We isolated a total of 3 3 10 8 guard cell protoplasts from 22,000 Arabidopsis thaliana plants and identified 1734 unique proteins using three complementary proteomic methods: protein spot identification from broad and narrow pH range twodimensional (2D) gels, and 2D liquid chromatography-matrix assisted laser desorption/ionization multidimensional protein identification technology. This extensive single-cell-type proteome includes 336 proteins not previously represented in transcriptome analyses of guard cells and 52 proteins classified as signaling proteins by Gene Ontology analysis, of which only two have been previously assessed in the context of guard cell function. THIOGLUCOSIDE GLUCOHYDROLASE1 (TGG1), a myrosinase that catalyzes the production of toxic isothiocyanates from glucosinolates, showed striking abundance in the guard cell proteome. tgg1 mutants were hyposensitive to abscisic acid (ABA) inhibition of guard cell inward K + channels and stomatal opening, revealing that the glucosinolate-myrosinase system, previously identified as a defense against biotic invaders, is required for key ABA responses of guard cells. Our results also suggest a mechanism whereby exposure to abiotic stresses may enhance plant defense against subsequent biotic stressors and exemplify how enhanced knowledge of the signaling networks of a specific cell type can be gained by proteomics approaches.
Nitrogen starvation induces a global stress response in microalgae that results in the accumulation of lipids as a potential source of biofuel. Using GC-MS-based metabolite and iTRAQ-labeled protein profiling, we examined and correlated the metabolic and proteomic response of Chlamydomonas reinhardtii under nitrogen stress. Key amino acids and metabolites involved in nitrogen sparing pathways, methyl group transfer reactions, and energy production were decreased in abundance, whereas certain fatty acids, citric acid, methionine, citramalic acid, triethanolamine, nicotianamine, trehalose, and sorbitol were increased in abundance. Proteins involved in nitrogen assimilation, amino acid metabolism, oxidative phosphorylation, glycolysis, TCA cycle, starch, and lipid metabolism were elevated compared with nonstressed cultures. In contrast, the enzymes of the glyoxylate cycle, one carbon metabolism, pentose phosphate pathway, the Calvin cycle, photosynthetic and light harvesting complex, and ribosomes were reduced. A noteworthy observation was that citrate accumulated during nitrogen stress coordinate with alterations in the enzymes that produce or utilize this metabolite, demonstrating the value of comparing protein and metabolite profiles to understand complex patterns of metabolic flow. Thus, the current study provides unique insight into the global metabolic adjustments leading to lipid storage during N starvation for application toward advanced biofuel production technologies.
S-Adenosylmethionine decarboxylase (AdoMetDC) is a pyruvoyl enzyme, and the pyruvate is formed in an intramolecular reaction that cleaves a proenzyme precursor and converts a serine residue into pyruvate. The wild type potato AdoMetDC proenzyme processed much faster than the human proenzyme and did not require putrescine for an optimal rate of processing despite the presence of three acidic residues (equivalent to ) is not present in the potato sequence. The site of potato AdoMetDC proenzyme processing was found to be Ser 73 in the conserved sequence, YVLSESS, which is the equivalent of Ser 68 in the human sequence. Replacement of the serine precursor with threonine or cysteine by site-directed mutagenesis in either the potato or the human AdoMetDC proenzyme did not prevent processing but caused a significant reduction in the rate. Although the COOH-terminal regions of the known eukaryotic AdoMetDCs are not conserved, only relatively small truncations of 8 residues from the human protein and 25 residues from the potato proenzyme were compatible with processing. The maximally truncated proteins show no similarity in COOHterminal amino acid sequence but each contained 46 amino acid residues after the last conserved sequence, suggesting that the length of this section of the protein is essential for maintaining the proenzyme conformation needed for autocatalytic processing. AdoMetDC1 is an essential enzyme for the biosynthesis of polyamines and is one of a small class of decarboxylases that uses a covalently bound pyruvate as a prosthetic group (1, 2). These pyruvoyl-dependent decarboxylases form amines such as histamine, decarboxylated S-adenosylmethionine, phosphatidylethanolamine (a component of membrane phospholipids), and -alanine (a precursor of coenzyme A), which are all of critical importance in cellular physiology and provide an important target for drug design. The mechanism of formation of the prosthetic group has been studied extensively using histidine decarboxylase from Lactobacillus (1, 3-6), and more preliminary studies with other decarboxylases including AdoMetDC (7-9) suggest that the mechanism is similar (Fig. 1). In all cases, the enzyme is synthesized as a proenzyme that then undergoes an intramolecular cleavage reaction forming the two subunits and generating the pyruvate at the amino terminus of the ␣ subunit from a serine precursor residue. Cleavage takes place via the formation of an intermediate ester resulting from a nucleophilic attack of this serine residue at the amide carbonyl group of the preceding amino acid. This is followed by -elimination to form the  subunit and the ␣ subunit containing a dehydroalanine at its amino terminus. The dehydroalanine then loses ammonia and is converted to pyruvate via the formation of imine and carbinolamine intermediates (1-3). The initial rearrangement step of this reaction to form a peptide ester linked to the hydroxyl side chain of serine is identical to that involved in protein splicing reactions (10, 11). Further information on such cleavage...
The structure and topology of AdoMetDC display internal symmetry, suggesting that this protein may be the product of an ancient gene duplication. The positions of conserved, functionally important residues suggest the location of the active site and a possible binding site for the effector molecule putrescine.
Mechanical damage caused by insect feeding along with components present in insect saliva and oral secretions are known to induce jasmonic acid-mediated defense responses in plants. This study investigated the effects of bacteria from oral secretions of the fall armyworm Spodoptera frugiperda on herbivore-induced defenses in tomato and maize plants. Using culture-dependent methods, we identified seven different bacterial isolates belonging to the family Enterobacteriacea from the oral secretions of field-collected caterpillars. Two isolates, Pantoea ananatis and Enterobacteriaceae-1, downregulated the activity of the plant defensive proteins polyphenol oxidase and trypsin proteinase inhibitors (trypsin PI) but upregulated peroxidase (POX) activity in tomato. A Raoultella sp. and a Klebsiella sp. downregulated POX but upregulated trypsin PI in this plant species. Conversely, all of these bacterial isolates upregulated the expression of the herbivore-induced maize proteinase inhibitor (mpi) gene in maize. Plant treatment with P. ananatis and Enterobacteriaceae-1 enhanced caterpillar growth on tomato but diminished their growth on maize plants. Our results highlight the importance of herbivore-associated microbes and their ability to mediate insect plant interactions differently in host plants fed on by the same herbivore.
We studied the effect of various doses of sodium iodide on thyroid radioiodine uptake in euthyroid volunteers by giving single doses of 10, 30, 50, and 100 mg and then daily doses of 10, 15, 30, 50, or 100 mg for 12 days thereafter. All single doses above 10 mg suppressed 24-hour thyroid uptake of 123I to 0.7 to 1.5 per cent. Continued daily administration of 15 mg of iodide or more resulted in values consistently below 2 per cent. A small but statistically significant fall in serum thyroxine (T4) and triiodothyronine (T3) and a rise in serum thyrotropin (TSH) concentrations were observed after eight and 12 days of iodide treatment. These data suggest that the thyroid uptake of radioactive iodine can be markedly suppressed by single-dose administration of 30 mg of stable iodide and that suppression can be maintained with daily doses of at least 15 mg. This study provides guidelines for stable iodide prophylaxis in the event of exposure to radioactive iodine.
Signaling cascades mediated by heterotrimeric G proteins are ubiquitous and important signal transduction mechanisms in both metazoans and plants. In the model plant Arabidopsis thaliana, the sole canonical G protein alpha subunit, GPA1, has been implicated in multiple signaling events, including guard cell movement regulated by the plant stress hormone abscisic acid (ABA). However, only a handful of proteins have been demonstrated to be involved in GPA1 signaling to date. Here, we compared the proteome composition of guard cells from wild type Col vs gpa1-4 null mutants with and without ABA treatment using iTRAQ technology to identify guard cell proteins whose abundance was affected by ABA and/or GPA1. After imposition of strict selection criteria, the abundance of two proteins in Col and six proteins in gpa1-4 was found to be affected by ABA in guard cells, and 18 guard cell proteins were quantitatively affected by the mutation of GPA1. On the basis of known functions of the differentially expressed proteins, our data suggest that GPA1 inhibits guard cell photosynthesis and promotes the availability of reactive oxygen species (ROS) in guard cells. These results exemplify how iTRAQ can be used to quantitatively study single cell signaling pathways in Arabidopsis.
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