Shotgun proteomic analysis of Arabidopsis thaliana leaves

Two shotgun tandem MS proteomics approaches, multidimensional protein identification technology (MudPIT) and 1-D gel-LC-MS/MS, were used to identify Arabidopsis thaliana leaf proteins. These methods utilize different protein/peptide separation strategies. Detergents not compatible with MudPIT were used with 1-D gel-LC-MS/MS to help enrich for the detection of membrane-spanning and hydrophobic proteins. By combining the data from all MudPIT and 1-D gel-LC-MS/MS experiments, 2342 nonredundant proteins spanning a broad range of molecular weights and pI values were detected. With the exception of unknown proteins, the distribution of gene ontology (GO) classifications for the detected proteins was similar to that encoded by the genome, which shows that these extraction and separation procedures are useful for a broad proteomic survey of plant cells. Unknown proteins will likely have to be targeted by using additional methods, some of which should be compatible with separation strategies taken here.     

Proteomic analysis of the Arabidopsis thaliana cell wall

With the completion of the Arabidopsis genome, many hypothetical proteins have been predicted without any information on their expression, subcellular localisation and function. We have performed proteomic analysis of proteins sequentially extracted from enriched Arabidopsis cell wall fractions and separated by two-dimensional gel electrophoresis (2-DE). The proteins were identified by peptide mass fingerprinting using matrix-assisted laser desorption/ionisation-time of flight (MALDI-TOF) mass spectrometry and genomic database searches. This is part of a targeted exercise to establish the entire Arabidopsis secretome database. We report evidence for new proteins of unknown function whose existence had been predicted from genomic sequences and, furthermore, localise them to the cell wall. In addition, we observed an unexpected presence in the cell wall preparations of proteins whose known biochemical activity has never been associated with this compartment hitherto. We discuss the implications of these findings and present results suggesting a possible involvement of cell wall kinases in plant responses to pathogen attack.     

Classifying substrate specificities of membrane transporters from Arabidopsis thaliana

Membrane transporters catalyze the active transport of molecules across biological barriers such as lipid bilayer membranes. Currently, the experimental annotation of which proteins transport which substrates is far from complete and will likely remain so for much longer. Therefore, it is highly desirable to develop computational methods that may aid in the substrate annotation of putative membrane transport proteins. Here, we measured the similarity of membrane transporters from Arabidopsis thaliana by their amino acid composition, higher sequence order information, amino acid characteristics, or sequence conservation. We considered the substrate classes amino acids, oligopeptides, phosphates, and hexoses. Substrate classification based on the amino acid frequency yielded an accuracy of 75% or higher. Integrating additional information improved the prediction performance to 90% and higher.     

Monitoring of isothiocyanates emanating from Arabidopsis thaliana upon paraquat spraying

Arabidopsis thaliana plants were sprayed with the superoxide-generating herbicide paraquat. The headspace of sprayed plants was characterized by a number of compounds, which were absent in the headspace of untreated plants. They were identified as isothiocyanates (ITCs) with 4-methylthiobutyl isothiocyanate as main compound. After identification, a GC-system, based on PDMS sorption, was used to continuously monitor the ITC emissions. The specificity of isothiocyanate emission was also determined by subjecting the Arabidopsis thaliana plants to in vitro mechanical wounding. Again, 4-methylthiobutyl isothiocyanate was the main component, but the emission profile was completely different since the compound was emitted immediately, i.e., during wounding itself.     

Hydrophilic interaction chromatography/electrospray mass spectrometry analysis of carbohydrate-related metabolites from Arabidopsis thaliana leaf tissue

This work describes the development and application of an on-line liquid chromatography/mass spectrometry (LC/MS) method using hydrophilic interaction chromatography (HILIC) coupled to negative ion mode electrospray ionisation ion trap mass spectrometry (ESI-MS) for the analysis of highly polar carbohydrate-related metabolites commonly found in plants, ranging from reducing and non-reducing sugars and sugar alcohols to sugar phosphates. Using this method, separation and detection of a mixture of eight authentic standard compounds containing glucose (Glc), sucrose (Suc), raffinose, verbascose, mannitol, maltitol, glucose-6-phosphate (Glc6P) and trehalose-6-phosphate (Tre6P) were achieved in less than 15 min. The method is rapid, robust, selective, and sensitive, with limits of detection (LODs) ranging from 0.2 microM obtained for neutral sugars, to 1.0 microM obtained for sugar alcohols, and 2.0 microM obtained for negatively charged sugar phosphates. We have studied the negative ion collision-induced dissociation (CID) fragmentation behaviour of the non-reducing raffinose family oligosaccharides (RFOs) raffinose, stachyose, and verbascose. Mainly Bi and Ci glycosidic and Ai cross-ring structurally informative cleavages are observed. We have applied this HILIC/ESI-MS method for the analysis of Arabidopsis thaliana wild-type Columbia-0 (Col-0) and its starchless phosphoglucomutase mutant (pgm1) leaf extracts. The method was used to quantify Glc, Suc, raffinose, and Glc6P in A. thaliana extracts. Data obtained using this HILIC/ESI-MS method were compared with those obtained using a comparable porous graphitic carbon-based LC/ESI-MS method.     

Ligand migration in nonsymbiotic hemoglobin AHb1 from Arabidopsis thaliana

AHb1 is a hexacoordinated type 1 nonsymbiotic hemoglobin recently discovered in Arabidopsis thaliana. To gain insight into the ligand migration inside the protein, we studied the CO rebinding kinetics of AHb1 encapsulated in silica gels, in the presence of glycerol. The CO rebinding kinetics after nanosecond laser flash photolysis exhibits complex ligand migration patterns, consistent with the existence of discrete docking sites in which ligands can temporarily be stored before rebinding to the heme at different times. This finding may be of relevance to the physiological NO dioxygenase activity of this protein, which requires sequential binding of two substrates, NO and O2, to the heme.     

Application of sequence Fourier analysis to a specific interaction in Arabidopsis thaliana

The sequence Fourier analysis reported previously seems to be applicable to elucidating a simple and concerted interaction in Arabidopsis thaliana, similar to that in Homo sapiens.     

Capillary electrophoresis-mass spectrometry analysis of trehalose-6-phosphate in Arabidopsis thaliana seedlings

Trehalose-6-phosphate (T6P) is an intermediate in the plant metabolic pathway that results in trehalose production. T6P has been shown to inhibit the sucrose nonfermenting-1-related protein kinase 1, which is a major regulator of metabolism. The quantitation of T6P has proven difficult due to the complexity of the plant matrix and the low abundance of T6P in plant tissues. The aim of this work was to develop a quantitation method for T6P present in Arabidopsis tissues, with capillary electrophoresis (CE) coupled to electrospray ionization-mass spectrometry (MS) with a sheath liquid (SL) interface. The CE-MS method was first optimized with respect to T6P signal intensity and separation of isomers by studying the composition of the background electrolyte (BGE) and SL. The use of triethylamine (TEA) in the BGE was favorable, providing separation of T6P from sucrose-6-phosphate and minimizing ionization suppression. Replacing ammonium acetate with TEA enhanced T6P signal intensities more than four times. The optimized method allowed quantification of T6P in plant extracts with good linearity (r ² > 0.99) within a biologically relevant concentration range. The limit of quantification was 80 nM in Arabidopsis extracts, corresponding to 33 pmol/g plant fresh weight. The CE-MS method was applied to the determination of T6P in seedlings from wild type (WT) Arabidopsis and mutants lacking the trehalase AtTRE1, tre1-1, challenged with trehalose or sorbitol. T6P accumulation in tre1-1 plants grown on sorbitol was about twice the level of T6P found in WT. CE-MS is shown to be a fast and reliable technique to analyze phosphodisaccharides for seedling extracts. The low sample volume requirement of CE and its direct MS coupling makes it an attractive alternative for anion-exchange liquid chromatography–MS.     

Absorption and fluorescence spectroscopic characterization of cryptochrome 3 from Arabidopsis thaliana

The blue light photoreceptor cryptochrome 3 (cry3) from Arabidopsis thaliana was characterized at room temperature in vitro in aqueous solution by optical absorption and emission spectroscopic studies. The protein non-covalently binds the chromophores flavin adenine dinucleotide (FAD) and N5,N10-methenyl-5,6,7,8-tetrahydrofolate (MTHF). In the dark-adapted state of cry3, the bound FAD is present in the oxidized form (FAD(ox), ca. 38.5%), in the semiquinone form (FADH., ca. 5%), and in the fully reduced neutral form (FAD(red)H2) or fully reduced anionic form (FAD(red)H-, ca. 55%). Some amount of FAD (ca. 1.5%) in the oxidized state remains unbound probably caused by chromophore release and/or denaturation. F?rster-type energy transfer from MTHF to FAD(ox) is observed. Photo-excitation reversibly modifies the protein conformation causing a slight rise of the MTHF absorption strength and an increase of the MTHF fluorescence efficiency (efficient protein conformation photo-cycle). Additionally there occurs reversible reduction of bound FAD(ox) to FAD(red)H2 (or FAD(red)H-, FAD(ox) photo-cycle of moderate efficiency), reversible reduction of FADH. to FAD(red)H2 (or FAD(red)H-, FADH. photo-cycle of high efficiency), and modification of re-oxidable FAD(red)H2 (or FAD(red)H-) to permanent FAD(red)H2 (or FAD(red)H-) with low quantum efficiency. Photo-excitation of MTHF causes the reversible formation of a MTHF species (MTHF', MTHF photo-cycle, moderate quantum efficiency) with slow recovery to the initial dark state, and also the formation of an irreversible photoproduct (MTHF').     

Identification of a light-regulated protein kinase activity from seedlings of Arabidopsis thaliana

Protein kinase transduction pathways are thought to be involved in light signaling in plants, but other than the photoreceptors, no protein kinase activity has been shown to be light-regulated in vivo. Using an in-gel protein kinase assay technique with histone H III SS as an exogenous substrate, we identified a light-regulated protein kinase activity with an apparent molecular weight ca 50 kDa. The kinase activity increased transiently after irradiation of dark-grown seedlings with continuous far red light (FR) and blue light (B) and decreased after irradiation with red light (R). The maximal activation was achieved after 30 min to 1 h with FR or B. After irradiation times longer than 2 h, the kinase activity decreased to below the sensitivity level of the assay. In Arabidopsis mutants lacking either the photoreceptors phytochrome A, phytochrome B or the blue-light receptor cryptochrome 1, kinase activity was undetectable, whereas in the photomorphogenic mutants cop1 and det1 the kinase activity was also observed in the absence of light signals, though still stimulated by B and FR. Interestingly, the R inhibition of the kinase activity was lost in the mutant hy5. Pretreatment with cycloheximide blocked the kinase activity.     

A new triterpene synthase from Arabidopsis thaliana produces a tricyclic triterpene with two hydroxyl groups

[structure: see text] Thirteen oxidosqualene cyclase homologues exist in the genome of Arabidopsis thaliana. One of these genes, At4g15340, was amplified by PCR and expressed in yeast. The yeast transformant accumulated tricyclic triterpene, (3S,13R)-malabarica-17,21-dien-3,14-diol (arabidiol), whose structure was determined by NMR and MS analyses. Its epoxide analogue, (3S,13R,21S)-malabarica-17-en-20,21-epoxy-3,14-diol (arabidiol 20,21-epoxide), was also isolated from the transformed yeast. This is the first example of a triterpene synthase that yields a tricyclic triterpene with two hydroxyl groups.     

Biochemical dissection of the mitochondrial proteome from Arabidopsis thaliana by three-dimensional gel electrophoresis

We report a subdivision of the mitochondrial proteome into defined sets of proteins, which is based on the combination of three different gel electrophoresis procedures. First, Blue-native polyacrylamide gel electrophoresis is employed to separate mitochondrial protein complexes. The protein complexes are electroeluted and completely detached from Coomasssie blue. Subsequently the subunits of the protein complexes are separated by isoelectric focusing and finally by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The resolution capacity of the procedure is demonstrated for the ATP synthase complex, the cytochrome c reductase complex and the preprotein translocase of the outer mitochondrial membrane (the TOM complex). The method allows the separation of isoforms of subunits forming part of protein complexes, whose occurrence seems to be rather a rule than an exception in higher eukaryotes. Furthermore, extremely hydrophobic proteins are detectable on the gels.     

Enantioselective hydrolysis of (RS)-2-fluoroarylacetonitriles using nitrilase from Arabidopsis thaliana

The enzymatic resolution of 2-fluoroarylacetonitriles (RS)-3 using nitrilase from the plant Arabidopsis thaliana is described. Racemic 2-fluoronitriles 3 are easily accessible from O-silylated aromatic cyanohydrins 2 by reaction with DAST. The nitriles (RS)-3 were hydrolysed with the nitrilase as a catalyst, not to the expected 2-fluoroarylacetic acids but to the corresponding (R)-2-fluoroarylacetamides (R)-5 as the main products. After optimization of reaction conditions (pH 9, 7°C), the enantiomeric excesses of (R)-5a,c and f (R=H, 3-Me, 3-OMe) could be improved to >99% by one recrystallization. The acid catalysed hydrolysis of (R)-5a,5c and 5f afforded the corresponding (R)-2-fluoroarylacetic acids (R)-4a,4c and 4f without racemization.     

A proteomic analysis of secreted proteins from xylan-induced Bacillus sp. strain K-1

The expression level of extracellular proteins in an alkaliphilic bacterium, Bacillus sp. strain K-1, grown in a xylan-containing medium, is significantly increased when compared with that grown in the nonxylan culture medium. A proteomic approach has been efficiently applied to separate and characterize these differentially expressed secretory proteins. Eight prominent protein spots were identified and subjected to N-terminal amino acid sequencing. The results show that three spots share considerable similarity with the xylanolytic enzymes and that two spots share considerable similarity with the GltC regulatory protein and 3-dehydroquinate dehydratase, respectively. In addition, the three other proteins show little similarity with the known proteins in the database. In conclusion, our results demonstrate that the proteomic approach is a highly efficient method to rapidly study the differential expression of the secreted proteins by Bacillus sp. strain K-1 grown under xylan-induced condition.     

ANALYSIS OF MULTIPLE PHOTORECEPTOR PIGMENTS FOR PHOTOTROPISM IN A MUTANT OF Arabidopsis thaliana

Abstract
The shape of the fluence-response relationship for the phototropic response of the JK224 strain of Arabidopsis thaliana depends on the fluence rate and wavelength of the actinic light. At low fluence rate (0.1 μmol m^-2s^-1), the response to 450-nm light is characterized by a single maximum at about 9 μmol m^-2. At higher fluence rate (0.4 μmol m^-2s^-1), the response shows two maxima, at 4.5 and 9 μmol m^-2. The response to 510-nm light shows a single maximum at 4.5 μmol m^-2. Unilateral preirradiation with high fluence rate (25 μmol m^-2s^-1) 510-nm light eliminates the maximum at 4.5 μmol m^-2 in the fluence response curve to a subsequent unilateral 450-nm irradiation, while the second maximum at 9 μmol m^-2 is unaffected. Based on these results, it is concluded that a single photoreceptor pigment has been altered in the JK224 strain of Arabidopsis thaliana.