Genetic Control of TCA Cycle During Seed Germination

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Mature oil seeds (cucumber, pumpkin, peanut, Brassica, etc) contain few mitochondria and have low levels of tricarboxylic acid (TCA) cycle enzymes. Following imbibition the TCA cycle enzymes are synthesized de novo. The biogenesis of the TCA cycle enzymes, however, is not uniform. The non-decarboxylative portion of the pathway (the reactions between succinate and oxaloacetate) is synthesized first. A few days later, the enzymes of the decarboxylative part of the cycle, the reactions between pyruvate (or citrate) and succinate, are formed. This switch results from changing metabolism in the cotyledons of these seeds. Early in germination the cotyledons are converting stored lipid to carbohydrate. While the non-decarboxylative portion of the TCA cycle is essential for this process the decarboxylative portion is not present thus preventing carbon loss through the decarboxylation reactions. Later as these tissues become photosynthetic, a full TCA cycle is needed and the enzymes of the decarboxylative portion are synthesized. In this proposal Dr. Oliver will examine the mechanism by which this differential expression of mitochondrial proteins is accomplished using fumarase, an enzyme specific for the non-decarboxylative part, and the NAD+-dependent isocitrate dehydrogenase, an enzyme found only in the decarboxylative portion of the TCA cycle. The proteins have been purified, antibody tools are being produced, and N-terminal sequence information is being obtained. The antibody and/or oligonucleotide probes will be used to isolate the cDNA (and then genomic) clones for these two proteins. The level of control of gene expression(transcriptional, translational, etc) will be determined by measuring mRNA levels and with run on transcription assays. Promoter-GUS contructs will be transformed into Brassica napus and these transgenic plants will be used to study, 1. the time course and tissue specificity of expression of both genes, 2. environmental and physiological factors that control the expression of these two genes, and 3. repression of TCA cycle proteins during seed maturation. Promoter analysis will be initiated. These studies may be augmented by the use of transient expression assays and rapid cycling Brassica or Arabidopsis.