Physiological studies on acid metabolism in green plants IX. The distribution of 14 C in malate of darkened Kalanchoё leaf fragments after infiltration with labelled pyruvate

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Abstract
When carboxyl-, carbonyl- or methyl-labelled pyruvates are supplied to darkened fragments of Kalanchog crenata Haw. leaves, malate usually becomes the most heavily labelled metabolite. To help in elucidating the process involved in the labelling, the distribution of C14 within the malate molecule has been determined. Investigations were made for leaves in which dark acidification led to a net synthesis of malate and for leaves in which dark deacidification led to a net consumption of malate. Some experiments were carried out in the presence of aerobic atmospheres containing 20% CO2. The results are consistent with the oxidation of the pyruvate by pyruvlc oxidase and enzymes associated with the conventional TCA cycle. In addition to such slight net synthesis of malate as may have occurred by this sequence there appeared to be two other distinct synthetic processes involving B-carboxylations. In one of these, C14O2 that arose from pyruvate or a labelled product thereof was fixed into malate by the enzymic system responsible for the massive CO2 fixation and resulting acidification in darkened Kalanchoe leaves. In this reaction, apparently, the carbon skeleton of pyruvate was not directly incorporated into the malate. In the other carboxylative synthesis the carbon skeleton of labelled pyruvate appeared to be incorporated as a whole into malic acid. Only a very small proportion of the total fixation of C 14O2 by the leaves was attributable to this synthesis. It is suggested that the first of these carboxylatlon involves phosphoenolpyruvlc carboxylase acting in conjunction with other enzymes whereas the second is mediated by malic enzyme. Support is provided for the concept that most of the malate synthesized when CO2 is fixed during dark acidification is transferred to a storage pool. The present analyses suggest that malate synthesized from the labelled pyruvate by action of malic enzyme or in oxidative reactions of the TCA cycle is relatively rapidly metabolized. The implications of the possible occurrence of the acids in Kalanchoe leaves partly in storage pools, where they are not subject to metabolic transformations, and partly in active pools, where they are rapidly transformed in metabolic reactions, are discussed briefly with respect to the interpretation of results observed when labelled metabolites are supplied to plant tissues.