The Journal of American Science, 2(2), 2006, Xi, et al, Developments of Arabidopsis thaliana Proteomics Research
1. Key Laboratory for Molecular Enzymology & Engineering of the Ministry of Education (Jilin University),
Changchun, Jilin 130021, China, Email: ydgl@tom.com
; hao@jlu.edu.cn
2. College of Plant Science, Jilin University, Changchun, Jilin 130062, China
3. School of Agriculture Northeast Agricultural University, Harbin, Heilongjiang 150030, China
Abstract: Since the completion of the first flowering plant Arabidopsis genome sequencing, more attention has been focused on determining the functions and functional networks of proteins by proteomics analysis. Proteomics is becoming a more powerful and indispensable technology in molecular biology. During the last decade an important progress has been made in the field of Arabidopsis proteomics research, many dedicated research groups have used this technology to systematically analyze the Arabidopsis proteome on the level of the organ, tissue, organelle and the whole plant. Many improvements in separation and identification of proteins, such as two-dimensional electrophoresis (2-DE) and mass spectrometry (MS), as well as some new techniques including tandem affinity, top-down mass spectrometry and protein chips have emerged. At the same time, proteomics bioinformatics is essential to cope with the huge information of proteome. In this review, we discuss the progress made in the field of Arabidopsis proteomics, limitations of current techniques and expatiate the perspectives for proteomics. [The Journal of American Science. 2006;2(2):50-57].
Keywords: Arabidopsis thaliana
；
Proteomics
；
Review
Introduction
The flowering plant Arabidopsis thaliana is an important model system for identifying genes and determining their functions [1, 2] . Because of its short life cycle and small size Arabidopsis was chosen by plant geneticists and made an ideal experimental organism. In
December of 2000, the Arabidopsis research group announced a major accomplishment: the completion of sequencing the flowering plant genome [3] . This is the first time that the scientists have sequenced all the genes necessary for a plant to function, knowledge unparalleled in the history of science. To date, functions can be hypothesized for only one third of the genes already sequenced in this species. The information of potential [4] . Proteome approaches present new perspectives to analyze the complex functions of model plants and crop species at different levels. Proteomics is a new tool used to identify and characterize all the proteins expressed in an organism or cell [5] . The essential method for proteomics is two dimensional gel electrophoresis (2-DE) which was started by by O’
Farrell more than twenty years ago [6] . Present proteomics research aims at both identifying new proteins in relation to their function and ultimately at explaining how their expressions are controlled within gene sequence is not sufficient to provide significant biology knowledge of the organisms.
In the post-genomic era, research will be focused on functional genomics, especially proteomics which plays an important role in the functional genomics field because proteins are directly related to their functions.
Thus the proteomics approach is helpful to answer questions of protein functional analysis. Tremendous progress has been made in the past few years in regulatory networks. Detailed discussions on these various aspects of proteomics and on some major technical advances can be found in recent reviews and articles. This article reviews the most recent developments in the various aspects of Arabidopsis thaliana proteome research.
1. Approach and technology of proteomics
( ⅰ
) Sample preparation generating large-scale data sets for protein-protein interactions, organelle composition, protein activity patterns and protein profiles in technological
Arabidopsis improvements,
. But further organization of international proteomics projects and open access to the results are necessary for proteomics to fulfill its
Proteins are physically and chemically much more diverse than nucleic acids, which hinders the quantitative analysis of complex samples of proteins.
To take advantage of the high resolution of 2-DE, proteins of the sample have to be denatured, disaggregated, reduced and solubilized to achieve complete disruption of inter- or intra-molecular interactions and to ensure that each spot represents an
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The Journal of American Science, 2(2), 2006, Xi, et al, Developments of Arabidopsis thaliana Proteomics Research individual polypeptide. TCA/acetone precipitation is very useful for minimizing protein degradation and removing interfering compounds, such as salt, or polyphenols. At present the most suitable approach towards this goal is separation and visualization of proteins from crude tissue extracts by 2-DE followed by the identification and characterization of the isolated proteins by mass spectrometric techniques [7] . A hydroponic cultivation system was developed for growing Arabidopsis plantlets under sterile in controlled conditions. By this way proteome and metabolite analyses were performed on root and shoot tissues and demonstrated excellent reproducibility, indicating that the system is advantageous when biological variation is minimized [8] . Now some new methods have been recommended about protein extracts of A. thaliana . Patrick reported a suitable method which not only avoids any loss of protein in the course of sample preparation, but the total number of protein spots to detect in 2-DE patterns exceeds the resolution commonly reported for plant tissue about threefold [9] . Currently there is great interest in the development of methods to simplify complex protein mixtures for analysis by proteomic strategies. Scientists develop and evaluate immobilized heparin chromatography to simplify protein mixtures and to enrich minor proteins [10] . This prefractionation technique has strong potential for incorporation into both qualitative and quantitative proteomics strategies.
Prefractionation of samples prior to 2-DE can create more discrete samples, allowing for further analysis.
Prefractionation methods include sequential extraction with increasingly stronger solubilization solution; subcellular fractionation [11] ; selective removal of the most abundant protein, such as Rubisco (ribulose
1,5-bisphosphate carboxylase/oxygenase) in plant leaf [12] for detecting less abundant proteins. SDS-PAGE
(sodium dodecyl sulphate-polyacrylamide gel electrophoresis) based size prefractionation provides improved separation and detection of high molecular weight or low abundance proteins [13] . The aim of sample preparation for 2-DE is to convert the native sample into a suitable physicochemical state for first dimension isoelectric focusing electrophoresis (IEF) while preserving the native charge and molecular weight of the constituent proteins [14] .
( ⅱ
) Separation and analysis of sample
The combination of 2-DE and MS has become an important analytical technique for the characterization of complex protein populations extracted from tissues, cells or subcellular fractions. The large-gel 2-DE technique developed and optimized by Klose and co-workers can separate and display more than 10 000 different proteins in a single experiment [15] . Application of this technique to crude proteome extracts from the plants A. thaliana and barley, and the identification of the fractionated proteins by the use of
MALDI-TOF-MS as many as possible is part of a research effort, large-scale automated plant proteomics.
2-DE with immobilized pH gradients (IPGs) combined with protein identification by mass spectrometry (MS) is currently the workhorse for proteomics. With the aim of increasing the resolution of plant tissue proteins in 2-DE by improving the protein extraction procedure, Giavalisco reported a protocol comprising sequences of steps for tissue desintegration, protein solubilization, and removal of insoluble material by ultracentrifugation, leading to three different fractions [16] .
Visualization of the separated proteins is achieved by different staining techniques. Colour density and size of the detected spots enable protein quantification.
Coomassie Brilliant Blue (CBB) and silver staining are usual methods. The accuracy of these methods, however, is limited due to the low dynamic range of most staining techniques. The recent development of fluorescent dyes for proteins may overcome this limit [17] . The task of detecting true changes in protein expression has been greatly simplified by the introduction of Difference Gel Electrophoresis by
Ünlü [18] . Difference gel electrophoresis (DIGE) is a prelabelling technique using separate Cy dyes for different samples which then can be analyzed on one gel, avoiding that shifts in gel patterns normally occur when samples conventionally separated on two gels are compared [19] .
2-DE is tedious to perform and has difficulty dealing with hydrophobic and basic proteins [20] . Poor reproducibility and limited protein spots number also are the disadvantage of 2-DE. Identification of the large numbers of proteins separated by 2-DE is the most commonly achieved by automated matrix-assisted laser desorption/ionization time-of-flight mass spectrometric
(MALDI TOF-MS) peptide mapping followed by extensive database searches [21] .
A recently developed methodology for the characterization of complex proteomes, top-down
Fourier transform mass spectrometry (FTMS), is applied for the first time to Arabidopsis proteome, of the 3000 proteins predicted by the genome sequence, 97 were recently identified in two separate bottom-up mass spectrometry studies [22] . Capillary electrophoresis (CE) mass spectrometry (MS), with its ability to separate compounds which are present in extremely small volume samples rapidly, with high separation efficiency, and with compound identification capability based on molecular weight, is an extremely valuable analytical technique for the analysis of complex proteins. Several
CE-MS interfacing techniques have recently been introduced which could potentially be capable of replacing or complimenting certain 2-DE gel
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The Journal of American Science, 2(2), 2006, Xi, et al, Developments of Arabidopsis thaliana Proteomics Research techniques [23] .
Biochemical studies of protein activity have traditionally focused on the analyses of single molecular species. The rapid pace of the discovery of new gene products by large-scale genomic and proteomic initiatives has required the development of high-throughput strategies to elucidate their functions [24] . Due to limitations of 2D-gel separation technology, increasing attention is being focused on a second approach, the development of protein microarrays as an alternative and complementary approach [25] .
( ⅲ ) Image analysis, database searching, and bioinformatics
2-DE gel images can be used for identifying and characterising many forms of a particular protein encoded by a single gene. 2-DE gel image analysis is very important in the field of proteomics. In order to carry out gel image analysis, one first needs to accurately detect and measure the protein spots in a gel image. In order to analyze the function of a number of proteins, it is important to develop the software for proteome analysis. A number of software packages for
2-DE pattern image analysis is available including the most widely used Image Master 2D Platinum 5.0
(Amersham Pharmacia Biotech, Uppsala, Sweden),
PDQuest Version 7.3.1 (Bio-Rad Laboratories, Hercules,
CA, USA), Phoretix2D (Nonlinear Company, UK). A standardized image analysis technique (and software) will be greatly helpful in the 2-DE gel images for easy and accurate comparison of A. thaliana proteins worldwide. However, there is no sophisticated software which, for example, can predict the function of proteins from the data of amino acid sequence, post translational modification, protein-protein interaction and higher order structure.
For amino acid sequence homology, the next important step leading to a good 2-DE separation, to image analysis and protein identification following
MS/MS analysis comparisons, a wide variety of nonredundant protein and translated nucleic acid databases are available to A. thaliana researchers. To identify proteins in sequence databases by the use of mass spectrometric peptide maps, the determined peptide molecular masses are compared with expected values computed from the database entries according to the enzyme’s cleavage specificity.
Since the development of proteomics, enormous information on proteome analysis has been produced.
However, some organizations have been making great efforts to design protein databases. Two dedicated plant proteome databases have already been introduced [26] .
The second database, computing data already published on the Arabidopsis plasma membrane proteome [27] , offers extended bioinformatic retrieval possibilities [28] .
Such developments are likely to play a crucial role in the exploitation of the enormous amount of data that is starting to be produced by functional proteomics programmes. The following database contain satisfactory information for Arabidopsis plant: The
Arabidopsis Information Resource: http://www.Arabidopsis.org/aboutarabidopsis.html
,
SWISS-PROT, TIGR Arabidopsis thaliana Database.
For protein identification by peptide mass fingerprinting, SWISS-PROT and NCBI protein databases can be accessed by using the search engines
Pro-Found ( http://www.proteometrics.com
), and
ProteinProbe (TIGR Arabidopsis Gene Index) databases. Bioinformatics is another essential tool that links the Arabidopsis proteome to its genome.
SWISS-2D-PAGE (http://www.expasy.ch/ch2d/ ) is an annotated two-dimensional polyacrylamide gel electrophoresis (2-DE) database established in 1993. Some functions to access the data have been provided through the ExPASy proteomics server [29] . This database contains data from a variety of human and mouse biological samples as well as from A. thaliana .
TrSDB - TranScout Database -
(http://ibb.uab.es/trsdb) is a proteome database of eukaryotic transcription factors based upon predicted motifs by TranScout and data sources such as InterPro and Gene Ontology Annotation. Nine eukaryotic proteomes (including Arabidopsis thaliana) are included in the current version [30] .
2. Proteome analysis of Arabidopsis thaliana organs and tissues
Protein expression varies depending on particular species, variety, growth stage, organ and tissue in particular environment. The expression profile is closely related to the function of proteins. When proteins are extracted from the tissues and organs under different conditions and are compared, it is called protein differential display analysis. With this analysis, the interspecies and varietal differences of plant proteins have been studied.
A study of the A. thaliana root proteome based on
2-DE and peptide mass fingerprinting for protein separation investigate the natural variation in the proteome among 8 Arabidopsis thaliana ecotypes, which displayed the biodiversity between ecotypes of a single plant species [31] . A survey of the proteome complement of total proteins extracted from developmental mutants of A. thaliana (L.) Heyhn. and from wild-type plants cultivated in the presence of various hormones were analyzed by 2-DE. Based on computer analysis of 2-D gels followed by a statistical treatment of data allowed us to build a phenogram that describes the biochemical distances between the different genotypes [32] . Analysis of the comparative proteome about normal and K + nutrient deficiencies for
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The Journal of American Science, 2(2), 2006, Xi, et al, Developments of Arabidopsis thaliana Proteomics Research proteins isolated from Arabidopsis seedlings was performed [33] also.
A proteome approach was used to compared the protein patterns of the Arabidopsis ecotypes Col-0 and
Ws-2 based on 2-DE. A pair of protein spots were found to be diagnostic for each of the lines. Both pairs of spots were identified as closely related germin-like proteins differing in only one amino acid by using peptide mass finger printing of tryptic digests and by gaining additional data from post-source decay spectra in the MALDI-TOF analysis. Western blot analysis and mass spectrometrical identification of the corresponding weakly stained protein in Coomassie blue-stained gels of the ecotype Col-0 also demonstrated for the first time the occurrence of
AtGER3 protein in root extracts. The results demonstrate the capacity of proteome analysis to distinguish closely related members of large protein families [34] .
Studies of the developmental processes occurring during seed germination were performed in Arabidopsis
[35] . Two-dimensional gel electrophoresis was used to resolve and analyze seed proteins and the changes during germination. 2-DE revealed about 1300 proteins in seeds, of which 74 showed different abundance during germination. In a subsequent paper, the role of gibberellins (GAs) during seed germination was investigated by comparing protein patterns of a
GA-deficient line, wildtype controls, and wild-type seeds treated with an inhibitor of GA biosynthesis during the germination of GAs [36] .
The identification of the most abundant
Arabidopsis pollen is essential to understand the role of the initiating pollination. Jacob indicated that the pollen extracellular matrix contains proteins mediating species specificity and components needed for efficient pollination. Peptide sequencing revealed the identity of all the detectable coat proteins with mobility exceeding
10 kD; protein identity was verified using mass spectroscopy in some cases. The depletion of these proteins in pollen coat mutants indicated they are extracellular [37] .
Georg et al. have taken a proteomics approach to identify GAPs from Arabidopsis Columbia callus cell.
Two-dimensional fluorescence difference gel electrophoresis and one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated the existence of a large number of phospholipase C-sensitive Arabidopsis proteins. The results provide strong support for the predictions of our previous bioinformatic analysis [38] .
3. Subcellular proteome analysis in Arabidopsis thaliana
Information of specific organelle proteome analysis will provide new insights into pathway compartmentalisation and the localization of proteins.
Proteomics is a powerful tool to reveal the protein complement of subcellular organelles and to obtain new insights into intracellular protein sorting and biochemical pathways. Progress has been made for the proteome analysis of plant mitochondria, peroxisomes [39] , and chloroplasts [40] .
( ⅰ
) Mitochondria
Mitochondria perform a variety of biochemical functions within the eukaryotic cell. Their primary roles are the oxidation of organic acids via the tricarboxylic acid cycle and the synthesis of ATP coupled to the transfer of electrons from reduced NAD + to oxygen via the electron transport chain. However, in plants, mitochondria perform many important secondary functions, such as the synthesis of nucleotides, amino acids, lipids, and vitamins [41] . The Arabidopsis mitochondrial proteome was analyzed by 2-DE and 650 different proteins were separated on single gels.
Fifty-two protein spots were identified by immunoblotting, direct protein sequencing, and mass spectrometry. Over 50 proteins were characterized, including 20% of unidentified proteins not previously described in plant mitochondria [42] .
Making use of blue-native PAGE to fractionate protein complexes of mitochondria in their native state, a total of 40 protein spots were reproducibly resolved, and 29 were identified by means of mass spectrometry, thus giving a map of the most abundant complexes in plant mitochondria [43] . The identification of divalent metal cation binding proteins in plant mitochondria is important to study in proteomics. Divalent metal binding proteins in the Arabidopsis mitochondrial proteome were analyzed by mobility shifts in the presence of divalent cations during two-dimensional diagonal SDS-PAGE. A series of proteins known to be divalent cation-binding proteins, or to catalyse divalent cation-dependent reactions, were identified. These included succinyl CoA ligase beta subunit,
Mn-superoxide dismutase (SOD), an Fe-S centred component of complex I and the REISKE iron-sulphur protein of the b/c (1) complex [44] .
A novel insight into Arabidopsis mitochondrial function was revealed from a large experimental proteome derived by liquid chromatography – tandem mass spectrometry. Within the experimental set of 416 identified proteins, a significant number of low-abundance proteins involved in DNA synthesis, transcriptional regulation, protein complex assembly, and cellular signaling were discovered [45] . These analysis results revealed new metabolic, regulation, and signaling pathways in plant mitochondria, providing a key data set for their future experimental analysis of mitochondrial biogenesis, regulation, and function in plants [46, 47] .
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The Journal of American Science, 2(2), 2006, Xi, et al, Developments of Arabidopsis thaliana Proteomics Research
( ⅱ ) Chloroplast
Chloroplasts are typical plant cell organelles that develop and differentiate from proplastids in a tissue specific and signal dependent manner. The chloroplast proteome is expected to contain 2000-3000 proteins.
From the view of biochemistry, the chloroplast can be divided into several compartments, with each compartment having their own specific subset of proteins or subproteome.
Proteomes of the inner envelope membrane, the thylakoid membrane, and the thylakoid lumen of chloroplasts from Arabidopsis were assembled based on published, well-documented localizations. These proteomes were evaluated for distribution of physical-chemical parameters, with the goal of extracting parameters for improved subcellular prediction and subsequent identification of additional components of each membrane system [48] . Bong-Kwan discussed the response of Arabidopsis chloroplast proteins to high light stress using 2-DE and matrix assisted laser desorption/ ionizationtime of flight-mass spectrometry (MALDI-TOF-MS). 64 protein spots were identified as candidate factors which responded to high light stress [49] . These proteins were then selected for analysis and 52 of these were successfully identified using MALDI-TOF-MS analysis. 35 of the 52 identified proteins were found to decrease their expression levels during high light stress and furthermore 14 of the candidate proteins had unregulated expression levels under these conditions.
Most of the proteins that were downregulated during high light stress are involved in photosynthesis pathways. However, many of the 14 unregulated proteins were identified as previously well-known high light stress-related proteins, such as heat shock proteins
(HSPs), dehydroascorbate reductase (DHAR), and superoxide dismutase (SOD).
By tandem mass spectrometry, Torsten identified
690 different proteins from purified Arabidopsis chloroplasts [50] . Most proteins could be assigned to known protein complexes and metabolic pathways, but more than 30% of the proteins have unknown functions, and many are not predicted to localize in the chloroplast.
Novel structure and function prediction methods provided more informative annotations for proteins of unknown functions.
( ⅲ ) Nucleus
The nucleus is an important subcellular organelle that contains genetic information, and is essential for gene expression and regulation. One of the principal features of eukaryotic organisms is the presence of the nucleus, the subcellular compartment containing the genetic materials. The architecture of the nucleus is thought to be composed of two mutually interrelated structures, both of which contain nucleic acids, chromatin and a nuclear matrix [51] . A group of nucleolar proteins engaged in other functions have been identified.
They include nucleolin, a ubiquitous MAR-binding nucleolar protein [52] . In thereinafter study, the comprehensive characterization of the Arabidopsis nuclear proteome was discussed. The nuclear proteins were identified and the changes of the nuclear proteome were evaluated in response to cold treatment. Of 54 proteins were either up- or downregulated by cold treatment. Six of these proteins were further identified.
Gene expression of these six proteins was also affected by cold stress, as revealed by Northern analyses.
Subcellular localization was examined by transformation and expression of the corresponding genes in BY-2 protoplasts. Two proteins were detected both in the cytoplasm and in the nucleus, while the other four proteins were exclusively observed in the nucleus [53] . This report presents the systematic analysis of the nuclear proteome in plants, where it has been possible to be functionally classified. The nuclear proteome is dynamic, changing its composition in response to intracellular and environmental stimuli.
This current analysis provides an insight into the complexity of the proteins in the nucleus.
The eukaryotic nucleus has been proposed to be organized by two interdependent nucleoprotein structures, the DNA-based chromatin and the
RNA-dependent nuclear matrix. The functional composition and molecular organization of the second component have not yet been resolved. Tomasz et al., described the isolation of the nuclear matrix from the model plant Arabidopsis , its initial characterization by confocal and electron microscopy, and the identification of 36 proteins by mass spectrometry [54] .
Electron microscopy of resinless samples confirmed a structure very similar to that described for the animal nuclear matrix. Two-dimensional gel electrophoresis resolved approximately 300 protein spots. Proteins were identified in batches by ESI tandem mass spectrometry. Among the identified proteins, there were a number of demonstrated or predicted Arabidopsis homologs of nucleolar proteins such as IMP4, Nop56,
Nop58, fibrillarins, nucleolin, as well as ribosomal components and a putative histone deacetylase. Others included homologs of eEF-1, HSP/HSC70, and DnaJ.
The analysis of proteins identified in the nuclear matrix fraction reveals a high enrichment of proteins associated with the nucleolus.
With the advent of the completely sequenced
Arabidopsis genome, numerous studies have characterized large-scale protein expression under various conditions as well as in specific organelles [55] .
Furthermore, many proteins encoded by the
Arabidopsis genome have no assigned or even putative functions [56] . Although much is known about the
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The Journal of American Science, 2(2), 2006, Xi, et al, Developments of Arabidopsis thaliana Proteomics Research general functions of the plant vacuole, a detailed knowledge of proteins targeted to the vacuole and their underlying molecular functions is lacking. Yet, this is an essential step for understanding the biology of this organelle. The diverse functions of the vacuole suggest that a large array of proteins is required to conduct all of these processes.
4. Prospect of proteomics
Many databases were built based on the 2-DE, and entered the Internet. The traditional 2-DE will be substituted by LE-MS-MS and HPLC MS/MS, which can carry out full automatization of sample, separation, analysis. Protein chip technique will make the proteome become possible for simpleness, economy and large-scale [57] . Another level of complexity arises when considering the potential number of protein-protein interactions in an organism modified by developmental events and physiological constraints.
The overall progress in Arabidopsis proteomics is encouraging, and it is becoming an active field with a large impact on plant biology. In particular, as
Arabidopsis is an excellent model plant and is very important for agricultural crop’s research. There are a number of issues that need to be addressed to improve and develop Arabidopsis proteomics to its full potential.
In our view, proteomics will be most useful when combined with other functional genomics tools and approaches. A combination of microarray and proteomics analysis will indicate whether gene regulation is controlled at the level of transcription, or translation and protein accumulation. Protein function can be further studied by a combination of reverse and forward genetics and proteomics [58] . About 40% of the predicted proteins in the Arabidopsis genome have no assigned function. Although reverse genetics will help to determine such functions, redundancy, lethality, and strong phenotypes can often prevent obtaining any insight in gene function. Most proteins have a transient or stable interaction with other proteins and the determination of these interacting proteins often can help to obtain more insight in gene function. Epitope tagging of transgenes, followed by affinity purification, will be very useful to identify protein complexes. For more high-throughput analysis, multidimensional purification schemes can possible be used for rapid identification of protein-protein interactions.
Acknowledgements
This work was supported by the National Natural
Science Foundation of China (Grant No.
30470159/C01020304).
Correspondence to:
Dongyun Hao
Key Laboratory for Molecular Enzymology &
Engineering of the Ministry of Education
(Jilin University)
Changchun, Jilin 130021, China
Email: hao@jlu.edu.cn
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