No abstract

Plant tissues contain highly conjugated forms of folate. Despite this, the ability of plant folate-dependent enzymes to utilize tetrahydrofolate polyglutamates has not been examined in detail. In leaf mitochondria, the glycine-cleavage system and serine hydroxymethyltransferase, present in large amounts in the matrix space and involved in the photorespiratory cycle, necessitate the presence of tetrahydrofolate as a cofactor. The aim of the present work was to determine whether glutamate chain length (one to six glutamate residues) influenced the affinity constant for tetrahydrofolate and the maximal velocities displayed by these two enzymes. The results show that the affinity constant decreased by at least one order of magnitude when the tetrahydrofolate substrate contained three or more glutamate residues. In contrast, maximal velocities were not altered in the presence of these substrates. These results are consistent with analyses of mitochondrial folates which revealed a pool of polyglutamates dominated by tetra and pentaglutamates. The equilibrium constant of the serine hydroxymethyltransferase suggests that, during photorespiration, the reaction must be permanently pushed toward the formation of serine (the unfavourable direction) to allow the recycling of tetrahydrofolate necessary for the operation of the glycine decarboxylase T-protein.

Leaf extracts of 14-d-old pea (Pisum safivum L. cv Homesteader) seedlings were examined for folate derivatives and for 10-formyltetrahydrofolate synthetase (SYN), 5,lO-methenyltetrahydrofolate cyclohydrolase (CYC), and 5,l O-methylenetetrahydrofolate dehydrogenase (DHY) activities. Microbiological and enzyme assays showed that leaf folates SYN, CYC, and DHY were predominantly cytosolic. Extracts of Percoll gradient-purified mitochondria contained less than 1 % of total leaf folate and less that 170 of each enzyme activity. Fractionation of whole-leaf homogenates resulted in the copurification of DHY and CYC (subunit 38 kD) and the isolation of a SYN protein (subunit 66 kD). Polyclonal antibodies were raised against purified cytosolic DHY-CYC (DHY-CYC-Ab) and cytosolic SYN (SYN-Ab), respectively. lmmunoblots showed that DHY-CYC-Ab cross-reacted with a mitochondrial protein band (38 kD). l w o mitochondrial protein bands (subunit M, = 40,000 and 44,000) crossreacted with SYN-Ab. lmmunoaffinity chromatography (DHY-CYC-Ab as the immobile ligand) indicated that the bulk of mitochondrial SYN activity was not associated with mitochondrial DHY or CYC. When 9-d-old etiolated pea seedlings were exposed to light for up to 3 d, the specific enzyme activities of DHY-CYC in whole-leaf extracts rose 2-fold and more DHY-CYC-Ab cross-reacting protein was detected. In contrast, the specific activity of SYN fel1 from 5 to 1 pnol min-' mg-' protein and less SYN-Ab cross-reacting protein was detected. l h e data suggest that in pea leaves, the bulk of one-carbon-substituted tetrahydrofolates and enzymes for the generation of 1 O-formyltetrahydrofolate are extra-mitochondrial.

MIany plant tissues are known to produce ethanol when they are subjected to experimental stresses such as low oxygen supply or a variety of respiratory inhibitors (2). Ethanol production has also been observed under natural conditions in such tissues as germinating seeds, fruits, and root tips (10). The question whether ethanol, particularly that accumulated in periods of natural or imposed anaerobiosis, can be metabolized when the tissue subsequently gains better access to 02 is still unanswered (10). In earlier experiments, Cossins and Turner (5, 7) showed that in a variety of germinating seedlings, previously accumulated ethanol was consumed by the tissues, and by providing ethanol-2-C14 (6) to pea cotyledons they were able to show extensive conversion to a variety of products, including acetaldehyde, acids of the tricarboxylic acid cycle, and amino acids. In this investigation, some twelve tissues, including storage organs, parts of seedlings, coleoptiles, fruits, roots, stems, and leaves have been examined for their ability to metabolize ethanol-C14 and the products of its utilization. Without exception, these materials converted part of the added ethanol to C02, organic acids, amino acids, and other products in periods of 4 hours or less. Distinctive differences in the patterns and rates of utilization were observed: in some tissues the rates of utilization were sufficiently high that the added ethanol (15 ILmoles) was completely metabolized. Materials & Methods Planit Materials. Peas, (Pisumiji sativuwn L. var. Alaska) castor beans (Ricinus coninunis L. var. Cimmaron), and corn (Zea mnays L. var. wf9 x 38-11 single cross hybrid) seeds were soaked overnight at 250 in tap water and then surface sterilized by washing in 0.1 % mercuric chloride solution w/v, followed by three successive washings in distilled water. The pea and corn seeds were sown in pots of garden soil and germinated at 250 in the greenhouse. For the experiments using 1 to 3-day old pea and corn seedlings, the seeds were germinated between layers of moist filter paper at 25°in the dark. The castor I

Methanol-C14was rapidly metabolized by carrot tissue slices, pea cotyledons, soybean cotyledons, castor bean endosperm, beet storage tissues, and mature beet leaves. With the exception of beet storage tissues, carbon dioxide was a chief product of methanol metabolism. In all tissues, methanol carbon was also incorporated in the organic acids, sugars, amino acids, and the insoluble residue. Serine, methionine, methionine sulfone, and methionine sulfoxide were important labelled components present in the amino acid fractions separated. Degradation of the serine-C14that was produced by carrot tissues metabolizing methanol-C14showed that the bulk of the label was present in the 3-position. The results are interpreted as indicating that methanol can act as a precursor of the carbon-1 units that are to be utilized in transmethylation reactions leading to serine and methionine biosynthesis. In addition, methanol can be oxidized to carbon dioxide by these tissues, and this reaction possibly involves dehydrogenase systems.