Planta (1984)161:314~319

Planta O Springer-Verlag 1984

Circadian rhythms in crassulacean acid metabolism: phase relationships between gas exchange, leaf water relations and malate metabolism in Kalancho dMgremontiana Irene C. Buchanan-Bollig and J. Andrew C. Smith Institut ffir Botanik, Technische Hochschule, Schnittspahnstrasse 3-5, D-6100 Darmstadt, Federal Republic of Germany

Abstract. Gas exchange, leaf water relations, malate content and phosphoenolpyruvate (PEP) carboxylase activity in crude extracts were examined for circadian rhythmicity in the crassulacean acid metabolism plant KalanchoY daigremontiana. At low irradiance (20 W m - z ) the rhythm in CO 2 uptake continued for several days with a period length of approx. 22 h, whereas the transpiration rhythm was no longer apparent after 24 h. This shows that the CO2 rhythm in continuous light (LL) is not under stomatal control. Circadian oscillations in malate content were detectable for up to 72 h j n LL but were of much reduced amplitude. This was reflected in the changes in leaf water relations, which quickly damped after transfer to LL. The activity of PEP carboxylase assayed immediately after extraction showed a rhythmicity for at least 18 h, but after 36 h, values from different plants were scattered. We suggest that the CO 2uptake rhythm is primarily the result of endogenous changes in the activity of PEP carboxylase, which competes to varying degrees with ribulose1,5-bisphosphate carboxylase for CO 2. Key words: Carbon dioxide exchange - Crassulacean acid metabolism - Kalancho6- Leaf water relations - Phosphoenolpyruvate carboxylase Transpiration.

Introduction Endogenous rhythms in the gas exchange of crassulacean acid metabolism (CAM) plants have been described on many occasions. Circadian changes in CO 2 fixation and transpiration have been measured in Kalancho6 daigrernontiana under continuAbbreviations and symbols." C A M - c r a s s u l a c e a n acid metabolism; LD = 12 h light: 12 h dark cycle; L L = c o n t i n u o u s light; PEP = phosphoenolpyruvate; P = turgor pressure; g t = water potential; ~ = osmotic pressure

ous light conditions (Nuernbergk 1961 ; BuchananBollig 1984), but these gas-exchange rhythms are not necessarily associated with concomitant changes in the malate content of the bulk leaf tissue (Lfittge and Ball 1978). Thus the question has emerged whether the gas-exchange rhythm in CAM plants is regulated merely by a rhythm in stomatal aperture or rather by metabolic events in the leaf mesophyll. In earlier experiments Wilkins and Holowinski (1965) observed a circadian rhythm of CO 2 exchange in an undifferentiated tissue culture of Bryophyllum fedtschenkoi. Kluge and Fischer (1967) also noted that leaves of K. daigremontiana from which the epidermis had been removed still showed a rhythm of CO 2 exchange under normal day-night conditions (see also Wilkins 1959; Wilkinson and Smith 1976). Both findings lead us to favour the latter hypothesis. But which events in the mesophyll determine the rhythm in CO z uptake? In continuous darkness, the rhythm in CO 2 output into CO2-free air (Warren and Wilkins 1961; Bollig and Wilkins 1979) has been shown to be correlated with changes in the extractable activity of phosphoenolpyruvate (PEP) carboxylase (Wilkinson and Smith 1976), the primary carboxylating enzyme in CO2 dark fixation. During the normal day-night cycle, PEP carboxylase is inhibited during those parts of the day when malic acid is decarboxylated. This inhibition is caused by day-night changes in enzyme characteristics, especially the affinity for the substrate and the sensitivity to malate inhibition (Winter 1980a, 1982; Kluge et al. 1981 b). In addition, leaf water relations may also be important in determining the degree of CAM activity. For example, malic-acid accumulation in Kalancho6 leaf slices is influenced by the osmotic pressure (~) of the bathing solution (Lfittge et al. 1975). The relationship between leaf water potential (gO, cell-sap ~ and malic-acid content of the tissue has brought possible turgor-pres-

I.C. Buchanan-Bollig and J.A.C. Smith: Circadian rhythms in crassulacean acid metabolism

sure (P) changes into the discussion of CAM regulation (Ltittge et al. 1975, 1982). In the present paper we describe experiments dealing with the time-course and phase relationships of the different CAM variables in K. daigremontiana. Malate levels, transpiration rates and leaf water relations have been followed together with net CO z uptake and PEP-carboxylase activity during the normal day-night cycle and for several days after transfer to continuous light (LL). The phase relationships and the dampening of the oscillations of the different CAM variables are considered in relation to the observed circadian rhythm of CO z uptake. Material and methods Plant material. Plants of Kalancho~ daigremontiana Hamet et Pert. were cultivated from plantlets in a greenhouse under natural light and a 12/12 h temperature cycle (23 25 ~ C during the day, 15 ~ C at night). During the winter season, light was supplemented to a 12-h day. One to three weeks before being used experimentally, the plants were transferred to a controlled-environment cabinet and exposed to 12/12 h light-dark cycles (LD) at 20~ and 6 5 + 5 % relative humidity. Light was provided by high-pressure mercury-vapour lamps (HQL-R 250 W / N P L ; Osram, Munich, F R G ) giving an irradiance of 80 W m - 2 at middle-leaf level as measured with a Li-190 SEB PI sensor (LiCor, Inc., Lincoln, Nebraska, USA). In experiments with reduced light, the irradiance was reduced by a layer of perspex (R6hm and Haas, Darmstadt, F R G ; Type umbra 802) and three layers of fine wire netting. For experiments, leaves or leaf discs from the middle of the plant were used (4-6th pair as counted from the top). Gas-exchange measurements. For recording gas exchange, 2-4 leaves of a plant were sealed in a Perspex chamber (volume 8.2 1) kept within the cabinet containing the other experimentally used plants. The chamber was flushed with humidity-controlled air from the growth cabinet at a flow rate of 0.06 m 3 h - 1 and a CO 2 content of 450-550 gl 1-1. The CO z and water content of the air entering and leaving the Perspex chamber were monitored with a two-channel infrared gas analyser (Binos, Heraeus G m b H , D-6450 Hanau, F R G ) in sequence (3 min in 10-min intervals). Leaf and air temperatures inside the Perspex chamber and the growth cabinet were monitored with a YSI-Tele thermometer and thermistor sensor (YSI 423, 427; Yellow Springs, USA). Leaf temperature was 23-24 ~ C at an irradiance of 20 W m -2, and the incoming air was 20-21 ~ C. At the end of each experiment, leaf area, fresh weight and chlorophyll content were measured. For comparison with values from the literature, a fresh weight of 1 kg corresponded to a leaf area of 0.53 +0.051 m z. The chlorophyll content of 1 kg fresh leaf material was 0.32+0.021 g (estimation after Ziegler and Egle 1965). Determination of leaf water relations. For the determination of leaf water relations three or four leaves from different plants were excised at the base of the petiole at various times during the day-night rhythm. Xylem tension was measured immediately using a pressure b o m b (C.W. Cook Div., Birmingham, U K ; Scholander et al. 1964). The first samples expelled from the cut petiole when the pressure in the b o m b was increased above

315

the balance pressure were used for the estimation of the osmotic pressure (~) of the xylem sap. The value for the xylem-sap was added to the xylem tension to give a value for bulk leaf water potential (g0- Samples of the leaf lamina (approx. 1.0 g) were taken after removing the leaf from the pressure b o m b and frozen; leaf-cell sap was obtained later by centrifugation of the thawed sample. Leaf-cell sap ~ was determined cryoscopically. Leaf-cell turgor pressure (P) was estimated from the algebraic sum of leaf-cell sap ~ and bulk leaf 7t.

Extraction and assay of P E P carboxylase Replicate samples of leaf discs were taken from leaves of eight to ten plants (per experiment) repeatedly during the light-dark cycle or in LL (five to six discs per sample, 0.50-0.89 g fresh weight). The tissue was homogenized in a microdismembrator (Braun, Melsungen, F R G ) at full speed for 0.5 rain. The extraction buffer (3.5ml per sample) contained 2 0 0 m M N,N'-bis(2-hydroxyethyl)glycine (Bicine), 10 m M MgCI2, 4 m M NaHCO3, 14.2 m M mercaptoethanol and 30 g 1-1 polyvinylpyrrolidone ( M W 10000) at pH 8.0. To remove foam, a droplet of octan-l-ol was added to the extract, which was then filtered through Miracloth (Calbiochem, Giessen, F R G ) and assayed immediately for PEP-carboxylase activity. The time between the addition of extraction buffer to the leaf tissue and the start of the photometric assay was 2-3 rain. The standard reaction mixture (1 ml) contained 50 m M Bicine, 50 m M 2-(N-morpholino)ethanesulfonic acid (Mes), 1 m M dithiothreitol, 5 m M MgCI2, 10 m M NaHCO3, 0.15 m M N A D H and 5 gg malate dehydrogenase (undialyzed) and 2 m M PEP at pH 7.0 and 25 ~ C. The reaction was started by adding 20 gl leaf extract. Repeated assays of PEP-carboxylase activity followed 1 2 h after extraction when the crude extracts were centrifuged (20 rain at 3500 g) and desalted on Sephadex G 25 M (prepacked PD ; Pharmacia, Uppsala, Sweden). At this time the enzyme showed optimum activity. The initial activity was expressed as a percentage of the activity measured after 2 h at 4 ~ C. Malate was estimated euzymatically in the crude extracts by the method of M611ering (1974).

Malate and p H in individual leaves. A small area of leaf tissue (approx. 2 x 2 ram) of five individual leaves still attached to the plants was mechanically abraded with forceps without disrupting the lower epidermis. Cell sap was sucked up with 2-gl microcaps, a volume which is sufficient for an enzymatic malate test; the pH was determined in the remaining sap with a microcombination pH probe (Microelectrodes, Inc., Londonderry, New Hampshire, USA). Samples on both sides of the midribs of the leaves were taken during the day-night cycle after transfer to LL at about 10-mm intervals. All experiments were repeated two to three times. Results obtained showed the same trends, but the absolute values differed between experiments. Thus we present results obtained from representative individual experiments.

Results

The light-dependency of the circadian rhythms has been studied in detail, especially with respect to the importance of leaf temperature (BuchananBollig 1984). Although air temperature entering the Perspex cuvette was kept constant, leaf temperature increased with applied irradiation by several

316

I.C. Buchanan-Bollig and J.A.C. Smith: Circadian rhythms in crassulacean acid metabolism

3oo ..% ~./i

......./"

'

.......,....................... .....

;i ii

-100 "~:z~ -o

::k

-150 zz E -100 "~

0.7 l i

o.6/ 0.5J "" 0 A q

-50

I,.+A,§

-0.1 -0.2 ~. = -o.3

\

'" -o.6 -0.5 L ~ ~" 9

21

-0

.

\,/ 9

~E

,4--.,..

+ 21

9

21

9

21

Time of day / h Fig. 1 a-e. Gas exchange, malate concentration and leaf water relations of Kalanchok"daigremontiana leaves after transfer from light-dark cycles to continuous light 20 W m -z, 20 ~ C, 65% relative humidity), a Net CO 2 uptake ( ), and transpiration ( ..... ) of a control plant, b Malate ( . - - . ) , osmotic pressure (~, o---o) of leaf-cell sap and estimated cell turgot pressure (P, A---A). Arithmetic mean of three to four leaves_+ SD. e Bulk leaf water potential, 7t ( a - - z x ) of the same leaves as in b. Means +_SD

degrees. For the experiments described below, we concentrated on 20 W m - 2 and an air temperature of 20 ~ C, conditions under which the CO 2 uptake showed a clear circadian rhythm in PEP-carboxylase activity and is accompanied by changes in malate and leaf water relations. We studied these variables after the transfer from LD to LL for approx. 72 h. Stomatal aperture as a regulatory factor for CO 2 uptake could be excluded because of the continuously high transpiration rates measured from the second day onwards in LL while CO 2 uptake oscillated significantly (compare Buchanan-Bollig 1984). Malate and leaf water relations in LL. Malate content of the leaf-cell sap increased by about 80 m M during the first 12 h in LL, which corresponded to approx. 70% of the value normally reached in darkness (Fig. I b). Parallel to the drop in CO 2 fixation (Fig. 1 a), malate accumulation was followed by some deacidification; during this phase there was in fact a net loss of CO 2 from the leaf. Increase in CO 2 uptake during the next phase (the second " n i g h t " ) was accompanied by only a slight increase in malate content. Comparing values ob-

tained in different experiments, the lowest values in malate content always coincided with the minima in CO 2 fixation. We therefore assume that there is a highly damped rhythm in malate corresponding in phase to the rhythm in CO 2 uptake. Figures 1 b and c give information on leaf water relations in LL. Parallel to the malate content, increased during the first 12 h in LL from 0.4 to nearly 0.7 MPa. It then decreased gradually to 0.35-0.40 M P a after approx. 36 h and stabilized at this value. The more rapid decrease in ~ as compared with malate indicates that other substances normally contributing to ~ also become metabolized in LL. Bulk leaf ~ decreased after transfer to LL from --0.37 to --0.58 M P a as expected in view of the transpiration rate. During the following normal "light" period, gt recovered again to - 0 . 3 9 M P a as the transpiration rate decreased. It stabilized around this value for the rest of the experiment. Leaf P showed a pattern similar to ~. At first it increased at the onset of LL from 0.03 M P a to 0.15 M P a but decreased thereafter. After 36 h of LL treatment, leaf P was close to zero and did not show any further significant changes during the course of the experiment. Generally the water-relation variables showed the expected behaviour during the first 24 h in LL; after that their changes were no longer significantly rhythmic. One possible reason for the highly damped rhythm in malate and water relations is that leaves sampled from different plants possess individual differences in period length of the CO2-uptake rhythm. The individuals may thus become more and more out of phase with continuing duration of LL, and a damped oscillation with small amplitude would be masked when measuring a population. Following malate and p H of cell sap f r o m individual leaves, we observed clear circadian oscillations over the first 24-36 h (Fig. 2). After one 24-h cycle in LL, however, the amplitude of these oscillations was reduced, not exceeding 20-30 m M in malate content or 0.3-0.5 p H units. Even in individual leaves, therefore, the rhythms in malate and pH are more damped than the rhythm of CO 2 fixation (Fig. 2). Activity of P E P carboxylase in L D and LL. Activity of PEP carboxylase in desalted crude extracts showed no significant day-night changes when assayed 2 h after extraction at pH 7.0. However, when assayed immediately after extraction, PEP carboxylase showed marked changes in activity (Fig. 3). The minimum "initial" activity, reached 3 h after the start of the light phase, was only 10%

I.C. Buchanan-Bollig and J.A.C. Smith: Circadian rhythms in crassulacean acid metabolism

317

a 100

.40 EL

100',

o-

/

' 5.0

930 "

>" 8 0 20: 100-

:.= 60-

',

.4.0

"

/ ojO." " o

,oo

,

?4 ~

o_o. \_./'.4.5 o-"

i

"o-5.0

N 20-

g m

"~ 60-

.-

(j

?-o,

c.

a- 20ILl 13_ Om 140~E E lOO-

"10 o

o'

o 45

o

:i \

~176 li

o'"

o.o.O-O-~

oo

'o

..~-.

1

d.

" t~176176176 i i

o-, -4.2 ,' ~ ""o -5.0 o'

0of

12

15

i~"~ 9

18 21

T i m e of -4.5 20-

9

21

9

2'1

9

~

Time of D a y /

2~1

- , - - - , - -4.2

9

\~

"~ 6020-

2060-

(3_ LU

% 2

b

\

5.0 u

V, 7o ,'.~ : f

40-

40-

~5 ~ 5

b.

7o

\

602

-20 ""

(J

"o,"

__

21

h

Fig. 2 a~l. Phase relationship between oscillation in cell-sap pH, (o---o) and malate ( o - - o ) from normal LD cycle to LL (20 W m-Z). Examples are given for single leaves (a, b, e, d) on four different plants

of the activity in the extract 2 h later; in the latter part of the dark phase the initial activity was nearly equal to the final activity. Comparing the initial PEP-carboxylase activity with malate content and the rate of CO 2 uptake by leaves (Fig. 3b), it is clear that the enzyme activity follows the course of CO2 uptake rather than malate content. The gradual decrease in the final activity of PEP carboxylase in the extracts over the day-night cycle, and the fact that less malate was accumulated by the start of the second light phase as compared with the first, may have been a result of the sampling procedure. Samples had to be taken from the same leaves during the whole experiment to avoid leaf-age effects described earlier (BuchananBollig et al. 1980; Jones et al. 1981). The time-course of PEP-carboxylase activity measured immediately after extraction and expressed as percent of final activity is shown for an extended LL treatment in Fig. 4. After the normal light phase and transfer to LL, an endogenous rhythm was apparent in PEP-carboxylase activity for about 18 h. After 36 h in LL, the values from individual plants were scattered and no longer reflected the time-course of the continuing rhythm of CO 2 fixation (compare Fig. 4 and Fig. I a).

2'4

3

6

9

day / h

Fig. 3 a, b. Phase relationship between PEP-carboxylase (PEP-c) activity, malate and net CO 2 uptake during the day-night cycle (12 h light/12 h dark), a Activity of PEP carboxylase in crude extracts. Activity 2-3 min after extraction ("initial" activity, o), activity 1-2 h after extraction (final activity, o) and initial activity expressed as % of final activity (+). (Arithmetic means of duplicate samples. Assay: pH 7.0; 2 mM PEP, 25 ~ C). b Example of the CO 2 uptake of an individual plant (o) and malate content of the leaf discs sampled above (o)

l

100o"~ 80 => 60*6

40-

c~ I

!!!

2-!t.J

o

"o

20 n

0

21

9

21 9 2'1 Time of d a y / h

9

2'1

9

21

Fig. 4. Activity of PEP carboxylase in crude extracts after transfer from a light-dark cycle to continuous light (20 W m-2, 20~ air temperature). Assay at pH 7.0, 2 mM PEP, 25 ~ C. All values are initial activities expressed as % of final activity, obtained during three experiments. (Each point represents the mean of duplicate samples)

Discussion

Our observation that the circadian rhythm of CO 2 fixation at low irradiance was not associated with a concomitant rhythm in transpiration (Fig. 1) indicates that fixation is not under stomatal control. Winter (1980b) also concluded from experiments with K. pinnata that CO 2 assimilation in prolonged light is controlled by nonstomatal components of

318

I.C. Buchanan-Bollig and J.A.C. Smith: Circadian rhythms in crassulacean acid metabolism

photosynthesis. It has even been shown that leaves of K. daigremontiana from which the epidermis has been stripped still show a day-night rhythm in CO 2 exchange (Kluge and Fischer 1967). Nevertheless, under certain environmental conditions the rhythm in CO 2 fixation can be associated with a concomitant rhythm in transpiration (Liittge and Ball 1978), and this may depend in some way on the water status of the plants. Our present results with well-watered K. daigremontiana (Fig. 1) confirm the findings of Lfittge and Ball (1977) that daynight changes in ~ and P are mainly determined by the course of malic-acid accumulation and decarboxylation. As in LD, the cell-sap g follows in LL the changes in malate content, whereas bulk leaf 7t is determined principally by transpiration rate. The changes in malate content of the leaves accompanying the circadian rhythm of CO2 fixation are also highly damped (Figs. 2, 3), but are discernible for up to 72 h after the onset of LL (see also Buchanan-Bollig 1984). We assume that during the phases of high CO 2 uptake in LL, both ribulose-l,5-bisphosphate (RuBP) carboxylase and PEP carboxylase are active. Evidence for this was obtained by Winter (1980b) for K. pinnata (though the light period was extended only for a single 12-h phase) and in labelling experiments during LL (Buchanan-Bollig et al. 1984). Our present results on the activity of PEP carboxylase assayed immediately after extraction (Fig. 4) show that a rhythm was detectable for about 18 h into LL but that after 36 h it was no longer clearly discernible. This is caused, at least in part, by differences in the period lengths shown by individual plants. In fact, Wilkinson and Smith (1976) were able to demonstrate a correlation between extractable PEP-carboxylase activity and the circadian rhythm of CO 2 output into CO2-free air in continuous darkness, but under these conditions there is no competition by RuBP carboxylase. Further experiments with K. daigremontiana using 14CO2 have provided clear evidence for circadian oscillations in the activity of PEP carboxylase in LL for at least 65 h (Buchanan-Bollig et al. 1984). Feedback inhibition of PEP carboxylase by malate together with day-night rhythms in the enzyme characteristics are thought to participate in the regulation of CO 2 fixation in CAM during an LD cycle (Winter 1980a, 1982; Kluge et al. 1981 b) and could also be important in LL. The relative rates of CO 2 fixation by RuBP carboxylase and PEP carboxylase might also be affected by temperature (e.g. Buchanan-Bollig and Kluge 1981), and this possibility should be investigated.

Whether changes in PEP carboxylase properties per se are solely responsible for the CO2-fixation rhythm is a matter of speculation. For instance, an endogenous rhythm in the rate of starch breakdown (e.g. Pongratz and Beck 1978) giving rise to oscillating substrate availability might contribute to the CO2-fixation pattern in LL. However, the lack of a persistent rhythm in starch and soluble-carbohydrate content (shown by Buchanan-Bollig 1984) is consistent with the assumption that the CO2-fixation rhythm is caused by an endogenous rhythm in PEP-carboxylase activity. The restriction of malate accumulation in LL may simply be the result of competition for substrates by both fixation systems (Osmond and A1laway 1974), although a direct effect of light on the transport properties of the tonoplast cannot be excluded (cf. Lfittge et al. 1982). At any rate, we can conclude that the circadian rhythm of CO 2 uptake in LL is not under stomatal control but is determined by a metabolic rhythm in the leaf mesophyll. This work was supported by grants from the Deutsche Forschungsgemeinschaft (Bonn-Bad Godesberg) and by a research fellowship to J.A.C.S. from the Royal Society, UK. We thank Professor U. Liittge for helpful and critical discussions and Sabine Heuer for technical assistance.

References Bollig, I.C., Wilkins, M.B. (1979) Inhibition of the circadian rhythm of CO2 metabolism in Bryophyllum leaves by cycloheximide and dinitrophenol. Planta 145, 105 112 Buchanan-Bollig, I.C. (1984) Circadian rhythms in Kalancho~: effects of irradiance and temperature on gas exchange and carbon metabolism. Planta 160, 264-271 Buchanan-Bollig, I.C., Fischer, A., Kluge, M. (1984) Circadian rhythms in Kalancho6: the pathway of 14CO2 fixation during prolonged light. Planta 161, 71-80 Buchanan-Bollig, I.C., Kluge, M. (1981) Crassulacean acid metabolism (CAM) in Kalancho6 daigremontiana: temperature response of phosphoenolpyruvate (PEP)-carboxylase in relation to allosteric effectors. Planta 152, 181-188 Buchanan-Bollig, I.C., Kluge, M., Liittge, U. (1980) PEP-carboxylase activities and the regulation of CAM: effects of extraction procedures and leaf age. Z. Pflanzenphysiol. 97, 457-470 Jones, R., Buchanan, I.C., Wilkins, M.B., Fewson, C.A., Malcolm, A.D.B. (1981) Phosphoenolpyruvate carboxylase from the Crassulacean plant Bryophyllum fedtschenkoi Hamet et Perrier. J. Exp. Bot. 32, 427-441 Kluge, M., B6hlke, C., Queiroz, O. (1981 a) Crassulacean acid metabolism (CAM) in Kalancho6: changes in intercellular CO 2 concentration during a normal CAM cycle and during cycles in continuous light or darkness. Planta 152, 87-92 Kluge, M., Brulfert, J., Queiroz, O. (1981b) Diurnal changes in the regulatory properties of PEP-carboxylase in crassulacean acid metabolism (CAM). Plant Cell Environ. 4, 251-256

I.C. Buchanan-Boltig and J.A.C. Smith: Circadian rhythms in crassulacean acid metabolism Kluge, M., Fischer, K. (1967) Uber Zusammenh/inge zwischen CO 2 Austausch nnd der Abgabe von Wasserdampf durch Bryophyllum daigremontianum. Planta 77, 212-223 Lfittge, U., Ball, E. (1977) Water relation parameters of the CAM plant Kalanchob"daigremontiana in relation to diurnal malate oscillations. Oecologia (Berlin) 31, 85 94 Ltittge, U., Ball, E. (1978) Free running oscillations of transpiration and CO 2 exchange in CAM plants without a concomitant rhythm of malate levels. Z. Pflanzenphysiol. 90, 69-77 L/ittge, U., Kluge, M., Ball, E. (1975) Effects of osmotic gradients on vacuolar malic acid storage. A basic principle in oscillatory behavior of crassulacean acid metabolism. Plant Physiol. 56, 613-616 Liittge, U., Smith, J.A.C., Marigo, G. (1982) Membrane transport, osmoregulation, and the control of CAM. In : Crassulacean acid metabolism, pp. 69-91, Ting, I.P., Gibbs, M., eds. American Society of Plant Physiologists, Maryland M611ering, H. (1974) L-(--)-Malat. Bestimmung mit Malat-Dehydrogenase and Glutamat-Oxalacetat-Transaminase. In: Methoden der enzymatischen Analyse, pp. 1636-1639, Bergmeyer, H.U., ed. Verlag Chemie, Weinheim Nuernbergk, E.L. (1961) Endogener Rhythmus und CO2-Stoffwechsel bei Pflanzen mit diurnalem Sfiurerhythmus. Planta 56, 28-70 Osmond, C.B., Allaway, W.G. (1974) Pathways of CO 2 fixation during photosynthesis in Kalancho~ daigremontiana. I. Patterns of ~4C fixation in the light. Aust. J. Plant Physiol. 1, 503 512 Pongratz, P., Beck, E. (1978) Diurnal oscillation of amylolytic activity in spinach chloroplasts. Plant Physiol. 62, 687-689 Scholander, P.F., Hammel, H.T., Hemmingsen, E.A., Bradstreet, E.D. (1964) Hydrostatic pressure and osmotic potential in leaves of mangroves and some other plants. Proc. Natl. Acad. Sci. USA 52, 119-129

319

Warren, D.M., Wilkins, M.B. (1961) An endogenous rhythm in the rate of dark fixation of carbon dioxide in leaves of BryophylIumfedtschenkoi. Nature (London) 191, 68(~688 Wilkins, M.B. (1959) An endogenous rhythm in the rate of carbon dioxide output of Bryophyllum. I. Some preliminary experiments. J. Exp. Bot. 10, 372390 Wilkins, M.B., Holowinski, A.W. (1965) The occurrence of an endogenous circadian rhythm in a plant tissue culture. Plant Physiol. 40, 907-909 Wilkinson, M.J., Smith, H. (1976) Properties of phosphoenol pyruvate carboxylase from Bryophyllumfedtschenkoi leaves and fluctuations in carboxylase activity during the endogenous rhythm of carbon dioxide output. Plant Sci. Lett. 6, 319-324 Winter, K. (I 980 a) Day/night changes in the sensitivity of phosphoenolpyruvate carboxylase to malate during Crassulacean acid metabolism. Plant Physiol. 65, 79~796 Winter, K. (1980b) Carbon dioxide and water vapor exchange in the Crassulacean acid metabolism plant KalanchoYpinndta during a prolonged light period. Metabolic and stomatal control of carbon metabolism. Plant Physiol. 66, 917-921 Winter, K. (1982) Properties of phosphoenolpyruvate carboxylase in rapidly prepared, desalted leaf extracts of the crassulacean acid metabolism plant Mesembryanthemum crystallinum L. Planta 154, 298-308 Ziegler, H., Egle, K. (1965) Zur quantitativen Analyse der Chloroplastenpigmente. I. Kritische Uberprfifung der spektralphotometrischen Chlorophyll-Bestimmung. Beitr. Biol. Pflanz. 41, 11-37

Received 28 November 1983; accepted 31 January 1984