Concurrent elevation of CO2, O3 and temperature severely affects oil quality and quantity in rapeseed.

Plant oil is an essential dietary and bio-energy resource. Despite this, the effects of climate change on plant oil quality remain to be elucidated. The present study is the first to show changes in oil quality and quantity of four rapeseed cultivars in climate scenarios with elevated [CO2], [O3] and temperature (T) combined and as single factors. The combination of environmental factors resembled IPCC's 'business as usual' emission scenario predicted for late this century. Generally, the climate scenarios reduced the average amounts of the six fatty acids (FAs) analysed, though in some treatments single FAs remained unchanged or even increased. Most reduced was the FA essential for human nutrition, C18:3-ω3, which decreased by 39% and 45% in the combined scenarios with elevated [CO2]+T+[O3] and [CO2]+T, respectively. Average oil content decreased 3-17%. When [CO2] and T were elevated concurrently, the seed biomass was reduced by half, doubling the losses in FAs and oil content. This corresponded to a 58% reduction in the oil yield per hectare, and C18:3-ω3 decreased by 77%. Furthermore, the polyunsaturated FAs were significantly decreased. The results indicate undesirable consequences for production and health benefits of rapeseed oil with future climate change. The results also showed strong interactive effects of CO2, T and O3 on oil quality, demonstrating why prediction of climate effects requires experiments with combined factors and should not be based on extrapolation from single factor experiments.

Mechanistic aspects of the nucleophilic substitution of pectin. On the formation of chloromethane.

Chloromethane, accounting for approximately 16% of the tropospheric chlorine, is mainly coming from natural sources. However anthropogenic activities, such as combustion of biomass may contribute significantly as well. The present study focuses on the thermal solid state reaction between pectin, an important constituent of biomass, and chloride ions as found in alkali metal chlorides. The formation of chloromethane is evident with the amount formed being linear with respect to chloride if pectin is in great excess. Thus the reaction is explained as a pseudo first order SN2 reaction between the chloride ion and the methyl ester moiety in pectin. It is suggested that the polymeric nature of pectin plays an active role by an enhanced transport of halides along the carbohydrate chain. Optimal reaction temperature is around 210°C. At higher temperatures the yield of chloromethane decreases due to a thermal decomposition of the pectin. The possible influence of the type of cation is discussed.

Neutral lipid accumulation at elevated temperature in conditional mutants of two microalgae species.

Triacylglycerols, an energy storage compound in microalgae, are known to be accumulated after nitrogen starvation of microalgae cells. Microalgae could be of importance for future biodiesel production due to their fast growth rate and high oil content. In collections of temperature sensitive mutants of Chlamydomonas reinhardtii and Chlorella vulgaris, nine out of fourty-one mutants in C. reinhardtii and eleven out of fifty-three mutants in C. vulgaris contained increased amounts of neutral lipids, predominantly as triacylglycerols. Upon temperature induced cell-cycle arrest, these mutants showed enlarged cellular volume compared with the wild type. The C. reinhardtii mutants were analyzed further and one type of mutants displayed a shift in lipid composition from polar membrane lipids to neutral lipids after a temperature up-shift, while the second type of mutants accumulated more total lipid per cell, predominantly as neutral lipids as compared with the wild type. Three C. reinhardtii mutants were analyzed further and found to be arrested after DNA synthesis but prior to cell division in the cell cycle. These mutants will be useful in order to further understand neutral lipid accumulation in microalgae and suggest possibilities for biodiesel production by specific induction of lipid accumulation in miroalgal cultures by cell-cycle inhibition.

Carbohydrate analysis of hemicelluloses by gas chromatography-mass spectrometry of acetylated methyl glycosides.

A method based on gas chromatography-mass spectrometry analysis of acetylated methyl glycosides was developed in order to analyze monosaccharides obtained from various hemicelluloses. The derivatives of monosaccharide standards, arabinose, glucose, and xylose were studied in detail and (13)C-labeled analogues were used for identification and quantitative analysis. Excellent chromatographic separation of the monosaccharide derivatives was found and identification of the anomeric configuration was feasible through a prepared and identified pure methyl 2,3,4,6-tetra-O-acetyl-ß-D-glucopyranoside. The electron ionization mass spectrum and fragmentation path was studied for each monosaccharide derivative. Fragment ion pairs of labeled and unlabeled monosaccharides were used for quantification; m/z 243/248 for glucose, 128/132 for xylose, and 217/218 for arabinose. Using the intensity ratios obtained from the extracted ion chromatograms, accurate quantification of monosaccharide constituents of selected hemicelluloses was demonstrated.

Determination of D/L-amino acids by zero needle voltage electrospray ionisation.

Ion formation may be made more efficient than in normal electrospray ionization (ESI) for certain classes of compounds, such as the polar amino acids Glu, Asn, His, Ser, Asp, Arg, Tyr and Lys, by adjusting the voltage of a normal ESI interface needle to zero voltage. For aspartic acid (Asp) the gain in signal-to-noise (S/N) ratio of the liquid chromatography/mass spectrometry (LC/MS) chromatograms obtained in the selective ion monitoring (SIM) mode (m/z 134) with zero needle potential was 40-50 times higher than detection at 4 kV. Ion formation at zero potential is likely to follow a mechanism related to sonic spray ionization. The utility of the zero needle voltage ESI was illustrated by determining the age of a human tooth by the aspartic acid epimerization method. The procedure involved separating the D- and L-aspartic acid of a tooth extract on a chiral HPLC column and detection by zero voltage ESI-MS3.

Enzymatic evidence for the key role of arginine in nitrogen translocation by arbuscular mycorrhizal fungi.

Key enzymes of the urea cycle and (15)N-labeling patterns of arginine (Arg) were measured to elucidate the involvement of Arg in nitrogen translocation by arbuscular mycorrhizal (AM) fungi. Mycorrhiza was established between transformed carrot (Daucus carota) roots and Glomus intraradices in two-compartment petri dishes and three ammonium levels were supplied to the compartment containing the extraradical mycelium (ERM), but no roots. Time courses of specific enzyme activity were obtained for glutamine synthetase, argininosuccinate synthetase, arginase, and urease in the ERM and AM roots. (15)NH(4)(+) was used to follow the dynamics of nitrogen incorporation into and turnover of Arg. Both the absence of external nitrogen and the presence of L-norvaline, an inhibitor of Arg synthesis, prevented the synthesis of Arg in the ERM and resulted in decreased activity of arginase and urease in the AM root. The catabolic activity of the urea cycle in the roots therefore depends on Arg translocation from the ERM. (15)N labeling of Arg in the ERM was very fast and analysis of its time course and isotopomer pattern allowed estimation of the translocation rate of Arg along the mycelium as 0.13 microg Arg mg(-1) fresh weight h(-1). The results highlight the synchronization of the spatially separated reactions involved in the anabolic and catabolic arms of the urea cycle. This synchronization is a prerequisite for Arg to be a key component in nitrogen translocation in the AM mycelium.

Direct analysis of 15N-label in amino and amide groups of glutamine and asparagine.

A novel method for on-line determination of the amount and position of 15N-labeling in complex mixtures of amino acids is presented. Underivatized amino acids were analyzed by ion-pair chromatography in combination with mass spectrometry. This enables the direct determination of the 15N label distribution. The fragmentation pathways of the nitrogen moieties of glutamine (Gln) and asparagine (Asn) were studied in detail using all mono 15N isotopomers, which led to a method for differentiating between 15N-amide and 15N-amino labeling. The fragmentation involving the amino and amide groups of Gln led to distinct ion structures. The equivalent fragmentation pattern was not observed for Asn. Instead, the amide group of Asn was eliminated as HNCO in a secondary process. The developed analytical method was evaluated by analysis of a range of standard mixtures taking into account different levels of 15N abundance and distribution between the amino and amide groups. The detection limit (3 SD) for the presence of a 15N label was 0.7 and 1.0% for Gln and Asn, respectively. The determination of the positional labeling follows a nonlinear function. A representative example at 30% 15N was used as a benchmark resulting in average relative standard deviations of 2.7 and 15% for Gln and Asn, respectively. The corresponding expectation windows for the positional labeling were found to be 2 and 12%, respectively.

Modification of jute fibers with polystyrene via atom transfer radical polymerization.

Atom transfer radical polymerization (ATRP) was investigated as a method of covalently bonding polystyrene to jute (Corchorus capsularis) and as a possible approach to fiber composites with enhanced properties. Jute fibers were modified with a brominated initiator and subsequently ATRP modified to attach polystyrene and then examined using SEM, DSC, TGA, FTIR, XPS, elemental analysis, and Py-GC-MS. These techniques confirmed that polystyrene had been covalently bound to the fibers and consequently ATRP-modified jute fiber mats were used to prepare hot-pressed polystyrene composites. Composite specimens were tensile tested and fracture surfaces examined using SEM. Although SEM examination suggested different fracture modes between unmodified fiber and ATRP-modified samples, the tensile strength of modified samples was slightly lower on average than that of unmodified samples. For fiber composite applications, we conclude that further optimization of the ATRP method is required, possibly targeting higher and more uniform loading of polystyrene on the fibers.

A proteomics study of in vitro cyst germination and appressoria formation in Phytophthora infestans.

A proteomics study using two-dimensional gel electrophoresis (2-DE) and mass spectrometry was performed on Phytophthora infestans. Proteins from cysts, germinated cysts and appressoria grown in vitro were isolated and separated by 2-DE. Statistical quantitative analysis of the protein spots from five independent experiments of each developmental stage revealed significant up-regulation of ten spots on gels from germinated cysts compared to cysts. Five spots were significantly up-regulated on gels from appressoria compared to germinated cysts and one of these up-regulated spots was not detectable on gels from cysts. In addition, one spot was significantly down-regulated and another spot not detectable on the gels from appressoria. The corresponding proteins to 13 of these spots were identified with high confidence using tandem mass spectrometry and database searches. The functions of the proteins that were up-regulated in germinated cysts and appressoria can be grouped into the following categories: protein synthesis (e.g. a DEAD box RNA helicase), amino acid metabolism, energy metabolism and reactive oxygen species scavenging. The spot not detected in appressoria was identified as the P. infestans crinkling- and necrosis-inducing protein CRN2. The identified proteins are most likely involved in the establishment of the infection of the host plant.

Identification of oxidised proteins in the matrix of rice leaf mitochondria by immunoprecipitation and two-dimensional liquid chromatography-tandem mass spectrometry.

Highly purified mitochondria were isolated from green 7-day-old rice leaves. The mitochondria were sonicated and the matrix fraction isolated as the 100,000g supernatant. Part of the matrix fraction was left untreated while the other part was subjected to a mild oxidative treatment (0.5 mM H2O2+0.2 mM CuSO4 for 10 min at room temperature). The oxidised proteins in both samples were tagged with dinitrophenylhydrazine (DNP), which forms a covalent bond with carbonyl groups. The DNP-tagged proteins were immunoprecipitated using anti-DNP antibodies and digested with trypsin. The mixture of peptides was analysed by nano-HPLC coupled online to an ESI-Quad-TOF mass spectrometer. The peptides were separated by stepwise ion exchange chromatography followed by reverse phase chromatography (2D-LC), and analysed by MS/MS. Proteins were identified by un-interpreted fragment ion database searches. Using this approach we identified 20 oxidised proteins in the control sample and a further 32 in the oxidised sample. Western blots of 2D-gels of the same samples prior to immunoprecipitation verified that the oxidation treatment increases protein oxidation also for specific proteins. Likewise Western blots showed that neither the isolation of mitochondria nor their subfractionation introduced carbonyl groups. We therefore conclude that a number of proteins are oxidised in the matrix of rice leaf mitochondria in vivo and further identify a group of proteins that are particularly susceptible to mild oxidation in vitro.

Identification of 14 new phosphoproteins involved in important plant mitochondrial processes.

Protein phosphorylation is a very important posttranslational modification the role of which is practically unexplored in mitochondria. Using two-dimensional gel electrophoresis followed by mass spectrometry, 14 new phosphoproteins are identified in potato tuber mitochondria, all household proteins also present in mammalian and fungal mitochondria. Seven of the new phosphoproteins are involved in the tricarboxylic acid cycle or associated reactions, four are subunits of respiratory complexes and involved in electron transport, ATP synthesis and protein processing, two are heat shock proteins and one is involved in defence against oxidative stress. These findings open up entirely new possibilities for the regulation and signal integration of mitochondrial processes.

Phosphorylation of formate dehydrogenase in potato tuber mitochondria.

Two highly phosphorylated proteins were detected after two-dimensional (blue native/SDS-PAGE) gel electrophoretic separation of the matrix fraction isolated from potato tuber mitochondria. These two phosphoproteins were identified by mass spectrometry as formate dehydrogenase (FDH) and the E1alpha-subunit of pyruvate dehydrogenase (PDH). Isoelectric focusing/SDS-PAGE two-dimensional gels separated FDH and PDH and resolved several different phosphorylated forms of FDH. By using combinations of matrix-assisted laser desorption/ionization mass spectrometry and electrospray ionization tandem mass spectrometry, several phosphorylation sites were identified for the first time in FDH and PDH. FDH was phosphorylated on Thr76 and Thr333, whereas PDH was phosphorylated on Ser294. Both Thr76 and Thr333 in FDH were accessible to protein kinases, as demonstrated by protein structure homology modeling. The extent of phosphorylation of both FDH and PDH was strongly decreased by NAD+, formate, and pyruvate, indicating that reversible phosphorylation of FDH and PDHs was regulated in a similar fashion. At low oxygen concentrations inside the intact potato tubers, FDH activity was strongly increased relative to cytochrome c oxidase activity pointing to a possible involvement of FDH in hypoxic metabolism. Computational sequence analysis indicated that a conserved local sequence motif of pyruvate formate-lyase is found in the Arabidopsis thaliana genome, and this enzyme might be the source of formate for FDH in plants.

Exploring symbiotic nitrogen fixation and assimilation in pea root nodules by in vivo 15N nuclear magnetic resonance spectroscopy and liquid chromatography-mass spectrometry.

Nitrogen (N) fixation and assimilation in pea (Pisum sativum) root nodules were studied by in vivo (15)N nuclear magnetic resonance (NMR) by exposing detached nodules to (15)N(2) via a perfusion medium, while recording a time course of spectra. In vivo (31)P NMR spectroscopy was used to monitor the physiological state of the metabolically active nodules. The nodules were extracted after the NMR studies and analyzed for total soluble amino acid pools and (15)N labeling of individual amino acids by liquid chromatography-mass spectrometry. A substantial pool of free ammonium was observed by (15)N NMR to be present in metabolically active, intact nodules. The ammonium ions were located in an intracellular environment that caused a remarkable change in the in vivo (15)N chemical shift. Alkalinity of the ammonium-containing compartment may explain the unusual chemical shift; thus, the observations could indicate that ammonium is located in the bacteroids. The observed (15)N-labeled amino acids, glutamine/glutamate and asparagine (Asn), apparently reside in a different compartment, presumably the plant cytoplasm, because no changes in the expected in vivo (15)N chemical shifts were observed. Extensive (15)N labeling of Asn was observed by liquid chromatography-mass spectrometry, which is consistent with the generally accepted role of Asn as the end product of primary N assimilation in pea nodules. However, the Asn (15)N amino signal was absent in in vivo (15)N NMR spectra, which could be because of an unfavorable nuclear Overhauser effect. gamma-Aminobutyric acid accumulated in the nodules during incubation, but newly synthesized (15)N gamma-aminobutyric acid seemed to be immobilized in metabolically active pea nodules, which made it NMR invisible.