Coordinate expression of transcriptionally regulated isocitrate lyase and malate synthase genes in Brassica napus L.

Abstract

We have analyzed the temporal and spatial expression of genes encoding the glyoxylate cycle enzymes isocitrate lyase and malate synthase in Brassica napus L. to determine whether they are coordinately expressed. Both enzymes participate in reactions associated with lipid mobilization in oilseed plant seedlings and are sequestered in a specialized organellel the glyoxysome. We have identified an isocitrate lyase cDNA clone containing the complete protein coding region. RNA blot and in situ hybridization studies with isocitrate lyase and malate synthase cDNA clones from B. napus showed that the genes exhibit similar expression patterns. The mRNAs begin to accumulate during late embryogeny, reach maximal levels in seedling cotyledons, are not detected at significant amounts in leaves, and are distributed similarly in cotyledons and axes of seedlings. Furthermore, transcription studies with isolated nuclei indicate that the genes are controlled primarily although not exclusively at the transcriptional level. We conclude that glyoxysome biogenesis is regulated in part through the coordinate expression of isocitrate lyase and malate synthase genes. INTRODUCTIONIsocitrate lyase (threo-D~-isocitrate glyoxylate-lyase, EC 4.1.3.1) and malate synthase (L-malate glyoxylate-lyase [CoA-acetylating], EC 4.1.3.2) are key enzymes involved in storage lipid mobilization during the growth of higher plant seedlings (reviewed by Trelease and Doman, 1984). They participate in reactions of the glyoxylate cycle, a pathway responsible for the net conversion of two molecules of acetyl coenzyme A into succinate. The metabolic intermediate is ultimately converted into sucrose to serve as a primary nutrient source for growing seedlings unti; photosynthetic activity commences.The two glyoxylate cycle enzymes are encoded by nuclear genes and compartmentalized in a specialized peroxisome, the glyoxysome, which contains glyoxylate cycle and/%oxidation enzymes (Breidenbach et al., 1967;Hutton and Stumpf, 1969). Biogenesis of the organelle is developmentally regulated; glyoxysomes are present primarily in developing seeds and in seedlings, although other peroxisomes are found in mature plant organs (reviewed by Huang et al., 1983;Trelease, 1984). In general, all peroxisomes appear to share a number of common characteristics: they are bounded by a single membrane, usually possess catalase and hydrogen peroxide-generating oxidases, and lack an organellar genome. However, the organelle fulfills different roles in distinct cell types that appear to be dictated by the environment or the differen-1 To whom correspondence should be addressed. tiated state of the cell. For example, in contrast to glyoxysomes, leaf-type peroxisomes possess a different set of prevalent enzymes that are involved in photorespiration. Therefore, biogenesis appears to be controlled partially by the regulated accumulation of constituents unique to a particular class of peroxisomes.To investigate the cellular processes controlling glyoxysome biogenesis, we have been studying the ...

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