHAM Dark Dark + SHAM Dark + CCCP
Cyt ox- (R4) + sE(n) .mg' Chl-h-' pmolNH4 247 ± 19 (3) 314 ± 22 (3) 208 ± 10 (3) 149 ± 7 (3) 205 ± 10 (4) 80 ± 9 (3) 146 ± 7 (5) 0 (3)
0 (2)
0 (2)
IV). The addition of ammonium to KCN-treated wild-type cells also resulted in a stimulation of 02 consumption and CO2 release, which was shown to be SHAM-sensitive (Fig. 2). These results imply that respiratory carbon flow to amino acid synthesis was supported by the Cyt pathway in wild-type cells. Only in the Cyt ox- mutant, or in wild-type cells in the presence of KCN, did the alternative pathway play a role in maintaining carbon flow for biosynthesis. These results do not support the hypothesis that the alternative pathway serves to maintain biosynthetic TCA cycle carbon flow. The use of inhibitors in evaluating the engagement of the alternative pathway may cause artefacts (13). Recently it has been shown that the alternative and Cyt pathways discriminate differentially against 1802 (7, 19). By examining the discrimination against 1802 by whole cell respiration it is possible to determine the partitioning of respiratory electron flow between the Cyt and alternative pathways in the absence of inhibitors. Use of this technique confirms the conclusions reached from the inhibitor-based work: the stimulation of mitochondrial 02 consumption during ammonium assimilation by wild-type cells was mediated entirely via the Cyt pathway, and that the alternative pathway was not engaged (Table I). Not surprisingly, the ammonium-induced stimulation of mitochondrial 02 consumption by Cyt ox- cells was mediated via the alternative pathway. TCA Cycle Carbon Flow during Photosynthetic Ammonium Assimilation
Although the above results have shown that the alternative pathway did not support respiratory carbon flow to amino acid synthesis in the dark unless the Cyt pathway was absent or inhibited, it was conceivable that the alternative pathway could play a role in supporting carbon flow for biosynthesis during photosynthesis. This apparently is not the case in wildtype cells as there was no SHAM-sensitive 02 consumption in the absence of ammonium (Table II) or during the ammonium assimilation-enhanced respiration (Fig. 3). These results imply that even during photosynthesis the Cyt pathway supports biosynthetic TCA cycle carbon flow. In the Cyt ox- strain, NH4' assimilation was nearly fourfold higher in the light than in the dark (Table IV), possibly due to inadequate ATP supply due to the absence of a functional Cyt pathway. Ammonium addition in the light resulted in both increased mitochondrial 02 consumption and TCA cycle CO2 release (Fig. 3B). As expected, this increase was SHAMsensitive, indicating that it was mediated by the alternative
Role of Altemative Pathway Respiration
The results presented in this paper indicate that the alternative pathway can indeed function to maintain TCA cycle carbon flow for the provision of intermediates during biosynthesis when the Cyt pathway is nonfunctional (+KCN, Cyt ox- cells). Other work with Euglena has shown that alternative pathway capacity and activity can be induced by growing cells in the presence of antimycin A, which inhibits Cyt pathway activity (3). However, in the presence of a functional Cyt pathway, we were unable to demonstrate engagement of the alternative pathway under any conditions tested. In wild-type cells, the continued reductant oxidation during ammonium assimilation-enhanced TCA cycle activity was mediated exclusively via Cyt pathway activity. This suggests that, under the conditions tested, the alternative pathway is not involved in the maintenance ofTCA cycle carbon flow for the provision of biosynthetic intermediates in wild-type cells. ACKNOWLEDGMENTS We wish to thank Dr. R. G. Smith for useful discussions. LITERATURE CITED 1. Ahmad I, Hellebust JA (1988) Enzymology of ammonium assimilation in three green flagellates. New Phytol 109: 415-421 2. 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 3. Benichou P, Calvayrac R, Claisse M (1988) Induction by antimycin A of cyanide-resistant respiration in heterotrophic Euglena gracilis: effects on growth, respiration and protein biosynthesis. Planta 175: 23-32 4. Elrifil IR, Turpin DH (1986) Nitrate and ammonium-induced
5. 6. 7.
8.
9. 10.
11.
12.
photosynthetic suppression in N-limited Selenastrum minutum. Plant Physiol 81: 273-279 Good NE, Brown AH (1960) The contribution of endogenous oxygen to the respiration of photosynthesizing Chlorella cells. Biochim Biophys Acta 50: 544-554 Goyal A, Tolbert NE (1990) Salicylhydroxamic acid (SHAM) inhibition of the dissolved inorganic carbon concentrating process in unicellular green algae. Plant Physiol 92: 630-636 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 Jeffrey SW, Humphrey GF (1975) New spectrophotometric equations for determining chlorophylls a, b, cl and c2 in higher plants, algae and natural phytoplankton. Biochem Physiol Pflanzen 167: 19 1-194 Joy KW (1988) Ammonia, glutamine, and asparagine: a carbonnitrogen interface. Can J Bot 66: 2103-2109 Kanazawa T, Kanazawa K, Kirk MR, Bassham JA (1972) Regulatory effects of ammonia on carbon metabolism in Chlorella pyrenoidosa during photosynthesis and respiration. Biochim Biophys Acta 226: 656-659 Lance C, Chauveau M, Dizengremel P (1985) The cyanideresistant pathway of plant mitochondria. In R Douce, DA Day, eds, Encyclopedia of Plant Physiology New Series Vol 18. Springer-Verlag, Berlin, pp 202-247 M0ller IM, Palmer JM (1982) Direct evidence for the presence of a rotenone-resistant NADH-dehydrogenase on the inner surface of the inner membrane of plant mitochondria. Physiol Plant 54: 267-274
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13. M0ller IM, Berczi A, van der Plas LHW, Lambers H (1988) Measurement of the activity and capacity of the alternative pathway in intact plant tissues: identification of problems and possible solutions. Physiol Plant 72: 642-649 14. Palmer JM (1976) The organization and regulation of electron transport in plant mitochondria. Annu Rev Plant Physiol 27: 133-157 15. Siedow JN, Berthold DA (1986) The alternative oxidase: a cyanide-resistant respiratory pathway in higher plants. Physiol Plant 66: 569-573 16. Turpin DH, Elrifi IR, Birch DG, Weger HG, Holmes JJ (1988) Interactions between photosynthesis, respiration, and nitrogen assimilation in microalgae. Can J Bot 66: 2083-2097 17. Weger HG, Turpin DH (1989) Mitochondrial respiration supports N03 and N02 reduction during photosynthesis: interactions between photosynthesis, respiration and N assimilation
18.
19.
20. 21.
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in the N-limited green alga Selenastrum minutum. Plant Physiol 89: 409-415 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 Weger HG, Guy RD, Turpin DH (1990) Cytochrome and alternative pathway respiration in green algae. Measurements using inhibitors and 1802 discrimination. Plant Physiol 93: 356-360 Williams TG, Turpin DH (1987) The role of external carbonic anhydrase in inorganic carbon acquisition by Chiamydomonas reinhardtii at alkaline pH. Plant Physiol 83: 92-96 Yokota A, Iwaki T, Miura K, Wadano A, Kitaoka S (1987) Model for the relationship between C02-concentrating mechanism, CO2 fixation, and glycolate synthesis during photosynthesis in Chlamydomonas reinhardtii. Plant Cell Physiol 28: 1363-1376
