14CO2 dark fixation in the halophytic species mesembryanthemum crystallinum

Biochirnica et Biophysica Acta, 343 (1974) 465--468 © Elsevier Scientific Publishing Company, Amsterdam - - Printed in The Netherlands B B A 27393

14 CO: D A R K FIXATION IN THE HALOPHYTIC SPECIES

MESEMBR YANTHEMUM CR Y S T A L L I N U M

KLAUS WINTER, ULRICH LiJTTGE and ERIKA BALL

Botanisches Institut, Faehbereieh Biologie der Technischen Hochschule Darmstadt, Darmstadt (Germany) (Received November 19th, 1973)

Summary The time course of ,4 CO2 dark fixation was studied in leaves of the facultatively halophytic plant species Mesembryanthemum crystallinum cultivated with and w i t h o u t 400 mM NaC1 in the nutrient medium. It is generally known from the literature t h a t plants grown under saline conditions incorporate ' 4 C predominately into amino acids. By contrast in leaves of M. crystallinum grown on NaC1 and exposed to ,4 CO2 in the dark, relatively more radioactivity is incorporated in the organic acids (especially malate) than in amino acids. The data obtained are discussed in relation to the NaC1 induced Crassulacean acid metabolism in M. crystallinum reported earlier.

Introduction

Plants capable of Crassulacean acid metabolism are characterised by massive net CO2 uptake in the dark [1,2], which becomes incorporated into organic acids. Thus after dark fixation of ' 4 CO2, 14 C is found mainly in Krebs cycle acids, especially in malate [3--5]. Plants grown under saline conditions may also fix ,4 CO2 in the dark [6--8], but now amino acids axe the major labelled compounds unlike plants capable of Crassulacean acid metabolism and glycophytically cultivated plants. When treated with high NaC1 concentrations in the nutrient medium, the halophytic species Mesembryanthemum crystallinum has been shown to have a decreased net CO2 uptake in the light, and to fix CO2 in the dark [9--11]. This dark fixation of CO2 results in a diurnal accumulation of malic acid in the leaf mesophyU. The present study was undertaken to investigate the effect of NaCl on the ratio of ,4 C-labelled amino acids to ,4 C-labelled organic acids after dark assimilation o f ' 4 CO2 in this species.

466 Materials and Methods 6-weeks-old plants were transferred from the greenhouse to a controlledenvironment room which operated on a 12-h light: 12-h dark rhythm. The light intensity was 8000 Lux (Philips HPL 400 Watt), the temperature 25°C during the light period and 15°C during the dark period and the relative air humidity averaged 90%. The NaC1 content of the nutrient solution [11] was increased daily by 100 mM up to the eventual level specified in the discussion of the results. After 18 days of treatment with NaC1 plants were used for the experiments. The ,4 CO2 dark fixation experiments were carried out 6 h after the beginning of the dark period. Prior to placing the plants into a chamber permitting exposure to ' 4 CO2, all the leaves were cut off except the 3rd foliar leaf pair. ,4 CO2 (100 pCi; specific activity 59 Ci/mole) was liberated by acidification of a Nail ~4 CO3 -solution and injected into the chamber. ' 4 CO2 assimilation was terminated by dropping the leaves into liquid nitrogen. The leaves were freeze-dried and homogenized in boiling 80% ethanol. The ethanol extracts were concentrated under reduced pressure and separated into organic acid, amino acid, neutral and phosphate fractions on ion exchange columns (Ionenaustauscher I, Merck, and Dowex 200--400 mesh, Type 1 × 8, Serva). Because of the relatively low amounts of ' 4 C radioactivity found in the neutral and phosphate fractions, they were not included in further analysis. The organic acids and the amino acids were analysed by one-dimensional thin-layer chromatography using cellulose-plates (MN 300, Machery, Nagel Co, Dtiren) and using the solvent suggested b y Feige et al. [12] (0.25 g EDTA, 20 ml 33% ammonia, 190 ml water, 70 ml n-propanol, 15 ml isopropanol, 15 ml nbutanol, 500 ml isobutyric acid). Kadioactive c o m p o u n d s were located by radioautograms made by exposing the chromatograms to X-ray film (Kx Fuji). Quantitative determinations of radioactivity were carried out in a liquid scintillation counter after scraping off the radioactive spots and collecting material in counting vials. The labelled compounds were identified by co-chromatography with authentic substances. Results and Discussion The data obtained confirm the results reported earlier [9--11] that

M. crystallinurn grown under high saline conditions exhibits Crassulacean acid metabolism. Dark 14 CO2 fixation of control plants is only approximately 1/10 of that found after NaC1 treatment of the plants (Fig. 1A). The patterns of labelled compounds found in the plants without added NaC1 (Fig. 1B) are n o t substantially different from those of other glycophytically grown angiosperms. By contrast, M. crystallinurn treated with 400 mM NaC1 incorporates ' 4 C predominately into the organic acids malate and citrate/isocitrate when exposed to ' 4 CO2 in the dark (Fig. 1C). Using a 15 min exposure time, 93.5% of total recovered ' 4 C radioactivity is found in the organic acid fraction and only 5.8% in the amino acids instead of 32.5% observed after 1 min ,4 CO2 dark fixation. The results of Fig. 1C are corroborated by a further experiment. When

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the NaC1 treated plants are exposed to a 1-min pulse of 14 CO2 in the dark, followed by a chase in normal atmosphere up to 10 min, nearly the whole ~ 4 C radioactivity recovered is detectable in the pool of malate and citrate/isocitrate after the 10-min chase (Fig. 2). The percentage distribution of labelled compounds after 1-min exposure to 14 CO2 is n o t completely identical with the values shown in Fig. 1C. But this difference, due to an inhomogenity of plants which can be hardly avoided from experiment to experiment, does not affect the conclusions in principle. The distribution of 14C radioactivity in the metabolic intermediates found in the NaC1 treated plants is typical for plants exhibiting Crassulacean acid metabolism, but entirely different from patterns of 14 C-labelled com-

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468 pounds known previously from plants grown under saline conditions (where incorporation of 14 C into amino acids predominates). The data described here and those already reported for M. crystallinum suggest a drastic increase of P-enolpyruvate carboxylase and malate dehydrogenase activity in vivo in saline conditions. By contrast earlier investigations using other plant species suggested an enhanced activity of aspartate transaminase and other transaminases or an inhibition of malate dehydrogenase [6]. Indeed a large stimulation of P-enolpyruvate carboxylase activity was recently reported for the halophytic species Aeluropus litoralis cultivated with 100 mM NaC1 in the nutrient medium [13]. In M. crystallinum a well developed Crassulacean acid metabolism is obtained particularly when the roots are exposed to high saline environment (for example 400 mM NaC1). However a diurnal malate accumulation in the leaves is also induced without NaC1 treatment by water potential deficit resulting in a wilting of the leaves [11]. Therefore it is suggested, that NaC1 per se does not affect the enzymatic regulation in M. crystallinum, but is altering the hormonal balance in the leaves which might be closely related to the activation of the key enzymes of CO2 dark assimilation. It is well known that abscisic acid and cytokinins are involved in such hormonal regulations which play an important role as a consequence of water stress [14--16]. Recent investigations (Winter, K., unpublished data) support our hypothesis of a hormonal control of CO~ fixation metabolism in M. crystallinum.

Acknowledgements We thank Frl. Elke Wiens for technical assistance. This work was supported by Deutsche Forschungsgemeinschaft.

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