I In the leaves of many succulent plants, organic acid is formed during the night and broken down in the day-time. This diurnal variation in acid content has been related to carbohydrate metabolism by various authors since the middle of the 19th century. Modern research qualified the variation in acidity to changes in the malic acid fraction. The carbohydrate content, particularly that of starch and sedoheptulose, increases during the day and decreases at night. A strict correlation was found to occur between the two opposed variations in malic acid and in carbohydrate content. Arguments both from biochemical and physiological research strongly plead in favour of malic acid production by way of /3-carboxylation of some intermediate of carbohydrate breakdown. Formation of carbohydrate from malic acid by the reversed way might occur. The diurnal variation in acid content is caused by the external factors temperature and illumination. A change in the atmospheric composition, too, can effect the acid metabolism. In the present paper it is submitted that temperature and illumination may influence the acid metabolism by means of the composition of the intercellular gas. The two factors affect the gas exchange of the leaf cells; if this gas exchange with the intercellular spaces surpasses that of the intercellular spaces with the outer atmosphere, a diurnal variation in intercellular gas composition will be caused by the diurnal alternation of assimilation and dissimilation. It is conceivable that this will happen in succulent leaves. Especially a variation in carbon dioxide content could be important, since this compound is known to be a metabolite in the acid variation. It is the intention of the present paper to investigate whether illumination affects the Crassulacean acid metabolism by means of the carbon dioxide tension in the intercellular spaces, or in a more direct manner. II In order to determine whether a considerable diurnal variation in the gas composition of intercellular spaces can occur, [indeed, in succulent leaves, the ability for gas exchanges with the outer atmosphere was studied on leaves—phyllodes, properly speaking—of the Crassulacean plant Bryophyllum tubiflorum Harv.. Respiration rates of excised leaves were compared with those of leaf sections, using the Warburg manometric method. With leaf sections, the rate of gas exchange with the outer atmosphere allowed for an unrestricted respiration at the optimum temperature, about 35° C, at even 5 % of oxygen. The respiration rate could neither be enhanced by the addition of suitable substrates, nor could it be influenced by carbon dioxide (Expts. 1, 2, 3 and 4). Excised leaves exhibited a smaller respiration rate than comparable sections under the same conditions. Leaf respiration could be accelerated by those changes in atmospheric composition, which caused stomatal aperture to widen (Expt. 5, Table i). Transpiration measurements and porometer experiments demonstrated, that stomata of ~ B. tubiflorum leaves are opened wider in 1 % of carbon dioxide than they are in 0 % or in 5 %. Oxygen, from zero concentration to 100 %, had a progressively greater closing effect on stomatal aperture (Expts. G, 7, 8 and 9). It is concluded that leaf respiration is limited by oxygen deficiency, even in a milieu of pure oxygen. The oxygen supply is controlled by stomatal aperture. Thus, the composition of the intercellular gas can differ greatly from that of the outer atmosphere. The diurnal alternation of light and darkness is expected, therefore, to cause a considerable diurnal variation in the intercellular gas composition by the alternating metabolic activities of the leaf cells. Ill The diurnal acid variation in B. tubiflorum phyllodes is comparable to that of plants, commonly used in this field of research: the amplitude is of the same order and the variation occurs in the free malic acid fraction (Expts. 10, II and 12). Weather conditions in the day-time affect both deacidification during that day and acidification in the subsequent night (Expt. 13). Rising temperature stimulates deacidification and retards acidification (Expt. 14). At a constant temperature, a variation in the acid content can be brought about by light-dark alternations (Expts 15 and 16). To study the effects of illumination and carbon dioxide tension separately and in any combination, leaf sections had to be used. These sections were previously kept under constant conditions as to temperature, illumination and atmospheric composition. Temperature was invariably kept at 20.0° C throughout the experiment. Under constant conditions, the acid content of sections becomes constant too. When sections had thus become adapted to their environmental conditions, illumination and/or carbon dioxide tension were changed and the response in the acid content determined. The acid content of adapted sections, 1 mm thick, was always lower than that of comparable excised leaves under the same conditions. No conclusive explanation could be given for this phenomenon (Expts 17, 18, 19, 20 and 21). In the dark, the acid content increases if the carbon dioxide tension rises between 0 % and 2 %; higher tensions do not further influence the acid level. The graph of the relation is a Blackman curve. At the light intensity of the compensation point, the optimum acid level is reached at about 3 % of carbon dioxide, at strong illumination at about 4 %. Since this optimum level is equal both in the light and in the dark, at carbon dioxide tensions surpassing 4 %, it is immaterial for the acid level, whether the sections are illuminated or not (Expts 22, 23, 24 and 25). IV An attempt to elucidate the biochemical pathways of the Crassulacean acid metabolism starts from three conditions. 1. The main acid to be concerned is malic acid. 2. The variation in the malic acid content is related to carbohydrate metabolism. 3. Only enzymes, known to occur generally in the plant kingdom, play a role. The following pathways are considered to be the most probable ones (Fig. 2): 1. Dark deacidification: oxidative decarboxylation of malic acid and subsequent respiration into carbon dioxide. 2. Dark acidification: carboxylative transformation of starch and sedoheptulose into phospho-glyceric acid, with the co-operation of the enzyme carboxydismutase; carboxylation of phospho-enolpyruvic acid, derived from phospho-glyceric acid, by the enzyme phospho-enolpyruvate carboxylase into oxaloacetic acid; reduction of this compound into malic acid (Fig. 4). 3. Light deacidification: oxidation and decarboxylation of malic acid into phosphoenolpyruvic acid; conversion of this compound into carbohydrates by reversal of the glycolysis pathway, with the aid of energy and reducing power from photolysis of water. The results of the Expts. 23, 24 and 25 fit well in this picture. The carbon dioxide tension is immaterial as to dark deacidificalion. It affects dark acidification and light deacidification. In dark acidification, carbon dioxide, up to a tension of 2 %, is the limiting factor in the carboxylative production of phospho-glyceric acid from pentose-diphosphate by the enzyme carboxydismutase. At tensions over 2 %, sufficient carbon dioxide is available to saturate the acidifying enzyme system with phospho-glyceric acid. Light deacidification occurs at carbon dioxide tensions up to 4 %. At these lower concentrations, the enzyme carboxydismutase falls short in supplying enough phospho-glyceric acid to saturate the carbohydrates synthesizing enzyme system. The gap is filled up by phospho-glyceric acid from malic acid: malic acid replaces carbon dioxide as a source in carbohydrate synthesis. If the carbon dioxide concentration rises between 0 % and 4 %, less and less malic acid is required. At low light intensities, e.g. at the compensation point, the carbohydrates synthesizing apparatus will be saturated sooner than at a strong illumination. If this apparatus is saturated with carboxylatively produced phospho-glyceric acid, and, in addition, part of this phospho-glyceric acid is available for the acidifying enzyme system, then the final acid level will be equal to that in the dark at that carbon dioxide tension. This is the case at the light intensity of the compensation point above about 3 %, in stronger light above about 4 % of carbon dioxide; at these concentrations, the horizontal parts of the Blackman curves are reached. A literature datum shows, that in vitro carboxydismutase is still unsaturated at r r\ / r\i i • /> i n • n r~% , i • n • * 1 1 5 % C02. Photosynthesis of leaf sections of B. tubiflorum is, under the usual experimental conditions and at strong illumination, not saturated untill a carbon dioxide tension of 2 % is applied. V During the relatively cool nights, succulent plants are able to produce malic acid out of carbohydrates, thanks to the preponderance of carboxylative enzyme systems over oxidative ones. Possibly, the composition of the intercellular gas plays a role in this relationship. In the day-time, at a normal composition of the outer atmosphere, succulent plants will use up their malic acid as a source for the energy-fixing synthesis of carbohydrates; partly, malic acid is directly converted into carbohydrates (Fig. 4). Plants, resisting against desiccating climatic conditions by developing succulence, which curtails their ability for gas exchange, are able to maintain a lively metabolic activity by the diurnal variation in their acid content; this variation allows of a continuous fixation of carbon dioxide, night and day.