The redistribution of N from vegetative plant parts to the developing seed in soybeans [Glycine max (L.) Merrill] may influence the duration of seed filling and yield. The objective of this study was to investigate the N redistribution characteristics of soybean cultivars of varying maturities and growth habit. Eight cultivars ranging from Maturity Group II to V and including indeterminate, determinate, and semi‐dwarf growth habits were grown in the field in 1977 and 1978 at Lexington, Ky. using conventional cultural practices. The soil type was a Lanton silt loam (Cumulic Haplaquolls) in 1977 and an Eagam silt loam (Cumulic Hapludolls) in 1978. Nitrogen redistribution was estimated by harvesting plants at beginning seed growth (R5) and at maturity. The abscised leaf blades and petioles were also collected and the dry weight and total N was measured in all plant parts. The vegetative dry weight at RS increased in cultivars of later maturity. There were no consistent culvar differences in N concentration at R5. The proportion of seed N that came from redistribution varied from 30 to essentially 100% and there were significant cultivar differences. The cultivar differences were positively correlated with the amount of N in the plant at R5 which was determined primarily by the vegetative dry weight at R5. Late maturing cultivars got more of their seed N from redistribution than early maturing cultivars. Although there were significant cultivar differences in yield and the duration of seed fill, they were not related to the amount of seed N that came from redistribution. Nitrogen redistribution does not appear to be an important factor determining the duration of seed filling or yield in soybeans.
Acetyl-coenzyme A (CoA) synthetase was purified 364-fold from leaves of spinach (Spinacia oleracea L.) using ammonium sulfate fractionation followed by ion exchange, dye-ligand, and gel permeation chromatography. The final specific activity was 2.77 units per (24) showed with isolated spinach chloroplasts that radiolabeled acetate at physiological concentrations preferentially labeled fatty acids whereas radiolabeled pyruvate showed preference for products of prenyl metabolism. Heintz et al. (9) reported that in developing plastids of barley leaves pyruvate was a precursor (via chloroplast PDC) for isoprenoids and fatty acids, but that as the tissue matured the chloroplast PDC declined as a precursor of C2 units. Heintz et al. (9) further showed that when acetate was provided to both developing and mature barley chloroplasts it was the primary source of C2 units for fatty acid biosynthesis.Significant levels of acetate have been measured in leaf tissue from several plant species (12, 27) and in a spinach leaves; 15 to 20% of the total cellular acetate coincided with the chloroplast fraction (27). Taken together, this evidence indicates that acetyl-CoA synthetase is at least partially responsible for chloroplast acetyl-CoA formation in most plant tissues.Despite the potential importance of acetyl-CoA synthetase in chloroplast acetyl-CoA metabolism, no attempt has been made to purify this enzyme from photosynthetic tissue. Millerd and Bonner (17) reported some properties of the enzyme using ammonium sulfate precipitated enzyme from spinach leaf extracts. This enriched spinach enzyme showed rather broad substrate specificity, with butyrate and succinate being 84 and 52%, respectively, as active as acetate, but showed no activity with propionate as substrate. Millerd and Bonner (17) attributed the substrate nonspecificity to the presence of several activating enzymes. Young and Anderson (32) identified a short chain fatty acyl-CoA synthetase in extracts of germinated seeds of Pinus radiata which had a greater affinity for butyrate than for acetate, but no butyryl-CoA synthetase activity has been reported for leaf tissue. In contrast, acetylCoA synthetase purified from several different sources (4, 7, 10, 29) activated acetate and propionate but not butyrate. Therefore, it was our intent to investigate whether acetyl-CoA synthetase isolated from spinach leaves is indeed an acetyl-
Leaf protoplasts of tobacco (Nicotlana tabacum L.) were employed for transfection of chimeric transcriptional gene fusions comprising the 35S promoter from cauliflower mosaic virus, the coding sequence of the G-protein from vesicular stomatitis virus (VSVG) and the transcriptional terminator from the Agrobacterium tumefaciens nopaline-synthetase gene. Transient expression of the chimeric gene was monitored through Northern analysis of total protoplast RNA using a labeled VSV cDNA probe, and through Western-blot analysis of protoplast proteins using a polyclonal and-VSV antiserum. Although a single species of mRNA was detected in the transfected protoplasts, two glycoproteins differing in mass by approx. 9 kDa were detected by the antiserum. Biosynthesis of the VSVG isoforms was not impeded by chemical inhibitors of cell-wall production or of proline hydroxylation. Transfection using mutant forms of the VSVG coding sequence in which either one or both consensus glycosylation sites were removed resulted in the production of progressively smaller VSVG proteins. Those proteins produced from the double mutant had mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis that were very similar to those produced from the wild-type construct in the presence of tunicamycin. Analysis of protoplast homogenates by differential centrifugation showed that the two VSVG isoforms were exclusively associated with cellular membranes. The larger protein co-localized with the plasma membrane and with the organelles of the endomembrane-secretory pathway leading to the plasma membrane. The smaller protein was associated with membranes of lower isopycnic densities which were not identical to the endoplasmic reticulum. The larger protein displayed greater sensitivity than did the smaller to degradation in vivo by exogenously added protease. Immunofluorescence microscopy demonstrated that the VSVG isoforms were present both within the protoplasts and at the surface of the plasma membrane. The intracellular distribution was either punctate or reticulate. These results are consistent with the progressive and accurate glycosylation of the newly synthesized VSVG polypeptide during its passage through the endomembrane-secretory pathway, the access of the larger isoform to the cell surface, and the conversion of the larger to the small isoform by selective proteolysis.
Acetyl coenzyme A (acetyl-CoA) synthetase and acetate kinase were localized within the soluble portion of Bradyrhizobium japonicum bacteroids, and no appreciable activity was found elsewhere in the nodule. The presence of each acetate-activating enzyme was confirmed by separation of the two enzyme activities on a hydroxylapatite column, by substrate dependence of each enzyme in both the forward and reverse directions, by substrate specificity, by inhibition patterns, and also by identification of the reaction products by C18 reverse-phase high-pressure liquid chromatography. Phosphotransacetylase activity, found in the soluble portion of the bacteroid, was dependent on the presence of potassium and was inhibited by added sodium. The greatest acetyl-CoA hydrolase activity was found in the root nodule cytosol, although appreciable activity also was found within the bacteroids. The combined specific activities of acetyl-CoA synthetase and acetate kinase-phosphotr,ansacetylase were approximate to that of the pyruvate dehydrogenase complex, thus providing B. japonicum with sufficient capacity to generate acetyl-CoA. Acetate metabolism performs a diversity of functions in the physiology of procaryotic and eucaryotic organisms. Acetyl coenzyme A (acetyl-CoA) is the most common metabolic form of acetate, since it is a key intermediate in the citric acid cycle, the glyoxylate cycle, amino acid metabolism, and the polyhydroxybutyrate cycle. Acetylphosphate is integral to the energy metabolism of many bacteria and has recently been shown to be involved in the phosphoenolpyruvate-dependent phosphotransferase system in Escherichia coli and Salmonella typhimiurium (4). Several reports indicate that in Bradyrhizobium japonicum, the nitrogen-fixing endophyte of soybean, acetate metabolism is active during symbiotic functioning. (i) The exopolysaccharides (EPS) produced by this organism are essential for recognition and subsequent infection of the host plant (3, 10, 21). Mutants defective in EPS synthesis, resulting in altered composition, form ineffective nodules, i.e., they are incapable of symbiotic nitrogen fixation (3, 10). EPS can account for up to 20% of the dry weight of B. japonicum, and when the constituents of EPS are considered on a molar basis, acetate is second only to glucose (21). Overall, acetate accounts for about 4% of the dry weight of the organism, and
Mitochondria from Pisum sativum seedlings purified free of peroxisomal and chlorophyll contamination were examined for acetyl-coenzyme A (CoA) hydrolase activity. Acetyl-CoA hydrolase activity was latent when assayed in isotonic media. The majority of the enzyme activity was found in the soluble matrix of the mitochondria. The products, acetate and CoA, were quantified by two independent methods and verified that the observed activity was an acetyl-CoA hydrolase. The pea mitochondrial acetyl-CoA hydrolase showed a Km for acetyl-CoA of 74 micromolar and a Vm.. of 6
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