Characterising the morphological characters and carbohydrate metabolism of oat culms and their association with lodging resistance

• Lodging resistance can be improved by enhancing the mechanical strength of culms, and culm carbohydrates could improve this mechanical strength. Culm carbohydrates can regulate development of the culm and affect its toughness.
• The present study determines the relationship between lodging and carbohydrate content in oat culms. Field experiments were conducted in alpine regions in 2017 and 2018 using three oat varieties with different lodging resistance. Lodging‐related morphological characteristics were directly determined and culm carbohydrate content and enzyme activity related to cellulose synthesis and sucrose metabolism were evaluated with ultraviolet spectrophotometry.
• Results showed that the lower the gravity height or the lower ratio of gravity height to plant height, the stronger the lodging resistance of the varieties. Higher culm nonstructural (NSC) and structural (SC) carbohydrate content contributed to the ability of culms to resist lodging, especially the content of cellulose and sucrose. PCA showed that sucrose metabolism and SC content were closely related to lodging resistance. Correlation analysis showed that the lodging index (LI) was significantly negatively correlated with NSC. Sucrose content was highly and significantly positively correlated with NSC. Additionally, the activities of sucrose phosphate synthase (SPS) and sucrose synthase (SS) were highly and significantly positively correlated with sucrose and cellulose content.
• The relationship between field characters and oat lodging, as well as the regulatory mechanism of carbohydrate content on lodging resistance of the culm are discussed.

     

Activity and localisation of sucrose synthase and invertase in ovules of sexual and apomicticBrachiaria decumbens

Summary Brachiaria decumbens has sexual and apomictic reproduction. Apomixis is facultative and of the aposporic type. In early stages of ovule development, differences in the pattern of callose deposition between sexual and apomictic plants were observed which points to possible differences in carbohydrate metabolism. Therefore, a comparative study on carbohydrate metabolism between a sexual diploid ecotype and an apomictic tetraploidB. decumbens was made. A histochemical determination of two enzymes responsible for sucrose degradation, sucrose synthase and invertase, was performed for all stages of ovule development. In addition, the concentrations of sucrose, glucose, and fructose were measured for each stage of ovule development, both for sexual and apomictic plants. The enzymes were localised by immunohistochemistry with heterologous antibodies. A distinct difference between sexual and apomictic plants was observed in the localisation of sucrose synthase activity as well as in the amount of activity, especially in the early stages of ovular development. Invertase activity localisation was comparable between ovules of the sexual and apomictic plants, but its activity is clearly higher in ovules of sexual plants. The localisation of the enzymes coincided with the place of activity. For both sexual and apomictic plants the amount of sucrose in the ovaries increased with the stage of ovule development. Differences in the amount of sucrose between sexual and apomictic plants in ovaries with ovules in comparable stages of development were detected. A delay in the onset of carbohydrate metabolism during early stages of ovule development characterises the apomictic plant.Abbreviations MMC megaspore mother cell - MC meiocyte - MS megaspore - AI apospore initial - CO coenocyte - MES mature embryo sac - SuSy sucrose synthase - INV invertase - BMM buthylmethyl methacrylate - DTT dithiothreitol - DAPI 4,6-diamidino-2-phenylindole - PBS phosphate-buffered saline     

Effect of a strong cold storage induced desiccation on metabolic solutes,stock quality and regrowth,in seedlings of two oak species

Bare-root seedlings of pedunculate oak (Quercus robur L.) and northern red oak (Quercus rubra L.) were lifted in January and stored at 1.8°C, at 82% relative humidity, until their fresh weight declined by 33%. Root growth potential (RGP), fine root electrolyte leakage (REL), fine root water content (RWC), shoot tip water content (SWC), starch and metabolic solute contents in root and shoot, were measured just after lifting and after treatment. Survival of treated seedlings was also assessed in a field trial. RWC, SWC, REL, RGP were dramatically affected by desiccation during cold storage. In both species, root soluble carbohydrate level, inositol level and isocitrate level increased, whereas root starch level and shoot soluble carbohydrate level decreased. In northern red oak, treated seedlings had higher root contents of soluble carbohydrates, inositol and proline than in pedunculate oak. Moreover, treatment induced proline accumulation only in northern red oak roots. These differences could explain why field survival of treated seedlings was significantly better in northern red oak than in pedunculate oak.     

Ecophysiological adaptations of black spruce (<Emphasis Type="Italic"> Picea mariana</Emphasis>) and tamarack (<Emphasis Type="Italic"> Larix laricina</Emphasis>) seedlings to flooding

Black spruce [ Picea mariana (Mill.) B.S.P.] and tamarack [ Larix laricina (Du Roi) K. Koch] are the predominant tree species in boreal peatlands. The effects of 34 days of flooding on morphological and physiological responses were investigated in the greenhouse for black spruce and tamarack seedlings in their second growing season (18 months old). Flooding resulted in reduced root hydraulic conductance, net assimilation rate and stomatal conductance and increased needle electrolyte leakage in both species. Flooded tamarack seedlings maintained a higher net assimilation rate and stomatal conductance compared to flooded black spruce. Flooded tamarack seedlings were also able to maintain higher root hydraulic conductance compared to flooded black spruce seedlings at a comparable time period of flooding. Root respiration declined in both species under flooding. Sugar concentration increased in shoots while decreasing in roots in both species under flooding. Needles of flooded black spruce appeared necrotic and electrolyte leakage increased over time with flooding and remained significantly higher than in flooded tamarack seedlings. No visible damage symptoms were observed in flooded tamarack seedlings. Flooded tamarack seedlings developed adventitious roots beginning 16 days after the start of flooding treatment. Adventitious roots exhibited significantly higher root hydraulic conductivity than similarly sized flooded tamarack roots. Flooded black spruce lacked any such morphological adaptation. These results suggest that tamarack is better able to adjust both morphologically and physiologically to prolonged soil flooding than black spruce seedlings.     

Production of recombinant xenotransplantation antigen in Escherichia coli

The synthesis of sufficient amounts of oligosaccharides is the bottleneck for the study of their biological function and their possible use as drug. As an alternative for chemical synthesis, we propose to use Escherichia coli as a "living factory." We have addressed the production of the Galp alpha(1-3)Galp beta(1-4)GlcNAc epitope, the major porcine antigen responsible for xenograft rejection. An E. coli strain was generated which simultaneously expresses NodC (to provide the chitin-pentaose acceptor), beta(1-4) galactosyltransferase LgtB, and bovine alpha(1-3) galactosyltransferase GstA. This strain produced 0.68 g/L of the heptasaccharide Galp alpha(1-3)Galp beta(1-4)(GlcNAc)(5), which harbours the xenoantigen at its non-reducing end, establishing the feasibility of this approach.     

Partial characterisation of carbohydrate-rich Echinococcus granulosus coproantigens

Coproantigen ELISA based tests for diagnosis of canine echinococcosis provide high specificity and sensitivity. However, the antigenic molecules present in faeces from infected dogs have not yet been characterised. While initial attempts to determine the molecular weights of Echinococcus granulosus coproantigens by SDS-PAGE and Western blotting with coproantigen reactive capture antibodies were equivocal, they suggested presence of a significant carbohydrate component. Periodate treatment of coproantigen positive faecal supernatants resulted in a significant reduction (53%) in ELISA activity, suggesting that carbohydrates are involved in the antigenic structure of E. granulosus coproantigens. Protease treatment of antigenic molecules resulted in an 11% reduction in absorbance in ELISA, indicating that protein components were also present which affected by enzyme activity. Lectin-binding ELISA assays indicated strong affinity of E. granulosus coproantigens to concanavalin agglutinin and Lens culinaris agglutinin, and moderate binding to wheat-germ agglutinin and peanut agglutinin. No binding was detectable to Ulex europaensis agglutinin-I, Bandeiraea simplicifolia or Dolichos biflorus agglutinin. These data indicate that E. granulosus coproantigens from infected dog faeces possibly contained alpha-D-mannose and/or alpha-D-glucose, beta-galactose and N-acetyl-beta-glucosamine residues. To verify the role of carbohydrate moieties in coproantigens, faecal samples were treated with exoglycosidase and tested in the coproantigen ELISA. Treatment with beta-galactosidase or N-acetyl-beta-glucosamine reduced ELISA activity by 44 and 30%, respectively. Incubation with a panel of other specific exoglycosidases including alpha-galactosidase as well as alpha-L-fucosidase, alpha-mannosidase, beta-mannosidase, alpha-glucosidase, beta-glucosidase, beta- fructosidase, or neuraminidase, did not alter coproantigen detection in ELISA. The results indicate that coproantigens present in faeces from E. granulosus naturally infected dogs were highly glycosylated and contain beta- galactose and N-acetyl-beta-glucosamine. The putative relationship of antigenic molecules with the tapeworm glycocalyx is discussed.     

Interaction between carbohydrate residues of alpha(1)-acid glycoprotein (orosomucoid) and progesterone. A fluorescence study

Interaction between progesterone and the carbohydrate residues of alpha(1)-acid glycoprotein was followed by fluorescence studies using calcofluor white. The fluorophore interacts with polysaccharides and is commonly used in clinical studies. Binding of progesterone to the protein induces a decrease in the fluorescence intensity of calcofluor white, accompanied by a shift to the short wavelengths of its emission maximum. The dissociation constant of the complex was found equal to 8.62 microM. Interaction between progesterone and free calcofluor in solution induces a low decrease in the fluorescence intensity of the fluorophore without any shift of the emission maximum. These results show that in alpha(1)-acid glycoprotein, the binding site of progesterone is very close to the carbohydrate residues. Fluorescence intensity quenching of free calcofluor in solution with cesium ion gives a bimolecular diffusion constant (k(q)) of 2.23 x 10(9) M(-1) s(-1). This value decreases to 0.19 x 10(9) M(-1) s(-1) when calcofluor white is bound to alpha(1)-acid glycoprotein. Binding of progesterone does not modify the value of k(q) of the cesium. Previous studies have shown that the terminal sialic acid residue is mobile, while the other glycannes are rigid [Albani, J. R.; Sillen, A.; Coddeville, B.; Plancke, Y. D.; Engelborghs, Y. Carbohydr. Res. 1999, 322, 87-94]. Red-edge excitation spectra and Perrin plot experiments performed on sialylated and asialylated alpha(1)-acid glycoprotein show that binding of progesterone to alpha(1)-acid glycoprotein does not modify the local dynamics of the carbohydrate residues of the protein.     

Carbohydrate cycling in micro-organisms: what can (13)C-NMR tell us?

The extension of (13)C-nuclear magnetic resonance (NMR) techniques to study cellular metabolism over recent years has provided valuable data supporting the occurrence, diversity and extent of carbon cycling in the carbohydrate metabolism of micro-organisms. The occurrence of such cycles, resulting from the simultaneous operation of different and sometimes opposite individual steps, is inherently related to the network organisation of cellular metabolism. These cycles are tentatively classified here as 'reversibility', 'metabolic' and 'substrate' cycles on the basis of their balance in carbon and cofactors. Current hypotheses concerning the physiological relevance of carbohydrate cycles are discussed in light of the (13)C-NMR data. They most likely represent system-level mechanisms for coherent and timely partitioning of carbon resources to fit with the various biosynthetic, energetic or redox needs of cells and/or additional strategies in the adaptive capacity of micro-organisms to face variation in environmental conditions.     

Exercise and recovery metabolism in the pacific spiny dogfish (<Emphasis Type="Italic">Squalus acanthias</Emphasis>)

We examined the effects of exhaustive exercise and post-exercise recovery on white muscle substrate depletion and metabolite distribution between white muscle and blood plasma in the Pacific spiny dogfish, both in vivo and in an electrically stimulated perfused tail-trunk preparation. Measurements of arterial-venous lactate, total ammonia, -hydroxybutyrate, glucose, and l-alanine concentrations in the perfused tail-trunk assessed white muscle metabolite fluxes. Exhaustive exercise was fuelled primarily by creatine phosphate hydrolysis and glycolysis as indicated by 62, 71, and 85% decreases in ATP, creatine phosphate, and glycogen, respectively. White muscle lactate production during exercise caused a sustained increase (~12 h post-exercise) in plasma lactate load and a short-lived increase (~4 h post-exercise) in plasma metabolic acid load during recovery. Exhaustive exercise and recovery did not affect arterial PO₂, PCO₂, or PNH₃ but the metabolic acidosis caused a decrease in arterial HCO₃^– immediately after exercise and during the first 8 h recovery. During recovery, lactate was retained in the white muscle at higher concentrations than in the plasma despite increased lactate efflux from the muscle. Pyruvate dehydrogenase activity was very low in dogfish white muscle at rest and during recovery (0.53±0.15 nmol g wet tissue^–1 min^–1; n=40) indicating that lactate oxidation is not the major fate of lactate during post-exercise recovery. The lack of change in white muscle free-carnitine and variable changes in short-chain fatty acyl-carnitine suggest that dogfish white muscle does not rely on lipid oxidation to fuel exhaustive exercise or recovery. These findings support the notion that extrahepatic tissues cannot utilize fatty acids as an oxidative fuel. Furthermore, our data strongly suggest that ketone body oxidation is important in fuelling recovery metabolism in dogfish white muscle and at least 20% of the ATP required for recovery could be supplied by uptake and oxidation of -hydroxybutyrate from the plasma.Abbreviations CoA-SH free coenzyme A - CPT-1 carnitine palmitoyltransferase-1 - CrP creatine phosphate - H⁺_m metabolic proton load - Lac lactate load - PDH pyruvate dehydrogenase - PVP polyvinylpyrrolidone - SCFA-carnitine short-chain fatty acyl-carnitine - TAG triacylglycerol - TENS trancutaneous electrical nerve stimulator Communicated by: L.C.-H. Wang     

Competitive inhibition analysis of the enzyme-substrate interaction in the carboxy-terminal processing of the precursor D1 protein of photosystem II reaction center using substituted oligopeptides

A clear parallelism was demonstrated between the efficiency as substrate of the substituted oligopeptides corresponding to the carboxy-terminal (C-terminal) sequence of the precursor D1 protein (pD1) in the in vitro enzymatic assay and their competitive inhibitory capacity toward the proteolytic C-terminal processing of the full-length pD1 integrated in the intact photosystem II complex embedded in the thylakoid membrane of Scenedesmus obliquus LF-1 mutant, as shown e.g. by the influence of L343A, A345G and A345V substitutions and the effect of C-terminal fragments. This suggests that the basic mechanism for substrate recognition by the processing protease elucidated in the enzymatic analysis using synthetic oligopeptides is also effective in vivo, although it can sometimes be difficult to detect the consequence of amino acid substitution in the integrated systems.