Plant Physiol. (1981) 67, 728-732 0032-0889/81/67/0728/05/$00.50/0 The Effects of Levulinic Acid and 4,6-Dioxoheptanoic Acid on the Metabolism of Etiolated and Greening Barley Leaves' Received for publication July 21, 1980 and in revised form October 8, 1980 ERNA MELLER AND MERRILL L. GASSMAN2 Department of Biological Sciences, University of Illinois at Chicago Circle, Chicago, Illinois 60680 ABSTRACT Application of levulinic acid (LA), a competitive inhibitor of 8-aminolev- ulinic acid (ALA) dehydratase, to greening plant tissues causes ALA to accumulate at the expense of chlorophyll. 4,6-Dioxoheptanoic acid (DA), which has been reported to be an effective inhibitor of this enzyme in animal systems, has a similar but more powerful effect on ALA and chlorophyll metabolism in greening leaves of Hordeum vulgare L. var. Larker. Both LA and DA also inhibit the uptake of l'4Clamino acids into etiolated and greening barley leaves and reduce their incorporation into protein. Treatment of etiolated and greening leaves with these compounds results in the inhibition of "CO2 evolution from labeled precursors, includ- ing amino and organic acids. Inhibition of "CO2 evolution by these compounds is more effective in greening leaves than in etiolated leaves when 14-14CIALA or il-14Ciglutamate are employed as precursors. Both LA and DA also inhibit the uptake and increase the incorporation of 32pi into organophosphorus by etiolated barley leaves. These results indicate that LA and DA have more far-reaching effects upon plant metabolism than was previously believed. ALA3 is the first committed intermediate in the biosynthetic pathway leading to porphyrins, and it is the rate-limiting substrate for the synthesis of Chl, heme, and bile pigments in living systems (1O). LA, a competitive inhibitor of ALA dehydratase (19), inhibits the conversion of ALA to PBG and tetrapyrroles, thereby causing ALA to accumulate at the expense of Chl (9, 11). The measure- ment of ALA accumulation in vivo in the presence of LA has provided an important technique for studying the physiology and biochemistry of ALA synthesis in higher plants and algae (1, 4, 11). In all of these studies, it has been assumed that LA specifically inhibits porphyrin biosynthesis (9). However, there are reports which indicate that LA has other effects upon plant metabolism. Klein et al. (14) have shown that LA significantly reduced the number and size of thylakoids in grana of mesophyll chloroplasts from maize leaves. Konis et al. (15) have shown that LA prevents the synthesis of CPII, a pigment-protein complex associated with PSII activity, in greening maize leaves. LA is also reported to inhibit respiration in Scenedesmus (21), to stimulate paramylum breakdown in Euglena (23), and to retard cell division in Skele- tonema (22). In a preliminary study, Duggan et al. (5) recently showed that [14C]LA is extensively metabolized by barley leaves. DA, a breakdown product of tyrosine, is excreted by humans ' This work was supported by National Science Foundation Grant PCM 79-01605 to M. L. G. 2To whom reprint requests should be addressed. 'Abbreviations: ALA, 8-aminolevulinic acid; LA, levulinic acid; DA, 4,6-dioxoheptanoic acid; PBG, porphobilinogen. afflicted with hereditary tyrosinemia (16). This compound has been reported to be an effective inhibitor of ALA dehydratase in animal systems (6, 16). Here, we report the inhibition of barley ALA dehydratase by DA, in vivo and in vitro. We also report evidence that LA and DA have effects upon plant metabolism other than those described for porphyrin synthesis. A preliminary report of this work has appeared (18). MATERIALS AND METHODS Growth and Manipulation of Plant Material. Barley (Hordeum vulgare L. var. "Larker") seeds, obtained from Field Seed Farm; Byron, Minn., were germinated in vermiculite in darkness at 22 C. All manipulations of plant material were carried out under a dim green safelight (7). The apical 5-cm portion of 7-day-old leaves was excised in the dark and divided into 1-cm segments. One g lots were placed into 125-ml Erlenmeyer flasks containing 1 to 5 ,uCi of "4C-labeled compound, plus any additions, in 1.0 ml of 50 mm K-phosphate buffer, pH 5.0, and incubated for up to 4 h in the dark or in the light. Incubations with 32Pi were carried out in 50 mm citrate buffer, pH 5.0. Irradiations. Irradiations were carried out at 22 C for the indi- cated time under GE Cool-White fluorescent lamps at an irradi- ance of 1.5 x 104 ergs/cm2. s. Assay of ALA Dehydratase. ALA dehydratase (EC 4.3.1.24) was assayed by a modification of the method of Nandi and Waygood (20). Ten g of etiolated leaves, irradiated for I h, were ground in a mortar and pestle at 4 C in 20 ml of 50 mm Tris-HCl, pH 7.6, containing 10 mm cysteine. After filtration through four layers of cheese cloth, the extract was centrifuged at 18,000g for 15 min, and the supernatant used as a source of enzyme. Incuba- tions were carried out at 30 C, with or without LA or DA, as indicated. The reaction mixture contained: ALA, 7.5 .tmol; Tris- HCI, 98 ,umol; MgCl2, 9.9 ,umol; GSH, 30,umol; and protein, 1.25 mg, in a final volume of 3.0 ml. The reaction was stopped by addition of 1.0 ml of 20% trichloroacetic acid, and porphobilino- gen was determined by the method of Mauzerall and Granick (17). Determination of ALA Accumulation in Vivo. ALA, which accumulated in irradiated barley leaves treated with LA or DA, was determined as described previously (12). Pigment Extraction and Determination. Chlorophyll content of greening tissue was determined from 80% acetone extracts with the aid of a Coleman Perkin-Elmer model 124 spectrophotometer equipped with a model 165 recorder. Concentrations were calcu- lated using the equation given by Withrow et al. (24). Measurement of the Uptake of '4C-labeled Compounds and the Evolution of "CO2. At the conclusion of an experiment, 50 ml of water was added to each incubation flask, and the shoot tissue was removed and washed with an excess of water. The washings were combined with the diluted incubation medium, the resulting volume made up to 250 ml, and aliquots removed for the deter- mination of radioactivity. Isotopic uptake was calculated by sub- 728