Microbodies in the cotyledons of cucumber seedlings perform two successive metabolic functions during early postgerminative development. During the first 4 or 5 d, glyoxylate cycle enzymes accumulate in microbodies called glyoxysomes. Beginning at about day 3, light-induced activities of enzymes involved in photorespiratory glycolate metabolism accumulate rapidly in microbodies. As the cotyledonary microbodies undergo a functional transition from glyoxysomal to peroxisomal metabolism, both sets of enzymes are present at the same time, either within two distinct populations of microbodies with different functions or within a single population of microbodies with a dual function.We have used protein A-gold immunoelectron microscopy to detect two glyoxylate cycle enzymes, isocitrate lyase (ICL) and malate synthase, and two glycolate pathway enzymes, serine:glyoxylate aminotransferase (SGAT) and hydroxypyruvate reductase, in microbodies of transition-stage (day 4) cotyledons. Double-label immunoelectron microscopy was used to demonstrate directly the co-existence of ICL and SGAT within individual microbodies, thereby discrediting the two-population hypothesis. Quantitation of protein A-gold labeling density confirmed that labeling was specific for microbodies. Quantitation of immunolabeling for ICL or SGAT in microbodies adjacent to lipid bodies, to chloroplasts, or to both organelles revealed very similar labeling densities in these three categories, suggesting that concentrations of glyoxysomal and peroxisomal enzymes in transition-stage microbodies probably cannot be predicted based on the apparent associations of microbodies with other organelles.

The development of peroxisomal enzymes in cotyledons of cucumber seedlings is strongly dependent on light. In light-grown seedlings, activities of two peroxisomal enzymes, hydroxypyruvate reductase (HPR) and serine: glyoxylate aminotransferase (SGAT), were barely detectable until three days postimbibition, after which time both activities increased rapidly and linearly for at least three days. In the dark, the activities of these enzymes increased slightly over the same time period, but only to about 5% to 10% of 7-day light-induced levels. When 51/2-day dark-grown seedlings were transferred into white light, activities of HPR and SGAT began to increase after approximately 8 h. HPR protein was shown by an immunoprecipitation assay to increase concurrently with enzymatic activity in both light- and dark-grown cotyledons. Immunoblotting results suggested that the amounts of SGAT-A and SGAT-B, the two subunits of SGAT, also developed along with SGAT activity. The relative levels of translatable mRNAs encoding HPR, SGAT-A, and SGAT-B were also light-dependent, and increased with a developmental pattern similar to enzyme activity and protein levels in light- and dark-grown cotyledons. In 51/2-day dark-grown cotyledons that were transferred to the light, translatable mRNAs for SGAT-A and SGAT-B began to increase within 1 h of illumination and continued of increase rapidly and linearly for the next 24 h in the light to a new steady-state level that was 45 times that of dark controls. Translatable HPR mRNA exhibited a biphasic pattern of accumulation, with a three-fold increase during the first 6 h of illumination, followed by an additional six-fold increase between 8 and 24 h. The accumulation of translationally active mRNA for both enzymes preceded the accumulation of the corresponding protein and enzyme activity by about 8 h. Our data suggest that the rise in enzyme activity depends on an increase in translatable mRNA for these enzymes and is regulated at a pretranslational level, most likely involving transcription of new mRNA.

Hydroxypyruvate reductase (HPR), a marker enzyme of peroxisomes, has been purified to homogeneity from cotyledons of light-grown cucumber seedlings (Cucumis sativus var. Improved Long Green). In addition, the peroxisomal location of both HPR and serine-glyoxylate aminotransferase has been confirmed in cucumber cotyledons. The isolation procedure involved Polymin-P precipitation, a two-step precipitation with ammonium sulfate (35 and Seedlings of fat-storing species undergo a postgerminative transition from chemotrophy to phototrophy that involves a change in the metabolic role of microbodies from fat utilization (glyoxysomes) to photorespiratory glycolate metabolism (peroxisomes) (2,4). In greening cotyledons, the increase in peroxisomal enzyme activities typically occurs at the same time as the decrease in glyoxysomal activities (4, 18), evidently in the absence of cell division (2). The mechanism underlying this changeover in microbody function has not been definitively established and has in fact generated considerable controversy (4,6,8,23,28).To date, we have focused on the regulation of glyoxysomal enzymes in cucumber cotyledons. We have isolated several glyoxysomal enzymes (16,20), raised antibodies to them, and used these to investigate both the dependence of enzyme appearance upon translatable mRNA levels (29) and the compartmentalization of these enzymes within glyoxysomes (3,20). To extend such studies to the biogenesis ofperoxisomes and thus to the mechanism underlying the transition from glyoxysomal to peroxisomal function, one or more peroxisomal marker enzymes need to be purified and used to prepare monospecific antibodies.

Monospecific antbodies raised against four glyoxysomal enzymes (isocitrate lyase, catalase, malate synthase, and malate dehydrogenase) have been used to detect these proteins among the products of in vitro translation in a wheat germ system programmed with cotyledonary RNA from cucumber seedigs. In vitro immunoprecipitates were compared electrophoretically with the same enzymes labeled in vivo and also with the purified proteins. Isocitrate lyase yields two bands on sodium dodecyl sulfatepolyacrylamide gels, as synthesized both in vitro (61.5K and 60K products) and in vivo (63K and 61.5K pobpeptides). Both the 63K and 61.5K subunits can also be demonstrated for the isolated enzyme. The two subunits are antigenically cross-reactive and yield similar electrophoretic profiles upon partial proteolytic digestion. A larger subuit is seen in vitro than in vivo for both malate dehydrogenase (38K versus 33K) and catalase (55K versus 54K); this suggests a need for processing which is often a characteristic of proteins that must be transported across or into membranes. Malate synthase has a molecular weight of 57K both in vitro and in vivo, but the isolated enzyme is a glycoprotein, containing N-acetyl glucosamine, mannose, and possibly also fucose and xylose. This indicates that the polypeptide portion of the isolated enzyme is smafler than the in vitro product and suggests processing of malate synthase also. None of the other three enzymes appears to be glycosylated. The implications of these size dffferences for the compartmentalization of matrix and membrane-bound glyoxysomal enzymes are discussed.Seed germination in fat-storing species such as cucumber requires a functional glyoxylate cycle to effect net gluconeogenesis from storage triglycerides (3). During early germination, glyoxylate cycle enzymes such as ICL4 (threo-D-isocitrate glyoxylate lyase, EC 4.1.3.1) and MS (L-malate glyoxylate lyase, CoA-acetylating, '

Serine:glyoxylate aminotransferase, a marker enzyme for leaf peroxisomes, has been purified to homogeneity from cucumber cotyledons (Cucamis sativus cv Improved Long Green). The isolation procedure involved precipitation with polyethyleneimine, a two-step ammonium sulfate fractionation (35 to 45%), gel filtration on Ultrogel AcA 34, and ion exchange chromatography on diethylaminoethyl-cellulose, first in the presence of pyridoxal-5-phosphate, and then in its absence. The enzyme was purified approximately 690-fold to a final specific activity of 34.4 units per milligram. Electrophoresis of the purified enzyme on sodium dodecyl sulfate-polyacrylamide gels revealed two polypeptide bands with apparent molecular weights of approximately 47,000 and 45,000. Both polypeptides coeluted with enzyme activity under all chromatographic conditions investigated, both were localized to the peroxisome, and both accumulated in cotyledons as enzyme activity increased during development. The two polypeptides appear not to be structurally related, since they showed little immunological cross-reactivity and gave rise to different peptide fragments when subjected to partial proteolytic digestion. Antiserum raised against either the denatured enzyme or the 45,000-dalton polypeptide did not react with any other polypeptides present in a crude cotyledonary homogenate. The purified enzyme also had alanine:glyoxylate aminotransferase activity, but was about twice as active with serine as the amino donor.In cucurbits and related plant species with fat-storing cotyledons, the cotyledons serve as the site of lipid mobilization during early germination, then emerge above ground and become photosynthetic (2). The microbodies present at early stages (glyoxysomes) play a central role in fat mobilization, whereas those present after the onset of photosynthesis (peroxisomes) are involved in the glycolate pathway of photorespiration (2,28,29). The decrease in glyoxysomal enzyme activities usually occurs concomitantly with the increase in peroxisomal activities in the greening cotyledon (1,12,19,29). Much interest has focused on this changeover in microbody function (2,4,7,23,29), but the mechanism is still unresolved.