Translational or post-translational processes affect differentially the accumulation of isocitrate lyase and malate synthase proteins and enzyme activities in embryos and seedlings of Brassica napus

We have analyzed the accumulation of the glyoxylate cycle enzymes isocitrate lyase and malate synthase in embryos and seedlings of Brassica napus L. The two enzyme activities and proteins begin to accumulate during late embryogeny, reach maximal levels in seedlings, and are not detected in young leaves of mature plants. We showed previously that mRNAs encoding the two enzymes exhibit similar qualitative patterns of accumulation during development and that the two mRNAs accumulate to different levels in both embryos and seedlings (L. Comai et al., 1989, Plant Cell 1, 293-300). In this report, we show that the relative accumulation of the proteins and activities do not correspond to these mRNA levels. In embryos and seedlings, the specific activities of isocitrate lyase and malate synthase are approximately constant. By contrast, the ratio of malate synthase protein to mRNA is 14-fold higher than that of isocitrate lyase. Differences in the translational efficiencies of the two mRNAs in vitro do not appear to account for the discrepancy between mRNA and protein levels. Our results suggest that translational and/or post-translational processes affect differentially the accumulation of the proteins.     

Characterization of a bifunctional glyoxylate cycle enzyme, malate synthase/isocitrate lyase, of Euglena gracilis

The glyoxylate cycle is a modified form of the tricarboxylic acid cycle, which enables organisms to synthesize carbohydrates from C2 compounds. In the protozoan Euglena gracilis, the key enzyme activities of the glyoxylate cycle, isocitrate lyase (ICL) and malate synthase (MS), are conferred by a single bifunctional protein named glyoxylate cycle enzyme (Euglena gracilis glyoxylate cycle enzyme [EgGCE]). We analyzed the enzymatic properties of recombinant EgGCE to determine the functions of its different domains. The 62-kDa N-terminal domain of EgGCE was sufficient to provide the MS activity as expected from an analysis of the deduced amino acid sequence. In contrast, expression of the 67-kDa C-terminal domain of EgGCE failed to yield ICL activity even though this domain was structurally similar to ICL family enzymes. Analyses of truncation mutants suggested that the N-terminal residues of EgGCE are critical for both the ICL and MS activities. The ICL activity of EgGCE increased in the presence of micro-molar concentrations of acetyl-coenzyme A (CoA). Acetyl-CoA also increased the activity in a mutant type EgGCE with a mutation at the acetyl-CoA binding site in the MS domain of EgGCE. This suggests that acetyl-CoA regulates the ICL reaction by binding to a site other than the catalytic center of the MS reaction.     

Determination of isocitrate lyase and malate synthase activities in a marine bivalve mollusk by a new method of assay

1. A new method for the assay of isocitrate lyase (EC 4.1.3.1) was developed, based on the isolation of 14C-glyoxylate semicarbazone by co-crystallization with authentic carrier. The method was easily adapted to measure malate synthase (EC 4.1.3.2). 2. Interfering reactions were avoided with this method, and isocitrate dehydrogenase (ID) was easily distinguished from isocitrate lyase (IL). Assay of IL in germinating pumpkin seeds gave rates proportional to the amount of extract, with greater sensitivity and less variability than the spectrophotometric method. 3. Six species of marine bivalve mollusks were tested for IL activity, and two species produced glyoxylate: Crassostrea virginica at 0.10 mumol/hr/g tissue, and Petricola pholadiformis at 0.85 mumol/hr/g. The other four species, and four other marine invertebrates from other phyla, lacked detectable IL activity. 4. The rate of disappearance of glyoxylate in the malate synthase (MS) reaction indicated that Petricola had an activity of 0.60 mumol/hr/g: this is the first demonstration of activities of both IL and MS in a marine invertebrate.     

Molecular characterization of a bifunctional glyoxylate cycle enzyme, malate synthase/isocitrate lyase, in Euglena gracilis

Euglena gracilis induced glyoxylate cycle enzymes when ethanol was fed as a sole carbon source. We purified, cloned and characterized a bifunctional glyoxylate cycle enzyme from E. gracilis (EgGCE). This enzyme consists of an N-terminal malate synthase (MS) domain fused to a C-terminal isocitrate lyase (ICL) domain in a single polypeptide chain. This domain order is inverted compared to the bifunctional glyoxylate cycle enzyme in Caenorhabditis elegans, an N-terminal ICL domain fused to a C-terminal MS domain. Purified EgGCE catalyzed the sequential ICL and MS reactions. ICL activity of purified EgGCE increased in the existence of acetyl-CoA at a concentration of micro-molar order. We discussed the physiological roles of the bifunctional glyoxylate cycle enzyme in these organisms as well as its molecular evolution.     

Regulation of glyoxysomal enzymes during germination of cucumber. Temporal changes in translatable mRNAs for isocitrate lyase and malate synthase

The relative levels of translatable messenger RNA for isocitrate lyase and malate synthase were determined in the dry seed and for the first seven days of development of cucumber cotyledons. After extraction and quantification of total and poly(A)-rich RNA each day, the RNA fractions were translated in an optimized wheat germ system and the specific polypeptides were immunoprecipitated quantitatively. The radiolabeled isocitrate lyase and malate synthase polypeptides were then fractionated on dodecylsulphate/polyacrylamide gels, visualized by exposure to X-ray film and quantified densitometrically. The relative levels of translatable messenger RNA for these enzymes rise and fall with a developmental program similar to the enzyme activities, but preceding the latter by about one day. This implies that the rise in enzyme activity is dependent upon a prior postgerminative increase in translatable messenger RNA for the enzymes. These studies also suggest that messenger RNA levels may be regulated, at least in part, by light.     

Purification and characterization of a cold-adapted isocitrate lyase and a malate synthase from Colwellia maris, a psychrophilic bacterium.

Isocitrate lyase (ICL) and malate synthase (MS) of a psychrophilic marine bacterium, Colwellia maris, were purified to electrophoretically homogeneous state. The molecular mass of the ICL was found to be 240 kDa, composed of four identical subunits of 64.7 kDa. MS was a dimeric enzyme composed of 76.3 kDa subunits. N-Terminal amino acid sequences of the ICL and MS were analyzed. Purified ICL had its maximum activity at 20 degrees C and was rapidly inactivated at the temperatures above 30 degrees C, but the optimum temperature for the activity of MS was 45 degrees C. NaCl was found to protect ICL from heat inactivation above 30 degrees C, but the salt did not stabilize MS. Effects of temperatures on the kinetic parameters of both the enzymes were examined. The Km for the substrate (isocitrate) of ICL was decreased with decreasing temperature. On the other hand, the Km for the substrate (glyoxylate) of MS was increased with decreasing temperature. The calculated value of free energy of activation of ICL was on the same level as that of MS.     

Differential expression of duplicated peroxidase genes in the allotetraploid Brassica napus

Gene redundancy due to polyploidization provides a selective advantage for plant adaptation. We examined the expression patterns of two peroxidase genes (BnPOX1 and BnPOX2) in the natural allotetraploid Brassica napus and the model diploid progenitors Brassica rapa (Br) and Brassica oleracea (Bo) in response to the fungal pathogen Sclerotinia sclerotiorum. We demonstrated that the Bo homeolog of BnPOX1 was up-regulated after infection, while both BnPOX2 homeologs were down-regulated. A bias toward reciprocal expression of the homeologs of BnPOX1 in different organs in the natural allotetraploid of B. napus was also observed. These results suggest that subfunctionalization of the duplicated BnPOX genes after B. napus polyploidization as well as subneofunctionalization of the homeologs in response to this specific biotic stress has occurred. Retention of expression patterns in the diploid progenitors and the natural allotetraploid in some organs indicates that the function of peroxidase genes has been conserved during evolution.     

Influence of cytokinins on growth of Phycomyces blakesleeanus and on the activities of the glyoxylate cycle enzymes isocitrate lyase and malate synthase

Treatment of the 1 + strain of Phycomyces blakesleeanus Bgff. with various cytokinins resulted in a stimulation of growth. The magnitude of growth stimulation depended on both the structure of the hormone used and the carbon source in the culture medium. Most of the cytokinin derivatives were active effect in glucose and oleic acid cultures. Benzyladenine (BA) and benzyladenosine stimulated the fungal growth only when oleic acid was the sole carbon source, while they had no effect in glucose cultures within the tested range of concentrations. [¹⁴C]-BA was accumulated by the mycelium of oleic acid cultures. Therefore, differences in BA uptake between glucose and oleic acid cultures could account mainly for the specific growth-promoting effect of BA. In oleic acid cultures isocitrate lyase (EC 4.1.3.1) and malate synthase (EC 4.1.3.2) activities were enhanced by 40 and 34%, respectively, in the presence of BA. A time course of the hormone effect suggests that BA is not involved in induction, but in the regulation of the mentioned enzymes in Phycocmyces. In contrast, acetate when presented as the sole carbon source or after addition to a glucose culture medium, induced isocitrate lyase activity. This enzyme induction was prevented by simultaneous addition of cycloheximide.     

Differentially regulated malate synthase genes participate in carbon and nitrogen metabolism of S. cerevisiae.

We have isolated a second gene (MLS1), which in addition to DAL7, encodes malate synthase from S. cerevisiae. Expression of the two genes is specific for their physiological roles in carbon and nitrogen metabolism. Expression of MLS1, which participates in the utilization of non-fermentable carbon sources, is sensitive to carbon catabolite repression, but nearly insensitive to nitrogen catabolite repression. DAL7, which participates in catabolism of the nitrogenous compound allantoin, is insensitive to carbon catabolite repression, but highly sensitive to nitrogen catabolite repression. Results obtained with null mutations in these genes suggest that S. cerevisiae contains at least one and perhaps two additional malate synthase genes.     

Regulation of glyoxylate cycle enzymes in Saccharomycopsis lipolytica. I. Effect of the carbon source on isocitrate lyase and malate synthase activity

Comparative studies on the activities of isocitrate lyase (ICL) and malate synthase (MS) were carried out with Saccharomycopsis lipolytica incubating the yeast on media with different carbon sources. When cells were incubated in minimal medium with glucose, the activities of both enzymes were very low. In contrast, in minimal medium with acetate enhanced enzyme activities could be demonstrated. It is probably that the synthesis of ICL is repressed in presence of glucose. Furthermore the activity of ICL was inhibited by tricarboxylic acid cycle intermediates like succinic acid and oxalacetic acid. It was concluded that the syntheses of enzymes are derepressed. When cells of Sm. lipolytica were incubated in minimal medium with acetate, a high enzyme activity is evident. Synthesis of ICL on acetate was inhibited by cycloheximide and actinomycin D. The results were discussed comparing them with data obtained from other organisms.     

Transformation of Brassica napus L. using Agrobacterium tumefaciens: developmentally regulated expression of a reintroduced napin gene

Summary Genetically transformed plants of Brassica napus L. (oilseed rape) were obtained from hypocotyl expiants using Agrobacterium tumefaciens vectors. Hypocotyl explants were inoculated with disarmed or oncogenic A. tumefaciens strains, EHA101 and A281, and then cultured on media containing kanamycin. The A. tumefaciens strains harbored a binary vector, which contained a neomycin phosphotransferase II (NPTII) gene driven by the 35S promoter of cauliflower mosaic virus and an engineered napin (seed storage protein) gene with its own promoter (300 nucleotides 5 to the start of translation). Transformation of B. napus plants was confirmed by detection of NPT II enzyme activity, Southern blot analysis and inheritance of the kanamycin-resistance trait (NPT II gene) in the progeny. Expression of the engineered napin gene in embryos but not in leaves of transgenic plants was observed by Northern analysis. These data demonstrate that morphologically normal, fertile transgenic B. napus plants can be obtained using Agrobacterium as a gene vector and that developmentally regulated expression of reintroduced genes can be achieved.     

Leaf senescence in Brassica napus: expression of genes encoding pathogenesis-related proteins

Genes that are expressed during leaf senescence in Brassica napus were identified by the isolation of representative cDNA clones. DNA sequence and deduced protein sequence from two senescence-related cDNAs, LSC94 and LSC222, representing genes that are expressed early in leaf senescence before any yellowing of the leaves is visible, showed similarities to genes for pathogenesis-related (PR) proteins: a PR-1a-like protein and a class IV chitinase, respectively. The LSC94 and LSC222 genes showed differential regulation with respect to each other; an increase in expression was detected at different times during development of healthy leaves. Expression of both genes was induced by salicylic acid treatment. These findings suggest that some PR genes, as well as being induced by pathogen infection, may have alternative functions during plant development, for example in the process of leaf senescence.