[Show abstract][Hide abstract] ABSTRACT: In higher plants, fatty acids (FAs) with 18 carbons (18C) represent about 70% of total FAs, the most abundant species being 18:2 and 18:3. These two polyunsaturated FAs (PUFAs) represent about 55% of total FAs in Arabidopsis cell suspension cultures, whereas 18:1 represents about 10%. The level of PUFAs may vary, depending on ill-defined factors. Here, we compared various sets of plant cell cultures and noticed a correlation between the growth rate of a cell population and the level of unsaturation of 18C FAs. These observations suggest that the final level of PUFAs might depend in part on the rate of cell division, and that FAD2 and FAD3 desaturases, which are respectively responsible for the formation of 18:2 and 18:3 on phospholipids, have limiting activities in fast-growing cultures. In plant cell culture, phosphate (Pi) deprivation is known to impair cell division and to trigger lipid remodeling. We observed that Pi starvation had no effect on the expression of FAD genes, and that the level of PUFAs in this situation was also correlated with the growth rate. Thus, the level of PUFAs appears as a hallmark in determining cell maturity and aging.
[Show abstract][Hide abstract] ABSTRACT: Biochemical processes rely on elaborate networks containing thousands of compounds participating in thousands of reaction. Rapid turnover of diverse metabolites and lipids in an organism is an essential part of homeostasis. It affects energy production and storage, two important processes utilized in bioengineering. Conventional approaches to simultaneously quantify a large number of turnover rates in biological systems are currently not feasible. Here we show that pulse-chase analysis followed by laser ablation electrospray ionization mass spectrometry (LAESI-MS) enable the simultaneous and rapid determination of metabolic turnover rates. The incorporation of ion mobility separation (IMS) allowed an additional dimension of analysis, i.e., the detection and identification of isotopologs based on their collision cross sections. We demonstrated these capabilities by determining metabolite, lipid, and peptide turnover in the photosynthetic green algae, Chlamydomonas reinhardtii, in the presence of 15N-labeled ammonium chloride as the main nitrogen source. Following the reversal of isotope patterns in the chase phase by LAESI-IMS-MS revealed the turnover rates and half-lives for biochemical species with a wide range of natural concentrations, e.g., chlorophyll metabolites, lipids, and peptides. For example, the half-lives of lyso-DGTS(16:0) and DGTS(18:3/16:0), t1/2 = 43.6 ± 4.5 h and 47.6 ± 2.2 h, respectively, provided insight into lipid synthesis and degradation in this organism. Within the same experiment, half-lives for chlorophyll a, t1/2 = 24.1 ± 2.2 h, and a 2.8 kDa peptide, t1/2 = 10.4 ± 3.6 h, were also determined.
[Show abstract][Hide abstract] ABSTRACT: The metabolome of an organism results from a complex biochemical network of thousands of enzymatic reaction and metabolites. Mass spectrometry (MS) in combination with extraction and separation methods has been successfully applied for the detection, identification, and quantitation of these chemical species. However, these methods are slow and provide limited information on the metabolic fluxes essential for the understanding of disease response to treatment and for mechanism of action studies. Stable isotope pulse-chase analysis can be used to study critical pathways in metabolic networks by monitoring the propagation of isotope tracers using MS.1 Pulse-chase analysis has been used in the clinical laboratory to determine the turnover rates of peptides in diseases such as amyloidosis. In the pulse phase of these experiments, cells assimilate the labeled molecules, whereas in the chase phase unlabeled molecules are reintroduced into the cells. Molecular turnover rates and half-lives of the affected molecules can be calculated by measuring the kinetics of these processes.
Mass Spectrometry Applications to the Clinical Lab, San Diego, CA; 03/2015
[Show abstract][Hide abstract] ABSTRACT: The diatom Phaeodactylum is rich in very long chain polyunsaturated fatty acids (PUFAs). Fatty acid (FA) synthesis, elongation, and desaturation have been studied in depth in plants including Arabidopsis, but for secondary endosymbionts the full picture remains unclear. FAs are synthesized up to a chain length of 18 carbons inside chloroplasts, where they can be incorporated into glycerolipids. They are also exported to the ER for phospho- and betaine lipid syntheses. Elongation of FAs up to 22 carbons occurs in the ER. PUFAs can be reimported into plastids to serve as precursors for glycerolipids. In both organelles, FA desaturases are present, introducing double bonds between carbon atoms and giving rise to a variety of molecular species. In addition to the four desaturases characterized in Phaeodactylum (FAD2, FAD6, PtD5, PtD6), we identified eight putative desaturase genes. Combining subcellular localization predictions and comparisons with desaturases from other organisms like Arabidopsis, we propose a scheme at the whole cell level, including features that are likely specific to secondary endosymbionts.
[Show abstract][Hide abstract] ABSTRACT: ] The pollen tube, a fast tip-growing cell, is an excellent model to study membrane and cell wall biosynthesis. Here, we describe a simple protocol using an easy to use device to perform immunofluorescence labelling of pollen tube membrane and cell wall. The use of the NucleoSpin column to perform all the steps of the immunolabelling procedure results in obtaining more intact pollen tubes.
[Show abstract][Hide abstract] ABSTRACT: Inorganic phosphate (Pi) is present in most soils at sub-optimal concentrations, strongly limiting plant development. Plants have the ability to sense and adapt to the surrounding ionic environment, and several genes involved in the response to Pi starvation have been identified. However, a global understanding of the regulatory mechanisms involved in this process is still elusive. Here, we have initiated a "chemical genetics" approach and isolated compounds that inhibit the response to Pi starvation in Arabidopsis thaliana. Molecules were screened for their ability to inhibit the expression of a Pi starvation marker gene (the high-affinity Pi transporter PHT1;4). A drug family named Phosphatin (Phosphate starvation inhibitor: PTN) was thus identified, whose members act as partial suppressors of Pi starvation responses. PTN addition also reduced various traits of Pi starvation such as phospholipid/glycolipid conversion, and the accumulation of starch and anthocyanins. A transcriptomic assay revealed a broad impact of PTN on the expression of many genes regulated by low Pi availability. Despite the reduced amount of Pi transporters and resulting reduced Pi uptake capacity, no reduction of Pi content was observed. In addition, PTN improved plant growth; this reveals that the developmental restrictions induced by Pi starvation are not a consequence of metabolic limitation, but also result from genetic regulation. This highlights the existence of signal transduction pathway(s) that limit plant development under the Pi starvation condition.
[Show abstract][Hide abstract] ABSTRACT: The metabolic profiling of various microalga species and their genetic variants, grown under varied environmental conditions, has become critical to accelerate the exploration of phytoplankton biodiversity and biology. The accumulation of valuable metabolites, such as glycerolipids, is also sought in microalgae for biotechnological applications ranging from food, feed, medicine, cosmetics to bioenergy and green chemistry. In this report we describe the direct analysis of metabolites and glycerolipids in small cell populations of the green alga Chlamydomonas reinhardtii, using laser ablation electrospray ionization (LAESI) mass spectrometry (MS) coupled with ion mobility separation (IMS). These microorganisms are capable of redirecting energy storage pathways from starch to neutral lipids depending on environmental conditions and nutrient availability. Metabolite and lipid production was monitored in wild type (WT), and genetically modified C. reinhardtii strains with an impaired starch pathway. Lipids, such as triacylglycerols (TAG) and diacylglyceryl-N,N,N-trimethylhomoserine (DGTS) were monitored over time under altered light conditions. More than 200 ions related to metabolites, e.g., arginine, cysteine, serine, palmitate, chlorophyll a, chlorophyll b, etc., were detected. The lipid profiles at different light intensities for strains with impaired starch pathway (Sta1 and Sta6) contained 26 glycerolipids, such as DGTS, monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), as well as 33 TAG species. Results were obtained over a 72-hour time period in high and low light conditions for the WT species and the two mutants. Our results indicate that LAESI-IMS-MS can be utilized for the rapid analysis of increased TAG production at elevated light intensities. Compared to WT, the Sta6 strain showed 2.5-times higher lipid production at 72 hours under high light conditions. The results demonstrate our ability to rapidly observe numerous changes in metabolite and lipid levels in microalgae populations. These capabilities are expected to facilitate the exploration of genetically altered microalgae strains for biofuel production.
The Analyst 09/2014; 139(22). DOI:10.1039/C4AN01368A · 4.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In Angiosperms, the biosynthesis of galactolipids involves enzymes localized in the inner envelope membrane (IEM) of chloroplasts, including a phosphatidic acid phosphatase (PAP), dephosphorylating phosphatidic acid (PA) into diacylglycerol (DAG), and MGD1, transferring a galactose onto DAG thus generating monogalactosyldiacylglycerol (MGDG). It has been shown that PA and DAG could be synthesized in the plastid via the so-called 'prokaryotic' pathway or imported from the endoplasmic reticulum via the 'eukaryotic' pathway. In vitro studies support the existence of (1) a negative regulation of the plastid PAP by DAG and (2) an activation of MGD1 by PA. We developed a mathematical model of the IEM galactolipid biosynthesis pathway to understand the properties of the system ruled by the presence of these two regulatory motifs. We demonstrated that the design of the system implies that PA should accumulate to levels that are not observed experimentally, regardless of its prokaryotic or eukaryotic origin. PA should therefore be used for other syntheses, such as that of phosphatidylglycerol. Whereas a massive influx of eukaryotic PA appears unlikely, an influx of eukaryotic DAG in the IEM is supported by simulations. The model also implies that DAG cannot transiently accumulate and that PA mainly acts as a signal switching the whole system on. Eventually, this analysis highlights the fact that the PAP enzyme could easily become dispensable and that the design of the system, with the two regulatory motifs, could precede the loss of the PAP gene or activity in this pathway, a phenomenon that occurred independently in most clades of Angiosperms.
[Show abstract][Hide abstract] ABSTRACT: The microalga Chlamydomonas reinhardtii has been extensively studied for its relevance in biofuel production. This fully sequenced model organism has applications in focused areas such as photosynthesis, metabolism, cell cycle, motility, and genetics.1 To improve the understanding of lipid biosynthesis and its implications for using Chlamydomonas as a biofuel source, factors such as environmental conditions and selection of strains with particular genotypes need to be further investigated. Depending on nutrient availability and environmental conditions C. reinhardtii has the ability to store energy as starch or as neutral lipids, e.g., triacylglycerols (TAG). Here we demonstrate the use of a robust ambient analytical tool, laser ablation electrospray ionization (LAESI) mass spectrometry (MS) with ion mobility separation (IMS), to better understand metabolic changes and adaptations at the biochemical pathway level under varied stress conditions.
C. reinhardtii mutants with an impaired starch pathway were investigated for the production of neutral storage lipids under altered light conditions. Starch synthesis is inhibited by disrupting the central enzyme of ADP-glucose pyrophosphorylase (AGPase-SS) that is responsible for the formation of glucosyl nucleotides from glucose-1-phosphate with ATP in the starch pathway. This enzyme is activated by 3-phosphoglyceric acid (3-PGA). The Sta1 mutant has a disrupted large catalytic subunit of AGPase-SS, and as a consequence it exhibits a reduced activation by 3-PGA. This allows the mutant to retain less than ~10% of its normal starch production.2 The Sta6 mutant has a disrupted small catalytic subunit of AGPase-SS and as a result only retains ~1% of the normal starch levels.3
Conventional lipid analysis of microalgae consists of extensive extraction protocols and/or derivatization, resulting in lengthy runs and the loss of some structural information. To reduce the complexity of the sample, a separation step that distinguishes the different classes of compounds and isobaric species is required. LAESI-MS is an ambient ionization technique that has been employed to detect metabolites, lipids, and peptides from diverse biological samples. This technique utilizes the natural water content of cells and tissues to facilitate laser energy deposition by the strong absorption of water at 2.94 μm wavelength and to produce an ablation plume. The plume is then ionized by an electrospray and sampled by a high-performance Q- TOF mass spectrometer (Synapt G2 S, Waters Co.). A typical LAESI mass spectrum of Chlamydomonas contains approximately ~250 detected ions but it does not differentiate between structural isomers and isobaric species. Combination of LAESI-MS with IMS has been shown to produce a 3 fold increase in molecular coverage.4 The resulting enhanced data includes m/z ratios, drift times (DT) related to the collision cross section of the ion, and peak intensities. To reduce spectral interferences from the medium and to prevent osmotic shock the cells are deposited in a silica membrane spin tube and the supernatant is separated from the cells by centrifuging at 2,000×g for 1 min. The laser pulses are directly coupled into the remaining pellet of microalgae on the filter.
Initially the wild type (WT) C. reinhardtii was studied under low and high light conditions, with photosynthetically active radiation (PAR) 0 and 150 μmolm-2sec-1, respectively. After a 72 h period, the cells were harvested and LAESI-IMS-MS was performed. In the lipid region of the spectra, normalized for the most abundant diacylglyceryl-N,N,N-trimethylhomoserine (DGTS) ion, DGTS(34:3), enhanced production of monogalactosyldiacylglycerols (MGDG), digalactosyldiacylglycerols (DGDG) and TAG lipids was observed under high light conditions.
The Sta1 and Sta6 mutants were investigated for lipid production in altered light conditions ranging from 0 to 200 μmolm-2sec-1 over a 72 h time period. When compared to the WT cells, significant levels of TAG and DGDG were observed in both mutants throughout the 72 h time period. Using LAESI-MS without IMS ~10 TAG lipid species were detected. The introduction of IMS improved the molecular coverage of TAG lipids to ~50 species in the DT range of 189 to 204 ms (see Figure 1a).
When compared to WT, the Sta1 mutant showed stronger ion signal for TAG lipids within 800 < m/z < 880 at 72 h under high light condition. Conversely, the Sta6 mutant presented the strongest ion signal for TAG lipids at 72 h in low light illumination. Under opposite lighting conditions, the two Sta mutants exhibited similar TAG lipid profiles.
To reduce spectral interferences and differentiate close to isobaric species, for example TAG(54:7) and the 13C peak of chlorophyll a, both at nominal m/z 894, two DT ranges were inspected. In the LAESI-IMS-MS spectrum, the DT range integrated between 201 and 207 ms revealed the TAG(54:7) species at m/z 894.7571, whereas the DT range integrated between 181 and 188 ms showed the 13C isotope peak of chlorophyll a at m/z 894.5512 (see Figure 1b).
These results demonstrate our ability to observe numerous changes in lipid levels in WT and genetically modified microalgae populations affected by light exposure. We found that impaired starch pathways C. reinhardtii result in energy storage redirected to neutral lipids. These capabilities are expected to accelerate the research on the utility of genetically altered microalgae strains for biofuel production.
the 62nd ASMS National Conference, Baltimore, MD; 07/2014
[Show abstract][Hide abstract] ABSTRACT: INTRODUCTION
Chlamydomonas reinhardtii is extensively used as a model organism for exploring fundamental processes in photosynthesis and lipid transesterification. Laser ablation electrospray ionization (LAESI) mass spectrometry (MS) has been applied for the in situ metabolic and lipid profiling of microalgae under varied environmental conditions. Direct analyses of live microalgae enable the evaluation of metabolic network models, and advance our understanding of cellular responses and cell differentiation in relation to environmental factors. Stable isotope pulse-chase analysis by MS allows for time course profiling of labeled metabolites and the determination of molecular turnover rates under varied stress conditions. In this study, pulse-chase experiments were monitored by LAESI-MS to determine the metabolic and lipid turnover rates in small cell populations of C. reinhardtii.
For the pulse phase, wild type C. reinhardtii was cultured in 15N-labeled tris-acetate phosphate (TAP) medium and incubated at 27°C in a shaker running at 80 rpm for 96 hours. The chase phase was initiated by replacing the 15N-labeled TAP with and excess of unlabeled TAP containing 14N compounds. During the chase phase, sampling was performed at various time points followed by LAESI-MS analysis. Mass spectra were acquired by a quadrupole time-of- flight system. For evaluating the abundances of labeled compounds, the overlapping natural isotope peak intensities were manually subtracted. Following the time course of exchanging the 15N-labeled metabolites and lipids in the sample by their 14N counterparts provided insight into the turnover rates of these molecules.
We first evaluated the effect of nitrogen isotopic composition on the growth and development of C. reinhardtii. By comparison with the 14N-TAP control, our observations indicated that C.
reinhardtii grew in the 15N-TAP medium without any apparent physiological stress responses. No morphological differences between cells grown in 15N-TAP and 14N-TAP media were observed and they showed similar growth rates.
To explore the turnover rates of the abundant diacylglyceryl-N,N,N-trimethylhomoserines (DGTS) in C. reinhardtii, we focused on the corresponding peaks present between m/z 700 and 750. These lipids contained a single nitrogen atom, and the mass spectra in the pulse phase demonstrated that the C. reinhardtii cells successfully incorporated 15N into these molecules from the 15N-TAP media. Identification of the acyl chain lengths and verification of the 15N incorporation was achieved by tandem MS. For example, the ion assigned as DGTS(16:0/18:3) had a m/z 735 molecular ion and related lyso-lipid fragments at m/z 497 and 475 in 15N-TAP media, and a m/z 734 molecular ion and related lyso-lipid fragments of m/z 496 and 474 in 14N- TAP media.
Peak area ratios for m/z 735 and 734 as a function of time showed a decay that followed first order kinetics. Half-lives for the conversion of 15N-labeled DGTS(16:0/18:3) and DGTS(34:4) back to their unlabeled forms were 4.35 h and 5.70 h, respectively. The corresponding rate coefficients were 6.4×10-5 s-1 and 4.9×10-5 s-1. We observed similar kinetics for other nitrogen containing compounds and their rate coefficients were determined. LAESI-MS in combination with stable isotope pulse-chase experiments promises to provide a rapid assessment of metabolic conversion rates in complex biological systems.
62nd ASMS National Conference, Baltimore, MD; 07/2014