A pyruvate kinase cDNA from soybean somatic embryos.

Pyruvate kinase (EC 2.7.1.40) is one of the key regulatory enzymes controlling glycolysis in plants and animals. It catalyzes the irreversible formation of pyruvate and ATP from phosphoenolpyruvate and ADP. In plants, pyruvate kinases can be either heteroor homotetramers depending on the source of the isozyme (Plaxton, 1989). Pyruvate kinase enzymes have been purified or partially purified from severa1 plant species (Plaxton, 1988); however, the gene encoding pyruvate kinase has been isolated only from Ricinus communis (Blakeley et al., 1991) and Solanum tuberosum (Blakeley et al., 1990). Here, we report the isolation of a pyruvate kinase cDNA clone from soybean (Glycine max [L.] Merr.) somatic embryos. A XgtlO cDNA library was constructed from poly(A’) RNA of proliferating globular-stage somatic embryos. The pyruvate kinase cDNA (Table I) was initially isolated by differential hybridization based on higher expression in globular embryos than in cotyledon embryos. Excluding the polyadenylation tail, the cDNA is 1795 b p long and contains a n open reading frame of 1533 nucleotides (predicted protein of 511 amino acids) and a 61-nucleotide 5’ and a 198-nucleotide 3’ untranslated region. In the 3‘ untranslated region, there is a putative polyadenylation signal, AAATAA, upstream of the polyadenylate sequence. Comparison of the derived protein with data base proteins revealed extensive homology with many nonplant pyruvate kinases. The soybean pyruvate kinase has 89.4% identity with potato pyruvate kinase (Blakeley et al., 1990) and 32.5% identity with pKpa, the castor bean pyruvate kinase (Blakeley et al., 1991). Southern hybridization analysis revealed two bands when genomic DNA was digested with HindIII. Because there is one HindIII site within the cDNA, there is probably a single gene for this pyruvate kinase in soybean. Although differential hybridization indicated that pyruvate kinase mRNA was expressed at a higher leve1 in globular-stage somatic embryos than cotyledon-stage embryos, this was not supported by extensive northem hybridization analysis using RNA from various staged somatic embryos of soybean.