L. cv Xanthi-nc was analyzed in liquid suspension cultures using 02 uptake and calorimetric measurements. In young cultures(4-8 d after transfer), cyanide ...
Plant Physiol. (1992) 100, 1921-1926 0032-0889/92/100/1921 /06/$01 .00/0
Received for publication May 11, 1992 Accepted August 7, 1992
Salicylic Acid Induces Cyanide-Resistant Respiration in Tobacco Cell-Suspension Cultures' Yoram Kapulnik, Nasser Yalpani, and Ilya Raskin* AgBiotech Center, Cook College, Rutgers University, P.O. Box 231, New Brunswick, New Jersey 08903-0231 (N.Y., I.R.); and Institute of Field and Garden Crops, Agricultural Research Organization, Volcani Center, Bet Dagan 50250, Israel (Y.K.) ABSTRACT
day of blooming of Sauromatum guttatum Schott, a member of the Arum lily family, a dramatic increase in alternative pathway activity induces heat production in the thermogenic organs (12). Thermogenesis is accompanied by the synthesis of a new cluster of mitochondrial alternative oxidase proteins (4) and the expression of nuclear-encoded alternative oxidase genes (18). In thermogenic plants, the alternative pathwaygenerated heat is used to volatilize insect attractants from the inflorescence (12), however, the function of the alternative pathway in nonovertly thermogenic species is not understood. Alternative pathway activity is affected by developmental stage, tissue type, physiological status, and environmental conditions (11). SA2 was recently identified as the natural trigger of heat production in thermogenic plants (16). A transient, nearly 100-fold increase in the levels of SA in thermogenic tissues results in higher altemative pathway activity and temperature increases of up to 120C (16, 17). Additional confirmation of the involvement of SA in the induction of altemative pathway comes from recent work by Rhoads and McIntosh (18), who demonstrated that the expression of a nuclear gene encoding the altemative oxidase protein in Sauromatum guttatum is induced by SA application. In the unicellular algae Chlamydomonas reinhardtii, altemative pathway capacity can be increased by incubation of cells with 1 mm SA (6). So far, an involvement of SA in the regulation of the altemative pathway in nonovertly thermogenic plants has not been demonstrated. An aging-related increase in the capacity of altemative pathway has been observed in cell-suspension cultures and mitochondria of Nicotiana glutinosa L. (8), soybean (Glycine max) (13), sycamore (Acer pseudoplatanus) (21), and petunia (Petunia hybrida) (1). In N. glutinosa, this phenomenon was thought to result from an abundance of carbohydrates relative to demand (8), supporting the 'overflow' hypothesis (9). The overflow hypothesis stipulates that altemative pathway becomes engaged only when the Cyt pathway is saturated. A more recent hypothesis suggests that in nonthermogenic plants, the altemative pathway is activated by a high redox potential of the ubiquinone pool (2, 3). Greater redox potential may result from an increase in substrate flux to the mitochondria and/or surge in the cytosolic phosphorylation potential. Specific inhibitors of the alternative pathway such
Cyanide-resistant, alternative respiration in Nicotiana tabacum L. cv Xanthi-nc was analyzed in liquid suspension cultures using 02 uptake and calorimetric measurements. In young cultures (4-8 d after transfer), cyanide inhibited 02 uptake by up to 40% as compared to controls. Application of 20 uM salicylic acid (SA) to young cells increased cyanide-resistant 02 uptake within 2 h. Development of KCN resistance did not affect total 02 uptake, but was accompanied by a 60% increase in the rate of heat evolution from cells as measured by calorimetry. This stimulation of heat evolution by SA was not significantly affected by 1 mm cyanide, but was reduced by 10 mm salicylhydroxamic acid (SHAM), an inhibitor of cyanide-resistant respiration. Treatment of SA-induced or uninduced cells with a combination of cyanide and SHAM blocked most of the 02 consumption and heat evolution. Fifty percent of the applied SA was taken up within 10 min, with most of the intracellular SA metabolized in 2 h. 2,6-Dihydroxybenzoic and 4-hydroxybenzoic acids also induced cyanide-resistant respiration. These data indicate that in tobacco cell-suspension culture, SA induces the activity and the capacity of cyanide-resistant respiration without affecting the capacity of the cytochrome c respiration pathway.
The cyanide-insensitive, altemative respiratory electron transport system is found in mitochondria of vascular plants, fems, mosses, algae, fungi, and protists (7, 10, 19). It is a
nonphosphorylating process that diverges from the Cyt respiratory chain at the level of ubiquinone and dissipates most of the chemical energy of respiratory substrates as heat. We have recently shown that this heat can be detected by calorimetry and used to estimate alternative pathway activity in vivo (15). In this report, we differentiate between the capacity of the altemative pathway, which defines the maximum 02 uptake possible with full utilization of the electron flow potential of this pathway (measured in the presence of KCN), and the activity of the altemative pathway, which is the actual engagement of the pathway in situ. The function and regulation of the altemative pathway has been studied most extensively in thermogenic plants. At the 1 Financial support was from the Division of Energy Biosciences of the U.S. Department of Energy, New Jersey Commission for Science and Technology, and U.S. Department of Agriculture/Competitive Research Grants Office.
'Abbreviations: SA, salicylic acid; SHAM, salicylhydroxamic acid; MS, Murashige Skoog. 1921
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SHAM were found to be effective in cell cultures as well in mitochondria isolated from cultured cells (8). This study describes the induction of the alternative pathway activity and capacity in tobacco cell-suspension culture by SA and its analogs. This is the first such report in nonthermogenic vascular plants. Newly developed calorimetric methods (15) allowed us to study the alternative pathway in tobacco cells in more detail than was previously possible.
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MATERIALS AND METHODS
Culture of Tobacco
Nicotiana tabacum cv Xanthi-nc calli were generated from leaf segments using a MU-1 medium (20). A cell-suspension culture was established by transferring 5 g of callus tissue to flasks containing 30 mL of liquid basal MS medium (14) supplemented with 2 4g/L 2,4-D. Flasks were incubated in the dark at 250C on an orbital shaker at 110 rpm. Cultures were subcultured every 3 weeks by 5-fold dilution of the suspensions in fresh medium. For experimental use, cells were harvested by gentle filtration and resuspended in sterile medium (pH 5.9). In all experiments, washed cells were resuspended in 10 mL of fresh medium and incubated in a 25-mL Erlenmeyer flask as above. Protein was determined using Bio-Rad protein assay dye reagent after hydrolysis in 1.2 M NaOH at 90°C for 1.5 h. Respiration Assays
Oxygen uptake was measured with a Rank Oxygen electrode (Digital Oxygen System, model 10, Rank Brothers, Cambridge, UK) at 250C in a total volume of 2.0 mL. Cultures were mixed (1:1) with freshly aerated MS medium before conducting the measurements. The capacity of the Cyt respiratory pathway was studied following application of 10 mm SHAM to the 02 electrode chamber. The capacity of the alternative pathway was measured following addition of 1 mM KCN. Respiration inhibitors were prepared as previously described (15). SA and its analogs were prepared as 1 mm stock solutions in MS medium containing 0.1% (v/v) methanol. The final methanol concentration in the assay mixture was less than 0.02% (v/v). We verified that at this concentration, methanol caused no significant changes in cell respiration. Calorimetric Measurements
Measurements of the rate of heat evolution were performed using a differential scanning microcalorimeter (model 7707, Hart Scientific, Pleasant Grove, UT). Rates of heat evolution (W = J -s- ) were measured in 1 -mL ampules (10 mm diameter). Cell layers of uniform thickness were obtained by collecting cell suspensions on glass fiber filter discs (7 mm diameter, Whatman GF/C). Each disc was placed in a separate ampule containing 100 ,uL of MS medium. All calorimetric measurements were performed at 200C. The inhibitors, SHAM and KCN, were added to the cells 15 min before cells were collected on filter discs.
SA Uptake and Metabolism
Uptake experiments were started with the addition of 0.6 ,uCi ["4C-ring labeled]SA (7.6 mCi/mmol) (Sigma) to washed cells (0.6 g fresh weight) to a final volume of 10.5 mL. Unlabeled SA was used for metabolism experiments. Incubation was terminated by collecting the cells on a glass fiber filter disc (2.5 cm diameter, Whatman GF/F) and immediately rinsing them three times with 10 mL of ice-cold MS medium. Filtration and washing were completed within 20 s. Triplicate samples were used for each time point. Cells were ground in 0.5 mL of 90% methanol using a Ten Broeck tissue grinder (Fisher Scientific). Free and conjugated SA in the homogenate was extracted and quantified as previously described (5). RESULTS
During the first 34 d after transfer, the maximum rate of 02 consumption by tobacco cell suspensions was observed at day 8, and thereafter gradually declined by 60% over the following 5 weeks (Fig. 1). The effect of culture age on the capacity of the cyanide-resistant respiration pathway was analyzed. Cyanide (1 mM) inhibited 02 uptake at the early logarithmic stage of culture growth (4-8 d) with a maximum inhibition of 38% observed on day 4 (Fig. 1). At earlier or later stages of culture growth, 02 uptake was not significantly affected by cyanide. Subcultured cells still retained almost complete KCN resistance, typical of the older cultures, for 2 d after transfer to the fresh medium. Therefore, all subsequent experiments were conducted with 4- to 7-d-old cell cultures. The effect of exogenous applications of SA on respiration was determined at concentrations ranging from 0.2 to 200 ,OM. Incubating cells with up to 20 uM SA for 90 min did not affect 02 consumption significantly (Table I). However, a significant increase in heat evolution was detected in cultures incubated with 2 or 20 /.M SA (Table I). The fact that SA increased the ratio of heat evolution per mole of 02 consumed 250 *
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34
Figure 1. Effect of culture age on respiration. Rate of 02 uptake in the absence (O) or presence (-) of 1 mm KCN. Values are the mean of four replicated treatments (±SE).
SALICYLIC ACID INDUCES ALTERNATIVE RESPIRATION
Table I. Heat Evolution and Oxygen Consumption of Tobacco Cell Suspensions in the Presence of Different SA Concentrations Seven-day-old cell cultures were incubated with SA for 1.5 h (W = J/s). Values are the mean of three replicates (±SE). SA Concentration
Rate of Heat Evolution
Rate of 02
Uptake
Heat Evolution per 02 Consumed
MLM
protin MW/mg protein
AMOI 02/M9 h protein.
J/Mol 02
Am
gWlmg
715 ± 40 31 ± 4 156 ± 12 0 933 ± 108a 0.2 42 ± 4 162 ± 19 1025 ± 68a 47 ± 2a 165 ± 14 2.0 1165 ± 166a 170 ± 21 55 ± 8a 20 17 ± 10 765 ± 123 200 80 ± 12a a < P Student's t test. SA effect significant at 0.05 using
suggests that the increased rate of heat evolution results from greater altemative pathway activity (15). At 200 ,UM SA, both 02 consumption and heat evolution were significantly reduced. Induction of alternative pathway by SA was transient. Eight hours after cells were exposed to 20 ,uM SA, the levels of cyanide-resistant 02 uptake and heat evolution in treated cells returned to that of the control cells (data not shown).
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Inhibitors of the alternative pathway (SHAM) and the Cyt pathway (KCN) were used to determine the effect of SA on the capacity of these pathways and to confirm that the SAinduced heat evolution is a consequence of greater alternative pathway activity. Neither KCN nor SHAM had a significant effect on the 02 uptake rates of SA-induced cells, but uninduced cells treated with 1 mm KCN showed a 39% reduction in respiration rate (Fig. 2A). However, a 2-h incubation with 20 AM SA increased the rate of heat evolution by 60% compared to uninduced controls (Fig. 2B). SA-induced heat increase was sensitive to 10 mM SHAM, which inhibited heat production by 47%, completely reversing the thermogenic effect of SA (Fig. 2B). SA-induced heat production was fully resistant to KCN. In contrast, heat evolution from uninduced cells was inhibited 38% by KCN, but was not significantly affected by SHAM. Treatment of SA-induced or uninduced cells with a combination of both KCN and SHAM blocked the 02 uptake by 90 and 89%, respectively, and the rate of heat evolution by 85 and 76%, respectively, as compared to the controls (Fig. 2), indicating that each inhibitor was effective at the concentration used. To determine the speed of the alternative pathway induction by SA, the sensitivity of 02 uptake to cyanide in SAtreated and control cells was monitored every hour. A significant reduction of sensitivity to cyanide was observed after 1.5 h of incubation with 20 ,tM SA (Fig. 3). After 4 h of incubation with SA, cyanide-resistant 02 uptake increased from 69 to 98% of total 02 uptake. In the absence of respiratory inhibitors, SA did not significantly affect 02 uptake by tobacco cells (Fig. 3). "4C-labeled SA was added to the culture medium to measure the kinetics of SA uptake by tobacco cells. Exogenous SA corresponding to a concentration of 7.5 ,uM was rapidly taken up, with cellular levels reaching a plateau after 30 min (Fig.
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Figure 2. The effect of 10 mm SHAM and 1 mm KCN on the rate of 02 uptake (A) and heat evolution (B) of SA-induced (-) and uninduced (O) cells. Five-day-old cells were incubated with 20 ,M SA for 2 h prior to the application of respiration inhibitors. Values are the mean of three replicated treatments (+SE).
Figure 3. The effect of incubation time on the percentage of total respiration sensitive to 1 mM KCN for 5-d-old cells treated with (0) and without (0) 20 ylM SA. At time 0, 02 uptake in SA-induced and uninduced cells was 94 ± 15 and 73 ± 8 Amol 02/mg protein-h, respectively. After 5 h, it was 96 ± 7 and 100 ± 12 ,umol 02/mg protein-h, respectively. Data given are the mean of three experiments, each conducted with three replicated treatments.
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4). Within 10 min, 50% of the applied SA could be recovered with the cells. Thus, SA uptake is rapid enough to account for induction of altemative pathway activity that occurs within 1.5 h of incubation with SA (Fig. 3). The ability of the cells to metabolize applied SA was studied in a similar experiment in which the concentration of free and conjugated SA in the cells was measured. The cellular SA levels declined after 1 h of incubation with 20 tLM SA, and reached background levels by 4 h (Fig. 5). Levels of a conjugated form of SA, assumed to be j-glucosylsalicylic acid by analogy with tobacco plants (5), showed a small increase after 2 h of incubation and remained constant until 4 h (Fig. 5). The decline in the endogenous levels of SA preceded the loss of KCN resistance and thermogenicity observed 8 h after the addition of SA. Four benzoic acids structurally related to SA were compared to SA for their ability to induce the capacity of alternative pathway in 5-d-old cultures. At 20 tM, 2,6 dihydroxybenzoic and 4-hydroxybenzoic acid were as effective as SA in reducing KCN sensitivity of the cultures (Table II). 2Fluorobenzoic acid stimulated total 02 uptake by 54% compared to untreated controls without significantly affecting the capacity of the alternative pathway. 2-Fluorobenzoic and 2,6difluorobenzoic acids did not significantly affect the KCNresistant 02 uptake by tobacco cells.
DISCUSSION In agreement with previous reports obtained with other plant species, we have observed that respiration in N. tabacum cell suspensions was most sensitive to KCN application at the early stages of the culture growth cycle (Fig. 1). Larger KCN sensitivity correlated with the highest rates of total respiration observed in 4- and 8-d-old cultures. The activity of alternative pathway in aged potato slices
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KAPU LN IK ET AL.
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Figure 5. The effect of incubation time on the levels of SA (0) and putative glucosylsalicylic acid (0) in tobacco cells fed 20 gM SA. Values are the mean of three replicated treatments (±SE).
can be correlated with calorimetric measurements of the rate of heat evolution (15). The alternative pathway produces more heat per electron transferred to 02 than the Cyt pathway. Therefore, simultaneous determinations of 02 consumption and heat production in the presence or absence of KCN and/or SHAM provides an opportunity to study the effect of SA on the capacities and activities of both pathways. Our results show that SA can make 4- to 7-d-old tobacco cell cultures more thermogenic and more resistant to KCN. In addition, heat evolution from SA-treated cells was more sensitive to SHAM than the heat evolution from the untreated cells (Figs. 2 and 3, Table I). Alternative pathway activity in tobacco cells was increased by concentrations of SA as low as 0.2 ytM, which correspond to 550 ng SA/g fresh weight of cells (Table I). Maximum response was observed at 20 jLM SA. SA at 200 gM seemed to be toxic to cells. Induction of alternative pathway in tobacco cells was greatest 3 to 5 h from the addition of SA (Fig. 3). Our data indicate that SA produces the following effects in tobacco cells: (a) SA increases the capacity of the alternative pathway judging from the effects of KCN on heat production and 02 consumption in the induced and uninduced cells (Fig. 2). (b) SA does not change the capacity of the Cyt pathway, because SHAM-resistant 02 uptake and heat evolution were the same in the presence and absence of SA (Fig. 2). (c) SA, without changing the number of electrons moving through the respiratory chain (Fig. 2A), redirects their flow from the Cyt pathway to the alternative pathway. The SA-produced increase in the ratio of heat evolution per mole of 02 consumed supports this hypothesis (Table I). (d) Because SA only produces a change in heat evolution and not in 02 consumption, the capacity of the Cyt pathway is not fully utilized in SA-treated cells, whereas both the alternative pathway activity and capacities are greater (Fig. 2, Table I). This conclusion suggests that in our experimental system, the 'overflow' hypothesis that the alternative pathway becomes engaged only when the Cyt pathway is saturated (9) may not apply.
SALICYLIC ACID INDUCES ALTERNATIVE RESPIRATION
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Table II. Cyanide-Sensitive Oxygen Uptake in the Presence of SA or Its Analogs Seven-day-old cell cultures were incubated with a 20-,M solution of test compound for 2.5 h. Values are the mean of three replicates (±SE). Rate of 02 Uptake
Compound -KCN
+KCN
Amol 02/mg protein. h 147 ± 13 Control 92 ± 11 182 ± 16 2-Hydroxybenzoic acid (SA) 156 ± 15 121 ± 11 4-Hydroxybenzoicacid 119 ± 12 125 ± 14 132 ± 12 2,6-Dihydroxybenzoic acid 87 ± 10 155 ± 20 2,6-Difluorobenzoic acid 227 ± 32 73 ± 11 2-Fluorobenzoic acid a Significant effect of KCN at P < 0.05 following arcsin transformation.
The possibility that the alternative pathway may function even when the Cyt pathway is not saturated was recently suggested by Dry et al. (3), who demonstrated that in soybean mitochondria, the alternative pathway becomes engaged when 35 to 40% of the ubiquinone pool is reduced. In contrast to mitochondria from thermogenic plants, soybean mitochondria showed marked nonlinearity between the levels of the alternative pathway activity and the redox potential of the ubiquinone pool (2, 3). Presently, we do not know whether SA exerts its effect on the alternative pathway by changing the redox potential of the ubiquinone pool. It is possible that SA acts through other mechanisms, e.g. higher expression of the altemative oxidase gene. SA has a pKa of 2.98 and is present mainly in the charged, dissociated form at the near neutral pH of the culture medium. Because cell membranes provide a good barrier to the diffusion of charged molecules, the rate of SA uptake by tobacco cells (Fig. 4) may indicate the presence of carriers on the cell surface. Movement of SA into tobacco cells precedes the induction of the alternative pathway, whereas metabolism depletes intracellular SA before the alternative pathway returns to levels characteristic of uninduced cells (Fig. 5). The disappearance of SA from the treated cells after 3 h may explain the transient nature of the SA-induced increase in the alternative pathway activity, which lasts approximately 8 h. In N. tabacum (cv Xanthi-nc) plants, which were also the source of our suspension cultures, exogenously applied SA is largely metabolized to form f3-O-D-glucosylsalicylic acid (5). In the cell suspensions, conjugated SA was detected only at low levels, and its accumulation did not account for the rapid disappearance of SA from cells (Fig. 5). It is possible that conjugation is not the major path of metabolism of SA in tobacco-suspension cultures or that the conjugate has a high turnover rate in this cell line. As had been observed in voodoo lilies (17), in tobacco cells, 2,6-dihydroxybenzoic acid was as effective as SA in stimulating the altemative pathway (Table II). However, in contrast to thermogenic plants, 4-hydroxybenzoic acid was also capable of inducing the alternative pathway in tobacco cells (Table II). Although exogenously supplied SA induces alternative pathway activity and capacity, the changes in cyanide-sensitive respiration observed during the cell growth
Cyanide-Resistant
02 Uptake % of total
63a 117 101 95 56a 32a
cycle (Fig. 1) did not correlate with the endogenous SA levels in the cells, which remained near 45 ng/g fresh weight for cultures of different ages. It is possible that the changes in the alternative pathway in aging tobacco cells are regulated developmentally or are caused by another regulatory molecule. Our findings demonstrate the alternative pathway and heat-inducing effects of SA in nonthermogenic vascular plants. It is also the first indication of the SA enhancement of the alternative pathway in vegetative plant tissues. Therefore, the difference between thermogenic and not 'overtly' thermogenic plants with regard to the SA effects on the alternative pathway are quantitative rather than qualitative. ACKNOWLEDGMENTS We are grateful to Dr. Lee McIntosh for useful discussions and to Dr. Peter Day for reviewing the manuscript.
LITERATURE CITED 1. Connett MB, Hanson MR (1990) Differential mitochondrial electron transport through the cyanide-sensitive and cyanideinsensitive pathways in isonuclear lines of cytoplasmic male sterile, male fertile, and restored Petunia. Plant Physiol 93: 1634-1640 2. Day DA, Dry IB, Soole KL, Wiskich JT, Moore AL (1991) Regulation of alternative pathway activity in plant mitochondria. Deviations from Q-pool behavior during oxidation of NADH and quinols. Plant Physiol 95: 948-953 3. Dry IB, Moore AQL, 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 4. Elthon TE, Nickels RL, Mclntosh L (1989) Mitochondrial events during the development of thermogenesis in Sauromatum guttatum (Schott). Planta 180: 82-89 5. Enyedi AJ, Yalpani N, Silverman P. Raskin 1 (1992) Localization, conjugation, and function of salicylic acid in tobacco during the hypersensitive reaction to tobacco mosaic virus. Proc Natl Acad Sci USA 89: 2480-2484 6. Goyal A, Tolbert NE (1989) Variations in the alternative oxidase in Chlamydomonas grown in air or high CO2. Plant Physiol 89: 958-962 7. Henry MF, Nyns EJ (1975) Cyanide-insensitive respiration, an alternative pathway. Subcell Biochem 4: 1-65 8. Horn ME, Mertz D (1982) Cyanide-resistant respiration in sus-
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pension cultured cells of Nicotiana glutinosa L. Plant Physiol 69: 1439-1443 Lambers H (1980) The physiological significance of cyanideresistant respiration in higher plants. Plant Cell Environ 3: 293-302 Lance C, Chauveau M, Dizengremel P (1985) The cyanideresistant pathway of plant mitochondria. In R Douce, DA Day, eds, Higher Plant Cell Respiration. Encyclopedia of Plant Physiology. New Series, Vol 18. Springer Verlag, Berlin, pp 202-247 Laties GG (1982) The cyanide-resistant, alternative path in higher plant respiration. Annu Rev Plant Physiol 33: 519-555 Meeuse BJD, Raskin I (1988) Sexual reproduction in the arum lily family, with emphasis on thermogenicity. Sex Plant Reprod 1: 3-15 Miller CO (1979) Cytokinin inhibition of respiration by cells and mitochondria of soybean, Glycine max (L.) Merril. Planta 146: 503-511 Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15: 473-497
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15. Ordentlich A, Linzer RA, Raskin 1 (1991) Alternative respiration and heat evolution in plants. Plant Physiol 97: 1545-1550 16. Raskin I, Ehman A, Melander WR, Meeuse BJD (1987) Salicylic acid: a natural inducer of heat production in Arum lilies. Science 237: 1601-1602 17. Raskin I, Turner IM, Melander WR (1989) Regulation of heat production in the inflorescences of an Arum lily by endogenous salicylic acid. Proc Natl Acad Sci USA 86: 2214-2218 18. Rhoads DM, McIntosh L (1991) Isolation and characterization of a cDNA clone encoding an alternative oxidase protein of Sauromatum guttatum (Schott). Proc Natl Acad Sci USA 88: 2122-2126 19. Siedow JN, Berthold DA (1986) The alternative oxidase: a cyanide-resistant respiratory pathway in higher plants. Physiol Plant 66: 569-573 20. Uchimiya H, Murashige T (1976) Influence of the nutrient medium on the recovery of dividing cells from tobacco protoplasts. Plant Physiol 57: 424-429 21. Wilson SB (1971) Studies on the growth in culture of plant cells. XIII. Properties of mitochondria isolated from batch cultures of Acer pseudoplatanus cells. J Exp Bot 22: 725-734
