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.     

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.     

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.     

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.     

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.     

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.     

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.     

Mitogen-activated protein kinases are developmentally regulated during stress-induced microspore embryogenesis in Brassica napus L

Plant mitogen-activated protein kinase (MAPK) cascades are involved in extracellular stress signalling pathways, leading to different cellular responses. Stress-induced microspore embryogenesis involves the internalization of an extracellular stress signal, generating a number of cellular responses where MAPK cascades might be involved. These responses include a change of the developmental programme, the entry into an early proliferative stage and, subsequently, into differentiation stages during haploid embryogenesis. In this work we studied the expression during microspore embryogenesis of several kinases, to assess their putative role in these events. The known Brassica napus MAP kinase kinase kinases (MAP3Ks BnMAP3K1, BnMAP3K1 and BnMAP3K, the BnBSK kinase and B. napus extracellular signal-regulated kinase (ERK) homologues were analysed by electron microscope (EM) in situ hybridization, immuno-gold labelling, immunofluorescence and western blotting. The differential in situ expression of these kinases suggests a role for them during embryogenesis. Two different expression patterns were observed, indicating a different regulation. BnMAP3K1, BnMAP3K, and the ERKs showed a pattern consistent with a role mainly in proliferative events. Conversely, BnMAP3K1 and BnBSK, presented a pattern that suggested an involvement in differentiation stages. In addition, ERK homologues migrate to the nucleus immediately after induction, being found in a phosphorylated state in a larger amount.     

Transformation of Brassica napus canola cultivars with Arabidopsis thaliana acetohydroxyacid synthase genes and analysis of herbicide resistance

Summary A survey of selected crop species and weeds was conducted to evaluate the inhibition of the enzyme acetohydroxyacid synthase (AHAS) and seedling growth in vitro by the sulfonylurea herbicides chlorsulfuron, DPX A7881, DPX L5300, DPX M6316 and the imidazolinone herbicides AC243,997, AC263,499, AC252,214. Particular attention was given to the Brassica species including canola cultivars and cruciferous weeds such as B. kaber (wild mustard) and Thlaspi arvense (stinkweed). Transgenic lines of B. napus cultivars Westar and Profit, which express the Arabidopsis thaliana wild-type AHAS gene or the mutant gene csr1-1 at levels similar to the resident AHAS genes, were generated and compared. The mutant gene was essential for resistance to the sulfonylurea chlorsulfuron but not to DPX A7881, which appeared to be tolerated by certain Brassica species. Cross-resistance to the imidazolinones did not occur. The level of resistance to chlorsulfuron in transgenic canola greatly exceeded the levels that were toxic to the Brassica species or cruciferous weeds. Direct selection of transgenic lines with chlorsulfuron sprayed at field levels under greenhouse conditions was achieved.     

Retention of triplicated phytoene synthase (PSY) genes in Brassica napus L. and its diploid progenitors during the evolution of the Brassiceae

The extent of genome redundancy exhibited by Brassica species provides a model to study the evolutionary fate of multi-copy genes and the effects of polyploidy in economically important crops. Phytoene synthase (PSY) catalyzes the first committed reaction of the carotenoid biosynthetic pathway, which has been shown to be rate-limiting in Brassica napus seeds. In Arabidopsis thaliana, a single PSY gene (AtPSY) regulates phytoene synthesis in all tissues. Considering that diploid Brassica genomes contain three Arabidopsis-like subgenomes, the objectives of the present work were to determine whether PSY gene families exist in B. napus (AACC) and its diploid progenitor species, Brassica rapa (AA) and Brassica oleracea (CC); to establish the level of retention of Brassica PSY genes; to map PSY gene family members in the A and C genomes and to compare Brassica PSY gene expression patterns. A total of 12 PSY homologues were identified, 6 in B. napus (BnaX.PSY.a-f) and 3 in B. rapa (BraA.PSY.a-c) and B. oleracea (BolC.PSY.a-c). Indeed, with six members, B. napus has the largest PSY gene family described to date. Sequence comparison between AtPSY and Brassica PSY genes revealed a highly conserved gene structure and identity percentages above 85% at the coding sequence (CDS) level. Altogether, our data indicate that PSY gene family expansion preceded the speciation of B. rapa and B. oleracea, dating back to the paralogous subgenome triplication event. In these three Brassica species, all PSY homologues are expressed, exhibiting overlapping redundancy and signs of subfunctionalization among photosynthetic and non-photosynthetic tissues. This evidence supports the hypothesis that functional divergence of PSY gene expression facilitates the accumulation of high levels of carotenoids in chromoplast-rich tissues. Thus, functional retention of triplicated Brassica PSY genes could be at least partially explained by the selective advantage provided by increased levels of gene product in floral organs. A better understanding of carotenogenesis in Brassica will aid in the future development of transgenic and conventional cultivars with carotenoid-enriched oil.