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In JoVE (1)
Other Publications (42)
- The Plant Journal : for Cell and Molecular Biology
- The Plant Journal : for Cell and Molecular Biology
- The Journal of Biological Chemistry
- Plant Physiology
- Journal of Proteome Research
- Journal of Experimental Botany
- The Journal of Biological Chemistry
- The FEBS Journal
- Plant Physiology
- Journal of Proteome Research
- Biochemistry and Molecular Biology Education : a Bimonthly Publication of the International Union of Biochemistry and Molecular Biology
- Plant & Cell Physiology
- Molecular & Cellular Proteomics : MCP
- The Journal of Biological Chemistry
- Physiologia Plantarum
- Journal of Proteomics
- The Plant Journal : for Cell and Molecular Biology
- Journal of Proteome Research
- Journal of Proteomics
- Proteomics. Clinical Applications
- Journal of Proteomics
- Journal of Proteome Research
- Plant & Cell Physiology
- Journal of Proteome Research
- Journal of Integrative Plant Biology
- Journal of Plant Physiology
- The Journal of Biological Chemistry
- Journal of Proteome Research
- Journal of Plant Physiology
- The New Phytologist
- Computational Biology and Chemistry
- Journal of Proteome Research
- Journal of Plant Physiology
- BMC Plant Biology
- Journal of Proteome Research
Articles by Sixue Chen in JoVE
Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry
Jennifer Parker1, Ning Zhu2, Mengmeng Zhu2, Sixue Chen1,2,3,4
1Plant Molecular and Cellular Biology Program, University of Florida, 2Department of Biology, University of Florida, 3Interdisciplinary Center for Biotechnology Research, University of Florida, 4Genetics Institute, University of Florida
Reactive oxygen species level is elevated when cells encounter stress conditions. Here we show the example of 3'-3' diaminobenzidine staining as well as cysTMT labeling and mass spectrometry to profile the redox proteome in Pseudomonas syringae treated tomato leaves.
Other articles by Sixue Chen on PubMed
Planta. Feb, 2002 | Pubmed ID: 11925040
The glucosinolate composition and content in various tissues of Arabidopsis thaliana (L.) Heynh. ecotype Columbia during development from seeds to bolting plants were determined in detail by high-performance liquid chromatography. Comparison of the glucosinolate profiles of leaves, roots and stems from mature plants with those of green siliques and mature seeds indicated that a majority of the seed glucosinolates were synthesized de novo in the silique. A comparison of the glucosinolate profile of mature seeds with that of cotyledons indicated that a major part of seed glucosinolates was retained in the cotyledons. Turnover of glucosinolates was studied by germination of seeds containing radiolabelled p-hydroxybenzylglucosinolate (p-OHBG). Approximately 70% of the content of [14C]p-OHBG in the seeds was detected in seedlings at the cotyledon stage and [14C]p-OHBG was barely detectable in young plants with rosettes of six to eight leaves. The turn-over of p-OHBG was found to coincide with the expression of the glucosinolate-degrading enzyme myrosinase, which was detectable at very low levels in seedlings at the cotyledon stage, but which dramatically increased in leaves from plants at later developmental stages. This indicates that there is a continuous turnover of glucosinolates during development and not only upon tissue disruption.
CYP79F1 and CYP79F2 Have Distinct Functions in the Biosynthesis of Aliphatic Glucosinolates in Arabidopsis
The Plant Journal : for Cell and Molecular Biology. Mar, 2003 | Pubmed ID: 12609033
Cytochromes P450 of the CYP79 family catalyze the conversion of amino acids to oximes in the biosynthesis of glucosinolates, a group of natural plant products known to be involved in plant defense and as a source of flavor compounds, cancer-preventing agents and bioherbicides. We report a detailed biochemical analysis of the substrate specificity and kinetics of CYP79F1 and CYP79F2, two cytochromes P450 involved in the biosynthesis of aliphatic glucosinolates in Arabidopsis thaliana. Using recombinant CYP79F1 and CYP79F2 expressed in Escherichia coli and Saccharomyces cerevisiae, respectively, we show that CYP79F1 metabolizes mono- to hexahomomethionine, resulting in both short- and long-chain aliphatic glucosinolates. In contrast, CYP79F2 exclusively metabolizes long-chain elongated penta- and hexahomomethionines. CYP79F1 and CYP79F2 are spatially and developmentally regulated, with different gene expression patterns. CYP79F2 is highly expressed in hypocotyl and roots, whereas CYP79F1 is strongly expressed in cotyledons, rosette leaves, stems, and siliques. A transposon-tagged CYP79F1 knockout mutant completely lacks short-chain aliphatic glucosinolates, but has an increased level of long-chain aliphatic glucosinolates, especially in leaves and seeds. The level of long-chain aliphatic glucosinolates in a transposon-tagged CYP79F2 knockout mutant is substantially reduced, whereas the level of short-chain aliphatic glucosinolates is not affected. Biochemical characterization of CYP79F1 and CYP79F2, and gene expression analysis, combined with glucosinolate profiling of knockout mutants demonstrate the functional role of these enzymes. This provides valuable insights into the metabolic network leading to the biosynthesis of aliphatic glucosinolates, and into metabolic engineering of altered aliphatic glucosinolate profiles to improve nutritional value and pest resistance.
Transcriptome Analysis of Root Transporters Reveals Participation of Multiple Gene Families in the Response to Cation Stress
The Plant Journal : for Cell and Molecular Biology. Sep, 2003 | Pubmed ID: 12969422
Plant nutrition critically depends on the activity of membrane transporters that translocate minerals from the soil into the plant and are responsible for their intra- and intercellular distribution. Most plant membrane transporters are encoded by multigene families whose members often exhibit overlapping expression patterns and a high degree of sequence homology. Furthermore, many inorganic nutrients are transported by more than one transporter family. These considerations, coupled with a large number of so-far non-annotated putative transporter genes, hamper our progress in understanding how the activity of specific transporters is integrated into a response to fluctuating conditions. We designed an oligonucleotide microarray representing 1096 Arabidopsis transporter genes and analysed the root transporter transcriptome over a 96-h period with respect to 80 mM NaCl, K+ starvation and Ca2+ starvation. Our data show that cation stress led to changes in transcript level of many genes across most transporter gene families. Analysis of transcriptionally modulated genes across all functional groups of transporters revealed families such as V-type ATPases and aquaporins that responded to all treatments, and families - which included putative non-selective cation channels for the NaCl treatment and metal transporters for Ca2+ starvation conditions - that responded to specific ionic environments. Several gene families including primary pumps, antiporters and aquaporins were analysed in detail with respect to the mRNA levels of different isoforms during ion stress. Cluster analysis allowed identification of distinct expression profiles, and several novel putative regulatory motifs were discovered within sets of co-expressed genes.
Arabidopsis Thaliana Glutamate-cysteine Ligase: Functional Properties, Kinetic Mechanism, and Regulation of Activity
The Journal of Biological Chemistry. Aug, 2004 | Pubmed ID: 15180996
In plants, glutathione accumulates in response to different stress stimuli as a protective mechanism, but only limited biochemical information is available on the plant enzymes that synthesize glutathione. Glutamatecysteine ligase (GCL) catalyzes the first step in glutathione biosynthesis and plays an important role in regulating the intracellular redox environment. Because the putative Arabidopsis thaliana GCL (AtGCL) displays no significant homology to the GCL from bacteria and other eukaryotes, the identity of this protein as a GCL has been debated. We have purified AtGCL from an Escherichia coli expression system and demonstrated that the recombinant enzyme catalyzes the ATP-dependent formation of gamma-glutamylcysteine from glutamate (Km = 9.1 mm) and cysteine (Km = 2.7 mm). Glutathione feedback inhibits AtGCL (Ki approximately 1.0 mm). As with other GCL, buthionine sulfoximine and cystamine inactivate the Arabidopsis enzyme but with inactivation rates much slower than those of the mammalian, bacterial, and nematode enzymes. The slower inactivation rates observed with AtGCL suggest that the active site differs structurally from that of other GCL. Global fitting analysis of initial velocity data indicates that a random terreactant mechanism with a preferred binding order best describes the kinetic mechanism of AtGCL. Unlike the mammalian GCL, which consists of a catalytic subunit and a regulatory subunit, AtGCL functions and is regulated as a monomeric protein. In response to redox environment, AtGCL undergoes a reversible conformational change that modulates the enzymatic activity of the monomer. These results explain the reported posttranslational change in AtGCL activity in response to oxidative stress.
Proteomics. Jan, 2006 | Pubmed ID: 16294305
Current protein identification techniques are largely based on MALDI-TOF mass fingerprinting and LC-ESI MS/MS sequence tag analysis. Here we describe an improved method for rapid protein identification that uses direct infusion nanoelectrospray quadrupole time-of-flight (nanoESI QTOF) MS. Protein digests were analyzed without LC separation using nanoESI on a QSTAR XL MS/MS system in information dependent data acquisition mode. The protein identification conditions and parameters were extensively evaluated with in-solution and in-gel digested protein samples. Rapid identification of proteins was achieved and compared directly to the results obtained on the same samples using nanoflow HPLC-MS/MS on the QSTAR system. The increased throughput, reproducibility, the high data quality, and the ease of use make the direct infusion system an efficient and affordable technique for protein identification analysis.
Cell Wall Proteome in the Maize Primary Root Elongation Zone. I. Extraction and Identification of Water-soluble and Lightly Ionically Bound Proteins
Plant Physiology. Jan, 2006 | Pubmed ID: 16377746
Cell wall proteins (CWPs) play important roles in various processes, including cell elongation. However, relatively little is known about the composition of CWPs in growing regions. We are using a proteomics approach to gain a comprehensive understanding of the identity of CWPs in the maize (Zea mays) primary root elongation zone. As the first step, we examined the effectiveness of a vacuum infiltration-centrifugation technique for extracting water-soluble and loosely ionically bound (fraction 1) CWPs from the root elongation zone. The purity of the CWP extract was evaluated by comparing with total soluble proteins extracted from homogenized tissue. Several lines of evidence indicated that the vacuum infiltration-centrifugation technique effectively enriched for CWPs. Protein identification revealed that 84% of the CWPs were different from the total soluble proteins. About 40% of the fraction 1 CWPs had traditional signal peptides and 33% were predicted to be nonclassical secretory proteins, whereas only 3% and 11%, respectively, of the total soluble proteins were in these categories. Many of the CWPs have previously been shown to be involved in cell wall metabolism and cell elongation. In addition, maize has type II cell walls, and several of the CWPs identified in this study have not been identified in previous cell wall proteomics studies that have focused only on type I walls. These proteins include endo-1,3;1,4-beta-D-glucanase and alpha-L-arabinofuranosidase, which act on the major polysaccharides only or mainly present in type II cell walls.
The Cassava (Manihot Esculenta Crantz) Root Proteome: Protein Identification and Differential Expression
Proteomics. Mar, 2006 | Pubmed ID: 16421938
Using high-resolution 2-DE, we resolved proteins extracted from fibrous and tuberous root tissues of 3-month-old cassava plants. Gel image analysis revealed an average of 1467 electrophoretically resolved spots on the fibrous gels and 1595 spots on the tuberous gels in pH 3-10 range. Protein spots from both sets of gels were digested with trypsin. The digests were subjected to nanoelectrospray quadrupole TOF tandem mass analysis. Currently, we have obtained 299 protein identifications for 292 gel spots corresponding to 237 proteins. The proteins span various functional categories from energy, primary and secondary metabolism, disease and defense, destination and storage, transport, signal transduction, protein synthesis, cell structure, and transcription to cell growth and division. Gel image analysis has shown unique, as well as up- and down-regulated proteins, present in the tuberous and the fibrous tissues. Quantitative and qualitative analysis of the cassava root proteome is an important step towards further characterization of differentially expressed proteins and the elucidation of the mechanisms underlying the development and biological functions of the two types of roots.
Journal of Proteome Research. Apr, 2006 | Pubmed ID: 16602704
The xylem in plants has mainly been described as a conduit for water and minerals, but emerging evidence also indicates that the xylem contains protein. To study the proteins in xylem sap, we characterized the identity and composition of the maize xylem sap proteome. The composition of the xylem sap proteome in maize revealed proteins related to different phases of xylem differentiation including cell wall metabolism, secondary cell wall synthesis, and programmed cell death. Many proteins were found to be present as multiple isoforms and some of these isoforms are glycosylated. Proteins involved in defense mechanisms were also present in xylem sap and the sap proteins were shown to have antifungal activity in bioassays.
Additional Freeze Hardiness in Wheat Acquired by Exposure to -3 DegreesC is Associated with Extensive Physiological, Morphological, and Molecular Changes
Journal of Experimental Botany. 2006 | Pubmed ID: 16968883
Cold-acclimated plants acquire an additional 3-5 degrees C increase in freezing tolerance when exposed to -3 degrees C for 12-18 h before a freezing test (LT50) is applied. The -3 degrees C treatment replicates soil freezing that can occur in the days or weeks leading to overwintering by freezing-tolerant plants. This additional freezing tolerance is called subzero acclimation (SZA) to differentiate it from cold acclimation (CA) that is acquired at above-freezing temperatures. Using wheat as a model, results have been obtained indicating that SZA is accompanied by changes in physiology, cellular structure, the transcriptome, and the proteome. Using a variety of assays, including DNA arrays, reverse transcription-polymerase chain reaction (RT-PCR), 2D gels with mass spectroscopic identification of proteins, and electron microscopy, changes were observed to occur as a consequence of SZA and the acquisition of added freezing tolerance. In contrast to CA, SZA induced the movement of intracellular water to the extracellular space. Many unknown and stress-related genes were upregulated by SZA including some with obvious roles in SZA. Many genes related to photosynthesis and plastids were downregulated. Changes resulting from SZA often appeared to be a loss of rather than an appearance of new proteins. From a cytological perspective, SZA resulted in alterations of organelle structure including the Golgi. The results indicate that the enhanced freezing tolerance of SZA is correlated with a wide diversity of changes, indicating that the additional freezing tolerance is the result of complex biological processes.
Proteomics. Oct, 2006 | Pubmed ID: 16972296
With the avalanche of genomic information and improvements in analytical technology, proteomics is becoming increasingly important for the study of many different aspects of plant functions. Since proteins serve as important components of major signaling and biochemical pathways, studies at protein levels are essential to reveal molecular mechanisms underlying plant growth, development, and interactions with the environment. The plant proteome is highly complex and dynamic. Although great strides need to be taken towards the ultimate goal of characterizing all the proteins in a proteome, current technologies have provided immense opportunities for high-throughput proteomic studies that have gone beyond simple protein identification to analyzing various functional aspects, such as quantification, PTM, subcellular localization, and protein-protein interactions. In this review of plant proteomics, advances in protein fractionation, separation, and MS will be outlined. Focus will be on recent development in functional analysis of plant proteins, which paves the way towards the comprehensive integration with transcriptomics, metabolomics, and other large scale "-omics" into systems biology.
Functional Characterization of AtATM1, AtATM2, and AtATM3, a Subfamily of Arabidopsis Half-molecule ATP-binding Cassette Transporters Implicated in Iron Homeostasis
The Journal of Biological Chemistry. Jul, 2007 | Pubmed ID: 17517886
The functional capabilities of one of the smallest subfamilies of ATP-binding cassette transporters from Arabidopsis thaliana, the AtATMs, are described. Designated AtATM1, AtAATM2, and AtATM3, these half-molecule ABC proteins are homologous to the yeast mitochondrial membrane protein ATM1 (ScATM1), which is clearly implicated in the export of mitochondrially synthesized iron/sulfur clusters. Yeast ATM1-deficient (atm1) mutants grow very slowly (have a petite phenotype), are respiration-deficient, accumulate toxic levels of iron in their mitochondria, and show enhanced compensatory high affinity iron uptake. Of the three Arabidopsis ATMs, AtATM3 bears the closest functional resemblance to ScATM1. Heterologously expressed AtATM3 is not only able to complement the yeast atm1 petite phenotype but is also able to suppress the constitutively high capacity for high affinity iron uptake associated with loss of the chromosomal copy of ScATM1, abrogate intra-mitochondrial iron hyperaccumulation, and restore mitochondrial respiratory function and cytochrome c levels. By comparison, AtATM1 only weakly suppresses the atm1 phenotype, and AtATM2 exerts little or no suppressive action but instead is toxic when expressed in this system. The differences between AtATM3 and AtATM1 are maintained after exchanging their target peptides, and these proteins as well as AtATM2 colocalize with the mitochondrial fluor MitoTracker Red when expressed in yeast as GFP fusions. Although its toxicity when heterologously expressed in yeast, except when fused with GFP, precludes the functional analysis of native AtATM2, a common function, mitochondrial export of Fe/S clusters or their precursors for the assembly of cytosolic Fe/S proteins, is inferred for AtATM3 and AtATM1.
The FEBS Journal. Aug, 2007 | Pubmed ID: 17651441
Despite its large size and the numerous processes in which it is implicated, neither the identity nor the functions of the proteins targeted to the yeast vacuole have been defined comprehensively. In order to establish a methodological platform and protein inventory to address this shortfall, we refined techniques for the purification of 'proteomics-grade' intact vacuoles. As confirmed by retention of the preloaded fluorescent conjugate glutathione-bimane throughout the fractionation procedure, the resistance of soluble proteins that copurify with this fraction to digestion by exogenous extravacuolar proteinase K, and the results of flow cytometric, western and marker enzyme activity analyses, vacuoles prepared in this way retain most of their protein content and are of high purity and integrity. Using this material, 360 polypeptides species associated with the soluble fraction of the vacuolar isolates were resolved reproducibly by 2D gel electrophoresis. Of these, 260 were identified by peptide mass fingerprinting and peptide sequencing by MALDI-MS and liquid chromatography coupled to ion trap or quadrupole TOF tandem MS, respectively. The polypeptides identified in this way, many of which correspond to alternate size and charge states of the same parent translation product, can be assigned to 117 unique ORFs. Most of the proteins identified are canonical vacuolar proteases, glycosidases, phosphohydrolases, lipid-binding proteins or established vacuolar proteins of unknown function, or other proteases, glycosidases, lipid-binding proteins, regulatory proteins or proteins involved in intermediary metabolism, protein synthesis, folding or targeting, or the alleviation of oxidative stress. On the basis of the high purity of the vacuolar preparations, the electrophoretic properties of the proteins identified and the results of quantitative proteinase K protection measurements, many of the noncanonical vacuolar proteins identified are concluded to have entered this compartment for breakdown, processing and/or salvage purposes.
Planta. Nov, 2007 | Pubmed ID: 17899172
Glucosinolates and their degradation products are known to play important roles in plant interaction with herbivores and micro-organisms. In addition, they are important for human life. For example, some degradation products are flavor compounds and some exhibit anticarcinogenic properties. Recent years have seen great progress made in the understanding of glucosinolate biosynthesis in Arabidopsis thaliana. The core glucosinolate biosynthetic pathway has been revealed using biochemical and reverse genetics approaches. Future research needs to focus on questions related to regulation and control of glucosinolate metabolism. Here we review current status of studies on the regulation of glucosinolate metabolism at different levels, and highlight future research towards elucidating the signaling and metabolic network that control glucosinolate metabolism.
Cell Wall Proteome in the Maize Primary Root Elongation Zone. II. Region-specific Changes in Water Soluble and Lightly Ionically Bound Proteins Under Water Deficit
Plant Physiology. Dec, 2007 | Pubmed ID: 17951457
Previous work on the adaptation of maize (Zea mays) primary roots to water deficit showed that cell elongation is maintained preferentially toward the apex, and that this response involves modification of cell wall extension properties. To gain a comprehensive understanding of how cell wall protein (CWP) composition changes in association with the differential growth responses to water deficit in different regions of the elongation zone, a proteomics approach was used to examine water soluble and loosely ionically bound CWPs. The results revealed major and predominantly region-specific changes in protein profiles between well-watered and water-stressed roots. In total, 152 water deficit-responsive proteins were identified and categorized into five groups based on their potential function in the cell wall: reactive oxygen species (ROS) metabolism, defense and detoxification, hydrolases, carbohydrate metabolism, and other/unknown. The results indicate that stress-induced changes in CWPs involve multiple processes that are likely to regulate the response of cell elongation. In particular, the changes in protein abundance related to ROS metabolism predicted an increase in apoplastic ROS production in the apical region of the elongation zone of water-stressed roots. This was verified by quantification of hydrogen peroxide content in extracted apoplastic fluid and by in situ imaging of apoplastic ROS levels. This response could contribute directly to the enhancement of wall loosening in this region. This large-scale proteomic analysis provides novel insights into the complexity of mechanisms that regulate root growth under water deficit conditions and highlights the spatial differences in CWP composition in the root elongation zone.
Journal of Proteome Research. Dec, 2007 | Pubmed ID: 17958392
In higher plants, pollen grains represent the vestiges of a highly reduced male gametophyte generation. After germination, the pollen tube delivers the sperm cells by tip-growing to the embryo sac for fertilization. Besides the intrinsic importance for sexual reproduction, pollen development and germination serve as an attractive system to address important questions related to cell division, cell differentiation, polar growth, cell-cell interaction, and cell fate. Recently, pollen functional specification has been well-studied using multidisciplinary approaches. Here, we review recent advances in proteomics of pollen development and germination.
Developing a New Interdisciplinary Lab Course for Undergraduate and Graduate Students: Plant Cells and Proteins
Biochemistry and Molecular Biology Education : a Bimonthly Publication of the International Union of Biochemistry and Molecular Biology. Nov, 2007 | Pubmed ID: 21591136
Studies of protein function increasingly use multifaceted approaches that span disciplines including recombinant DNA technology, cell biology, and analytical biochemistry. These studies rely on sophisticated equipment and methodologies including confocal fluorescence microscopy, mass spectrometry, and X-ray crystallography that are beyond the scope of traditional laboratory courses. To equip the advanced undergraduate and beginning graduate students with an enabling base of knowledge and initial experience with advanced protein research methodologies, a laboratory course entitled Plant Cells and Proteins was developed in a partnership between Washington University and the Donald Danforth Plant Science Center in St. Louis. In this one semester course, 10-12 students obtain hands-on experience with plant tissue culture, gene transformation, subcellular localization of fluorescent recombinant proteins using confocal microscopy, purification of affinity-tagged recombinant proteins, isolation of total protein extracts, enzymatic assays, one- and two-dimensional gel electrophoresis, MALDI-TOF and ESI-Q-TOF mass spectrometry, protein crystallization, and X-ray diffraction. The course is taught as a series of modules, each led by an expert researcher. Students are evaluated based on a series of graded written reports and tests of their mastery of key concepts, interpretations, and the limitations of the experimental methods.
Comparative Investigations of the Glucosinolate-myrosinase System in Arabidopsis Suspension Cells and Hypocotyls
Plant & Cell Physiology. Mar, 2008 | Pubmed ID: 18202003
Glucosinolates are secondary metabolites derived from amino acids. Upon hydrolysis by myrosinases, they produce a variety of biologically active compounds. In this study, the glucosinolate-myrosinase system was characterized in Arabidopsis suspension cells. A total of seven glucosinolates were identified and the myrosinase activity was determined. Plant suspension cells have been used as model systems in many areas of study. To investigate whether the glucosinolate-myrosinase system in suspension cells works similarly to that in planta, 10-day-old seedling hypocotyls were used for comparative studies. A total of 16 glucosinolates were identified in hypocotyls. The two types of samples were also treated with methyljasmonate (MeJA)--a signaling compound induced by herbivore attack and wounding to initiate plant defense processes. The glucosinolate levels and their responses to MeJA varied greatly with the age of the cells. Two-day-old cells were most responsive, with the levels of all seven glucosinolates induced by MeJA, while in 4-day-old cells only the levels of indole glucosinolates were increased. In hypocotyls, the levels of indole glucosinolates and aliphatic glucosinolates (especially 4-methylsulfinylbutyl- and 8-methylsulfinyloctylglucosinolates) were significantly increased by MeJA treatment. The transcript levels of several genes involved in glucosinolate biosynthesis were induced in both suspension cells and hypocotyls after MeJA treatment. Myrosinase levels and activities were also monitored. The molecular bases underlying the differences of glucosinolate metabolism in the suspension cells and hypocotyls were discussed.
Functional Differentiation of Brassica Napus Guard Cells and Mesophyll Cells Revealed by Comparative Proteomics
Molecular & Cellular Proteomics : MCP. Apr, 2009 | Pubmed ID: 19106087
Guard cells are highly specialized cells that form tiny pores called stomata on the leaf surface. The opening and closing of stomata control leaf gas exchange and water transpiration as well as allow plants to quickly respond and adjust to new environmental conditions. Mesophyll cells are specialized for photosynthesis. Despite the phenotypic and obvious functional differences between the two types of cells, the full protein components and their functions have not been explored but are addressed here through a global comparative proteomics analysis of purified guard cells and mesophyll cells. With the use of isobaric tags for relative and absolute quantification (iTRAQ) tagging and two-dimensional liquid chromatography mass spectrometry, we identified 1458 non-redundant proteins in both guard cells and mesophyll cells of Brassica napus leaves. Based on stringent statistical criteria, a total of 427 proteins were quantified, and 74 proteins were found to be enriched in guard cells. Proteins involved in energy (respiration), transport, transcription (nucleosome), cell structure, and signaling are preferentially expressed in guard cells. We observed several well characterized guard cell proteins. By contrast, proteins involved in photosynthesis, starch synthesis, disease/defense/stress, and other metabolisms are preferentially represented in mesophyll cells. Of the identified proteins, 110 have corresponding microarray data obtained from Arabidopsis guard cells and mesophyll cells. About 72% of these proteins follow the same trend of expression at the transcript and protein levels. For the rest of proteins, the correlation between proteomics data and the microarray data is poor. This highlights the importance of quantitative profiling at the protein level. Collectively this work represents the most extensive proteomic description of B. napus guard cells and has improved our knowledge of the functional specification of guard cells and mesophyll cells.
Plant Vacuolar ATP-binding Cassette Transporters That Translocate Folates and Antifolates in Vitro and Contribute to Antifolate Tolerance in Vivo
The Journal of Biological Chemistry. Mar, 2009 | Pubmed ID: 19136566
The vacuoles of pea (Pisum sativum) leaves and red beet (Beta vulgaris) storage root are major sites for the intracellular compartmentation of folates. In the light of these findings and preliminary experiments indicating that some plant multidrug resistance-associated protein (MRP) subfamily ATP-binding cassette transporters are able to transport compounds of this type, the Arabidopsis thaliana vacuolar MRP, AtMRP1 (AtABCC1), and its functional equivalent(s) in vacuolar membrane vesicles purified from red beet storage root were studied. In so doing, it has been determined that heterologously expressed AtMRP1 and its equivalents in red beet vacuolar membranes are not only competent in the transport of glutathione conjugates but also folate monoglutamates and antifolates as exemplified by pteroyl-l-glutamic acid and methotrexate (MTX), respectively. In agreement with the results of these in vitro transport measurements, analyses of atmrp1 T-DNA insertion mutants of Arabidopsis ecotypes Wassilewskia and Columbia disclose an MTX-hypersensitive phenotype. atmrp1 knock-out mutants are more sensitive than wild-type plants to growth retardation by nanomolar concentrations of MTX, and this is associated with impaired vacuolar antifolate sequestration. The vacuoles of protoplasts isolated from the leaves of Wassilewskia atmrp1 mutants accumulate 50% less [(3)H]MTX than the vacuoles of protoplasts from wild-type plants when incubated in media containing nanomolar concentrations of this antifolate, and vacuolar membrane-enriched vesicles purified from the mutant catalyze MgATP-dependent [(3)H]MTX uptake at only 40% of the capacity of the equivalent membrane fraction from wild-type plants. AtMRP1 and its counterparts in other plant species therefore have the potential for participating in the vacuolar accumulation of folates and related compounds.
Characterization of Glucosinolate--myrosinase System in Developing Salt Cress Thellungiella Halophila
Physiologia Plantarum. May, 2009 | Pubmed ID: 19508363
Glucosinolates are specialized plant metabolites derived from amino acids. They can be hydrolyzed by myrosinases into different degradation products, which have a variety of biological activities. In this study, the compositions and contents of glucosinolates in salt cress (Thellungiella halophila) at different developmental stages were analyzed by high performance liquid chromatography and mass spectrometry (HPLC-MS). Myrosinase activities were also measured. Seven glucosinolates were identified in T. halophila throughout its life cycle. The glucosinolate profiles varied significantly among different tissues. The roots at stage 4 contained the highest concentrations of total, aromatic and indole glucosinolates among all tissues. Whereas roots, flowers and siliques contained all seven glucosinolates, seeds contained only four aliphatic glucosinolates. During development, the concentrations also displayed significant changes. From seeds to cotyledons and from stage 4 roots to stage 5 roots, there were dramatic declines of glucosinolates, which correlated well with changes in myrosinase activities. In other tissues, myrosinase activity alone could not explain the glucosinolate concentration changes. Certain tissues of T. halophila contained Arabidopsis myrosinase TGG1 and TGG2 orthologs. The molecular basis and functional significance of our findings are discussed here.
Journal of Proteomics. Nov, 2009 | Pubmed ID: 19628057
Protein redox regulation is increasingly recognized as an important switch of protein activity in yeast, bacteria, mammals and plants. In this study, we identified proteins with potential thiol switches involved in jasmonate signaling, which is essential for plant defense. Methyl jasmonate (MeJA) treatment led to enhanced production of hydrogen peroxide in Arabidopsis leaves and roots, indicating in vivo oxidative stress. With monobromobimane (mBBr) labeling to capture oxidized sulfhydryl groups and 2D gel separation, a total of 35 protein spots that displayed significant redox and/or total protein expression changes were isolated. Using LC-MS/MS, the proteins in 33 spots were identified in both control and MeJA-treated samples. By comparative analysis of mBBr and SyproRuby gel images, we were able to determine many proteins that were redox responsive and proteins that displayed abundance changes in response to MeJA. Interestingly, stress and defense proteins constitute a large group that responded to MeJA. In addition, many cysteine residues involved in the disulfide dynamics were mapped based on tandem MS data. Identification of redox proteins and their cysteine residues involved in the redox regulation allows for a deeper understanding of the jasmonate signaling networks.
A Redox-active Isopropylmalate Dehydrogenase Functions in the Biosynthesis of Glucosinolates and Leucine in Arabidopsis
The Plant Journal : for Cell and Molecular Biology. Nov, 2009 | Pubmed ID: 19674406
We report a detailed functional characterization of an Arabidopsis isopropylmalate dehydrogenase (AtIPMDH1) that is involved in both glucosinolate biosynthesis and leucine biosynthesis. AtIPMDH1 shares high homology with enzymes from bacteria and yeast that are known to function in leucine biosynthesis. In plants, AtIPMDH1 is co-expressed with nearly all the genes known to be involved in aliphatic glucosinolate biosynthesis. Mutation of AtIPMDH1 leads to a significant reduction in the levels of free leucine and of glucosinolates with side chains of four or more carbons. Complementation of the mutant phenotype by ectopic expression of AtIPMDH1, together with the enzyme's substrate specificity, implicates AtIPMDH1 in both glucosinolate and leucine biosynthesis. This functional assignment is substantiated by subcellular localization of the protein in the chloroplast stroma, and the gene expression patterns in various tissues. Interestingly, AtIPMDH1 activity is regulated by a thiol-based redox modification. This work characterized an enzyme in plants that catalyzes the oxidative decarboxylation step in both leucine biosynthesis (primary metabolism) and methionine chain elongation of glucosinolates (specialized metabolism). It provides evidence for the hypothesis that the two pathways share a common origin, and suggests a role for redox regulation of glucosinolate and leucine synthesis in plants.
Comparative Interactomics: Analysis of Arabidopsis 14-3-3 Complexes Reveals Highly Conserved 14-3-3 Interactions Between Humans and Plants
Journal of Proteome Research. Apr, 2009 | Pubmed ID: 19714877
As a first step in the broad characterization of plant 14-3-3 multiprotein complexes in vivo, stringent and specific antibody affinity purification was used to capture 14-3-3s together with their interacting proteins from extracts of Arabidopsis cell suspension cultures. Approximately 120 proteins were identified as potential in vivo 14-3-3 interacting proteins by mass spectrometry of the recovered complexes. Comparison of the proteins in this data set with the 14-3-3 interacting proteins from a similar study in human embryonic kidney cell cultures revealed eight interacting proteins that likely represent reasonably abundant, fundamental 14-3-3 interaction complexes that are highly conserved across all eukaryotes. The Arabidopsis 14-3-3 interaction data set was also compared to a yeast in vivo 14-3-3 interaction data set. Four 14-3-3 interacting proteins are conserved in yeast, humans, and Arabidopsis. Comparisons of the data sets based on biochemical function revealed many additional similarities in the human and Arabidopsis data sets that represent conserved functional interactions, while also leaving many proteins uniquely identified in either Arabidopsis or human cells. In particular, the Arabidopsis interaction data set is enriched for proteins involved in metabolism.
Journal of Proteomics. Dec, 2009 | Pubmed ID: 19782777
Apomixis in plants holds great promise for agriculture because of its vigor associated with heterozygosity and superior genotype. Despite the significance of apomictic reproductive process, our knowledge of proteins and their functions in apomictic development is limited. Here we report a comparative proteomic and transcriptomic analysis of sexual and apomictic processes in sugar beet. A total of 71 differentially expressed protein spots were successfully identified in the course of apomictic reproductive development using high-resolution 2-DE and MS analysis. The differentially expressed proteins were involved in several processes that might work cooperatively to lead to apomictic reproduction. This study has generated potential protein markers important for apomictic development.
Proteomic Analysis of Circulating Immune Complexes in Juvenile Idiopathic Arthritis Reveals Disease-associated Proteins
Proteomics. Clinical Applications. Jul, 2009 | Pubmed ID: 21136990
Juvenile idiopathic arthritis reflects a group of clinically heterogeneous arthritides hallmarked by elevated concentrations of circulating immune complexes. In this study, the circulating immune complex proteome was examined to elucidate disease-associated proteins that are overexpressed in patients with an aggressive, and at times destructive, disease phenotype. To solve this proteome, circulating immune complexes were isolated from the sera of patients with chronic, erosive or early-onset, aggressive disease and from patients in medical remission or healthy controls subsequent to protein separation by 2-DE. Thirty-seven protein spots were overexpressed in the circulating immune complexes of the aggressive disease groups as compared to controls, 28 of which have been confidently identified to date. Proteolytic fragments of glyceraldehyde-3-phosphate dehydrogenase, serotransferrin, and α-1-antitrypsin have been identified among others. In total, these 28 putative disease-associated proteins most definitely contribute to immune complex formation and likely have a significant role in disease etiology and pathogenesis. Moreover, these proteins represent markers of aggressive disease, which could aid in diagnosis and management strategies, and potential therapeutic targets to prevent or control disease outcome. This is the first in-depth analysis of the circulating immune complex proteome in juvenile idiopathic arthritis.
Analysis of Abscisic Acid Responsive Proteins in Brassica Napus Guard Cells by Multiplexed Isobaric Tagging
Journal of Proteomics. Feb, 2010 | Pubmed ID: 19913118
Guard cells, which form stomata on the leaf epidermis, play important roles in plant gas exchange and defense against pathogens. Abscisic acid (ABA) is a phytohormone that can be induced by drought and leads to stomatal closure. Guard cells have been a premier model system for studying ABA signal transduction. Despite significant progress on the identification of molecular components in the ABA signaling pathway, our knowledge of the protein components is very limited. Here, we employ a recently developed multiplexed isobaric tagging technology to identify ABA-responsive proteins in Brassica napus guard cells. A total of 431 unique proteins were identified with relative quantitative information in control and ABA-treated samples. Proteins involved in stress and defense constituted a major group among the 66 proteins with increased abundance. Thirty-eight proteins were decreased in abundance and fell into several functional groups including metabolism and protein synthesis. Many of the proteins have not been reported as being ABA responsive or involved in stomatal movement. A large percentage of the protein-coding genes contained ABA-responsive elements. This study not only established a comprehensive inventory of ABA-responsive proteins, but also identified new proteins for further investigation of their functions in guard cell ABA signaling.
Nature. Jan, 2010 | Pubmed ID: 20054389
Archaea, one of three major evolutionary lineages of life, encode proteasomes highly related to those of eukaryotes. In contrast, archaeal ubiquitin-like proteins are less conserved and not known to function in protein conjugation. This has complicated our understanding of the origins of ubiquitination and its connection to proteasomes. Here we report two small archaeal modifier proteins, SAMP1 and SAMP2, with a beta-grasp fold and carboxy-terminal diglycine motif similar to ubiquitin, that form protein conjugates in the archaeon Haloferax volcanii. The levels of SAMP-conjugates were altered by nitrogen-limitation and proteasomal gene knockout and spanned various functions including components of the Urm1 pathway. LC-MS/MS-based collision-induced dissociation demonstrated isopeptide bonds between the C-terminal glycine of SAMP2 and the epsilon-amino group of lysines from a number of protein targets and Lys 58 of SAMP2 itself, revealing poly-SAMP chains. The widespread distribution and diversity of pathways modified by SAMPylation suggest that this type of protein conjugation is central to the archaeal lineage.
Journal of Proteome Research. May, 2010 | Pubmed ID: 20377188
Salinity is a major abiotic stress affecting plant cultivation and productivity. Thellungiella halophila is a halophyte and has been used as a model for studying plant salt tolerance. Understanding the molecular mechanisms of salinity tolerance will facilitate the generation of salt tolerant crops. Here we report comparative leaf proteomics of Arabidopsis, a glycophyte, and its close relative Thellungiella, a halophyte, under different salt stress conditions. Proteins from control and NaCl treated Arabidopsis and Thellungiella leaf samples were extracted and separated by two-dimensional gel electrophoresis. A total of 88 protein spots from Arabidopsis gels and 37 protein spots from Thellungiella gels showed significant changes. Out of these spots, a total of 79 and 32 proteins were identified by mass spectrometry in Arabidopsis and Thellungiella, respectively. Most of the identified proteins were involved in photosynthesis, energy metabolism, and stress response in Arabidopsis and Thellungiella. As a complementary approach, isobaric tag for relative and absolute quantification (iTRAQ) LC-MS was used to identify crude microsomal proteins. A total of 31 and 32 differentially expressed proteins were identified in Arabidopsis and Thellungiella under salt treatment, respectively. Overall, there were more proteins changed in abundance in Arabidopsis than in Thellungiella. Distinct patterns of protein changes in the two species were observed. Collectively, this work represents the most extensive proteomic description of salinity responses of Arabidopsis and Thellungiella and has improved our knowledge of salt tolerance in glycophytes and halophytes.
Functional Specification of Arabidopsis Isopropylmalate Isomerases in Glucosinolate and Leucine Biosynthesis
Plant & Cell Physiology. Sep, 2010 | Pubmed ID: 20663849
The Arabidopsis genome encodes one potential isopropylmalate isomerase (IPMI) large subunit and three potential IPMI small subunits, which in bacteria and archaea form heterodimers to catalyze the isomerization of 2-isopropylmalate to 3-isopropylmalate in leucine biosynthesis. We demonstrate here that AtLeuC physically interacts with AtLeuD proteins to form functional IPMIs. The IPMIs are localized to chloroplast stroma. Tissue-specific expression analysis revealed that the patterns of AtLeuD1 and AtLeuD2 expression are similar, but distinct from that of AtLeuD3. This result indicates functional redundancy of AtLeuD1 and AtLeuD2, and functional specification of AtLeuD3. Reverse genetics and metabolite profiling showed that AtLeuD1 and AtLeuD2 function redundantly in aliphatic glucosinolate biosynthesis, but AtLeuD3 is not likely to be involved in this pathway. The lethal phenotype of the atleud3 mutant suggests functional specification of AtLeuD3 in leucine biosynthesis. A defect in female gametophyte development was found to contribute to the mutant lethality, suggesting the important role of AtLeuD3 in female gametophyte development.
Desiccation Tolerance Mechanism in Resurrection Fern-ally Selaginella Tamariscina Revealed by Physiological and Proteomic Analysis
Journal of Proteome Research. Dec, 2010 | Pubmed ID: 20923197
Drought is one of the most severe limitations to plant growth and productivity. Resurrection plants have evolved a unique capability to tolerate desiccation in vegetative tissues. Fern-ally Selaginella tamariscina (Beauv.) is one of the most primitive vascular resurrection plants, which can survive a desiccated state and recover when water becomes available. To better understand the mechanism of desiccation tolerance, we have applied physiological and proteomic analysis. Samples of S. tamariscina were water-deprived for up to seven days followed by 12 h of rewatering. Our results showed that endogenous abscisic acid (ABA) increased to regulate dehydration-responsive genes/proteins and physiological processes. In the course of dehydration, the contents of osmolytes represented by soluble sugars and proline were increased to maintain cell structure integrity. The activities of four antioxidant enzymes (superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and glutathione reductase (GR)) also increased. In contrast, both the rate of photosynthesis and the chlorophyll content decreased, and plasma membrane integrity was lost. We identified 138 desiccation-responsive two-dimensional electrophoresis (2-DE) spots, representing 103 unique proteins. Hierarchical clustering analysis revealed that 83% of the proteins were down-regulated upon dehydration. They were mainly involved in photosynthesis, carbohydrate and energy metabolism, stress and defense, protein metabolism, signaling, membrane/transport, cell structure, and cell division. The dynamic expression changes of the desiccation-responsive proteins provide strong evidence that cell structure modification, photosynthesis reduction, antioxidant system activation, and protein post-transcriptional/translational modifications are essential to the poikilochlorophyllous fern-ally S. tamariscina in response to dehydration. In addition, our comparative analysis of dehydration-responsive proteins in vegetative tissues from 19 desiccation tolerant and nontolerant plant species suggests that resurrection S. tamariscina has developed a specific desiccation tolerant mechanism. To our knowledge, this study constitutes the first detailed investigation of the protein complement in fern/fern-allies.
Isobaric Tags for Relative and Absolute Quantification- Based Comparative Proteomics Reveals the Features of Plasma Membrane-associated Proteomes of Pollen Grains and Pollen Tubes from Lilium Davidii
Journal of Integrative Plant Biology. Dec, 2010 | Pubmed ID: 21106004
Mature pollen grains (PGs) from most plant species are metabolically quiescent. However, once pollinated onto stigma, they quickly hydrate and germinate. A PG can give rise to a vegetative cell-derived polarized pollen tube (PT), which represents a specialized polar cell. The polarized PT grows by the tip and requires interaction of different signaling molecules localized in the apical plasma membrane and active membrane trafficking. The mechanisms underlying the interaction and membrane trafficking are not well understood. In this work, we purified PG and PT plasma-membrane vesicles from Lilium davidii Duch. using the aqueous two-phase partition technique, then enriched plasma membrane proteins by using Brij58 and KCl to remove loosely bound contaminants. We identified 223 integral and membrane-associated proteins in the plasma membrane of PGs and PTs by using isobaric tags for relative and absolute quantification (iTRAQ) and 2-D high-performance liquid chromatography-tandem mass spectrometry. More than 68% of the proteins have putative transmembrane domains and/or lipid-modified motifs. Proteins involved in signal transduction, membrane trafficking and transport are predominant in the plasma-membrane proteome. We revealed most components of the clathrin-dependent endocytosis pathway. Statistical analysis revealed 14 proteins differentially expressed in the two development stages: in PTs, six upregulated and eight downregulated are mainly involved in signaling, transport and membrane trafficking. These results provide novel insights into polarized PT growth.
Nuclear Localization of NPR1 is Required for Regulation of Salicylate Tolerance, Isochorismate Synthase 1 Expression and Salicylate Accumulation in Arabidopsis
Journal of Plant Physiology. Jan, 2010 | Pubmed ID: 19716624
Plant systemic acquired resistance (SAR) is a broad-spectrum immune response in which pathogen infection in local tissue induces resistance in systemic leaves. Activation of SAR requires the signal molecule salicylic acid (SA), which is primarily synthesized from chorismate via isochorismate through the action of isochorismate synthase 1 (ICS1) and a putative isochorismate pyruvate lyase. The Arabidopsis transcription coactivator NPR1 is a key regulator of SAR, which functions at multiple nodes in the SA signaling network. NPR1 not only acts downstream of SA to activate SAR, but also upstream of SA to suppress the expression of ICS1, thus inhibiting SA biosynthesis. NPR1 also positively regulates SA tolerance and plays a role in SA-mediated negative regulation of jasmonic acid (JA) signaling. The NPR1 protein contains a functional bipartite nuclear localization signal (NLS). It has been shown that the NLS and nuclear localization of NPR1 are required for activation of pathogenesis-related gene expression, whereas modulation of the crosstalk between SA- and JA-dependent defense pathways is mediated by cytosolic NPR1. In this study we used two transgenic lines, one expressing a mutated npr1 with a dysfunctional NLS and the other in which NPR1 nuclear localization can be induced by dexamethasone treatment, to test whether nuclear localization is required for other functions of NPR1. We found that prevention of NPR1 nuclear localization renders transgenic seedlings sensitive to the toxicity of high levels of SA and causes over-accumulation of ICS1 transcripts and SA in response to pathogen infection. Induction of NPR1 nuclear localization restores SA tolerance and normal accumulation of ICS1 transcripts and SA. These results indicate that the NLS and nuclear localization of NPR1 are required for regulation of SA tolerance, ICS1 expression and SA accumulation.
Structural and Functional Evolution of Isopropylmalate Dehydrogenases in the Leucine and Glucosinolate Pathways of Arabidopsis Thaliana
The Journal of Biological Chemistry. Aug, 2011 | Pubmed ID: 21697089
The methionine chain-elongation pathway is required for aliphatic glucosinolate biosynthesis in plants and evolved from leucine biosynthesis. In Arabidopsis thaliana, three 3-isopropylmalate dehydrogenases (AtIPMDHs) play key roles in methionine chain-elongation for the synthesis of aliphatic glucosinolates (e.g. AtIPMDH1) and leucine (e.g. AtIPMDH2 and AtIPMDH3). Here we elucidate the molecular basis underlying the metabolic specialization of these enzymes. The 2.25 Å resolution crystal structure of AtIPMDH2 was solved to provide the first detailed molecular architecture of a plant IPMDH. Modeling of 3-isopropylmalate binding in the AtIPMDH2 active site and sequence comparisons of prokaryotic and eukaryotic IPMDH suggest that substitution of one active site residue may lead to altered substrate specificity and metabolic function. Site-directed mutagenesis of Phe-137 to a leucine in AtIPMDH1 (AtIPMDH1-F137L) reduced activity toward 3-(2'-methylthio)ethylmalate by 200-fold, but enhanced catalytic efficiency with 3-isopropylmalate to levels observed with AtIPMDH2 and AtIPMDH3. Conversely, the AtIPMDH2-L134F and AtIPMDH3-L133F mutants enhanced catalytic efficiency with 3-(2'-methylthio)ethylmalate ∼100-fold and reduced activity for 3-isopropylmalate. Furthermore, the altered in vivo glucosinolate profile of an Arabidopsis ipmdh1 T-DNA knock-out mutant could be restored to wild-type levels by constructs expressing AtIPMDH1, AtIPMDH2-L134F, or AtIPMDH3-L133F, but not by AtIPMDH1-F137L. These results indicate that a single amino acid substitution results in functional divergence of IPMDH in planta to affect substrate specificity and contributes to the evolution of specialized glucosinolate biosynthesis from the ancestral leucine pathway.
Journal of Proteome Research. Sep, 2011 | Pubmed ID: 21732589
Soil salinity poses a serious threat to agriculture productivity throughout the world. Studying mechanisms of salinity tolerance in halophytic plants will provide valuable information for engineering plants for enhanced salt tolerance. Monocotyledonous Puccinellia tenuiflora is a halophytic species that widely distributed in the saline-alkali soil of the Songnen plain in northeastern China. Here we investigate the molecular mechanisms underlying moderate salt tolerance of P. tenuiflora using a combined physiological and proteomic approach. The changes in biomass, inorganic ion content, osmolytes, photosynthesis, defense-related enzyme activities, and metabolites in the course of salt treatment were analyzed in the leaves. Comparative proteomic analysis revealed 107 identities (representing 93 unique proteins) differentially expressed in P. tenuiflora leaves under saline conditions. These proteins were mainly involved in photosynthesis, stress and defense, carbohydrate and energy metabolism, protein metabolism, signaling, membrane, and transport. Our results showed that reduction of photosynthesis under salt treatment was attributed to the down-regulation of the light-harvesting complex (LHC) and Calvin cycle enzymes. Selective uptake of inorganic ions, high K(+)/Na(+) ratio, Ca(2+) concentration changes, and an accumulation of osmolytes contributed to ion balance and osmotic adjustment in leaf cells. Importantly, P. tenuiflora plants developed diverse reactive oxygen species (ROS) scavenging mechanisms in their leaves to cope with moderate salinity, including enhancement of the photorespiration pathway and thermal dissipation, synthesis of the low-molecular-weight antioxidant α-tocopherol, and an accumulation of compatible solutes. This study provides important information toward improving salt tolerance of cereals.
Identification of a Sugar Beet BvM14-MADS Box Gene Through Differential Gene Expression Analysis of Monosomic Addition Line M14
Journal of Plant Physiology. Nov, 2011 | Pubmed ID: 21807438
Monosomic addition line M14 carrying an additional chromosome 9 from Beta corolliflora Zosimovic ex Buttler was obtained through hybridization between the wild species B. corolliflora and a cultivated species Beta vulgaris L. var Saccharifera Alef. The M14 line showed diplosporic reproduction and stress tolerance. To identify differentially expressed genes in M14, a subtractive cDNA library was prepared by suppression subtractive hybridization (SSH) between M14 (2n=18+1) and B. vulgaris (2n=18). A total of 190 unique sequences were identified in the library and their putative functions were analyzed using Gene Ontology (GO). One of the genes, designated as BvM14-MADS box, encodes a MADS box transcription factor. It was cloned from M14 and over-expressed in transgenic tobacco plants. Interestingly, this gene was located on chromosome 2 of B. vulgaris, not on the additional chromosome 9. Overexpression of BvM14-MADS box led to significant phenotypic changes in tobacco. The differential expression of BvM14-MADS box gene in M14 may be caused by the interaction between the additional chromosome 9 from B. corolliflora and the B. vulgaris chromosomes in M14.
Functional Characterization of Arabidopsis Thaliana Isopropylmalate Dehydrogenases Reveals Their Important Roles in Gametophyte Development
The New Phytologist. Jan, 2011 | Pubmed ID: 20840499
• Isopropylmalate dehydrogenases (IPMDHs) catalyze the oxidative decarboxylation of 3-isopropylmalate (3-IPM) in leucine biosynthesis in microorganisms. The Arabidopsis thaliana genome contains three putative IPMDH genes. • IPMDH2 and IPMDH3 proteins exhibited significantly higher activity toward 3-IPM than IPMDH1, which is indicative of a pivotal role in leucine biosynthesis. Single mutants of IPMDH2 or IPMDH3 lacked a discernible phenotype. Genetic analysis showed that ipmdh2 ipmdh3 was lethal in male gametophytes and had reduced transmission through female gametophytes. The aborted pollen grains were small, abnormal in cellular structure, and arrested in germination. In addition, half of the double mutant embryo sacs exhibited slowed development. • The IPMDH2/ipmdh2 ipmdh3/ipmdh3 genotype exhibited abnormal vegetative phenotypes, suggesting haplo-insufficiency of IPMDH2 in the ipmdh3 background. This mutant and a triple mutant containing one allele of IPMDH2 or IPMDH3 had decreased leucine biosynthetic enzyme activities and lower free leucine concentrations. The latter mutant showed changes in glucosinolate profiles different from those in the ipmdh1 mutant. • The results demonstrate that IPMDH2 and IPMDH3 primarily function in leucine biosynthesis, are essential for pollen development and are needed for proper embryo sac development.
Computational Biology and Chemistry. Feb, 2011 | Pubmed ID: 21247808
Glucosinolates constitute a major group of secondary metabolites in Arabidopsis, which play an important role in plant interaction with pathogens and insects. Advances in glucosinolate research have defined the biosynthetic pathways. However, cross-talk and interaction between glucosinolate pathway and other molecular pathways are largely unknown. Here three bioinformatics tools were used to explore novel components and pathway connections in glucosinolate network. Although none of the software tools were prefect to predict glucosinolate genes, combination of results generated by all the tools led to successful prediction of all known glucosinolate genes. This approach was used to predict new genes in glucosinolate network. A total of 330 genes were found with high potential to relate to glucosinolate biosynthesis. Among them 64 genes were selected to construct glucosinolate network because their individual connection to at least one known glucosinolate gene was predicted by all the software tools. Microarray data of candidate gene mutants were used for validation of the results. The mutants of nine genes predicted by glucosinolate seed genes all exhibited changes in the expression of glucosinolate genes. Four of the genes have been well-known to functionally interact with glucosinolate biosynthesis. These results indicate that the approach we took provides a powerful way to reveal new players in glucosinolate networks. Creation of an in silico network of glucosinolate biosynthesis will allow the generation of many testable hypotheses and ultimately enable predictive biology.
Journal of Proteome Research. Apr, 2011 | Pubmed ID: 21338050
Due to the increasing prevalence of nosocomial and community-acquired antibiotic resistant Staphylococcus aureus (SA), understanding the determinants of SA nasal carriage has become a major imperative. Previous research has revealed many host and bacterial factors that contribute to SA nasal carriage. To assess bacterial factors that facilitate nasal carriage, we compared the exoproteome of a nasal carrier strain of SA to a genetically similar noncarrier strain. Additionally, the carrier strain biofilm exoproteome was also compared against its planktonic counterpart. Using high throughput proteomics, it was observed that the carrier strain of SA secretes a greater number of proteins that may promote successful colonization of the human nose, including cell attachment and immunoevasive proteins, than the noncarrier strain. Similarly, SA carrier strain biofilm exoproteome contains a greater number of immunoevasive proteins than its planktonic counterpart. Analysis of the most abundant immunoevasive proteins revealed that Staphylococcal protein A was present at significantly higher levels in carrier than in noncarrier strains of SA, suggesting an association with nasal carriage. While further analyses of specific differences between carrier and noncarrier strains of SA are required, many of the differentially expressed proteins identified can be considered to be putative determinants of nasal carriage.
Proteomic Identification of Differentially Expressed Proteins in Arabidopsis in Response to Methyl Jasmonate
Journal of Plant Physiology. Jul, 2011 | Pubmed ID: 21377756
Jasmonates (JAs) are the well characterized fatty acid-derived cyclopentanone signals involved in the plant response to biotic and abiotic stresses. JAs have been shown to regulate many aspects of plant metabolism, including glucosinolate biosynthesis. Glucosinolates are natural plant products that function in defense against herbivores and pathogens. In this study, we applied a proteomic approach to gain insight into the physiological processes, including glucosinolate metabolism, in response to methyl jasmonate (MeJA). We identified 194 differentially expressed protein spots that contained proteins that participated in a wide range of physiological processes. Functional classification analysis showed that photosynthesis and carbohydrate anabolism were repressed after MeJA treatment, while carbohydrate catabolism was up-regulated. Additionally, proteins related to the JA biosynthesis pathway, stress and defense, and secondary metabolism were up-regulated. Among the differentially expressed proteins, many were involved in oxidative tolerance. The results indicate that MeJA elicited a defense response at the proteome level through a mechanism of redirecting growth-related metabolism to defense-related metabolism.
BMC Plant Biology. 2011 | Pubmed ID: 21569438
Allergic reactions to peanuts (Arachis hypogaea L.) can cause severe symptoms and in some cases can be fatal, but avoidance is difficult due to the prevalence of peanut-derived products in processed foods. One strategy of reducing the allergenicity of peanuts is to alter or eliminate the allergenic proteins through mutagenesis. Other seed quality traits could be improved by altering biosynthetic enzyme activities. Targeting Induced Local Lesions in Genomes (TILLING), a reverse-genetics approach, was used to identify mutations affecting seed traits in peanut.
Journal of Proteome Research. Jan, 2012 | Pubmed ID: 22017755
Soil salinity is a major abiotic stress that limits plant growth and agriculture productivity. To cope with salt stress, plants have evolved complex salt-responsive signaling and metabolic processes at the cellular, organ, and whole-plant levels. Investigation of the physiological and molecular mechanisms underlying plant salinity tolerance will provide valuable information for effective engineering strategies. Current proteomics provides a high-throughput approach to study sophisticated molecular networks in plants. In this review, we describe a salt-responsive protein database by an integrated analysis of proteomics-based studies. The database contains 2171 salt-responsive protein identities representing 561 unique proteins. These proteins have been identified from leaves, roots, shoots, seedlings, unicells, grains, hypocotyls, radicles, and panicles from 34 plant species. The identified proteins provide invaluable information toward understanding the complex and fine-tuned plant salt-tolerance mechanisms in photosynthesis, reactive oxygen species (ROS) scavenging, ion homeostasis, osmotic modulation, signaling transduction, transcription, protein synthesis/turnover, cytoskeleton dynamics, and cross-tolerance to different stress conditions.
Boosting the Globalization of Plant Proteomics Through INPPO: Current Developments and Future Prospects
Proteomics. Feb, 2012 | Pubmed ID: 22290804
The International Plant Proteomics Organization (INPPO) is a non-profit-organization consisting of people who are involved or interested in plant proteomics. INPPO is constantly growing in volume and activity, which is mostly due to the realization among plant proteomics researchers worldwide for the need of such a global platform. Their active participation resulted in the rapid growth within the first year of INPPO's official launch in 2011 via its website (www.inppo.com) and publication of the 'Viewpoint paper' in a special issue of PROTEOMICS (May 2011). Here, we will be highlighting the progress achieved in the year 2011 and the future targets for the year 2012 and onwards. INPPO has achieved a successful administrative structure, the Core Committee (CC; composed of President, Vice-President, and General Secretaries), Executive Council (EC), and General Body (GB) to achieve INPPO objectives. Various committees and subcommittees are in the process of being functionalized via discussion amongst scientists around the globe. INPPO's primary aim to popularize the plant proteomics research in biological sciences has also been recognized by PROTEOMICS where a section dedicated to plant proteomics has been introduced starting January 2012, following the very first issue of this journal devoted to plant proteomics in May 2011. To disseminate organizational activities to the scientific community, INPPO has launched a biannual (in January and July) newsletter entitled 'INPPO Express: News & Views' with the first issue published in January 2012. INPPO is also planning to have several activities in 2012, including programs within the Education Outreach committee in different countries, and the development of research ideas and proposals with priority on crop and horticultural plants, while keeping tight interactions with proteomics programs on model plants such as Arabidopsis thaliana, rice, and Medicago truncatula. Altogether, the INPPO progress and upcoming activities are because of immense support, dedication, and hard work of all members of the INPPO community, and also due to the wide encouragement and support from the communities (scientific and non-scientific).