Schematic presentation of proteins and metabolites in the <i>IPMDH</i> mutants.

<p>Proteins and metabolites in red indicate increased and in green decreased in the mutants compared to wild-type. AH, aconitate hydratase; ALPase, aleurain-like protease; APase, aminopeptidase; APX, ascorbate peroxidase; ArgJ, arginine biosynthesis protein ArgJ; AsA, ascorbate acid; ASDH, aspartate semialdehyde dehydrogenase; ATCase, aspartate transcarbamylase; BCAT, branched-chain aminotransferase; CAT, catalase; CHSP70, chloroplast heat shock protein 70; CRNP, chloroplast 29 kDa ribonucleoprotein; CRR, chlororespiratory reduction; CS, citrate synthase; CYP, luminal cyclophilin; DBP, DNA-binding protein; DHA, dehydroascorbate; DHAD, dihydroxyacid dehydratase; DHAR, dehydroascorbate reductase; FBA, fructose-bisphosphate aldolase; GABA, γ-aminobutyrate; GCSH, glycine cleavage system H protein; GGAT, glutamate:glyoxylate aminotransferase; GOX, glyoxalase; GRX, glutaredoxin; GS, glutamate synthase; GSH, glutathione; GSSG, oxidized glutathione; GST, glutathione S transferase; HSC, heat shock cognate; HSK, homoserine kinase; IDH, isocitrate dehydrogenase; IPMDH, 3-isopropylmalate dehydrogenase; KARI, ketol-acid reductoisomerase; LeuC, isopropylmalate isomerase large subunit; LeuD, isopropylmalate isomerase small subunit; MAM, methylthioalkylmalate synthase; MDH, malate dehydrogenase; MDHA, monohydroascorbate; MUR, GDP-D-mannose-4,6-dehydratase; MS, methionine synthase; OAS-TL, O-acetylserine (thiol) lyase; PGM, phosphoglucomutase; PHS, alpha-glucan phosphorylase; PIFI, post-illumination chlorophyll fluorescence increase; Prx, 2-cys peroxiredoxin; PSI, photosystem I; PSII, photosystem II; RBP, RNA binding protein; SOD, superoxide dismutase; ST5B, desulfoglucosinolate sulfotransferase; SUR, superroot; TCA, tricarboxylic acid; TF, transcription factor; TIL, temperature-induced lipocalin; TLP, thylakoid lumenal protein; Trx, thioredoxin; TSP, thylakoid soluble phosphoprotein.</p

Integrated Proteomics and Metabolomics of Arabidopsis Acclimation to Gene-Dosage Dependent Perturbation of Isopropylmalate Dehydrogenases

<div><p>Maintaining metabolic homeostasis is critical for plant growth and development. Here we report proteome and metabolome changes when the metabolic homeostasis is perturbed due to gene-dosage dependent mutation of <i>Arabidopsis</i> isopropylmalate dehydrogenases (<i>IPMDHs</i>). By integrating complementary quantitative proteomics and metabolomics approaches, we discovered that gradual ablation of the oxidative decarboxylation step in leucine biosynthesis caused imbalance of amino acid homeostasis, redox changes and oxidative stress, increased protein synthesis, as well as a decline in photosynthesis, which led to rearrangement of central metabolism and growth retardation. Disruption of <i>IPMDHs</i> involved in aliphatic glucosinolate biosynthesis led to synchronized increase of both upstream and downstream biosynthetic enzymes, and concomitant repression of the degradation pathway, indicating metabolic regulatory mechanisms in controlling glucosinolate biosynthesis.</p> </div

Representative 2D-DIGE map of differentially expressed proteins in the <i>IPMDH</i> mutants and wild-type plants.

<p>The proteins samples from the mutants of <i>IPMDH1</i>, <i>IMPDH2</i>, and <i>IPMDH3</i>, and wild-type were labeled with Cy2, Cy3, and Cy5 and then separated on 24 cm IPG strips (pH 4–7 linear gradient) through isoelectric focusing (IEF) in the first dimension, followed by 12.5% SDS-PAGE gels in the second dimension. Molecular weight (MW) in kilodaltons and pI of proteins are indicated on the right and top of gel, respectively. A total of 84 differentially expressed proteins marked with spot numbers were identified by MS/MS. For detailed information, please refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057118#pone.0057118.s002" target="_blank">Table S2</a>.</p

Morphological phenotype, total chlorophyll contents, and fresh weight of the Arabidopsis isopropylmalate dehydrogenase (<i>IPMDH</i>) mutants.

<p>(A) Phenotypes of the four-week-old <i>IPMDH</i> mutants. (B) Chlorophyll contents (green columns) and leaf fresh weight (orange columns) of the <i>IPMDH</i> mutants. The bars showed standard errors of seven different plants. A, <i>IPMDH1</i>; a, <i>ipmdh1</i>; B, <i>IPMDH2</i>; b, <i>ipmdh2</i>; D, <i>IPMDH3</i>; d, <i>ipmdh3</i>; FW, fresh weight. Please note that the phenotype data for the <i>AABbdd</i> and <i>aaBbdd</i> mutants based on another experiment were published in He et al., 2011a. New Phytologist 189: 160–175.</p

Clusters of differentially expressed proteins in the <i>IPMDH</i> mutants identified by iTRAQ.

<p>Plotted are the log2 ratios of the protein levels in the mutants over in wild-type. Cluster 1 includes proteins induced in the triple mutant, but reduced in the double mutant; Cluster 2 has proteins with significant increase in abundance in the double mutant, but the increase is not further enhanced in the triple mutant; Cluster 3 includes proteins with significant increase in levels only in the triple mutant. Cluster 4 shows proteins induced in the double mutant and further enhanced in the triple mutant; Cluster 5 shows proteins with the trend of decrease in the single and triple mutants; Cluster 6 includes proteins with decreased levels in the double and triple mutants; Cluster 7 shows progressive protein decreases in the double and triple mutants. A, <i>IPMDH1</i>; a, <i>ipmdh1</i>; B, <i>IPMDH2</i>; b, <i>ipmdh2</i>; D, <i>IPMDH3</i>; d, <i>ipmdh3</i>.</p

Table_2_Metabolomics of Early Stage Plant Cell–Microbe Interaction Using Stable Isotope Labeling.pdf

<p>Metabolomics has been used in unraveling metabolites that play essential roles in plant–microbe (including pathogen) interactions. However, the problem of profiling a plant metabolome with potential contaminating metabolites from the coexisting microbes has been largely ignored. To address this problem, we implemented an effective stable isotope labeling approach, where the metabolome of a plant bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000 was labeled with heavy isotopes. The labeled bacterial cells were incubated with Arabidopsis thaliana epidermal peels (EPs) with guard cells, and excessive bacterial cells were subsequently removed from the plant tissues by washing. The plant metabolites were characterized by liquid chromatography mass spectrometry using multiple reactions monitoring, which can differentiate plant and bacterial metabolites. Targeted metabolomic analysis suggested that Pst DC3000 infection may modulate stomatal movement by reprograming plant signaling and primary metabolic pathways. This proof-of-concept study demonstrates the utility of this strategy in differentiation of the plant and microbe metabolomes, and it has broad applications in studying metabolic interactions between microbes and other organisms.</p

Clustering of proteins identified by 2D-DIGE with similar trends of differential expression in the <i>IPMDH</i> mutants.

<p>Plotted are the log2 ratios of the protein levels in the mutants over in wild-type. Cluster 1 includes proteins induced in the triple mutant, but reduced in the double mutant; Cluster 2 has proteins with significant increase in abundance in the double mutant, but the increase is not further enhanced in the triple mutant; Cluster 3 includes proteins with significant increase in levels only in the triple mutant. Cluster 4 shows proteins increased in the double mutant and further enhanced in the triple mutant; Cluster 5 shows proteins with the trend of decrease in the single and triple mutants; Cluster 6 includes proteins with decreased levels in the double and triple mutants; Cluster 7 shows progressive protein decreases in the double and triple mutants. A, <i>IPMDH1</i>; a, <i>ipmdh1</i>; B, <i>IPMDH2</i>; b, <i>ipmdh2</i>; D, <i>IPMDH3</i>; d, <i>ipmdh3</i>.</p

Clusters of metabolites with similar trends of changes in the <i>IPMDH</i> mutants.

<p>Plotted are the log2 ratios of the metabolite levels in the mutants over in wild-type. Cluster 1 shows metabolites decreased in levels in either the double or triple mutant; Cluster 2 includes metabolites decreased only in the triple mutant; Cluster 3 shows metabolites reduced in the double mutant, but incresed in the triple mutant; Cluster 4 includes metabolites increased in the double mutant, but the increase was not further enhanced in the triple mutant; Cluster 5 shows metabolites induced in the double mutant and further enhanced in the triple mutant. A, <i>IPMDH1</i>; a, <i>ipmdh1</i>; B, <i>IPMDH2</i>; b, <i>ipmdh2</i>; D, <i>IPMDH3</i>; d, <i>ipmdh3</i>.</p

Proteomic Investigation into Betulinic Acid-Induced Apoptosis of Human Cervical Cancer HeLa Cells

<div><p>Betulinic acid is a pentacyclic triterpenoid that exhibits anticancer functions in human cancer cells. This study provides evidence that betulinic acid is highly effective against the human cervical cancer cell line HeLa by inducing dose- and time-dependent apoptosis. The apoptotic process was further investigated using a proteomics approach to reveal protein expression changes in HeLa cells following betulinic acid treatment. Proteomic analysis revealed that there were six up- and thirty down-regulated proteins in betulinic acid-induced HeLa cells, and these proteins were then subjected to functional pathway analysis using multiple analysis software. UDP-glucose 6-dehydrogenase, 6-phosphogluconate dehydrogenase decarboxylating, chain A Horf6-a novel human peroxidase enzyme that involved in redox process, was found to be down-regulated during the apoptosis process of the oxidative stress response pathway. Consistent with our results at the protein level, an increase in intracellular reactive oxygen species was observed in betulinic acid-treated cells. The proteins glucose-regulated protein and cargo-selection protein TIP47, which are involved in the endoplasmic reticulum pathway, were up-regulated by betulinic acid treatment. Meanwhile, 14-3-3 family proteins, including 14-3-3β and 14-3-3ε, were down-regulated in response to betulinic acid treatment, which is consistent with the decrease in expression of the target genes <i>14-3-3β</i> and <i>14-3-3ε</i>. Furthermore, it was found that the antiapoptotic <i>bcl-2</i> gene was down-regulated while the proapoptotic <i>bax</i> gene was up-regulated after betulinic acid treatment in HeLa cells. These results suggest that betulinic acid induces apoptosis of HeLa cells by triggering both the endoplasmic reticulum pathway and the ROS-mediated mitochondrial pathway.</p></div

Flow cytometric analysis of reactive oxygen species (ROS) in BA-treated cells.

<p>HeLa cells were treated with 15 µmol/L, 30 µmol/L BA for 48 h and then incubated with 10 mmol/L DCFH-DA for 40 min. The fluorescent intensity of DCFH was measured by flow cytometry. (A) Actual spectra from a representative single experiment. (B) The fluorescence intensity of stained cells was determined by flow cytometry. Columns show mean values of three experiments (±SD). **<i>p</i><0.01 compared with the control group (0 µmol/L BA).</p