Quantum Yields of CAM Plants Measured by Photosynthetic 02 Exchange' Received for Publication October 22, 1985 and in Revised Form February 7, 1986

Plant Physiol. (1986) 81, 297-300 0032-0889/86/81/0297/04/$0 1.00/0

Short Communication

Quantum Yields of CAM Plants Measured by Photosynthetic 02 Exchange' Received for publication October 22, 1985 and in revised form February 7, 1986

WILLIAM W. ADAMS III, KoJIRO NISHIDA, AND C. BARRY OSMOND* Biological Sciences Center, Desert Research Institute, University ofNevada, P. 0. Box 60220, Reno, Nevada 89506 (W.W.A. III, C.B.O.); Department ofEnvironmental Biology, Research School ofBiological Sciences, Australian National University, Box 475, Canberra 2601 Australia (W.W.A. III, K.N., C.B.O.); and Department ofBiological Sciences, Kanazawa University, Kanazawa 920 Japan (K.N.)

ABSTRACT plants varies with time of day and/or with subtype of deacidification biochemistry. Our data show unexpectedly high and rel- The quantum yield of photosynthetic 02 exchange was measured in atively constant values of quantum yields in these plants, and eight species of leaf succulents representative of both malic enzyme type these are discussed in terms of our current understandings of and phosphoenolpyruvate carboxykinase type CAM plants. Measure- biochemical compartmentation in CAM plants. ments were made at 25°C and CO2 saturation using a leaf disc 02 electrode system, either during or after deacidification. The mean quantum yield was 0.095 1 0.012 (SD) moles 02 per mole quanta, which compared with 0.094 1 0.006 (SD) moles 02 per mole quanta for spinach MATERIALS AND METHODS leaf discs measured under the same conditions. There were no consistent differences in quantum yield between decarboxylation types or during Plants were grown from cuttings or plantlets in sandy loam different phases of CAM metabolism. On the basis of current notions of potting mix in temperature controlled glasshouses (27°C d/ 8°C compartmentation of CAM biochemistry, our observations are inter- night) which transmitted 85% of incident solar radiation. They preted to indicate that CO2 refixation is energetically independent of were irrigated daily with water and three times weekly with half- gluconeogenesis during deacidification. strength Hoagland nutrient solution. Shade plants ofKalanchoe daigremontiana were grown in the same glasshouse under black shade cloth which transmitted 15% of incident solar radiation. Nocturnal acidification was measured by titration of leaf and tissue samples collected at 6:00 PM and 7 to 8:00 AM, using 0.02 N NaoH. Tissue samples were killed in boiling 80% ethanol, extracted in boiling water and cooled before titration to pH 7.0. Plants which engage in CAM display a complex pattern of Photosynthetic 02 exchange and dark respiration were meas- daily CO2 exchange based on at least four distinct phases of ured using a Hansatech leaf disc electrode system (Decagon autotrophic and heterotrophic CO2 fixation biochemistry (11, Devices, Pullman WA) similar to that described by Delieu and 13, 18). Because dark C02 fixation processes are likely to accel- Walker (4). A disc of leaf tissue (10 cm2) was placed in the erate at low light intensities, it is sometimes difficult to measure electrode chamber and the volume ofthe chamber calibrated as the light-limited rate of photosynthesis by means of net CO2 described by Delieu and Walker (3). The concentration of02 in fixation (14, 20). Stomata also tend to be closed during deacidi- the chamber prior to charging with CO2 was calculated assuming fication, one of the more interesting phases of CAM from a 8.73 10-' mol L` at STP and correcting for temperature and biochemical point ofview, resulting in negligible CO2 fluxes. For average atmospheric pressure at each elevation (Canberra and these and other reasons, measurement of quantum yield of Reno). The chamber was filled with 5 to 6% CO2 saturated with photosynthesis in CAM plants by means of 02 exchange, rather water vapor by the operator breathing gently through the cham- than by CO2 fixation, seemed eminently desirable. ber until the electrode output indicated a decline from 21% 02 Methodological difficulties aside, the quantum yields so far to 15 to 16% 02. It should be noted that, when quantum yields reported for photosynthesis in CAM plants are variable, and are measured during the deacidification phase ofthe CAM cycle, difficult to interpret in biochemical terms. Overall quantum it is not necessary to charge the chamber with 5 to 6% CO2. As yields, based on total daily CO2 exchange and quantum flux are has been demonstrated frequently, internal CO2 concentrations low (10). Quantum yields estimated by light dependence of the of 1 to 4% are generated and sustained in CAM tissues during rate ofdeacidification (photosynthesis under CO2 saturation) are deacidification (2). Leaf discs in the chamber were maintained substantially higher than those measured during net CO2 ex- at 25°C for all measurements reported here. They were illumi- change after deacidification (17). We have used a more direct nated with a quartz iodide light source designed by 0. Bjorkman approach based on photosynthetic 02 exchange using a leafdisc and manufactured by the Biological Sciences Center, Desert electrode (4) to establish whether the quantum yield in CAM Research Institute. (An equivalent light source is now available from Decagon Devices). This light source delivered uniform light ' This research was supported by National Science Foundation grant (±5%) over the leaf disc in the chamber illuminating 8.85 cm2 PCM-8314980 and by a grant to K. N. under the Australian Academy ofthe disc. Rates of02 exchange were calculated on the basis of ofScience-Japan Society for Promotion ofScience Exchange Agreement. the illuminated area of the disc. No corrections for respiratory Downloaded from on January 15, 2972020 - Published by www.plantphysiol.org Copyright © 1986 American Society of Plant Biologists. All rights reserved. 298 ADAMS ET AL. Plant Physiol. Vol. 81, 1986 activity have been applied. The PFD2 was reproducibly con- 401 trolled by the use of neutral density filters (Mellis Griot, Vista, CA) which were exchanged in the slots provided in the light 35 [ source housing. 8:30 am The leaf discs were first illuminated with full intensity white 30 light (max 800 umol quantam 2 s-') for two periods of 5 to 10 min until steady state photosynthetic 02 evolution was achieved. 25 The chamber was then recharged with CO2 if necessary, and dark respiration allowed to attain steady rates. Appropriate filter com- 20 binations were then inserted and the rateof 02 exchange allowed to attain new steady rates (usually 2-5 min) at each light intensity. 15F .0/ Oscillations in 02 exchange were not encountered in the linear portion of the light response curve with the CAM plant tissues 10 or spinach used in these experiments. Light intensity incident on the leaf disc was measured with an . ! integrating quantum meter (LiCor Li 188B with Li190SB sensor, 5 LiCor, Inc., Lincoln, NE) which measured photosynthetically active radiation, 400 to 700 nm. In experiments done in Canberra 0 .. --/ reflectance and transmission of the leaf disc used for photosyn- cM thetic 02 evolution were measured in a small Ulbricht sphere E -5 using the same sensor and light source. The system was calibrated n with standardized reflecance cards (Kodak) of 93, 17.8, and E -11Ut- 4.3%. Leaf absorptance was calculated by difference and used to convert incident PFD to absorbed PFD. In experiments done in 0) 251 Reno, discs from comparable leaves from the same plant were c placed wholly within a larger Ulbricht sphere and illuminated 0 3:00 pm co 20 with a narrow beam of light from a quartz iodide source. The x absorptance spectrum was measured at 25 nm intervals between 0 CM 1sF[ ./ 400 to 700 nm and the overall absorptance computed. a)0 20 .2 101[ RESULTS AND DISCUSSION 15 10 Light response curves for photosynthetic 02 exchange at CO2 -c 5 a1 saturation in two representative CAM plants, Hoya carnosa (PEP 0 5 carboxykinase type) and Kalanchoe daigremontiana (malic en- ia 0 zyme type) (8), are shown in Figures 1 and 2. In each case the initial slope of the 02 exchange curve, measured below the light compensation point, was greater than that measured above. This Kok effect (9) resembles that recently described by pronounced 20- Sharp et al. (16) in sunflower, when measured at CO2 saturation. Although the light-saturated rate of photosynthesis sometimes declined throughout the d (e.g. Hoya), this was by no means a 15 9:00 pm consistent response (e.g. Kalanchoe). The principal features of Figures 1 and 2 are that the quantum 10 yield ofCAM plant photosynthesis does not vary throughout the day, that the quantum yield is similar in both classes of CAM 5 plants, and that the quantum yield is remarkably high. These features are sustained in a survey ofeight species ofleafsucculents (Table I), all of which exhibited nocturnal acid accumulation indicative of CAM. In some of these, CAM was not well ex- -5r pressed (cf Cotyledon with Kalanchoe), and the quantum yields 0 100 200 300 400 *500 600 700 800 were not diminished in species with high reflectance (e.g. Coty- Absorbed quanta, pmol m 1 ledon young leaves of Ananas). The quantum yields for photosynthetic 02 exchange measured FIG. 1. Light response curves for photosynthetic 02 exchange in leaf here are higher than those measured by rates of deacidification discs from H. carnosa (PEP carboxykinase type) measured at 25°C and or CO2 uptake. Spalding et al. (17) estimated a quantum yield CO2 saturation at different times of the d. of 0.062 mol malate mol' quanta during deacidification in Sedum praealtum. The data of Barrow and Cockburn (1) for photosynthetic 02 evolution by deacidifying tissue slices in so- light dependence of deacidification can be converted, on the lution at CO2 saturation, we estimate quantum yields for Kalan- basis of our fresh weight to leaf area ratio for Kalanchoe, daigre- choe of0.023 mol CO2 molr' quanta (in 654 and 617 nm light). montiana, to yield a value of0.086 mol malate mol' quanta (in Recent measurements ofthe quantum yields of C3 photosyn- 654 and 617 nm light). After deacidification, Spalding et al. (17) thesis in the absence ofphotorespiration range from 0.081 (7) to estimated quantum yields in Sedum of 0.053 mol CO2 mol-' 0.087 (16) when measured by CO2 exchange in white light. Discs quanta in low 02 (no photorespiration) and 0.024 mol CO2 of spinach leaves measured in the leaf disc 02 electrode by the mol-' quanta in air. Using data of Barrow and Cockburn (1) for same procedures as described here show quantum yields of0.094 mol 02 mol' quanta (Table I). Shading during growth does not 2Abbreviations: PFD, photon flux density; PEP, phosphoenolpyru- significantly alter quantum yields ofKalanchoe?, and similar data vate. have been obtained with spinach, using these techniques (19). Downloaded from on January 15, 2020 - Published by www.plantphysiol.org Copyright © 1986 American Society of Plant Biologists. All rights reserved. QUANTUM YIELD IN CAM PLANTS 299

glasshouse grown plants have been confirmed in field experiments with epiphytic C3 and CAM plants (21). We had expected that the quantum yield of CAM plant photosynthesis would vary throughout the d as one phase of C 15 / 8:00 am carbon metabolism gave way to the other. In particular, the E complex requirements for ATP and NADPH balance during _ 10 deacidification (6, 13) led us to expect lower quantum yields 0 E during deacidification in the morning than during CO2 saturated same 5 / C3 photosynthesis in the afternoon. By the reasoning, malic ./ enzyme type CAM plants, in which gluconeogenic recovery of the C3 decarboxylation product involves additional ATP expend- co @, iture via chloroplast localized pyruvate, Pi dikinase (6, 8), were expted to have lower quantum yields than PEP carboxykinase 0 5 type CAM plants. As shown in Table I, such a trend was not observed. 0 20 It is not difficult to rationalize our observations that, after deacidification, the quantum yield of CO2 saturated photosyn- pm thesis in CAM plants should be the same as that of C3 plants. It C is widely held that CAM plants, especially leaf succulents, carry co 10 / out normal C3 photosynthesis after deacidification (6, 12). It is 0 o more difficult to explain the similarly high quantum yield of a 5 photosynthetic 02 exchange during deacidification. The precise fate of malate during the deacidification, in terms of cytoplasmic decarboxylation and mitochondrial oxidation, is not known. However, in PEP carboxykinase type CAM plants -5 at least, it is reasonable to consider that deacidification proceeds 0 100 200 300 400 500 600 700 via chloroplastic CO2 fixation (at CO2 saturation) and cyto- Absorbed quanta, ,umol m 2 s 1 plasmic gluconeogenesis. The energy requirements ofthe former are those ofC3 photosynthesis, and those ofthe latter are met by FIG. 2. Light response curves for photosynthetic 02 exchange in leaf regulated metabolism in the cytoplasm and mitochondria (6, discs of K daigremontiana (malic enzyme type, grown in full sunlight) 13). measured at 25C and CO2 saturation at different times ofthe d. At 8:00 In malic enzyme type CAM plants the apparently obligatory AM tissue acidity was 300 ueq g-' fresh wt and at 1:00 PM it was 95 teq recovery of pyruvate in chloroplasts during gluconeogenesis (8) g-' fresh wt. must be considered. It is clear that pyruvate competes for pho- Using this technique with leaves of other C3 plants, quantum tosynthetically derived ATP in Mesembryanthemum chloroplasts yields have ranged from 0.087 to 0.115 mol 02 mol-' quanta isolated from tissues in the CAM mode, but not in the C3 mode (CB Osmond et al., unpublished data; 0 Bjorkman, B Demmig, (5). Moreover, chloroplasts isolated from another malic enzyme unpublished data). Thus the quantum yield ofphotosynthetic 02 type CAM plant, Sedum praealtum, like those of pea, are also exchange in the CAM plants examined here is within the range quite permeable to adenylates (15). In this category of CAM of values found for C3 photosynthesis. These observations with plants, therefore, it is possible that the additional ATP require-

Table I. Quantum Yields ofPhotosynthetic 02 Exchange in LeafDiscs ofCAM Plants and Spinach Data shown are mean and SD of measurements made morning and evening in Reno (1) and Canberra (2). Nocturnal Species Acid Absorptance Quantum Yield Accumulation Meq g-' mol 02 mo/-' fresh wt quanta Kalanchoe daigremontiana (ME)' Full sunlight 305 (1) 86.2 0.100 ± 0.001 (3) 15% Sunlight 158 (1) 86.1 0.106 ± 0.007 (2) Kalanchoe~marmorata (ME) 206 (1) 68.2 0.086 ± 0.006 (2) Senecio amaniensis (ME) 178 (1) 73.9 0.115 ± 0.013 (3) Cotyledon orbiculata (?) 13 (2) 46.6 0.104 ± 0.009 (3) Cereus spp. (?) ND (2) 84.4 0.094 ± 0.005 (2) Hoya carnosa (PEPCK) 130 (2) 90.4 0.107 ± 0.003 (3) Hoya australis (PEPCK) 115 (1) 71.0 0.086 ± 0.001 (3) Ananas comosus (PEPCK) Old leaves 35 (2) 83.1 0.077 ± 0.006 (5) New leaves 235 (1) 63.6 0.090 ± 0.011 (2) Spinacia oleracea (C3) ND (2) 84.7 0.094 ± 0.006 (3) Mean value for CAM 0.095 ± 0.012 *Known pathway ofdeacidification, or that inferred on the basis ofphylogeny: ME, malic enzyme; PEPCK, PEP carboxykinase; ?, uncertain. Downloaded from on January 15, 2020 - Published by www.plantphysiol.org Copyright © 1986 American Society of Plant Biologists. All rights reserved. 300 ADAMS ET AL. Plant Physiol. Vol. 81, 1986 ments for chloroplastic conversion of pyruvate to PEP are met during deacidification in plants with crassulacean acid metabolism. Aust J Plant Physiol 8: 31-44 by ATP generated from mitchondrial oxidation of a small por- 9. KOK B 1948 A critical consideration of the quantum yield of Chlorella tion ofthe malate pool (6). photosynthesis. Enzymologia 13: 1-56 These considerations suggest that the 02 exchange of photo- 10. NOBEL PS, TL HARTSOCK 1983 Relationships between photosynthetically synthesis in CAM plants during deacidification are identical to active radiation, nocturnal acid accumulation, and CO2 uptake for a crassulacean acid metabolism plant, Opuntiaficus-indica. Plant Physiol 71: 71- that of CO2 saturated C3 photosynthesis. The quantum yield 75 measurements reported here, and previously estimated quantum 11. OSMOND CB 1978 Crassulacean acid metabolism: a curiosity in context. Annu yields for malate consumption, support this interpretation. Rev Plant Physiol 29: 379-414 12. OSMOND CB, 0 BJORKMAN 1975 Pathways of CO2 fixation in the CAM plant Acknowledgments-We are grateful to Dr. 0. Bjorkman for sharing his design Kalanchoe daigremontiana II Effects of 02 and CO2 concentration on light for the light source and for access to instruments for absorptance measurements. and dark CO2 fixation. Aust J Plant Physiol 2: 155-162 We also acknowledge helpful discussions with Dr. K. Winter and Dr. J. A. Berry, 13. OSMOND CB, JAM HOLTUM 1981 Crassulacean acid metabolism. In MD and are grateful to Dr. I. P. Ting for providing cuttings of some of the plants, and Hatch, NK Boardman, eds, Photosynthesis, Vol 8 The Biochemistry of to Mr. I. Telford, Canberra Botanic Gardens for identification of some specimens. Plants. Academic Press, New York, pp 283-328 14. OSMOND CB, MM LUDLOW, R DAVEs, IR COWAN, SB POWLES, K WINTER LITERATURE CITED 1979 Stomatal responses to humidity in Opuntia inermis in relation to control of CO2 and H20 exchange patterns. Oecologia 41: 65-76 1. BARROW SR, W COCKBURN 1982 Effects of light quantity and quality on the 15. PIAZZA GJ, M GIBBS 1983 Influence of adenosine phosphates and magnesium decarboxylation of malic acid in crassulacean acid metabolism photosyn- on photosynthesis in chloroplasts from peas, Sedum and spinach. Plant thesis. Plant Physiol 69: 568-571 Physiol 71: 680-687 2. COCKBURN W, IP TING, LO STERNBERG 1979 Relationship between stomatal 16. SHARP RE, MA MATTHEWS, JS BOYER 1984 Kok effect and the quantum yield behavior and internal carbon dioxide concentration in CAM plants. Plant ofphotosynthesis. Plant Physiol 75: 95-101 Physiol 63: 1029-1032 17. SPALDING MH, GE EDWARDS, MSB Ku 1980 Quantum requirement for 3. DELIEU T, DA WALKER 1981 Polarographic measurement of photosynthetic photosynthesis in Sedum praealtum during two phases ofCrassulacean acid 02 evolution by leaf discs. New Phytol 89: 165-175 metabolism. Plant Physiol 66: 463-465 4. DELIEU T, DA WALKER 1983 Simultaneous measurement ofoxygen evolution 18. TING IP, M GiBBs 1982 Crassulacean acid metabolism. American Society of and chlorophyll fluorescence from leafpieces. Plant Physiol 73: 534-541 Plant Physiologists, Rockville, MD, pp 316 5. DEMMIG B, K WINTER 1983 Photosynthetic characteristics of chloroplasts 19. WALKER DA, CB OSMOND 1986 Measurements ofphotosynthesis in vivo with isolated from Mesembryanthemum crystallinum L., a halophilic plant capa- a leaf disc electrode: correlations between light dependence of steady state ble ofcrassulacean acid metabolism. Planta 159: 66-76 photosynthetic 02 evolution and chlorophyll a fluorescence transients. Proc 6. EDWARDS GE, JG FosTER, K WINTER 1982 Activity and intercellular com- R Soc Lond B Biol Sci. In press partmentation ofenzymes ofcarbon metabolism in CAM plants. In IP Ting, 20. WINTER K 1980 Carbon dioxide and water vapor exchange in the crassulacean M Gibbs, eds, Crassulacean Acid Metabolism. American Society of Plant acid metabolism plant Kalanchoe pinnata during a prolonged light period. Physiologists, Rockville, MD, pp 92-1 11 Plant Physiol 66: 917-921. 7. EHLERINGER J, RW PEARCY 1983 Variation in quantum yield for CO2 uptake 21. WINTER K, CB OSMOND, KT HUBICK 1985 Crassulacean acid metabolism in among C3 and C4 plants. Plant Physiol 73: 555-559 the shade studies on an epiphyte fern, Pyrrosia longifolia, and other rainforest 8. HOLTUM JAM, CB OSMOND 1981 The gluconeogenic metabolism of pyruvate species from Austrlia. Oecologia. In press

Vol. 81: 297-300 Page 297, line 17 under "Materials and Methods," should read: The concentration of02 in the chamber was calculated assum- W. W. Adams III, Kojiro Nishida, and C. Barry Osmond. ing 9.37 x 10-3 mol L' 02 in air at STP (3). Quantum Yields of CAM Plants Measured by Photosynthetic 02 Exchange.