The plastidic 3-phosphoglycerate ? acetyl-CoA pathway in barley leaves and its involvement in the synthesis of amino acids, plastidic isoprenoids and fatty acids during chloroplast development

Abstract

Abstract.Earlier studies on the synthesis of C3-derived amino acids, plastidic isoprenoids and fatty acids from CO 2 by isolated chloroplasts in the light indicate the presence of a complete, but low-capacity, chloroplast (chip) 3-phosphoglycerate ~ acetyl-CoA pathway which is predominantely active in immature (developing) chloroplasts (A. Heintze et al., 1990, Plant Physiol. 93, 1121 1127. In this paper, we demonstrate the activity of the enzymes involved i.e. chip phosphoglycerate mutase, chip enolase, chlp pyruvate kinase and chip pyruvate-dehydrogenase complex (PDC), in the stroma of purified barley (Hordeum sativum L.) chloroplasts of different developmental stages. The chip phosphoglycerate mutase was partially purified for the first time. The activities of the enzymes of this chlp pathway (except PDC) were about a magnitude lower than those of the cytosolic enzymes. The chlp PDC of barley was more active than that of spinach. The apparent K m values of the enzymes of this pathway were about 100 IJM or lower except for the chip phosphoglycerate mutase which had a K m of 1.6-1.8 mM for 3-phospho-D-glycerate. Interestingly, no appreciable change in the activity of these enzymes was observed during maturation of the chloroplasts. In contrast, the activity of the reversible NADP +-glyceraldehyde 3-phosphate dehydrogenase increased about five times (from 140 to 590 nkat per g leaf dry weight). The following hypothesis is put forward to explain the regulation of carbon metabolism during chloroplast development: 3-phospho-D-glycerate is withdrawn from a common pool by the actions of 3-phosphoglycerate kinase and NADP +-glyceraldehyde-3-phosphate dehydrogenase, the activity of which increases considerably during matuDedicated to Prof. Dr. M.H. Zenk, Munich, on the occasion of his 60th birthday Abbreviations: C3 --* C2 pathway = 3-phospho-D-glycerate ~ acetylCoA pathway; Chl = chlorophyll; chip = chloroplast(ic); GAP = Dglyceraldehyde-3-phosphate; GAPDH = glyceraldehyde-3-phosphate dehydrogenase; PDC=pyruvate dehydrogenase complex; PEP = phosphoenolpyruvate; 2-and 3-PGA = 2-and 3-phospho-Dglycerate; U=unit; mmol.min ~(= 16.67 nkat)Correspondence to: G. Schultz; FAX: 49(511) 9 538584 ration of chloroplasts. This leads to an insufficient supply of 3-phospho-glycerate for the chip phosphoglycerate mutase, which has a low affinity for its substrate.

Distribution of Pyruvate Dehydrogenase Complex Activities between Chloroplasts and Mitochondria from Leaves of Different Species

“…In the literature there are some uncertainties whether or not the chloroplast in a11 species is fully selfprovidmg with respect to acetyl-COA by its PDC isoform or if acetyl-COA is supplied from other compartments of the cell (Givan, 1983). The substrate pyruvate for cpPDC could be provided by a stromal glycolytic pathway starting with 3-phosphoglycerate (Murphy and Leech, 1978;Hoppe et al, 1993). Altematively, malate could be imported from the cytosol and converted into pyruvate by a stromal NADPmalic enzyme (Smith et al, 1992), or pyruvate could be imported directly to the stroma (Smith et al, 1992).…”

“…cpPDC is also regulated by product inhibition but is not phosphorylated (Elias and Givan, 1979; Camp and Randall, 1985; Liedvogel and Bauerle, 1986). The PDC isoforms have similar substrate dependencies but differ in pH and magnesium optima (Williams and Randall, 1979; Treede and Heise, 1986; Hoppe et al, 1993). The requirement of cpPDC for higher pH and Mg2+ concentrations is in accord with an indirect regulation by light/dark transitions.…”

“…Assay conditions were not changed for the species studied, since published pH and magnesium optima for different species have not shown any significant differences (Williams and Randall, 1979;Treede and Heise, 1986;Hoppe et al, 1993). All of the measured PDC capacities were fully dependent on pyruvate, NAD, and COA.…”

“…In pea, approximately 80% of the cellular PDC activity was confined to the mitochondria (Williams and Randall, 1979; Budde and Randall, 1990). A lower mtPDC compared to cpPDC activity was found in barley (Hoppe et al, 1993; Kromer et al, 1994). In spinach, a 10-fold higher mtPDC activity was found compared to cpPDC (Murphy and Stumpf, 1981).…”

“…This increased activity is proposed to correlate with high demands of lipids for membrane synthesis in the expanding cells. However, the developmental pattem of cpPDC is still uncertain, since severa1 studies base enzyme activities on parameters that change significantly during leaf expansion (Murphy and Stumpf, 1981;Heintze et al, 1990;Hoppe et al, 1993).…”