Hydroponic isotope labelling of entire plants (HILEP) for quantitative plant proteomics; an oxidative stress case study

Hydroponic isotope labelling of entire plants (HILEP) is a cost-effective method enabling metabolic labelling of whole and mature plants with a stable isotope such as N-15. By utilising hydroponic media that contain N-15 inorganic salts as the sole nitrogen source, near to 100% N-15-labelling of proteins can be achieved. In this study, it is shown that HILEP, in combination with mass spectrometry, is suitable for relative protein quantitation of seven week-old Arabidopsis plants submitted to oxidative stress. Protein extracts from pooled N-14- and N-15-hydroponically grown plants were fractionated by SDS-PAGE, digested and analysed by liquid chromatography electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS). Proteins were identified and the spectra of N-14/N-15 peptide pairs were extracted using their m/z chromatographic retention time, isotopic distributions, and the m/z difference between the N-14 and N-15 peptides. Relative amounts were calculated as the ratio of the sum of the peak areas of the two distinct N-14 and N-15 peptide isotope envelopes. Using Mascot and the open source trans-proteomic pipeline (TPP), the data processing was automated for global proteome quantitation down to the isoform level by extracting isoform specific peptides. With this combination of metabolic labelling and mass spectrometry it was possible to show differential protein expression in the apoplast of plants submitted to oxidative stress. Moreover, it was possible to discriminate between differentially expressed isoforms belonging to the same protein family, such as isoforms of xylanases and pathogen-related glucanases (PR 2). (C) 2008 Elsevier Ltd. All rights reserved.

In vivo transformations of dihydroartemisinic acid in Artemisia annua plants

[15-(CH3)-C-13-H-2]-dihydroartemisinic acid (2a) and [15-(CH3)-H-2]-dihydroartemisinic acid (2b) have been fed via the root to intact Artemisia annua plants and their transformations studied in vivo by one-dimensional H-2 NMR spectroscopy and two-dimensional, C-13-H-2 correlation NMR spectroscopy (C-13-(2) H COSY). Labelled dihydroartemisinic acid was transformed into 16 12-carboxy-amorphane and cadinane sesquiterpenes within a few days in the aerial parts of A. annua, although transformations in the root were much slower and more limited. Fifteen of these 16 metabolites have been reported previously as natural products from A. annua. Evidence is presented that the first step in the transformation of dihydroartemisinic acid in vivo is the formation of allylic hydroperoxides by the reaction of molecular oxygen with the Delta(4,5)-double bond in this compound. The origin of all 16 secondary metabolites might then be explained by the known further reactions of such hydroperoxides. The qualitative pattern for the transformations of dihydroartemisinic acid in vivo was essentially unaltered when a comparison was made between plants, which had been kept alive and plants which were allowed to die after feeding of the labelled precursor. This, coupled with the observation that the pattern of transformations of 2 in vivo demonstrated very close parallels with the spontaneous autoxidation chemistry for 2, which we have recently demonstrated in vitro, has lead us to conclude that the main 'metabolic route' for dihydroartemisinic acid in A. annua involves its spontaneous autoxidation and the subsequent spontaneous reactions of allylic hydroperoxides which are derived from 2. There may be no need to invoke the participation of enzymes in any of the later biogenetic steps leading to all 16 of the labelled 11,13-dihydro-amorphane sesquiterpenes which are found in A. annua as natural products. (C) 2003 Elsevier Ltd. All rights reserved.

In vivo transformations of artemisinic acid in Artemisia annua plants

Artemisinic acid labeled with both C-13 and H-2 at the 15-position has been fed to intact plants of Artemisia annua via the cut stem, and its in vivo transformations studied by 1D- and 2D-NMR spectroscopy. Seven labeled metabolites have been isolated, all of which are known as natural products from this species. The transformations of artemisinic acid-as observed both for a group of plants, which was kept alive by hydroponic administration of water and for a group, which was allowed to die by desiccation-closely paralleled those, which have been recently described for its 11,13-dihydro analog, dihydroartemisinic acid. It seems likely therefore that similar mechanisms, involving spontaneous autoxidation of the Delta(4,5) double bond in both artemisinic acid and dihydroartemisinic acid and subsequent rearrangements of the resultant allylic hydroperoxides, may be involved in the biological transformations, which are undergone by both compounds. All of the sesquiterpene metabolites, which were obtained from in vivo transformations of artemisinic acid retained their unsaturation at the 11,13-position, and there was no evidence for conversion into any 11,13-dihydro metabolite, including artemisinin, the antimalarial drug, which is produced by A. annua. This observation led to the proposal of a unified biosynthetic scheme, which accounts for the biogenesis of many of the amorphane and cadinane sesquiterpenes that have been isolated as natural products from A. annua. In this scheme, there is a bifurcation in the biosynthetic pathway starting from amorpha-4,11-diene leading to either artemisinic acid or dihydroartemisinic acid; these two committed precursors are then, respectively, the parents for the two large families of highly oxygenated 11,13-dehydro and 11,13-dihydro sesquiterpene metabolites, which are known from this species. (C) 2007 Elsevier Ltd. All rights reserved.

In vivo transformations of dihydro-epi-deoxyarteannuin B in Artemisia annua plants

[15-(CH3)-C-13-H-2]-dihydro-epi-deoxyarteannuin B (4a) has been fed to intact Artemisia annua plants via the root and three labeled metabolites (17a-19a) have been identified by 1D- and 2D-NMR spectroscopies. The in vivo transformations of 4a in A. annua are proposed to involve enzymatically-mediated processes in addition to possible spontaneous autoxidation. In the hypothetical spontaneous autoxidation pathway, the tri-substituted double bond in 4a appears to have undergone 'ene-type' reaction with oxygen to form an allylic hydroperoxide, which subsequently rearranges to the allylic hydroxyl group in the metabolite 3 alpha-hydroxy-dihydro-epi-deoxyarteannuin B (17a). In the enzymatically-mediated pathways, compound 17a has then been converted to its acetyl derivative, 3 alpha-acetoxy-dihydro-epi-deoxyarteannuin B (18a), while oxidation of 4a at the 'unactivated' 9-position has yielded 9 beta-hydroxy-dihydro-epi-deoxyarteannuin B (19a). Although all of the natural products artemisinin ( 1), arteannuin K ( 7), arteannuin L ( 8), and arteannuin M ( 9) have been suggested previously as hypothetical metabolites from dihydro-epi-deoxyarteannuin B in A. annua, none were isolated in labeled form in this study. It is argued that the nature of the transformations undergone by compound 4a are more consistent with a degradative metabolism, designed to eliminate this compound from the plant, rather than with a role as a late precursor in the biosynthesis of artemisinin or other natural products from A. annua. (C) 2007 Elsevier Ltd. All rights reserved.

Life cycle analysis of UK coal fired power plants

This paper examines the life cycle GHG emissions from existing UK pulverized coal power plants. The life cycle of the electricity Generation plant includes construction, operation and decommissioning. The operation phase is extended to upstream and downstream processes. Upstream processes include the mining and transport of coal including methane leakage and the production and transport of limestone and ammonia, which are necessary for flue gas clean up. Downstream processes, on the other hand, include waste disposal and the recovery of land used for surface mining. The methodology used is material based process analysis that allows calculation of the total emissions for each process involved. A simple model for predicting the energy and material requirements of the power plant is developed. Preliminary calculations reveal that for a typical UK coal fired plant, the life cycle emissions amount to 990 g CO2-e/kWh of electricity generated, which compares well with previous UK studies. The majority of these emissions result from direct fuel combustion (882 g/kWh 89%) with methane leakage from mining operations accounting for 60% of indirect emissions. In total, mining operations (including methane leakage) account for 67.4% of indirect emissions, while limestone and other material production and transport account for 31.5%. The methodology developed is also applied to a typical IGCC power plant. It is found that IGCC life cycle emissions are 15% less than those from PC power plants. Furthermore, upon investigating the influence of power plant parameters on life cycle emissions, it is determined that, while the effect of changing the load factor is negligible, increasing efficiency from 35% to 38% can reduce emissions by 7.6%. The current study is funded by the UK National Environment Research Council (NERC) and is undertaken as part of the UK Carbon Capture and Storage Consortium (UKCCSC). Future work will investigate the life cycle emissions from other power generation technologies with and without carbon capture and storage. The current paper reveals that it might be possible that, when CCS is employed. the emissions during generation decrease to a level where the emissions from upstream processes (i.e. coal production and transport) become dominant, and so, the life cycle efficiency of the CCS system can be significantly reduced. The location of coal, coal composition and mining method are important in determining the overall impacts. In addition to studying the net emissions from CCS systems, future work will also investigate the feasibility and technoeconomics of these systems as a means of carbon abatement.

Life cycle GHG assessment of fossil fuel power plants with carbon capture and storage

The evaluation of life cycle greenhouse gas emissions from power generation with carbon capture and storage (CCS) is a critical factor in energy and policy analysis. The current paper examines life cycle emissions from three types of fossil-fuel-based power plants, namely supercritical pulverized coal (super-PC), natural gas combined cycle (NGCC) and integrated gasification combined cycle (IGCC), with and without CCS. Results show that, for a 90% CO2 capture efficiency, life cycle GHG emissions are reduced by 75-84% depending on what technology is used. With GHG emissions less than 170 g/kWh, IGCC technology is found to be favorable to NGCC with CCS. Sensitivity analysis reveals that, for coal power plants, varying the CO2 capture efficiency and the coal transport distance has a more pronounced effect on life cycle GHG emissions than changing the length of CO2 transport pipeline. Finally, it is concluded from the current study that while the global warming potential is reduced when MEA-based CO2 capture is employed, the increase in other air pollutants such as NOx and NH3 leads to higher eutrophication and acidification potentials.

Predictions of some product parameters based on the processing conditions of ultra-high-temperature milk plants

The temperature-time profiles of 22 Australian industrial ultra-high-temperature (UHT) plants and 3 pilot plants, using both indirect and direct heating, were surveyed. From these data, the operating parameters of each plant, the chemical index C*, the bacteriological index B* and the predicted changes in the levels of beta-lactoglobulin, alpha-lactalbumin, lactulose, furosine and browning were determined using a simulation program based on published formulae and reaction kinetics data. There was a wide spread of heating conditions used, some of which resulted in a large margin of bacteriological safety and high chemical indices. However, no conditions were severe enough to cause browning during processing. The data showed a clear distinction between the indirect and direct heating plants. They also indicated that degree of denaturation of alpha-lactalbumin varied over a wide range and may be a useful discriminatory index of heat treatment. Application of the program to pilot plants illustrated its value in determining processing conditions in these plants to simulate the conditions in industrial UHT plants. (C) 2008 Elsevier Ltd. All rights reserved.

Spatial offset of test field elements from surround elements affects the strength of motion aftereffects

Static movement aftereffects (MAEs) were measured after adaptation to vertical square-wave luminance gratings drifting horizontally within a central window in a surrounding stationary vertical grating. The relationship between the stationary test grating and the surround was manipulated by varying the alignment of the stationary stripes in the window and those in the surround, and the type of outline separating the window and the surround [no outline, black outline (invisible on black stripes), and red outline (visible throughout its length)]. Offsetting the stripes in the window significantly increased both the duration and ratings of the strength of MAEs. Manipulating the outline had no significant effect on either measure of MAE strength. In a second experiment, in which the stationary test fields alone were presented, participants judged how segregated the test field appeared from its surround. In contrast to the MAE measures, outline as well as offset contributed to judged segregation. In a third experiment, in which test-stripe offset wits systematically manipulated, segregation ratings rose with offset. However, MAE strength was greater at medium than at either small or large (180 degrees phase shift) offsets. The effects of these manipulations on the MAE are interpreted in terms of a spatial mechanism which integrates motion signals along collinear contours of the test field and surround, and so causes a reduction of motion contrast at the edges of the test field.

Hydroponic isotope labeling of entire plants (HILEP) for quantitative plant proteomics

Quantitative analysis by mass spectrometry (MS) is a major challenge in proteomics as the correlation between analyte concentration and signal intensity is often poor due to varying ionisation efficiencies in the presence of molecular competitors. However, relative quantitation methods that utilise differential stable isotope labelling and mass spectrometric detection are available. Many drawbacks inherent to chemical labelling methods (ICAT, iTRAQ) can be overcome by metabolic labelling with amino acids containing stable isotopes (e.g. 13C and/or 15N) in methods such as Stable Isotope Labelling with Amino acids in Cell culture (SILAC). SILAC has also been used for labelling of proteins in plant cell cultures (1) but is not suitable for whole plant labelling. Plants are usually autotrophic (fixing carbon from atmospheric CO2) and, thus, labelling with carbon isotopes becomes impractical. In addition, SILAC is expensive. Recently, Arabidopsis cell cultures were labelled with 15N in a medium containing nitrate as sole nitrogen source. This was shown to be suitable for quantifying proteins and nitrogen-containing metabolites from this cell culture (2,3). Labelling whole plants, however, offers the advantage of studying quantitatively the response to stimulation or disease of a whole multicellular organism or multi-organism systems at the molecular level. Furthermore, plant metabolism enables the use of inexpensive labelling media without introducing additional stress to the organism. And finally, hydroponics is ideal to undertake metabolic labelling under extremely well-controlled conditions. We demonstrate the suitability of metabolic 15N hydroponic isotope labelling of entire plants (HILEP) for relative quantitative proteomic analysis by mass spectrometry. To evaluate this methodology, Arabidopsis plants were grown hydroponically in 14N and 15N media and subjected to oxidative stress.

Progressive erosion of genetic and epigenetic variation in callus-derived cocoa (Theobroma cacao) plants

Relatively little is known about the timing of genetic and epigenetic forms of somaclonal variation arising from callus growth. We surveyed for both types of change in cocoa (Theobroma cacao) plants regenerated from calli of various ages, and also between tissues from the source trees. For genetic change, we used 15 single sequence repeat (SSR) markers from four source trees and from 233 regenerated plants. For epigenetic change, we used 386 methylation-sensitive amplified polymorphism (MSAP) markers on leaf and explant (staminode) DNA from two source trees and on leaf DNA from 114 regenerants. Genetic variation within source trees was limited to one slippage mutation in one leaf. Regenerants were far more variable, with 35% exhibiting at least one mutation. Genetic variation initially accumulated with culture age but subsequently declined. MSAP (epigenetic) profiles diverged between leaf and staminode samples from source trees. Multivariate analysis revealed that leaves from regenerants occupied intermediate eigenspace between leaves and staminodes of source plants but became progressively more similar to source tree leaves with culture age. Statistical analysis confirmed this rather counterintuitive finding that leaves of ‘late regenerants’ exhibited significantly less genetic and epigenetic divergence from source leaves than those exposed to short periods of callus growth.

A study on the performance of the partial PIC CDMA detector in the presence of time offset errors

This paper investigates the effect of time offset errors on the partial parallel interference canceller (PIC) and compares the performance of it against that of the standard PIC. The BER performances of the standard and partial interference cancellers are simulated in a near far environment with varying time offset errors. These simulations indicate that whilst timing errors significantly affect the performance of both these schemes, they do not diminish the gains that are realised by the partial PIC over that of the standard PIC.