Fluxes of Reserve-Derived and Currently Assimilated Carbon and Nitrogen in Perennial Ryegrass Recovering from Defoliation. The Regrowing Tiller and Its Component Functionally Distinct Zones1

The quantitative significance of reserves and current assimilates in regrowing tillers of severely defoliated plants of perennial ryegrass (Lolium perenne L.) was assessed by a new approach, comprising 13C/12C and 15N/14N steady-state labeling and separation of sink and source zones. The functionally distinct zones showed large differences in the kinetics of currently assimilated C and N. These are interpreted in terms of ”substrate” and ”tissue” flux among zones and C and N turnover within zones. Tillers refoliated rapidly, although C and N supply was initially decreased. Rapid refoliation was associated with (a) transient depletion of water-soluble carbohydrates and dilution of structural biomass in the immature zone of expanding leaves, (b) rapid transition to current assimilation-derived growth, and (c) rapid reestablishment of a balanced C:N ratio in growth substrate. This balance (C:N, approximately 8.9 [w/w] in new biomass) indicated coregulation of growth by C and N supply and resulted from complementary fluxes of reserve- and current assimilation-derived C and N. Reserves were the dominant N source until approximately 3 d after defoliation. Amino-C constituted approximately 60% of the net influx of reserve C during the first 2 d. Carbohydrate reserves were an insignificant source of C for tiller growth after d 1. We discuss the physiological mechanisms contributing to defoliation tolerance.

Effects of Short- and Long-Term Elevated CO2 on the Expression of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Genes and Carbohydrate Accumulation in Leaves of Arabidopsis thaliana (L.) Heynh.1

To investigate the proposed molecular characteristics of sugar-mediated repression of photosynthetic genes during plant acclimation to elevated CO2, we examined the relationship between the accumulation and metabolism of nonstructural carbohydrates and changes in ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) gene expression in leaves of Arabidopsis thaliana exposed to elevated CO2. Long-term growth of Arabidopsis at high CO2 (1000 μL L−1) resulted in a 2-fold increase in nonstructural carbohydrates, a large decrease in the expression of Rubisco protein and in the transcript of rbcL, the gene encoding the large subunit of Rubisco (approximately 35–40%), and an even greater decline in mRNA of rbcS, the gene encoding the small subunit (approximately 60%). This differential response of protein and mRNAs suggests that transcriptional/posttranscriptional processes and protein turnover may determine the final amount of leaf Rubisco protein at high CO2. Analysis of mRNA levels of individual rbcS genes indicated that reduction in total rbcS transcripts was caused by decreased expression of all four rbcS genes. Short-term transfer of Arabidopsis plants grown at ambient CO2 to high CO2 resulted in a decrease in total rbcS mRNA by d 6, whereas Rubisco content and rbcL mRNA decreased by d 9. Transfer to high CO2 reduced the maximum expression level of the primary rbcS genes (1A and, particularly, 3B) by limiting their normal pattern of accumulation through the night period. The decreased nighttime levels of rbcS mRNA were associated with a nocturnal increase in leaf hexoses. We suggest that prolonged nighttime hexose metabolism resulting from exposure to elevated CO2 affects rbcS transcript accumulation and, ultimately, the level of Rubisco protein.

Health care, information needs, and outreach: reaching Ohio's rural citizens

As a rural state, Ohio has a vital interest in addressing rural health and information needs. NetWellness is a Web-based consumer health information service that focuses on the needs of the residents of Ohio. Health sciences faculty from the state's three Carnegie Research I universities—University of Cincinnati, Case Western Reserve University, and The Ohio State University—create and evaluate content and provide Ask an Expert service to all visitors. Through partnerships at the state and local levels, involving public, private, commercial, and noncommercial organizations, NetWellness has grown from a regional demonstration project in 1995 to a key statewide service. Collaboration with public libraries, complemented by alliances with kindergarten through twelfth grade agencies, makes NetWellness Ohio's essential health information resource.

Pyruvate ferredoxin oxidoreductase from the hyperthermophilic archaeon, Pyrococcus furiosus, functions as a CoA-dependent pyruvate decarboxylase

Pyruvate ferredoxin oxidoreductase (POR) has been previously purified from the hyperthermophilic archaeon, Pyrococcus furiosus, an organism that grows optimally at 100°C by fermenting carbohydrates and peptides. The enzyme contains thiamine pyrophosphate and catalyzes the oxidative decarboxylation of pyruvate to acetyl-CoA and CO2 and reduces P. furiosus ferredoxin. Here we show that this enzyme also catalyzes the formation of acetaldehyde from pyruvate in a CoA-dependent reaction. Desulfocoenzyme A substituted for CoA showing that the cofactor plays a structural rather than a catalytic role. Ferredoxin was not necessary for the pyruvate decarboxylase activity of POR, nor did it inhibit acetaldehyde production. The apparent Km values for CoA and pyruvate were 0.11 mM and 1.1 mM, respectively, and the optimal temperature for acetaldehyde formation was above 90°C. These data are comparable to those previously determined for the pyruvate oxidation reaction of POR. At 80°C (pH 8.0), the apparent Vm value for pyruvate decarboxylation was about 40% of the apparent Vm value for pyruvate oxidation rate (using P. furiosus ferredoxin as the electron acceptor). Tentative catalytic mechanisms for these two reactions are presented. In addition to POR, three other 2-keto acid ferredoxin oxidoreductases are involved in peptide fermentation by hyperthermophilic archaea. It is proposed that the various aldehydes produced by these oxidoreductases in vivo are used by two aldehyde-utilizing enzymes, alcohol dehydrogenase and aldehyde ferredoxin oxidoreductase, the physiological roles of which were previously unknown.

Conformational influences of glycosylation of a peptide: A possible model for the effect of glycosylation on the rate of protein folding

Improved strategies for synthesis make it possible to expand the range of glycopeptides available for detailed conformational studies. The glycopeptide 1 was synthesized using a new solid phase synthesis of carbohydrates and a convergent coupling to peptide followed by deprotection. Its conformational properties were subjected to NMR analysis and compared with a control peptide 2 prepared by conventional solid phase methods. Whereas peptide 2 fails to manifest any appreciable secondary structure, the glycopeptide 1 does show considerable conformational bias suggestive of an equilibrium between an ordered and a random state. The implications of this ordering effect for the larger issue of protein folding are considered.

Plant histochemistry by correlation peak imaging.

Using a new NMR correlation-peak imaging technique, we were able to investigate noninvasively the spatial distribution of carbohydrates and amino acids in the hypocotyl of castor bean seedlings. In addition to the expected high sucrose concentration in the phloem area of the vascular bundles, we could also observe high levels of sucrose in the cortex parenchyma, but low levels in the pith parenchyma. In contrast, the glucose concentration was found to be lower in the cortex parenchyma than in the pith parenchyma. Glutamine and/or glutamate was detected in the cortex parenchyma and in the vascular bundles. Lysine and arginine were mainly visible in the vascular bundles, whereas valine was observed in the cortex parenchyma, but not in the vascular bundles. Although the physiological significance of these metabolite distribution patterns is not known, they demonstrate the potential of spectroscopic NMR imaging to study noninvasively the physiology and spatial metabolic heterogeneity of living plants.

Carbohydrates in Individual Cells of Epidermis, Mesophyll, and Bundle Sheath in Barley Leaves with Changed Export or Photosynthetic Rate1

Carbohydrate metabolism of barley (Hordeum vulgare) leaves induced to accumulate sucrose (Suc) and fructans was investigated at the single-cell level using single-cell sampling and analysis. Cooling of the root and shoot apical meristem of barley plants led to the accumulation of Suc and fructan in leaf tissue. Suc and fructan accumulated in both mesophyll and parenchymatous bundle-sheath (PBS) cells because of the reduced export of sugars from leaves under cooling and to increased photosynthesis under high photon fluence rates. The general trends of Suc and fructan accumulation were similar for mesophyll and PBS cells. The fructan-to-Suc ratio was higher for PBS cells than for mesophyll cells, suggesting that the threshold Suc concentration needed for the initiation of fructan synthesis was lower for PBS cells. Epidermal cells contained very low concentrations of sugar throughout the cooling experiment. The difference in Suc concentration between control and treated plants was much less if compared at the single-cell level rather than the whole-tissue level, suggesting that the vascular tissue contains a significant proportion of total leaf Suc. We discuss the importance of analyzing complex tissues at the resolution of individual cells to assign molecular mechanisms to phenomena observed at the whole-plant level.

Nitric oxide inhibits creatine kinase and regulates rat heart contractile reserve.

Cardiac myocytes express both constitutive and cytokine-inducible nitric oxide syntheses (NOS). NO and its congeners have been implicated in the regulation of cardiac contractile function. To determine whether NO could affect myocardial energetics, 31P NMR spectroscopy was used to evaluate high-energy phosphate metabolism in isolated rat hearts perfused with the NO donor S-nitrosoacetylcysteine (SNAC). All hearts were exposed to an initial high Ca2+ (3.5 mM) challenge followed by a recovery period, and then, either in the presence or absence of SNAC, to a second high Ca2+ challenge. This protocol allowed us to monitor simultaneously the effect of SNAC infusion on both contractile reserve (i.e., baseline versus high workload contractile function) and high-energy phosphate metabolism. The initial high Ca2+ challenge caused the rate-pressure product to increase by 74 +/- 5% in all hearts. As expected, ATP was maintained as phosphocreatine (PCr) content briefly dropped and then returned to baseline during the subsequent recovery period. Control hearts responded similarLy to the second high Ca2+ challenge, but SNAC-treated hearts did not demonstrate the expected increase in rate-pressure product. In these hearts, ATP declined significantly during the second high Ca2+ challenge, whereas phosphocreatine did not differ from controls, suggesting that phosphoryl transfer by creatine kinase (CK) was inhibited. CK activity, measured biochemically, was decreased by 61 +/- 13% in SNAC-treated hearts compared to controls. Purified CK in solution was also inhibited by SNAC, and reversal could be accomplished with DTT, a sulfhydryl reducing agent. Thus, NO can regulate contractile reserve, possibly by reversible nitrosothiol modification of CK.