Planta

Planta (1988) 174:453-461

9 Springer-Verlag 1988

Temperature effects on malic-acid efflux from the vacuoles and on the carboxylation pathways in Crassulacean-acid-metabolism plants * V. Friemert I, D. Heininger 2, M. Kluge 1' **, and H. Ziegler 2 1 Institut fiir Botanik der Technischen Hochschule Darmstadt, Schnittspahnstrage 3, D-6100 Darmstadt, and 2 Institut ffir Botanik der Technischen Universit/it Miinchen, Arcisstrasse 21, D-8000 Miinchen, Federal Republic of Germany Abstract. The studies described in the paper were conducted with tissue slices of Crassulacean acid metabolism (CAM) plants floating in isotonic buffer. In a first series of experiments, temperature effects on the efflux of [14C]malate and 14CO2 were studied. An increase of temperature increased the efflux from the tissue in a non-linear manner. The efflux was markedly influenced also by the temperatures applied during the pretreatment. The rates of label export in response to the temperature and the relative contributions of 14C02 and [14C]malate to the label export were different in the two studied C A M plants (Kalancho6 daigremontiana, Sempervivum montanum). In further experiments, temperature response of the labelling patterns produced by 14CO2 fixation and light and darkness were studied. In tissue which had accumulated malate (acidified state) an increase of temperature decreased the rates of dark CO2 fixation whilst the rates of CO2 fixation in light remained largely unaffected. An increase of temperature shifted the labelling patterns from a C4-type (malate being the mainly labelled compound) into a C3-type (label in carbohydrates). N o such shift in the labelling patterns could be observed in the tissue which had depleted the previously stored malate (deacidified state). The results indicate that in the acidified tissue the increase of temperature increases the efflux of malate from the vacuole by changing the properties of the tonoplast. It is assumed that the increased export of malic acid lowers the in-vivo activity of phosphoenol pyruvate carboxylase by feedback inhibition. Abbreviations." CAM=Crassulacean acid metabolism; FWfresh weight; PEPCase- phosphoenolpyruvate carboxylase * Dedicated to Professor O.L. Lange, Wfirzburg, on the occasion of his 60th birthday ** To whom correspondence should be addressed

Key words: Crassulacean acid metabolism - Kalanc h o 6 - Phosphoenolpyruvate carboxylase - Sempervivum - Temperature and C A M

Introduction It is well documented in the literature that temperature influences Crassulacean acid metabolism (CAM) in various ways (for reviews, see Kluge and Ting 1978; Osmond 1978; Winter 1985; Ting 1985). Temperature affects the C A M processes of the dark period as much as those of the light period. Net CO2 fixation and malic-acid accumulation during the night are favoured by lower and inhibited by higher night temperature (Queiroz 1965; Kluge 1968; Neales 1973; Kaplan etal. 1976; Meyer 1979; Medina and Osmond 1981). The temperature optimum for the nocturnal C A M process was found to be between 10 ~ and 20 ~ C in the majority of plants studied so far. The C A M processes during the day, i.e. malic-acid depletion and photosynthetic reassimilation of the malic-acid-derived 0 2 , are also influenced by temperature: high temperatures favour these processes, while temperatures lower than 20 ~ C are markedly inhibitory (see the literature mentioned above). It is unlikely that temperature affects C A M by a single, general mechanism. Rather, several probably more or less independent temperature effects can be distinguished. For instance, in many cases the temperature acts on C A M indirectly by influencing stomatal behaviour via the water-potential differences between the plant and the ambient air (Lange and Medina 1979; Osmond et al. 1979; Medina 1982; von Willert et al. 1983; Friemert et al. 1986). The temperature can, on the other hand, also affect directly the metabolic processes of CAM. A typical example is the temperature de-

454

V. Friemert et al. : Temperature effects on malic-acid efflux in CAM plants

Table 1. Pretreatment of the plants prior the experiments. PAR = photosynthetically active radiation Series of expts,

A

B

Precultivation in glasshouse (months)

5-7

6-7

Precnltivation in growth chamber Duration (weeks)

Light regime

Temperature regime (~

Rel. air humidity (%)

1

12 h light 20 W . m -2 PAR from fluorescent lamps

15 (darkness)

40 (day)

12 h darkness

25 (light)

70 (night)

10 h light 16 W . m - 2 PAR from HQL-lamps, 400 W; 14 h darkness

19 (darkness)

30 (day)

30 (light)

60 (night)

3

pendence of the balance between respiration and malic-acid accumulation during the night (Kaplan et al. 1976; Wagner and Larcher 1981; Winter and Tenhunen 1982; Ritz et al. 1987). Finally, Wilkins (1983) concluded from temperature-related phase shifts of the endogenous CO2-exchange rhythms shown by CAM plants that alterations of temperature can influence the permeability of the tonoplast, thus controlling malic-acid transport across this membrane. The hypothesis put forward by Wilkins is interesting for understanding the mechanisms which control CAM, but it requires further experimental proof. Our present study has to be seen in this context. Firstly, it was our aim to obtain more quantitative data about temperature effects on malate efflux from the vacuoles of CAM-plant cells. Secondly, we were interested to find out whether or not the efflux characteristics can be correlated with temperature effects on the labelling pattern occurring during 14CO2 fixation in light. If the hypothesis by Wilkins is valid, an increase of temperature should shift these labelling patterns from a C4-type (substantial label trapped in products of the phosphoenol pyruvate carboxylase (PEPCase) pathway) to a C3-type (mainly products of the photosynthetic pathway being labelled). This prediction is based on the assumption that an increase of malate export from the vacuole into the cytoplasm by increasing temperature should lower the activity of PEPCase by feedback inhibition. We have chosen for our studies leaf tissue slices of CAM plants suspended in isotonic buffer. This system was introduced by Kluge and Heininger (1973) into the CAM research and was later studied in the context of the effects of temperature, pH and osmotic conditions on malic-acid transport across the tonoplast (Liittge and Ball 1974a, b; 1977; Lfittge et al. 1975). It is worth mentioning that the experimental approaches reported in the

present paper were initiated independently at two laboratories (Technical Universities of Darmstadt and Munich, FRG). Because of the close interrelationships of the studied problem we then decided to combine the results in a single paper. This history may explain certain deviations in the manner the plants were pretreated and some of the experiments were designed. Material and methods Plant material. Clones of the obligatory CAM plants Kalancho6 daigremontiana Hamet et Perr. and Sempervivum montanum L. were cultivated in glasshouses under natural conditions of light and temperature. The plants grew in clay pots with horticultural "standard soil". When used, the plants were about six months old. Before an experiment was initiated, the plants were transferred for acclimatization into a growth cabinet. The details of the growth conditions applied to the plants are outlined in Table 1. It was made sure by infrared gas analyzer measurements according to Kluge and Fischer (1967) that the plants showed the net CO2 exchange typical for CAM (see Kluge and Ting 1978; Osmond 1978) when used in the experiments.

Preparation of leaf-tissue slices. Fully expanded leaves were harvested from the plants either at the end of the night when they had accumulated malic acid (" acidified" state), or at the end of the day when the previously stored malic acid was depleted ("deacidified" state). About 1 g of leaf material was quickly frozen and thawed, and ceil sap was squeezed out by a garIic press. In the obtained sap the osmotically related water potential (~u~) was determined kryoscopically. According to tile measured ~u~, an isotonic buffer (" suspension medium") was prepared (N,N-bis(2-hydroxyethyl)glycine, 5 0 m M ; CaSO, 0.1 mM; pH 7; sorbitol as required). From the laminae of mature leaves (without mid-rib), tissue slices (1 mm thick) were cut as described by Kluge and Heininger (1973) and immediately suspended in the isotonic buffer. The buffer was kept at the temperature required later in the experiment and aerated vigorously by streaming air. About 20 g of tissue were suspended in 1 1 of buffer and washed for about 30 min, while the buffer was repeatedly changed. Experimental design Efflux experiments (series A). The aim of the following experiments was to study temperature effects on [14C]malate effiux

V. Friemert et al. : Temperature effects on malic-acid efflux in CAM plants from prelabelled leaf tissue into the suspension medium. With the applied conditions and timescales of the experiments, this effiux represents mainly malate efflux from the vacuoles (Kluge and Heininger 1973). Since, in the efflux experiments, large numbers of samples had to be handled, the tracer method was more convenient than the analytical estimation of malate contents in tissue extracts and suspension media. Therefore, such analyses were conducted only with a few selected samples. The detailed experimental procedure was as follows: 1 g of the above-mentioned washed tissue (acidified state) was suspended in 50-ml beakers containing 10 ml of the isotonic suspension medium. The samples were gently shaken on a water bath held in darkness at the temperature shown in the relevant figures. After 10 rain, NaH14CO3 (7.4.10 z kBq; specific activity 2.22 GBq.mmo1-1) was added and the tissue slices were allowed to incorporate ~4C in darkness for 30 rain. This phase of the experiments is denoted in the following as "preincubation" (compare Figs. 1 and 2). After preincubation, the labelled tissue was rapidly separated from the incubation medium by filtration through nylon nets. The labelled tissue was then rinsed with non-radioactive suspension medium, and resuspended with suspension medium (10 ml.g -~ tissue) in closed Erlenmeyer flasks (50 ml). The flasks were connected with washing flasks containing 10% KOH and were held at the desired temperatures (water bath). All manipulations were performed in light weak enough to exclude the possibility that the prelabelled tissue performed photosynthesis while being treated. The labelled samples were then incubated for 1 h in darkness, while a permanent stream of air was passed first through the suspension medium with the samples and then through the KOH trap. This phase of the experiment will be denoted in the following as "incubation". At the end of the incubation, the tissue was separated from the incubation medium, and immediately transferred into boiling 70% methanol. The suspension medium remaining in the Erlenmeyer flasks was acidified by acetic acid while still connected to the K O H traps to recover the final dissolved 14CO 2 . Aliquots of the KOH in the traps were then counted for radioactivity. The values found represent the release of 14CO2 by the tissue during incubation. Also, aliquots of the suspension media (remaining from the preincubation and incubation) were counted for radioactivity. The values found represent the effiux of labelled compounds into the suspension medium during the relevant phase of the experiment. Since, under the described conditions, practically only [14C]malate was found (not shown in figures), the ~4Cefflux into the suspension medium is denoted in the following as "malate effiux". To obtain the radioactivity remaining at the end of the incubation in the tissue, the samples were first extracted by boiling 70% methanol (10 min), followed by extractions with 40% methanol and finally with water (10 min each). The extracts were pooled, brought to a final volume, and the radioactivity was counted in aliquots. Separation of the labelled compounds in the extracts by thin-layer chromatography showed that between 90 and 95% of the tissue-label was in [*4C]malate (not shown in figures). [14C]Malate was isolated from the tissue extracts as from the incubation medium, and the intramolecular label distribution in this [~4C]malate was estimated according to Kluge et al. (1974).

Fixation of J4C02 in the light (series B). The aim of the following experiments was to study, with suspended leaf tissue, temperature effects on label distribution brought about by 14CO 2 fixation in light. The preparation and pretreatment of the tissue slices were the same as already described for series A. For each

455

temperature treatment, two parallel samples were illuminated from above by Argaphot B11 500-W lamps (Philips, Eindhoyen, The Netherlands) providing a photosynthetically active radiation (PAR) of 56 W . m -2 at the level of the tissue, while two further parallel samples were kept in darkness (but otherwise identical conditions) by wrapping them in alluminum foil. After preincubation for 10 min, NaH14CO3 (7.4.102 kBq; specific activity 2.22 GBq-mmol-1) was added to each sample and the tissue was allowed to fix 1~CO 2 . The 14CO 2 incorporation into the tissue was stopped by quickly decanting the radioactive suspension medium and replacing it by boiling methanol. Aliquots of the suspension medium were acidified with acetic acid, dried, and redissolved in known volumes of water. In aliquots of these samples, radioactivity and malate content were determined. The data represent the efflnx of 14C-labelled malate from the tissue into the suspension medium during the time of incubation. The labelled tissue was extracted according to the method of Kluge et al. (1973). The measurement of radioactivity in the soluble extract and in starch followed the procedures by B6cher and Kluge (1978). Label distribution among soluble products was studied by two-dimensional thin-layer chromatography as already described (B6cher and Kluge 1978).

Analytical procedures. Radioactivity was measured by means of liquid scintillation counting. The data were corrected for quenching. L-Malate was measured enzymatically according to Hohorst (1970).

Results

Temperature effects on efflux of [14C]malate and ~4C02. In a first series of experiments (Series A) it was aimed to study the interrelationship between temperature and malate export from the vacuole. As an indicator of such an export we used the release of radioactivity from suspended tissue slices prelabelled by dark CO2 fixation. Under the given experimental conditions, the [14C]malate released from the vacuoles appeared either as [l*C]malate in the suspension medium, or it was decarboxylated, thus appearing outside in the form of 14COz trapped in KOH flasks. Figure 1 (KalanchoY daigremontiana) and Fig. 2 (Sempervivum montanum) show that the temperature during preincubation (i.e. during the time of label application) as well as that afterwards (during incubation) influenced the export of [14C]malate and also the release of 14CO2. The general trends were similar in both plants studied. That is, the export of radioactivity during the incubation increased when the temperature was increased. At 35 ~ C, during 1 h incubation, most of the previously incorporated label was released from the tissue. The Qlo values of label efflux were not equal over all of the temperature ranges studied so far (Table 2). The highest values were observed between 15-25~ whilst between 5-15~ and 25-35 ~ C the Qlo was clearly lower.

456

V. Friemert et al. : Temperature effects on malic-acid efflux in CAM plants

1

o~ x x ! !

(.9

(O

Fig. 1. Tissue slices of KalanehoY daigremontiana prelabelled by 14CO2 fixation in darkness: effects of temperature applied during preincubation and incubation on the effiux of [14C]malate and l~COz during incubation

Fig. 2. Tissue slices of Sempervivum montanum prelabelled by 14CO2 fixation in darkness: Effects of temperature applied during preincubation and incubation on the efflux of 14C-malate and 14C0z during incubation

The export of [14C]malate and 14C02 depended also on the temperature applied during preincubation. Again, the efflux increased with increasing temperature. The fact that the temperature during preincubation was also important is interesting, because it indicates a kind of memory effect. As a consequence of the observed temperature responses the highest values of label export from the tissue occurred when the temperatures both during preincubation and during incubation were high,

whereas the lowest values were observed when the temperature was low both during preincubation and incubation. There were differences between the two studied plants in the Qlo values (Table 2), in the relative amount of 14C released and in the relative proportion of ~4C02 and [~'~C]malate in the efflux (Figs. 1, 2). Sempervivum exhibited lower Qlo values of label export (Table 2), a proportionally higher contribution of ~4CO2 to the total release

V. Friemert et al. : Temperature effects on malic-acid efflux in C A M plants

of label, and a clearly higher label release in the low-temperature range (5-15 ~ C). In the experiments with KalanchoO, at the various temperature regimes, the intramolecular label distribution was determined for [14C]malate released into the medium and for that remaining in the tissue. The label distribution in malate from the suspension medium and from the tissue was practically the same (r=0.041), and was independent of the temperature. That is, about 70% of the label was located in the C4 atom of p4C]malate, about 30% in the Cj atom. It is worth mentioning that we obtained similar effiux responses to temperature when, instead of the tracer, the malate contents were considered. Since, however, as mentioned in Materials and methods, malate contents were not estimated for all the treatments shown in Figs. i and 2, and since, moreover, the estimation of malate contents provided no additional information, these data were not included in this paper. The following values may, however, show the orders of magnitudes of changes in the malate contents occurring in some efflux experiments. For instance, in K. daigremontiana, with preincubation at 15 ~ C, the malate content in the tissue decreased from initially 165 gmol.g -a fresh weight (FW) to 110 p m o l ' g 1FW during 1 h incubation at 35 ~ C. This difference could be explained by the appearance of 9 pmol.g - t F W malate in the efflux, whilst 46 pmol malate vanished otherwise, probably by decarboxylation. In an identical experiment with S. montanum, the malate content decreased from initially 30 p m o l - g - ~ F W to 9.7 g m o l . g - 1 FW, with 4.4 gmol appearing in the efflux and about 16 gmol vanishing by decarboxylation.

Patterns of ~ 4 C - l a b e l l i n g in light and darkness. Figure 3 shows the temperature responses of t'CO2 fixation in light and darkness. There was a large difference in the behaviour of acidified and deacidified tissues. In the acidified tissue, a'CO2 fixation in light was at each temperature at least tenfold higher than in darkness. The temperature response of light 14CO2 fixation showed a broad optimum between 20 ~ and 35 ~ C, but even at 40 ~ C the 14CO2 incorporation was still high. Dark '4CO2 fixation in the acidified tissue decreased steeply with increased temperature. That is, even with temperatures higher than 20~ only traces of label were incorporated. In the deacidified tissue the t4CO2-fixation rates in light and darkness were, in the lower temperature range, nearly identical. U p to 20 ~ C both rates increased, but at higher temperatures CO2

457

Table 2. Qt0 values of total [*4C]malate, 14C02 and total label &flux from tissue slices of K. daigremontiana and S. montanum during incubation. The data derive from the experiments shown in Figs. 1 and 2 Effiux

Temperature ranges during incubation 5-15~ C

K. daigremontiana : [t4C]malate 2.1 14CO2 2 Total 2 S. montanum : [14C]malate

1.3

14C02

1

Total

1.1

15-25~ C

25-35~ C

2.8 2.5 2.8

1.5 2.0 1.9

t.5 2.8 2.1

1.5 1.2 1.5

10

\ 0/0"~0_0/0~

\.o 6

\

LL 4 "7

o

0

LL

0.2 E

acidified .....

/

o

\

0~0_ o~O

o_o /

--9

\o.O

O

~6

9p

o,

0.6 0.4 ;

E 284 o. "o

0.8

.6/

"1o

0 o

8 _g Nx

6 ~-

"o

c7

\

\

4 o

~-2

g

deacidified