Comparative Proteomic Analysis Provides New Insights into the Fiber

Comparative Proteomic Analysis Provides New Insights into the Fiber Elongating Process in Cotton Yi-Wei Yang,† Shao-Min Bian,† Yuan Yao, and Jin-Yuan Liu* Laboratory of Molecular Biology and MOE Laboratory of Protein Science, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, P. R. China Received December 22, 2007

A comparative proteomic analysis was performed to explore the mechanism of cell elongation in developing cotton fibers. The temporal changes of global proteomes at five representative development stages (5-25 days post-anthesis [dpa]) were examined using 2-D electrophoresis. Among ∼1800 stained protein spots reproducibly detected on each gel, 235 spots were differentially expressed with significant dynamics in elongating fibers. Of these, 120 spots showed a more than 2-fold change in at least one stage point, and 21 spots appeared to be specific to developmental stages. Furthermore, 106 differentially expressed proteins were identified from mass spectrometry to match 66 unique protein species. These proteins involve different cellular and metabolic processes with obvious functional tendencies toward energy/carbohydrate metabolism, protein turnover, cytoskeleton dynamics, cellular responses and redox homeostasis, indicating a good correlation between development-dependent proteins and fiber biochemical processes, as well as morphogenesis. Newly identified proteins such as phospholipase D alpha, vf14-3-3 protein, small ras-related protein, and GDP dissociation inhibitor will advance our knowledge of the complicated regulatory network. Identification of these proteins, combined with their changes in abundance, provides a global view of the development-dependent protein changes in cotton fibers, and offers a framework for further functional research of target proteins associated with fiber development. Keywords: Cotton • Differentially expressed proteins • Dynamic changes • Fiber cell elongation • Proteomic analysis • Network

Introduction Lint fibers of cotton (Ghirsutum L. and other Gossypium species) are the most prevalent natural fibers used in the textile industry, and their economic impact is estimated to be approximately $500 billion per year worldwide.1 In addition to their economic importance, cotton fibers provide an excellent single-celled model for the study of many basic biological processes in plants. Each cotton fiber is a single and phenomenally elongated cell that originates from the epidermal layer of the ovule.1,2 These single fiber cells, developing nearsynchronously, are dramatically elongated after anthesis and typically reach up to 3.0 cm in length in most commercially important species, being 1000-3000 times greater in length than in diameter. Thus, cotton fibers are both the fastest growing and the longest plant cells in the plant kingdom.2,3 Meanwhile, the primary wall elongation (within 20 days postanthesis [dpa]) and secondary wall thickening (from 20-35 dpa) require the synthesis of large amounts of polysaccharide components, finally producing a thick cell wall consisting of more than 94% cellulose.3,4 It is therefore of great importance * Corresponding author: Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, P. R. China. Fax: +86-1062772243. E-mail: [email protected]. † These authors contributed equally to this work and share the first authorship. 10.1021/pr800550q CCC: $40.75

 2008 American Chemical Society

to characterize the fiber transcriptome or proteome, as well as to determine the number and species of genes or proteins required to make a fiber. It is well-established that various fiber-specific and fiberenriched genes are involved in the different stages of cotton fiber development, such as sucrose synthase,5 cellulose synthase,6 β-galactosidase,7 endo-1,4-β-glucanase and expansin,8 phosphoenopyruvate carboxylase,9 glucuronosyltranferase,10 reversibly glycosylated polypeptide,11 R-tublin,12 β-tublin,13 actin, 14 and MYB protein.15 However, the relatively few fiber genes characterized to date provide only a snapshot view of the molecular mechanisms underlying fiber development. Recent transcriptomic analyses using microarrays have also led to the identification of many candidate genes involved in fiber cell initiation and elongation.2,16-18 Arpat et al. were the first to adopt a transcriptome approach to fiber development in order to investigate the increasing numbers of cotton expressed tags (ESTs) from elongating fibers of a cultivated diploid cotton species (Gossypium arboretum L.). They revealed that approximately 14 000 unique genes were expressed in rapidly elongating fiber cells, which represents as much as 35-40% of the whole genome, and identified 2553 elongation stagespecific genes with down-regulation in 24 dpa fibers undergoing secondary cell wall thickening and 81 secondary cell wall thickening stage-specific genes that were up-regulated relative Journal of Proteome Research 2008, 7, 4623–4637 4623 Published on Web 09/30/2008

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to 10 dpa fibers. This work provides the first in-depth view of the genetic complexity of the transcriptome of an elongation cell and interesting targets for characterization and functional verification. Wu et al. used mRNA from early stage fertilized ovules of wild-type and six reduced fiber or fiberless mutants of the allotetraploid cotton species (Gosypium hirsutum L.) to probe a cotton cDNA microarray containing about 10 000 ovule cDNA clones and identified 13 candidate genes, including two types of transcription factors, a cell cycle gene and other genes with known or presumed roles in cell growth.16 Shi et al. reported that the RNA samples from wild-type ovules (G. hirsutum L. cv Xuzhou 142) and its fiberless fl mutant were employed to probe a cotton cDNA microarray containing 12 233 unique ESTs from fast elongation fiber cells and found that ethylene biosynthesis is one of the most significantly upregulated biochemical pathways during fiber elongation.17 A recent study also indicated that there is preferential accumulation of genes encoding putative transcription factors such as MYB and WRKY and genes encoding predicted proteins involved in auxin, brassinosteroid (BR), gibberellic acid (GA), abscisic acid (ABA) and ethylene signal pathways during early stages of fiber development.18 However, although DNA microarray analysis is useful for examining the physiological state of fiber cells, mRNA expression patterns may not precisely recapitulate the biological system because mRNA expression levels are not necessarily consistent with the activities of vitally important protein. Therefore, transcriptome analysis is often combined with proteome analysis since mRNA levels combined with protein abundance and activity data are thought to faithfully represent biological systems.19 Proteomics is the study of protein properties on a large scale to obtain a global, integrated view of cellular processes and networks at the protein level. Since most of the functional targets are proteins that translate genome sequence information into cellular functions, a study to efficiently explore the genome at the protein level is of great value. Nevertheless, there have been no reports to investigate proteome profiles during cotton fiber elongation to date. Weighing the advantages and disadvantages of currently available methods, we have embarked on a two-dimensional gel electrophoresis-based approach for comparative proteomic analysis of cell elongation in developing cotton fibers. In our previous work, an efficient proteomic workflow has been established for developing cotton fibers by two-dimensional gel electrophoresis and mass identification (2-DE/MS).3 Recently, Li et al. identified an ascorbate peroxidase (APX) by proteomic analysis of cotton ovules between G. hirsutum L cv. Xuzhou 142 and its fiberless mutant, and this enzyme is involved in hydrogen peroxide homeostasis during cell development.20 In this study, we analyzed the proteome profiles of cotton fibers during the elongation stage (5-25 dpa). A wide range of proteins relevant to cotton fiber elongation were separated by 2-DE. A total of 106 proteins, which showed significantly dynamic changes, were identified by a combination of mass/tandem mass spectrometry technology and protein/EST database searching. Identification of these proteins combined with the change of the protein abundance and the impact of the development on the physiological and morphological events occurring during cotton fiber elongation revealed the linkages between the changes in specific protein abundance and the overall response to fiber elongation, gives a global view of the proteome changes under fiber elongation process, and shows possibly the first reported protein network accompanying fiber elongation. The data presented in this 4624

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Yang et al. study also provide a framework for further functional studies of each member of this network in cotton fiber development.

Materials and Methods Chemicals. All chemicals used were of the highest obtainable grade and are outlined in our previous report3 unless otherwise stated. Deionized water (Millipore, Bedford, MA) with resistance greater than 18 MΩ cm was used throughout. Plant Materials, Growth, and Sampling. Cotton (G. hirsutum) cultivar CRI 35 was grown in a normal agronomic field during the period from May to August. The seeds were a gift from the Cotton Research Institute, Chinese Academy of Agricultural Sciences. The ages of fibers selected for total protein extraction were 5, 10, 15, 20, and 25 dpa. All 5 stages of fibers were used with an additional three stages (30, 35, and 40 dpa) to quantify the amount of total proteins in unit fibers. All of the collected fibers were frozen in liquid nitrogen and then stored at -80 °C until use. Preparation of Cotton Fiber Proteins. Protein extraction was performed according to Yao et al.3 About 2 g of frozen cotton fiber was ground with 10% PVPP (w/w) and 10% quartz sand (w/w) in liquid nitrogen using a mortal and pestle. Twenty milliliters of ice-cold acetone (adding 2% β-mercaptoethanol) was added to the powder to wash away impurities, and this step was repeated twice. The freeze-dried powder was homogenized in 5 mL of extraction buffer containing 50 mM TrisHCl, pH 8.6, 2% SDS, 2% (w/w) β-mercaptoethanol, 1 mM PMSF, and then an equal volume of Tris saturated phenol (pH 8.0) was added. The mixture was vortexed thoroughly for 5 min, and the phenol phase was collected and precipitated with 5 vol of 0.1 M ammonium acetate in methanol at -20 °C for 30 min. After centrifuging at 12 000g for 15 min, the collected protein pellets were washed three times with cold 0.1 M ammonium acetate in methanol, and then washed three times with cold 80% acetone in water. The lyophilized pellets were dissolved in rehydration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 1% IPG buffer, 40 mM DTT) and stored at -80 °C until use. Prior to 2-DE, the fiber proteins were diluted with the rehydration buffer, and centrifuged at 12 000g for 15 min to remove insoluble materials. The concentration of extracted proteins was quantified according to the method of Bradford.21 Albumin Bovine V (BSA) (Biotechnology Grade, Amresco, Solon, OH) was used as a standard in protein quantification. Protein contents in cotton fibers were measured by a ninhydrin-based assay described by Starcher.22 Samples (5 mg) of extracted proteins were hydrolyzed in 500 mL of 6 N HCl at 100 °C for 24 h, and then were dried in vacuum. The residues were redissolved in a known volume of water, vortexed, and then centrifuged. Ninhydrin reagent (100 mL)22 was added to 1-10 mg of protein hydrolysate in an Eppendorf tube. The reaction was carried out in a boiling water bath for 10 min. The absorption was then measured at 575 nm. The protein yield was defined as the protein quantity extracted from 1 g of sample (frozen weight). 2-DE and Spot Quantification. 2-DE was performed essentially as reported previously.3 One milligram of proteins in 450 µL was loaded onto a nonlinear IPG strip (pH 3-10, 24 cm, GE Healthcare, Buckinghamshire, U.K.), followed with IEF as 100 V for 40 min, 500 V for 40 min, 1000 V for 1 h, 4000 V for 2 h, and 8000 V for 8 h until total voltage hours of 75 000 was achieved. Before SDS-PAGE analysis, strips were incubated for 2 × 15 min in equilibration buffer (6 M urea, 50 mM pH 8.8 Tris-HCl, 30% (v/v) glycerol, 2% (w/v) SDS, a trace of

Proteomics Study of Elongating Cotton Fibers bromophenol blue). One percent DTT (w/v) was added to the above for the first 15 min and 2.5% iodoacetamide (w/v) instead for the second 15 min. After equilibration, strips were placed on top of a vertical 12.5% SDS-polyacrylamide self-cast gel prepared according to Laemmli,23 and electrophoresis was performed at 6 °C and 5 W/gel for 45 min, and then 17 W/gel for 5 h until the dye front reached the bottom of gels. After electrophoresis, 2-D gels were stained by Coomassie R-250.24 The 2-D gels were scanned at 600 dpi resolution using a UMAX PowerLook 2100XL scanner (Willich, Germany) and image analysis was performed with ImageMaster Platinum software version 5.0 (GE Healthcare). At least triplicates were applied to each sample, and a total of 16 CBB-stained 2-D gel images were obtained. The spots were quantified using the % volume criterion. The match analysis was done in automatic mode, and further manual editing was performed to correct the mismatched and unmatched spots. The relative volume of each spot was assumed to represent its expression level. A significant difference was defined by the criterion p < 0.05 when analyzing parallel spots between groups with one-way ANOVA and Student-NewmanKeuls test using the SAS software package version 8.2 (SAS Institute).25 Protein Identification and Database Search. Protein spots showing significant differences in abundance during fiber elongation were selected and excised manually for protein identification. In-gel digestion of protein spots was performed according to Yao et al.3 The digested protein sample for peptide map fingerprinting (PMF) was mixed with 5 µL R-cyano-4hydroxycinnamic acid (CHCA) matrix, and analyzed on the Ultraflex MALDI-TOF-TOF mass spectrometer with Flexcontrol 2.2 software (Bruker). The obtained spectrum was analyzed with the FlexAnalysis 2.2 (Bruker) and Biotool 2.2 (Bruker) software. The standard peptide mixtures (angiotensin II, angiotensin I, substance P, bombesin, ACTH clip 1-17, ACTH clip 18-39, and somatostatin 28) were used for external mass calibration, while self-degraded fragments of trypsin were used for internal calibration. With the use of Sophisticated Numerical Annotation Procedure (SNAP) algorithm, those peaks with S/N > 3 were picked out for PMF at resolution of 8000. For each MS/MS spectrum, after being smoothed and subtracted, no more than 250 peaks with S/N > 2 were picked with SNAP algorithm. Matrix peaks (833.090, 855.072, 871.046, 907.994, 1060.088, 1082.070 and 1098.044) and porcine trypsin autolysis peaks (842.510, 870.541, 1045.564, 2211.105, 2225.120, 2283.181 and 3337.758) were excluded as contaminant. The mass spectrum data were submitted to an online database (MASCOT version 2.1, http://www.matrixscience.com/) for protein identification. The searching parameters were set as follows: NCBInr database (051121, including 3 044 223 sequences; 1 047 289 308 residues); enzyme-trypsin (cleavage at the C-term side of Lys and Arg unless the next residue was Pro); taxonomy on viridiplantae (217 962 sequences); fixed modifications-carbamidomethyl (C); variable modifications-oxidation (M); no restrictions on protein mass; allow up to 1 missed cleavage. The MS tolerance was set as (100 ppm and MS/MS (0.7 Da. For PMF, only those proteins with score >69 (p < 0.05) were accepted as identified, while for MS/MS individual ion cutoff score was set as 35 (p < 0.05). As to those peptides matched to multiple members of a protein family in different species, the one with the highest score was reported, unless its species was cotton, when the one from cotton was reported. All identified proteins from other species were further checked in cotton EST by submitting the

research articles peptide sequences from MASCOT to an online database (WUBLAST version 2.0) (http://compbio.dfci.harvard.edu/tgi/cgibin/tgi/Blast/index.cgi)againstcottonESTdatabase(GUDB.cotton; including 40 348 sequences; 31 581 061 total letters). The accepted ones should cover at least 10 peptides of the PMF, each of which can be matched with one of the theoretical peptides resulting from the best matched EST sequence, with a mass error of (0.2 Da. Only those proteins with theoretical mass and isoelectric points approximately equal to the experimentally obtained values were accepted as successfully identified. All the theoretical molecular weight and pI of identified proteins were calculated using the PeptideMass program (http://au.expasy.org/tools/peptide-mass.html). Semiquantitative RT-PCR. Total RNA was prepared from 5, 10, 15, 20, 25 dpa fibers using a Purescript RNA Purification kit (Genetra Systems, Minneapolis, MN). First-strand cDNA was synthesized from 1 µg of total RNA using the TaKaRa RNA PCR kit (AMV) for RT-PCR (TaKaRa, Dalian, China). One-fifteenth of the first-strand cDNA was used as templates in 50 µL PCR reactions. Gene-specific PCR primers were designed according to the cDNA sequences and synthesized commercially (Sangon, Shanghai, China). Primers Pf1 (5′-AAGAGTGTCCGTTGGTCAC3′) and Pr1 (5′-ACGTGCTTGGGATACGTGTC-3′) were used to detect the transcript for acyltransferase-like protein (AAL67994), Pf2 (5′-AGATGGCCGATACTGATGATATT-3′) and Pr2 (5′-GCTGGAAGAGGACTTCTGGACAAC-3′) for actin 8 (AAP73455), Pf3 (5′ATGGATCCTTTCCCACAAATCCTC-3′) and Pr3 (5′-TCCTAGCTTCCTCAACAGCATCTCT-3′) for late embryogenesis-like protein (AAC24588), and Pf4 (5′-GAGACTGTCCATTCTCCCAGA-3′) and Pr4 (5′-TGTTCATGCATTCACCTTAGGTG-3′) for dehydroascorbate reductase (AAL71857). Parallel reactions using cotton ubiquitin UBQ7 (DQ116441) primers Pf5 (5′-TCTTTGCGCATCTTCGCC-3′) and Pr5 (5′-ACCAGCCTTCTGGTAAACG-3′) served to normalize the amount of template added. Equal volumes of the PCR products for the same gene were electrophoresed in a 1.5% agarose gel. DNA bands were visualized by staining the gel with ethidium bromide and analyzed by Band Leader 3.0 (Magnitec, Tel Aviv, Israel). At least triplicates were applied to each sample. Enzyme Assays. Enzyme assays were performed essentially as reported previously.26 Frozen cotton fiber was ground in liquid nitrogen using a mortar and pestle. The powder (about 150 mg) was homogenized in 2 mL of 50 mM pH 7.0 potassium phosphate buffer containing 1 mM EDTA and 1% PVPP, with the addition of 1 mM ascorbic acid in the case of the APX assay. After centrifugation at 14 000g for 30 min at 4 °C, the supernatant was directly used for enzyme assays. Protein content was determined according to the method of Bradford21 using BSA as a standard. All spectrophotometric analyses were conducted with an Ultrospec 3300 Spectrophotometer (GE Healthcare). Ascorbate peroxidase (APX) activity was determined by following the consumption of ascorbic acid (extinction coefficient 2.8 mM-1 · cm-1) at 290 nm for 3 min. The reaction solution contained 50 mM potassium phosphate buffer (pH 7.0), 0.1 mM EDTA, 0.5 mM ascorbic acid, 0.1 mM H2O2 and 10 µg of protein extract in a 1 mL volume. Measurement of H2O2. H2O2 was measured according to the method described by Okuda et al.27 Briefly, frozen cotton fiber was ground in liquid nitrogen, and the powder (about 150 mg) was homogenized in 2 mL of 0.2 N HClO4 for 2 min. After centrifugation at 20 000g for 5 min at 4 °C, the supernatant was neutralized to pH 7.5 with 4 N KOH and then the solution was centrifuged at 1000g for 1 min. An aliquot (200 µL) of Journal of Proteome Research • Vol. 7, No. 11, 2008 4625

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Figure 1. Dynamic changes of total protein percentage during cotton fiber development. This graph represents the dynamic changes of total protein percentage from 5 to 40 dpa. The abundance of total protein in each sample was quantified by a ninhydrin-based assay (detailed in Materials and Methods) with BSA as standard.

supernatant was applied to a 1 mL column of DEAE 52, and the column was washed with 1600 µL of distilled water. The eluate was used for the assay of H2O2. The reaction solution contained 200 µL of the eluate, 80 µL of 12.5 mM DMAB in 0.375 M phosphate buffer (pH 6.5), 20 µL of MBTH and 4 µL of peroxidase (0.25 unit) in a total volume of 304 µL. The reaction was started by the addition of peroxidase at 25 °C. The absorbance increase at 590 nm was monitored with an Ultrospec 3300 Spectrophotometer (GE Healthcare).

Results Dynamic Changes in Levels of Total Proteins in Developing Cotton Fibers. The most distinctive feature of the cell wall of the developing cotton fiber is that the secondary wall is almost pure cellulose. However, very little is known about the total protein content of the developing cotton fiber throughout the course of elongation and secondary wall formation. Therefore, it was necessary to measure the total protein content during the developing stages. For this purpose, the total protein content of the developing cotton fibers was measured by a ninhydrin-based assay (detailed in Materials and Methods) and expressed as a percentage of the weight of the frozen dried fiber. As shown in Figure 1, a dynamic change of cotton fiber proteins during development was recorded. The highest content of the total protein among the eight development stages is about 10.79% at 5 dpa, the youngest stage examined, but noticeably declines later in development (Figure 1). The percentage of total protein remains at a level of approximately 3.6% during the period from 15 to 30 dpa, followed by another remarkable decrease to approximately 2% (about 2.05% at 35 dpa and 2.01% at 40 dpa). This result generally coincides with the physiological changes during cotton fiber development: rapidly elongated fibers associate with a sharp increase of fiber mass between 5 and 15 dpa, while cotton fibers begin to enter the maturation stage after 30 dpa, in which the expression of majority genes must be gradually down regulated. Therefore, the first decrease (from 5 to 15 dpa) was mainly caused by the increase of fiber mass, and the second one (after 30 dpa) mostly resulted in the decrease of total protein. This measurement of total protein percentage offers the first overview of dynamic changes of fiber protein abundance. However, further illumination is needed to determine what kinds of proteins are changed during the fiber development process. To investigate the 2-DE profile of the developing fiber proteome, an efficient protein preparation method3 was used to isolate total proteins from developing fiber tissues. The 4626

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Figure 2. 2-DE map of total proteins in 5 dpa cotton fibers (IPG strip pH 3-10 NL, 12.5% SDS-PAGE, blue silver staining). A total of 1624 proteins were detected in the gel. All identified differentially displayed protein spots are shown in the top map; all nonidentified differentially displayed protein spots are shown in the bottom map. The framed regions are enlarged to clearly show the spot numbers.

highest protein yield was at a level of approximately 6 mg/g (6.05 ( 1.17 mg protein/1 g fiber) at 5 dpa, then decreased to approximately 4 mg/g (3.90 ( 0.40 mg protein /1 g fiber) at 10 dpa, and maintained at a level of approximately 2.5 mg/g at 15, 20, and 25 dpa (2.52 ( 0.59 mg protein/1 g fiber, 2.68 ( 0.91 mg protein/1 g fiber, 2.57 ( 0.55 mg protein/1 g fiber, respectively). These different protein yields coincide with the tendency of the dynamic changes of protein abundance, indicating that the decrease of protein yield was caused mainly by the decrease of protein abundance in unit mass of cotton fiber. The Proteome Profiles of Cotton Fibers at Different Elongation Stages. The fiber proteome profiles in the pH range of 3-10 were established for five important stages (5, 10, 15, 20 and 25 dpa). For each sample, at least triplicate gels were performed with three biological replicates. The 2-DE proteome gel maps of developing fibers are shown in Figure S1 of Supporting Information. Approximately 1800 stained spots were reproducibly detected by image analysis on CBB stained gels across the pH range 3-10 (Figure S1 in Supporting Information). The proteome map of an equal mixture of the total proteins from 5 differential stages was designated as our reference map, and other maps were compared to the reference map to screen differentially expressed protein spots. Quantitative image analysis revealed a total of 235 protein spots (the number of each spot is shown in Figure 2) with significantly

Proteomics Study of Elongating Cotton Fibers

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Figure 3. Cluster analysis of expression profiles of development-dependent proteins in elongating cotton fibers. By normalizing the sum of protein-abundance squares of 5 stage points to 1, the differentially expressed 235 proteins during fiber elongation (5-25 dpa) were clustered into 4 types (A) based on their expression abundance (Table S1 in Supporting Information). On the left are their ids, and on the right are the types to which they belong. The 5 columns represent the normalized abundances of 5 stage points (from 5 to 25 dpa), with different colors representing their values. Of these, the changed patterns of 8 representing spots are shown in (B).

changed intensities (p < 0.05), most of which (226 spots) showed more than 1.5-fold change in at least one stage point; 120 of these spots exhibit a greater than 2-fold enhancement (Figures 2, 3A and Table S1 in Supporting Information). Although most spots showed abundance changes, some spots showed stage-dependent changes. Twenty-one spots in response to differential development stages could not be detected in at least one stage point (Figure 3A and Table S1 in Supporting Information), showing they were specific to certain developmental stages. All 235 dynamically changed spots were grouped using clustering, enabling us to rapidly view their relatively changed patterns (Figure 3A). On the basis of quantitative changes of each spot, these spots are clustered into four types (type A, B, C, D), showing the increase and decrease of up- and downregulated proteins during the development process from 5 to 25 dpa. When the sum of protein-abundance squares of 5 stage points were normalized to 1, the accumulation profiles of all differentially changed spots were summarized according to their types (Table S1 in Supporting Information, Figure 3A). Type A includes 39 spots from s001 to s039 (16.6% in total), and is characterized by a similar dynamic change profile showing a gradual increase in abundance of each spot during the development process, such as s021 and s038 (Figure 3B). These two typical spots began to accumulate as early as the initiation of fiber elongation, and maintained at a relatively constant level during the process of fiber elongation, followed by a further accumulation when entering the stage of second cell wall synthesis (Figure 3B). In contrast, the abundance of proteins in type B (72 spots from s164 to s235, 30.6%) shows a gradual decrease, in which s171 and s224 are two typical examples (Figure 3B). Type C, composed of 44 spots (s120 to s163, 18.7%), represents a category with the characteristic of a transitional decrease in abundance during fiber elongation. Two members of this type, s140 and s147 (Figure 3B), function

at a relatively low abundance during fiber elongation, compared to the stages of initiation end and secondary cell wall synthesis. The 80 spots of type D (s040 to s119, 34.1%) show a transitional increase during fiber elongation. Taking s045 and s109 as examples, a vaulted abundance profile could be observed during fiber development (Figure 3B). Taken together, all of the different displayed proteins documented their abundance changes in response to different developing stages (Figure 2, 3, and Supporting Information Figure S1 and Table S1). This suggests that fiber cells were able to respond to different development signals by modulating corresponding protein expression, and also implies that an active proteome quality control system inside the fiber cells plays an important role in the fiber development process. Identification of Differentially Displayed Proteins. A total of 235 spots of the differentially displayed proteins were analyzed by MALDI-TOF/TOF-MS after excision from CCBstained 2-DE gels and digestion with trypsin. In total, 106 proteins were successfully identified, either by PMF or MS/ MS analysis (listed in Table 1 and Table S2 in Supporting Information, supplemental_spectra_msms, supplemental_spectra_PMF). As examples, the results of s093 and s112 are shown in Figures S2 and S3 in Supporting Information, respectively. There are 68 proteins identified by MALDI-TOF (Table 1 and Supplemental_spectra_PMF), while 38 proteins successfully identified by MS/MS analysis (Table S2 and Supplemental_spectra_msms in Supporting Information). For those peptides who matched to several members of a protein family in different species, the one with the highest score was reported if there was no one belongs to cotton; while the one from cotton was reported if there were some belong to cotton. The protein redundancy of each spot was listed in Supporting Information Table S3. Furthermore, for those identified spots whose species were not cotton, their homologues were searched for in the cotton EST database with Journal of Proteome Research • Vol. 7, No. 11, 2008 4627

4628

NCBI accession no.

CAA41115 AAQ18140 AAQ18140 AAQ18140 AAQ18140 P34921 CAA42904 CAD79700 AAM65189 NP_001047348 XP_482675 AAU44104 AAM13090 CAD24779 CAA26620 CAA52636 ACC17840 AAP54723 AAD28641 BAA89049 AAM64651 NP_200446 BAA33802 BAA33802 BAB62078 AAG52124 CAB93681 AAW56877

CAB09900 CAB09900 AAB67607 CAA45104 BAA20878 AAG53647 XP_478927 CAA50218 AAL59960 CAA52149 CAA47345 AAA66365 CAA44816 AAM62830 O24362 O24362

spot ID

117 135 204 233 234 023 024 091 143 144 056 207 208 209 065 201 129 163 171 172 067 198 082 085 002 146 054 130

173 147 205 109 231 229 070 189 050 177 180 183 141 111 150 151

Journal of Proteome Research • Vol. 7, No. 11, 2008 93.8 94.25 46.89 17.53 17.53 17.32 49.09 61.13 76.51 75.41 72.54 63.29 40.21 16.53 27.29 27.29

elongation factor 2 elongation factor 2 eukaryotic translation initiation factor 4A eukaryotic translation initiation factor 5A eukaryotic translation initiation factor 5A eukaryotic translation initiation factor 5A-I eukaryotic peptide chain release factor subunit 1-3 heat shock protein 60 heat shock protein 70 heat shock protein 70 heat shock protein 70 chaperonin precursor endopeptidase Putative ubiquitin-conjugating enzyme proteasome subunit alpha type 3 proteasome subunit alpha type 3

Protein Metabolism Beta vulgaris subsp. Beta vulgaris subsp. Zea mays Nicotiana plumbaginifolia Lycopersicon esculentum Lycopersicon esculentum Oryza sativa Cucurbita cv. Arabidopsis thaliana Cucumis sativus Phaseolus vulgaris Pisum sativum Phaseolus vulgaris Arabidopsis thaliana Spinacia oleracea Spinacia oleracea

Mr

47.8 47.73 47.73 47.73 47.73 36.91 36.91 36.91 36.91 36.91 64.56 50.22 50.22 50.22 58.93 58.93 68.52 55.18 92.76 92.76 54.41 54.41 42.49 42.49 80.11 34.47 63.12 60.82

organism

5.93 5.89 5.57 5.60 5.61 5.71 5.46 6.28 5.07 5.15 5.95 5.85 5.96 6.20 6.11 6.11

5.68 6.16 6.16 6.16 6.16 6.62 6.62 6.62 6.62 6.62 6.50 6.25 6.25 6.25 5.95 5.95 5.36 5.95 6.14 6.14 6.43 6.43 5.84 5.84 6.16 6.41 5.49 5.52

pI

Theoretical

Energy/Carbohydrate Metabolism enolase Solanum lycopersicum enolase Gossypium barbadense enolase Gossypium barbadense enolase Gossypium barbadense enolase Gossypium barbadense glyceraldehyde-3-phosphate dehydrogenase Dianthus caryophyllus glyceraldehyde-3-phosphate dehydrogenase Petunia x hybrida glyceraldehyde-3-phosphate dehydrogenase Oryza sativa glyceraldehyde-3-phosphate dehydrogenase C subunit Arabidopsis thaliana glyceraldehyde-3-phosphate dehydrogenase Oryza sativa putative phosphoglycerate dehydrogenase Oryza sativa isocitrate dehydrogenase Oryza sativa isocitrate dehydrogenase Arabidopsis thaliana isocitrate dehydrogenase Cucumis sativus ATP synthase beta subunit Nicotiana plumbaginifolia ATP synthase beta subunit Triticum aestivum vacuolar ATP synthase catalytic subunit A Gossypium hirsutum ATP synthase alpha subunit Oryza sativa sucrose synthase Gossypium hirsutum sucrose synthase Citrus unshiu pyruvate kinase Arabidopsis thaliana pyruvate kinase Arabidopsis thaliana cytosolic phosphoglycerate kinase 1 Populus nigra cytosolic phosphoglycerate kinase 1 Populus nigra transketolase Polygonum tinctorium GDP-4-keto-6-deoxy-D-mannose-3,5-epimerase-4-reductase Arabidopsis thaliana cytosolicphosphoglucomutase Solanum tuberosum phosphoglycerate mutase Oryza sativa

protein name

Table 1. Differentially Displayed Proteins Identified by PMF or MS/MS

102.8 33.57 49.93 17.60 17.47 17.73 53.33 63.40 81.13 77.73 72.53 62.40 43.33 16.40 30.33 30.33

52.67 53.8 52.00 51.00 52.00 39.40 39.40 39.71 42.33 39.73 63.33 45.80 45.80 45.87 56.33 54.53 69.20 61.86 95.07 93.93 59.80 59.60 42.47 42.07 81.93 35.20 69.60 67.67

Mr

6.03 5.48 5.36 5.49 5.67 5.68 5.67 5.41 4.85 4.75 5.39 5.30 6.31 6.17 5.78 5.95

5.51 5.59 5.68 5.68 5.59 7.18 7.87 7.66 7.68 7.45 5.68 6.53 6.31 6.61 5.39 5.39 5.49 5.37 6.27 6.04 6.20 6.29 5.86 5.76 5.88 6.94 5.83 5.72

pI

Observed

121 102 135 95 82 87 93 90 99 122 69 86 42 80 118 57

55 271 140 104 119 114 114 100 83 92 75 117 116 109 117 92 402 116 153 84 98 132 113 107 79 87 86 78

score

a

26/99 19/44 14/26 3* 6/13 9/79 18/98 14/41 18/98 21/98 2* 8/14 1* 8/21 1* 2*

1* 25/40 17/39 10/21 14/41 10/18 14/35 9/22 7/14 8/18 1* 16/43 16/42 14/40 14/35 13/43 48/98 18/98 27/95 13/32 3* 2* 12/37 9/18 1* 1* 9/18 12/34

M

b

30 15 33 13 37 52 38 25 22 2 5 19 6 64 6 12

3 66 48 35 40 30 37 30 27 22 3 33 33 36 33 30 78 38 34 15 10 6 37 27 2 3 20 15

C

B/2.04 C/7.49 B/2.62 D/4.81 B/2.58 B/2.30 D/1.43 B/2.44 D/2.91 B/4.73 B/1.80 B/4.61 C/6.94 D/2.01 C/1.64 C/2.16

D/3.14 C/1.43 B/2.05 B/3.06 B/2.79 A/1.83 A/3.03 D/1.37 C/5.53 C/2.28 D/2.24 B/1.51 B/1.76 B/1.92 D/1.80 B/3.25 C/1.70 C.3.88 B/3.68 B/2.77 D/2.67 B/2.59 D/10.09 D/2.27 A/2.31 C/3.61 D/1.75 C/2.35

Tc

research articles Yang et al.

NCBI accession no.

CAC43325 AAM61658

CAA58474 AAF26735 AAF74983 CAA42689 P08281 CAA42689 AAK29410 AAK29410 XP_479171 AAG52429

AAB62881 AAP73460 AAP73455 AAP73460 AAP73453 AAB62881 AAR13288 AAB67994 AAC33305 AAC33305 AAB67993 AAC33305 AAL92118 AAL92118 AAL92118

CAA52414 CAA52414 AAY54006 AAY54006 AAY54006 AAY54006 AAB80717 AAB80717 AAB80717 AAA32852 CAB78283 AAC24588 BAB17821 AAP34364

spot ID

105 131

048 123 174 212 213 218 078 080 059 168

075 076 079 083 092 140 093 095 025 027 028 029 008 068 069

112 159 042 043 044 045 136 196 200 155 052 021 153 038

Table 1. Continued

cyclophilin cyclophilin phospholipase D alpha phospholipase D alpha phospholipase D alpha phospholipase D alpha GDP dissociation inhibitor GDP dissociation inhibitor GDP dissociation inhibitor small ras-related protein stress-induced protein sti1-like protein late embryogenesis-like protein Vf14-3-3c protein major latex-like protein

actin actin actin actin actin actin annexin annexin fiber annexin fiber annexin fiber annexin fiber annexin beta-tubulin beta-tubulin beta-tubulin

methionine synthase methionine synthase methionine synthase glutamine synthetase glutamine synthetase glutamine synthetase S-adenosyl-L-methionine synthetase S-adenosyl-L-methionine synthetase alanine aminotransferase putative aminopeptidase

putative beta4 proteasome subunit T-complex protein 1, beta subunit

protein name

Phaseolus vulgaris Phaseolus vulgaris Arachis hypogaea Arachis hypogaea Arachis hypogaea Arachis hypogaea Nicotiana tabacum Nicotiana tabacum Nicotiana tabacum Arabidopsis thaliana Arabidopsis thaliana Prunus armeniaca Vicia faba Gossypium barbadense

Cell Response/Signal

Podocarpus macrophyllus Gossypium hirsutum Gossypium hirsutum Gossypium hirsutum Gossypium hirsutum Podocarpus macrophyllus Gossypium hirsutum Gossypium hirsutum Gossypium hirsutum Gossypium hirsutum Gossypium hirsutum Gossypium hirsutum Gossypium hirsutum Gossypium hirsutum Gossypium hirsutum

Cytoskeleton

Catharanthus roseus Coffea arabica Solanum tuberosum Lactuca sativa Pisum sativum Lactuca sativa Elaeagnus umbellata Elaeagnus umbellata Oryza sativa Arabidopsis thaliana

Amino Acid Metabolism

Nicotiana tabacum Arabidopsis thaliana

organism

18.16 18.16 91.62 91.62 91.62 91.62 49.7 49.7 49.7 22.96 63.71 33.10 28.14 14.62

41.67 41.72 41.73 37.17 41.72 41.67 36.06 35.83 36.00 36.00 36.00 36.00 50.00 50.00 50.00

84.67 24.45 84.67 39.47 41.01 39.47 43.14 43.14 59.51 108.10

14.54 57.29

Mr

8.36 8.36 5.46 5.46 5.46 5.46 5.44 5.44 5.44 6.71 6.00 4.91 4.98 5.87

5.31 5.31 5.31 5.38 5.31 5.31 6.19 6.38 6.34 6.34 6.34 6.34 4.76 4.76 4.76

5.93 5.69 5.93 5.24 5.32 5.24 5.50 5.50 5.95 5.97

6.40 5.59

pI

Theoretical

16.93 17.00 95.60 96.58 95.20 95.93 54.47 58.80 55.29 29.53 78.80 39.00 30.33 16.07

44.40 45.33 45.27 42.67 36.27 42.60 36.07 35.60 36.47 36.20 36.67 36.36 55.93 56.40 55.47

85.53 88.80 86.00 42.93 42.00 35.08 46.87 47.14 59.73 100.50

24.50 61.13

Mr

pI

7.89 7.25 5.56 5.59 5.49 5.53 5.78 5.77 5.70 6.47 6.2 4.57 4.62 5.74

5.37 5.30 5.42 5.50 5.50 5.70 6.10 6.22 6.29 6.35 6.21 6.43 5.11 4.82 5.06

6.02 6.48 6.10 5.66 5.56 5.42 5.54 5.56 5.49 5.69

5.84 5.85

Observed

54 56 77 138 125 148 96 122 100 137 80 93 55 122

141 111 107 108 87 174 321 121 244 195 130 133 223 144 214

81 108 98 82 39 80 121 92 87 78

106 53

score

a

1* 1* 1* 1* 1* 2* 9/20 20/98 13/33 15/88 1* 1* 1* 18/92

12/29 13/48 14/56 18/98 9/29 12/18 35/99 15/98 23/51 18/26 15/57 16/59 23/47 24/98 26/59

11/36 2* 19/98 1* 1* 1* 18/98 13/29 15/58 1*

1* 2*

M

b

8 8 10 10 10 16 30 38 35 56 20 5 17 64

54 44 43 48 36 54 86 58 61 49 50 54 55 42 55

19 14 28 5 4 5 44 39 31 2

11 3

C

D/2.01 C/3.17 D/3.58 D/inf. D/2.83 D/2.23 C/1.98 B/5.41 B/1.63 C/2.54 D/2.47 A/5.02 C/2.41 A/11.76

D/1.68 D/2.51 D/5.31 D/3.33 D/6.33 C/4.48 D/1.37 D/3.69 A/2.06 A/2.64 A/4.45 A/5.03 A/3.14 D/inf. D/5.14

D/2.01 C/2.54 B/1.92 B/1.88 B/3.97 B/3.74 D/4.06 D/3.06 D/2.15 B/2.98

D/inf. C/2.07

Tc

Proteomics Study of Elongating Cotton Fibers

research articles

Journal of Proteome Research • Vol. 7, No. 11, 2008 4629

4630

Journal of Proteome Research • Vol. 7, No. 11, 2008

AAR23816

AAC08576 CAA36380 CAA36380 AAL71857 AAL71857 AAR07601 AAM78225 AAM61756

P68172 AAB41814 AAA33856 CAA81078 CAB10172 CAB10172

AAS00533 AAU14831

AAF23260 P54774 CAA70703

AAL67994

055

102 013 203 156 224 018 103 108

064 197 074 137 012 071

086 211

041 121 062

061

acyltransferase-like protein

Transitional endoplasmic reticulum ATPase cell division cycle protein 48 homologue kap alpha protein

putative adenosine kinase adenosine kinase isoform 1T

adenosylhomocysteinase adenosylhomocysteinase S-adenosylhomocysteine hydrolase hydroxymethyltransferase hydroxymethyltransferase hydroxymethyltransferase

Zantedeschia aethiopica Gossypium hirsutum Gossypium hirsutum Nicotiana tabacum Nicotiana tabacum Gossypium barbadense Gossypium raimondii Arabidopsis thaliana

Redox Homeostasis

Gossypium hirsutum

organism

Gossypium hirsutum

Lipid Metabolism

Arabidopsis thaliana Glycine max Arabidopsis thaliana

Transport

Populus alba Nicotiana tabacum

Nucleotide Metabolism

Nicotiana sylvestris Medicago sativa Petroselinum crispum Flaveria prin Arabidopsis thaliana Arabidopsis thaliana

One-Carbon Compound Metabolism

ascorbate peroxidase catalase isozyme 1 catalase isozyme 1 dehydroascorbate reductase dehydroascorbate reductase fiber quinone-oxidoreductase putative quinone oxidoreductase S-adenosylmethionine: 2-demethylmenaquinone methyltransferase

betaine-aldehyde dehydrogenase

protein name

47.97

90.08 89.77 58.63

9.42 37.42

53.10 53.38 53.18 57.15 51.72 51.72

27.74 56.86 56.86 23.80 23.66 33.99 33.99 17.89

54.72

5.67

5.13 5.18 4.80

6.75 5.13

5.51 5.66 5.60 8.40 6.80 6.80

5.93 6.58 6.58 8.42 7.70 8.35 9.31 5.85

5.60

pI

Theoretical Mr

59.33

104.93 104.33 60.00

41.00 34.79

57.27 56.60 49.78 54.64 55.53 56.20

29.27 55.60 55.73 27.00 26.47 42.29 25.73 17.47

64.27

pI

5.55

5.30 5.34 5.44

5.49 5.42

5.90 5.71 5.85 7.11 7.05 7.61

6.02 6.91 7.18 7.32 6.75 8.71 5.90 5.42

5.48

Observed Mr

103

81 158 75

80 94

155 87 45 83 90 82

92 131 88 74 100 67 79 80

81

score

a

2*

19/31 33/51 1*

2* 2*

25/98 11/38 1* 10/23 8/16 12/36

1* 21//32 12/45 2* 1* 1* 2* 1*

13/27

M

b

7

24 44 2

14 8

42 26 8 19 19 23

8 47 27 17 8 4 38 7

38

C

D/3.26

D/3.15 C/2.40 D/2.11

D/1.98 B/1.50

D/2.28 B/2.59 D/inf. C/4.82 A/2.92 D/1.82

D/2.23 A/2.44 B/4.23 C/inf. B/3.18 A/24.84 D/1.62 D/1.58

D/1.57

Tc

a Number of mass value matched (matched/searched), the number marked by * indicates the peptide fragment(s) sequenced by MS/MS. Detailed amino acid sequence of each fragment is listed in Supporting InformationTable S2. b Sequence coverage. c Amount dynamic changes. The first capital letter indicates the type (See the Figure 3 for details), as A for gradual increase, B for gradual decrease, C for transitional decrease, D for transitional increase. The number followed the letter is the ratio of the maximum amount to the minimum amount among the five developmental stages (See the Table S1 in Supporting Information for details). Inf. Indicates the ratio is infinity.

NCBI accession no.

spot ID

Table 1. Continued

research articles Yang et al.

Proteomics Study of Elongating Cotton Fibers tBLASTn (http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/Blast/ index.cgi) using their peptide sequences resulting from MS/ MS analysis as queries. Finally, the peptide sequence of each spot was found to match to at least one nucleotide sequence in the cotton EST database. However, the observed molecular mass values of nine differentially expressed proteins (spots 021, 042, 043, 044, 045, 086,103,105 and 123) were much higher than the theoretical values of peptides resulting from MS analysis. To further annotate these queried proteins, we compared their homologues in the cotton EST database with their observed values and peptides resulting from MS. These nine corresponding regions from cotton EST homologues show more than 80% identities with the queried proteins at the amino acid level. Interestingly, all nine spots shared almost the same observed molecular mass values as the homologous theoretical proteins in cotton, indicating that they should really be the annotated proteins (listed in Table S4 in Supporting Information). For example, using two peptide sequence tags (SIDGGAAFGFPETPEDAAR, RNYLTFFCLGNR) generated from s045, an Arachis hypogaea homologue of ca. 21 kDa phospholipase D alpha (AAY54006) was found in the MASCOT with 148 MOWSE score. However, its molecular mass value was much smaller than the observed ca. 96 kDa of the spots. The cotton homologue in cotton EST database, phospholipase D (TC32226), shows almost the same molecular mass values with the observed 045 spot, and its corresponding region shares a 91% match with phospholipase D alpha (Table S4 in Supporting Information). Moreover, by protein blast (http://www.ncbi.nlm.nih.gov/ BLAST/) against NCBInr using the peptide sequence of AAY54006, we found a homologue from cotton, phospholipase D alpha (ABN51235), whose corresponding region shares 91% identity with AAY54006. Therefore, we annotate the cotton protein s045 as phospholipase D alpha (Figure S4 in Supporting Information). On the basis of the present study, a combination of MALDITOF and MALDI-TOF/TOF analysis, cross species identification, and cotton EST database utilization might further enhance identification efficiency and accuracy. Among the 106 proteins identified from this study, 28 proteins have previously been reported in cotton. Therefore, this is the first time that the other 78 proteins have been reported to be related to fiber development at the protein level. Although MALDI-TOF and MALDITOF/TOF were successfully executed, 129 spots still could not be identified because no matches were found in the databases. This is an inevitable disadvantage in species whose genomes have been not fully sequenced. However, their unique 2-DE locations and PMFs were annotated to some extent and will be helpful in the identification of these unknown proteins in further studies. Characterization of Development-Dependent Proteins in Cotton Fibers. A total of 106 detected and identified protein sequences were searched against GoFigure (http://www.geneontology.org) for functional classification, and were grouped into 10 major categories including energy/carbohydrate metabolism (28 spots), protein metabolism (18 spots), cytoskeleton (15 spots), cell response/signal (15 spots), redox homeostasis (8 spots), metabolisms of amino acid (10 spots), one-carbon compound (6 spots), nucleotide (2 spots), lipid (1 spot) and transport (3 spots) (Table 1 and Figure S5 in Supporting Information). Analysis of their relative abundance in each category documented that the first four cell process-related proteins were overrepresented among the identified differen-

research articles tially displayed proteins, either in number and/or expression level, suggesting the functional importance of these processes in fiber development (Table S1 in Supporting Information). Of 65 identified proteins responsible for metabolism (Table 1), 22 identities are involved in carbohydrate metabolism such as glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase, transketolase, pyruvate kinase, GDP-4keto-6-deoxy-D-mannose-3,5-epimerase-4-reductaseandisocitrate dehydrogenase, 18 identified proteins are related to synthesis, assembly and degradation of proteins including elongation factor 2, eukaryotic translation initiation factor, peptide chain release factor, chaperonins, proteasome subunits, endopeptidase, aminopeptidase, E2 ubiquitin-conjugating enzyme and T-complex protein, and 12 proteins are linked to synthesis of amino acids and nucleotide (adenosylhomocysteinase, Sadenosylhomocysteine hydrolase, methionine synthase, Sadenosyl-L-methionine synthetase, glutamine synthetase, alanine aminotransferase and adenosine kinase) (Table 1). Interestingly, among 29 identified proteins categorized as involved in protein, amino acid and lipid metabolism, almost all of them display the similar expression responses during fiber development. These protein expressions were found to reach higher volumes during the earlier elongation stages (5, 10, or 15 dpa), whereas their expression levels declined during later stages (20 and 25 dpa), showing attenuation of their expression with onset of secondary wall synthesis (Figure 3, Tables 1 and S1 in Supporting Information). These results indicate that the biochemical processes mentioned above are sensitive to fiber development and are prepared at the appropriate time to provide energy and building blocks for the biosynthesis of fiber cellular components. Our analysis also revealed 15 differentially expressed identities involved in cytoskeletal dynamics and 23 identities related to cell responses and redox homeostasis. All 15 identities, including actin with 5 isoforms, beta-tublin having 2 isoforms and annexin with 5 isoforms, were up-regulated during the fiber development process (Table S1 in Supporting Information). Previous studies have demonstrated that these proteins play a critical role in cell cytoskeleton architectures.14,28,29 Suppression of cotton actin 1 gene (GhACT1) expression dramatically reduces fiber elongation,14 while β-tubulins are well-known to be the major structural component of microtubules and play a key role in cotton fiber morphogenesis.28 Therefore, the cytoskeleton-related proteins detected in this work mirror that the formation of fiber may depend on new synthesis of these proteins. It is also worth noting that 22 proteins with an important role in cell responses and redox homeostasis were identified, including GDP dissociation inhibitor, phospholipase D alpha, vf14-3-3 protein, small ras-related protein, late embryogenesis-like protein, betaine-aldehyde dehydrogenase, ascorbate peroxidase, dehydroascorbate reductase, catalase isozyme 1, quinine oxidoreductase, and menaquinone methyltransferase. It is clear that the functions of these proteins are essential for the normal morphogenesis of fibers during the development process. For instance, small ras-related protein (s155) and its negative regulator, GDP dissociation inhibitor (s136, s196 and s200), regulate a diverse array of cellular functions, including signal transduction, cell proliferation, differentiation, cytoskeletal organization, and vesicle transports,30,31 while ascorbate peroxidase (s102) and catalase isozyme 1 (s013 and s203) play a vital role in scavenging reactive oxygen species (ROS).32,33 The greater than 2-fold upJournal of Proteome Research • Vol. 7, No. 11, 2008 4631

research articles regulation of antioxidant enzymes might indeed be consistent with a role in redox regulation during fiber developmental process. It is most interesting that 5 fiber proteins are expressed mainly at specific development stages, and thus, these development-phase specific proteins can be considered as possible biomarkers to define fiber development. For example, an S-adenosylhomocysteine hydrolase (AAA33856) and a betatubulin (AAL92118) were found from the 15 dpa to 25 dpa stage, but were not detected from 2-DE images at the 5 dpa and 10 dpa stages (Table S1 in Supporting Information), respectively. Moreover, a putative proteasome subunit beta type-4 protein (CAC43325) was only detected at the 15 dpa stage. These findings suggest that some fiber proteins function specifically at specific stages during the development process. Gene Expression and Enzyme Activity Analyses for Several Key Proteins. It is generally accepted that the mRNA level does not correlate well with the protein level, partly due to differential half-lives of transcripts and proteins or translation-on-demand.19 To investigate the changes of gene expression at the mRNA level, semiquantitative RT-PCR was used to analyze the transcripts encoding 4 different proteins with different abundance patterns during the fiber elongation process. As shown in Figure 4, the mRNA abundance of the two genes of acyltransferase-like protein (GhACY [AAL67994]) and actin 8 (ACT8 [AAP73455]) reached a maximum level at 10 dpa and then declined, showing similar trends as a whole between mRNA and protein. Nevertheless, no transcripts were detected for GhACY at the 20 and 25 dpa stages. Similar patterns were also observed between mRNA and protein for late embryogenesis-like protein (LEA [AAC24588]) and dehydroascorbate reductase (DHAR [AAL71857]), although the transcripts of LEA were hardly detected during the early stages (5 and 10 dpa). Taken together, these results indicate that the dynamic tendency of these genes in abundance appeared to have no large differences between the transcripts detected by RT-PCR and protein spots on the 2-DE images. Therefore, we designed an RT-PCR analysis to confirm the results drawn from 2-DE. Another important identified protein is ascorbate peroxidase (APX), which is an H2O2-scavenging enzyme capable of utilizing ascorbate as its specific electron donor.33 Moreover, it has been reported that H2O2 may function as a developmental signal in the differentiation of secondary walls in cotton fibers and may presumably be involved in dimerization of cotton fiber cellulose synthase catalytic subunits.34,35 In this study, the abundance of the GhAPX protein in various development stages was detected on the 2-DE images, and was found to increase to greater than 2-fold in 10 dpa fibers compared to 5 dpa fibers, whereas the expression level of APX was attenuated in the later stages (Table S1 in Supporting Information). The oxidation rate of ascorbate was assayed using protein extracts from the developing fibers with various stages indicated. The total APX activity reached a peak value at 10 dpa (Figure 5), which matched well to the proteomic level of the gene. The results are also supported by the report that cotton APX expression is up-regulated during earlier stages (5-10 dpa) of cotton fiber development in G. hirsutum L cv. Xuzhou 142.20 To investigate the possible correlation between APX activity and H2O2 accumulation, the concentration of H2O2 was quantitatively assessed in fibers. The level of H2O2 in young fibers (5 dpa) was low but increased strongly at 15-20 dpa (Figure 5). H2O2 accumulation corresponded to the time of of SCW biosynthesis 4632

Journal of Proteome Research • Vol. 7, No. 11, 2008

Yang et al.

Figure 4. Comparison of expression profiles of 4 selected genes during different elongation stages (from 5 to 25 dpa) of cotton fibers. Semiquantitative RT-PCR analysis was performed using gene-specific primers (detailed in Materials and Methods) and taking cotton ubiquitin (UBQ7, DQ116441) as the reference gene (A). The relative expression levels were analyzed by Band Leader 3.0 (B). The relative expression levels of the same protein at different elongation stages are shown in (C) based on their 2-DE images (Figure S1 and Table S1 in Supporting Information). Abbreviations for genes: GhACY, acyltransferase-like protein (AAL67994); ACT8, actin 8 (AAP73455); LEA, late embryogenesislike protein (AAC24588) and DHAR, dehydroascorbate reductase (AAL71857).

and sharply declined afterward. It is obvious that the decline was not resulted from increase of APX (Figure 5 and Table S1 in Supporting Information) or/and CAT (Table S1 in Supporting Information), since both antioxidant enzymes were decreased at that time point (25 dpa). Thus, the specific function of APX in the context of fiber development remains to be determined.

Discussion Fiber growth is a key developmental process in the cotton life cycle, which undergoes four major discrete developmental

Proteomics Study of Elongating Cotton Fibers

Figure 5. The dynamic changes of the APX enzyme activity and H2O2 content during different cotton fiber elongation stages (from 5 to 25 dpa).

stages (differentiation, elongation/primary cell wall synthesis, secondary cell wall synthesis and maturity), and eventually produces a thick cell wall consisting of more than 94% cellulose.4 In the present study, we initiated a carefully performed comparative proteomic analysis of the single-cell developmental process using this model cotton fiber. Our main aim is to define the proteome of elongating cotton fibers, and to identify characteristic proteins in response to developmental programs terminating cell elongation. These identified proteins from the various elongation phases will help elucidate the biochemical and molecular processes underlying fiber elongation. For the elongating cotton fibers, 235 differentially displayed spots were highlighted on the reproducible 2-DE gel images from a total of about 1800 spots. Of these, 106 development-dependent proteins were successfully identified by the use of MALDI-TOF MS coupled with database searching (Table 1). The possible functions together with their abundance changes in response to elongation stages depict a dynamic network of events that accompany cotton fiber elongation. Highly Active Metabolisms of Carbohydrate and Proteins Are the Vital Basis of Fast Fiber Elongation. Fiber elongation commences on the day of anthesis (0 dpa) and continues for a period of ∼24 days in the case of Ghirsutum species, while transition from primary to secondary cell wall synthesis occurs during the latter stage of 16-21 dpa.11 Since the fibers at 0 dpa are difficult to be stripped from the epidermal layer of the ovule, fiber samples from 5 to 25 dpa were selected for this proteomic analysis. On the basis of the present proteomic analysis, 59 out of 106 identities are involved in various metabolic processes such as glycolysis, TCA cycle, and protein turnover, suggesting that the energetic metabolisms might be a basis of the normal process of early fiber development. Carbohydrate metabolism is a principal process to provide energy and intermediates for cell wall synthesis, as well as for cell elongation. Our results show that 28 differentially expressed identities are related to energy/carbohydrate metabolism (Table 1). These enzymes exhibit different expression profiles during the fiber elongation stages (Figure 3 and Table S1 in Supporting Information) and participate in the glycolytic pathway, the TCA cycle, the pentose-phosphate pathway and energy metabolism, respectively (Figure S6 in Supporting Information). Sucrose synthase (s171 and s172) in fiber cells catalyzes a reversible

research articles reaction but preferentially converts sucrose into fructose and UDP-glucose, which supplies the intermediates and energy for cell expansion and cell wall synthesis.36,37 The expression level of two isoforms was higher at 5 dpa and down-regulated in the later stages, which is believed to coincide with the requirements of the beginning of fiber elongation.38,39 Glycolysis is the set of reactions that converts glucose into pyruvate. In the fiber cells, glycolysis should be the prelude to the citric acid cycle and the electron-transport chain, where most of the free energy in glucose is harvested for PCW synthesis and cell elongation. In the present work, 6 key enzymes were detected to relate to six main reactions in elongation fibers, which are cytosolic phosphoglucomutase (s054), glyceraldehyde-3-phosphate dehydrogenase (s023, s024, s091, s143 and s144), cytosolic phosphoglycerate kinase (s082 and s085), phosphoglycerate mutase (s130), enolase (spot 117, 135, 204, 233 and 234), and pyruvate kinase (s067 and s198) (Figure S6 in Supporting Information). The identification of these enzymes, together with their abundance changes, documented the presence of the energetic sugar metabolism in the fiber elongation process. Furthermore, NADP-dependent isocitrate dehydrogenase (s207, s208 and s209) belongs to one multienzymatic family whose members are widely distributed in living organisms and is localized in different cell compartments such as cytosol and mitochondria.40,41 This enzyme catalyzes the oxidative decarboxylation of isocitrate to yield R-ketoglutarate, with the production of the reduced coenzyme NADPH, which is an essential electron donor in numerous biosynthetic and detoxification reactions (Figure S6 in Supporting Information). Next, glutamate is synthesized from NH4+ and R-ketoglutarate, and then an ammonium ion is incorporated into glutamine by the action of glutamine synthetase (s212, s213 and s218) on glutamate (Figure S6 in Supporting Information). The regulation of glutamine synthetase plays a critical role in controlling nitrogen metabolism.42 Thus, R-ketoglutarate is a critical R-keto acid for the aminotransferase reaction connecting the TCA cycle (for carbon metabolism) and nitrogen metabolism (Figure S6 in Supporting Information). Another critical enzyme, transketolase (s002), was found to be up-regulated more than 2-fold during fiber elongation. This enzyme catalyzes reactions in the glycolysis and in the oxidative pentose phosphate pathway and produces erythrose-4-phosphate that is a precursor for the shikimate pathway leading to phenylpropanoid metabolism (Figure S6 in Supporting Information).43 Additionally, GDP-4keto-6-deoxy-D-mannose-3,5-epimerase-4-reductase (s146) reduces the keto group at the C4 position of GDP-4-keto-6-deoxyL-galactose in de novo biosynthesis of GDP-fucose (Figure S6 in Supporting Information), which is incorporated into various cell wall glycoconjugates, including xyloglucan, rhamnogalacturonan, and arabinogalactan-proteins.44-46 This enzyme was found to be up-regulated with a dramatic increase in the transition from PCW to SCW synthesis, suggesting that more fucosylation may take place during this process. It is wellknown that plants can utilize dihydroxyacetone phosphate as a starting point for synthesis of phosphatidic acid required for the biosynthesis of phospholipids (Figure S6 in Supporting Information). In this process, glycerol-3-phosphate acyltransferase can acylate the glycerol-3-phosphate at the 1-position to eventually yield phosphatidic acid.47 In our proteomic analysis, this enzyme (s061) was found to accumulate at higher levels during the early stages (from 5 to 15 dpa), suggesting the importance of this enzyme for the early development of cotton fibers. Taken together, these findings are the first to Journal of Proteome Research • Vol. 7, No. 11, 2008 4633

research articles show an energetic sugar metabolism network linked to nitrogen and lipid metabolisms in cotton fibers. The proteomic analysis in this study also revealed that 18 identities are related to protein metabolism. Not surprisingly, seven proteins related to protein translation were downregulated in level at the late elongation stage with a decrease of the fiber elongation rate (Table 1 and Table S1 in Supporting Information). Protein elongation initiation factor 4A (eIF4A: s205) is involved in protein translation regulation at the level of ribosome recruitment. The eIF4A polypeptide is a subunit of eIF4F, but it also has an independent existence and an independent function with ATP-dependent RNA helicase activity. The eukaryotic translation initiation factor 5A (eIF5A: s109, s229 and s231) is a small (∼17 kDa) acidic protein, highly conserved and essential in almost organisms.48 Despite being essential and highly conserved, the critical cellular role of eIF5A is not known. In agreement with the hypothetical role for eIF5A in translation, a recent study in yeast revealed that eIF5A physically interacts with protein components of the translational machinery components, not only with structural components of the ribosome, but also with elongation factors, suggesting that eIF5A specifically binds to actively translating ribosomes.49 The elongation factor 2 (EF-2: s147 and s173) catalyzes the GTP-dependent translocation of peptidyl-tRNA from the A site to the P site on the ribosome, resulting in the translocation of the ribosome by one codon during the elongation phase.50 Moreover the eukaryotic class 1 peptide chain release factor (eRF1: s070) is involved in the termination of translation, the final stage of polypeptide biosynthesis. The two major functions of eRF1 are recognition of one of the three stop codons in the decoding center of the small ribosomal subunit, and participation in the subsequent hydrolysis of the ester bond in peptidyl-tRNA.51 The high abundance of these seven translation-related proteins in at least one of the early elongation stages (from 5 to 15 dpa), together with high expression of five molecular chaperones and 10 animo acid metabolism-associated proteins (Table 1 and Table S1 in Supporting Information), suggests higher translational efficiency in fast elongating fibers (from 5 to 15 dpa), which is consistent with the view that novel protein biosynthesis was required for fast fiber elongation. The Important Roles of Methionine Recycling-Related Proteins in Elongating Cotton Fibers. Methionine recycling in developing cotton fibers has not been reported yet to date, and nothing is known about the function of distinct methyltransferases for regulation of fiber development.52,53 In the present study, proteomic analysis revealed for the first time the differential accumulation during fiber elongation of several methionine cycle-related enzymes, such as methionine synthase (s048, s143 and s174), S-adenosylmethionine (AdoMet) synthase (s078 and s080), S-adenosylhomocysteinase (s074 and s179) and S-adenosylmethionine-dependent methyltransferases (s108) (Table S1 in Supporting Information). As shown in Figure S7A in Supporting Information, the terminal step in the methionine biosynthesis is catalyzed by methionine synthase, while AdoMet synthase catalyzes the reaction of methionine with ATP to form S-adenosylmethionine (SAM). S-adenosylhomocysteine is then hydrolyzed by S-adenosylhomocysteinase (s074 and s197) to homocysteine and adenosine. It is wellestablished that methionine is not only a building block of protein biosynthesis, but also occupies a central position in plant cell metabolism in which about 80% of methionine converts into SAM via AdoMet synthase.53 In the elongating 4634

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Yang et al. cotton fibers, three isoforms of methionine synthase (s048, s143 and s174) showed different accumulation patterns and were displayed at their highest levels from 5 to 15 dpa (Figure S7A in Supporting Information), suggesting that methionine has an important role during the earlier stages of cotton fiber development. The key enzyme S-adenosylmethionine synthetase was detected in the form of two isozymes (s074 and s197) with different pI and Mr (Table 1). Both proteins were significantly accumulated at the 10 dpa stage, but declined in the late stages of cotton fiber elongation. Furthermore, the resulting metabolite SAM is the major methyl-group donor in transmethylation reactions and an intermediate in the biosynthesis of polyamines and of the phytohormone ethylene.53 Moreover, the observed temporal patterns of accumulation of these proteins are consistent with an essential role of endogenous ethylene in elongating fibers.17 Our results, together with other published data, indicate that methionine synthase and S-adenosylmethionine synthetase are fundamental components controlling metabolism during cotton fiber elongation. In addition, the other key enzymes adenosine kinase and methyltransferase (Table 1) were also first detected in elongating cotton fibers. Two isoforms of adenosine kinase (s086 and s211) showed down-regulation, while an S-adenosylmethionine: 2-demethylmenaqunon methyltransferase (s108) was upregulated during fiber elongation (Table S1 and Figure S7A in Supporting Information). Adenosine kinase catalyzes the salvage synthesis of adenine monophosphate (AMP) from adenosine and ATP, and thereby contributes to the maintenance of cellular energy charge and to the synthesis of a variety of biomolecules, including nucleotide cofactors and nucleic acids.54 Moreover, a recent study demonstrated that adenosine kinase deficiency in Arabidopsis is associated with developmental abnormalities and reduced transmethylation,55 indicating the importance of this enzyme in plant growth and development. The S-adenosylmethionine:2-demethylmenaquinone methyltransferase catalyzes the conversion of demethylmenaquinone to menaquinone (vitamin K2) that serves as a redox mediator in several anaerobic electron transport systems.56,57 Vitamin K is known as a critical nutrient required for bone homeostasis and blood coagulation, and it is clinically used as a therapeutic agent for osteoporosis. However, its precise function in elongating fiber cells requires further investigation. The High Correlation of H2O2 with the Elongation Process in Cotton Fibers. It is well-established that plants are not only able to achieve a high degree of control over H2O2 toxicity, but also to use H2O2 as a signaling molecule.58 Therefore, the delicate redox homeostasis between H2O2producing and H2O2-scavenging systems is expected to occur during plant growth and development. In the present study, a set of redox homeostasis-related proteins were found in elongating cotton fibers, including ascorbate peroxidase (APX) (s102), dehydroascorbate reductase (DHAR) (s156 and s224) and NADP-isocitrate dehydrogenase (NADP-ICDH) (s207, s208 and s209), catalase (CAT) (s013) and phospholipase D alpha (PLDR) (s011, s043, s044 and s045). The dynamics of these protein abundance and H2O2 contents mirror a partial scenario of redox homeostasis occurring in the elongating cotton fibers. NADPH is an essential electron donor in numerous biosynthetic and detoxification reactions. The NADP-dependent isocitrate dehydrogenase (NADP-ICDH), which catalyzes the production of NADPH, is being recognized as an essential component of the antioxidative defense mechanisms.42 In cotton fibers, there

research articles

Proteomics Study of Elongating Cotton Fibers is little information on the antioxidant properties of NADPICDH. The analyses of comparative proteomics revealed a down-regulation of three NADP-ICDH isoforms (Figure S7B in Supporting Information) at translational level depending on the elongation process, suggesting they have important functions during the early stages of fiber elongation. Furthermore, H2O2 scavenging is accomplished by catalase, various peroxidases, and an ascorbate-glutathione cycle.59 Our analysis revealed that three key enzymes, namely, APX (s102), DHAR (s156 and s224), and CAT (s013 and s203), exhibited different regulation patterns in the elongating cotton fibers. In the ascorbate-glutathione cycle, scavenging of H2O2 by APX (s102) is the first step in which its enzymatic action produces monodehydroascorbate (MDHA), which can dismutate spontaneously to ascorbate (AsA) and dehydroascorbate (DHA). DHAR (s156 and s224) also participates in this cycle and mediates in reducing DHA to AsA enzymatically, using GSH as an electron donor. The resulting oxidized glutathione (GSSG) is then converted back to the reduced form (GSH) by a NADPHdependent glutathione reductase (GR) (Figure S7B in Supporting Information). Alternatively, DHA is unstable and it has been reported that DHA can be degraded via several intermediates to oxalate and L-threonate, with some of these reactions possibly generating H2O2 in plants.60,61 The abundance and activity of APX peaks at 10 dpa (Table S1 in Supporting Information, Figure 5 and Figure S7B in Supporting Information), while two isoforms of DHAR accumulated highest level at 5 dpa (Table S1 and Figure S7B in Supporting Information), suggesting that these two enzymes might be able to play a major role in maintaining lower levels of H2O2 in elongating fiber cells (Figure 5). It was reported that MDHA stimulates redox reactions in the plasmalemma of onion roots, resulting in enhanced water and nutrient transport through the plasma membrane, thus contributing to cell growth.62,63 Therefore, it is reasonable to expect that the high accumulation of APX at the fiber cell rapid elongation stage is required for production of MDHA, thereby promoting cell elongation in cotton fibers. In addition, catalase is directly involved in cellular oxidative metabolism by degradation of H2O2 using NADPH as an electron donor in plants. In this study, the accumulation of catalase (s013) is consistent with H2O2 content during elongation (Figure S7B in Supporting Information), and both of them exhibit their maximum during the transition from SCW to PCW, a stage of oxidative burst.36 Thus, it is suggested that the catalase activity may contribute to removal of H2O2 and regulation of redox homeostasis during the elongation process in cotton fibers. Apart from these enzymes, AsA and GSH can directly interact with and detoxify reactive oxygen species (ROS), including H2O2, and thus contribute significantly to nonenzymatic ROS scavenging.58,59 Therefore, an understanding of the interplay between enzymatic and nonenzymatic ROS scavenging machinery is crucial for identifying key components involved in redox homeostasis in cotton fibers. It is also interesting to note that four isoforms of PLDR were successfully detected on the 2-DE gel image from the elongating cotton fiber sample. Furthermore, the accumulated patterns were characterized by a peak at 20 dpa, which is consistent with that of H2O2 content measured in elongating cotton fibers (Figure S7B in Supporting Information). PLD hydrolyzes structural phospholipids of biological membranes to produce phosphatidic acid (PA) and a free-headgroup, both of which are thought to serve directly as cellular messengers.64 Activation of PLD is involved in mediating cellular processes such as

oxidative burst, vesicle trafficking, cytoskeletal organization, and cell proliferation.65 It is also reported that PLDR, with its lipid product PA, is responsible for a key event in signal transduction that leads to the release of superoxide in Arabidopsis and the potential target is NADPH-oxidase.66 Thus, it is assumed that four isoforms of PLDR might participate in signal transduction for the release of ROS, particularly H2O2 via NADPH-oxidase in elongating cotton fiber cells (Figure S7A in Supporting Information). Loosening of plant cell walls, the most important reaction in the cell elongation process, is not only initiated by expansins,67 ROS such as superoxide radicals, H2O2 and hydroxyl radical were also found to participate in plant cell elongation.33,68 Moreover, there was some sufficient evidence to document that H2O2 may function as a developmental signal in the differentiation of second walls in cotton fibers.36 Our findings presented above, together with previously published data, strongly suggest that H2O2-mediated cell expansion may be one important mechanism that regulates cotton fiber elongation.

Concluding Remarks Elongation events in cotton fibers are known to be accompanied by major changes of proteins and/or regulators at different elongation stages. In the present study, changes of global proteins in elongating cells were investigated for the first time at the proteomic level in cotton fibers. A total of 235 protein spots were revealed to be differentially expressed in elongating fiber cells by a 2-DE-based differential proteomic approach, and 106 of them were further identified by MS analysis. These proteins might work cooperatively to establish a complex cellular network for fiber elongation and morphogenesis. The network covers a broad metabolic process, including the carbohydrate metabolism system, energy production, the protein biosynthesis and quality control system, biosynthesis of cell wall components, cellular redox homeostasis, and metabolite supply persisting to fiber growth. Cotton fiber elongation, metabolism and environmental responses, as well as other vital functions, should depend on the correct regulation of protein expression. The results presented in this study provide not only new insights into fiber elongation, but also a solid basis for further functional research of each member of this network during fiber elongation.

Acknowledgment. This work was supported by the State Key Basic Research and Development Plan of China (2004CB117303), and the Specialized Research Fund for the Doctoral Program of Higher Education (20050003066) to J.Y.L. We are grateful to Prof. Jian-guo Ji, College of Life Sciences, Peking University, Beijing, P.R. China, for their kind assistance in MS/MS analysis and database searching of proteins. Supporting Information Available: A supplementary Table S1 shows relative abundance of 235 differentially displayed protein spot. Table S2 shows the peptide tags of proteins identified by MS/MS. Table S3 shows redundancy of some identified differentially displayed proteins identified by PMF, and Table S4 shows the cotton homologues of the queried proteins. Figure S1 shows the 2-DE analysis of the cotton fiber proteome during different elongation stages. Figure S2 shows the PMF pattern of spot 025. Figure S3 shows the process of identification of spot 112 based on peptide sequence derived from MALDI-TOF/TOF MS. Figure S4 shows sequence alignJournal of Proteome Research • Vol. 7, No. 11, 2008 4635

research articles ment of phospholipase D alpha. Figure S5 shows function cataloging of 106 differentially expressed proteins in elongation cotton fibers. Figure S6 shows pathways involved in carbohydrate metabolism under cotton fiber elongation, and Figure S7 shows the putative models involved in methionine recycling (A) and redox homeostasis (B) in elongating cotton fibers. This material is available free of charge via the Internet at http:// pubs.acs.org.

References (1) Udall, J. A.; Swanson, J. M.; Haller, K.; Rapp, R. A.; Sparks, M. E.; Hatfield, J.; Yu, Y.; Wu, Y.; Dowd, C.; Arpat, A. B.; Sickler, B. A.; Wilkins, T. A.; Guo, J. Y.; Chen, X. Y.; Scheffler, J.; Taliercio, E.; Turley, R.; McFadden, H.; Payton, P.; Klueva, N.; Allen, R.; Zhang, D.; Haigler, C.; Wilkerson, C.; Suo, J.; Schulze, S. R.; Pierce, M. L.; Essenberg, M.; Kim, H.; Llewellyn, D. J.; Dennis, E. S.; Kudrna, D.; Wing, R.; Paterson, A. H.; Soderlund, C.; Wendel, J. F. A global assembly of cotton ESTs. Genome Res. 2006, 16, 441–450. (2) Arpat, A. B.; Waugh, M.; Sullivan, J. P.; Gonzales, M.; Frisch, D.; Main, D.; Wood, T.; Leslie, A.; Wing, R. A.; Wilkins, T. A. Functional genomics of cell elongation in developing cotton fibers. Plant Mol. Biol. 2004, 54, 911–929. (3) Yao, Y.; Yang, Y. W.; Liu, J. Y. An efficient protein preparation for proteomic analysis of developing cotton fibers by 2-DE. Electrophoresis 2006, 27, 4559–4569. (4) Kim, H. J.; Triplett, B. A. Cotton fiber growth in plant and in vitro, models for plant cell elongation and cell wall biogenesis. Plant Physiol. 2001, 127, 1361–1366. (5) Ruan, Y. L.; Chourey, P. S. A fiberless seed mutation in cotton is associated with lack of fibre cell initiation in ovule epidermis and alterations in sucrose synthase expression and carbon partitioning in developing seeds. Plant Physiol. 1998, 118, 399–406. (6) Pear, J. R.; Kawagoe, Y.; Schreckengost, W. E.; Delmer, D. P.; Stalker, D. M. Higher plants contain homologs of the bacterial celA genes encoding the catalytic subunit of cellulose synthase. Proc. Natl Acad. Sci. U.S.A. 1996, 93, 12637–12642. (7) Zhang, H. M.; Liu, J. Y. Molecular cloning and characterization of a β-galactosidase gene expressed preferentially in cotton fibers. J. Integr. Plant Biol. 2005, 47, 223–232. (8) Shimisu, Y.; Aotsuka, S.; Hasegawa, O.; Kawada, T.; Sakuno, T.; Sakai, F.; Hayashi, T. Changes in levels of mRNAs for cell wallrelated enzymes in growing cotton fibre cells. Plant Cell Physiol. 1997, 38, 375–378. (9) Vojdani, F.; Kim, W.; Wilkins, T. A. Phosphoenolpyruvate carboxylase cDNA from developing cotton (G.hirsutum L.) fibres (accession nos.AF0089393 and AF008940) (PGR97-135). Plant Physiol. 1997, 115, 315. (10) Wu, Y. T.; Liu, J. Y. Molecular cloning and characterization of a cotton glucuronosyltranferase gene. J. Plant Physiol. 2005, 162, 573–582. (11) Zhao, G. R.; Liu, J. Y. Isolation of a cotton RGP gene: a homolog of reversibly glycosylated polypeptide highly expressed during fiber development. Biochim. Biophys. Acta 2002, 1574, 370–374. (12) Whittaker, D. J.; Triplett, B. A. Gene specific changes in R-tubulin transcript accumulation in developing cotton fibres. Plant Physiol. 1999, 121, 181–188. (13) Li, X. B.; Cai, L.; Cheng, N. H.; Liu, J. W. Molecular characterization of the cotton GhTUB1 gene that is preferentially expressed in fibre. Plant Physiol. 2002, 130, 666–674. (14) Li, X. B.; Fan, X. P.; Wang, X. L.; Cai, L.; Yang, W. C. The cotton ACTIN1 gene is functionally expressed in fibers and participates in fiber elongation. Plant Cell 2005, 17, 859–875. (15) Wang, S.; Wang, J. W.; Yu, N.; Li, C. H.; Luo, B.; Gou, J. Y.; Wang, L. J.; Chen, X. Y. Control of plant trichome development by a cotton fiber MYB gene. Plant Cell 2004, 16, 2323–2334. (16) Wu, Y.; Machado, A. C.; White, R. G.; Llewellyn, D. J.; Dennis, E. S. Expression profiling identifies genes expressed early during lint fibre initiation in cotton. Plant Cell Physiol. 2006, 47, 107–127. (17) Shi, Y. H.; Zhu, S. W.; Mao, X. Z.; Feng, J. X.; Qin, Y. M.; Zhang, L.; Cheng, J.; Wei, L. P.; Wang, Z. Y.; Zhu, Y. X. Transcriptome profiling, molecular biological and physiological studies reveal a major role for ethylene in cotton fiber cell elongation. Plant Cell 2006, 18, 651–664. (18) Samuel Yang, S.; Cheung, F.; Lee, J. J.; Ha, M.; Wei, N. E.; Sze, S. H.; Stelly, D. M.; Thaxton, P.; Triplett, B.; Town, C. D.; Jeffrey Chen, Z. Accumulation of genome-specific transcripts, transcription factors and phytohormonal regulators during early stages of fiber cell development in allotetraploid cotton. Plant J. 2006, 47, 761–775.

4636

Journal of Proteome Research • Vol. 7, No. 11, 2008

Yang et al. (19) Schmidt, M. W.; Houseman, A.; Ivanov, A. R.; Wolf, D. A. Comparative proteomic and transcriptomic profiling of the fission yeast Schizosaccharomyces pombe. Mol. Syst. Biol. 2007, 3, 79. (20) Li, H. B.; Qin, Y. M.; Pang, Y.; Song, W. Q.; Mei, W. Q.; Zhu, Y. X. A cotton ascorbate peroxidase is involved in hydrogen peroxide homeostasis during fibre cell development. New Phytol. 2007, 175, 462–471. (21) Bradford, M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248–254. (22) Starcher, B. A ninhydrin-based assay to quantitate the total protein content of tissue samples. Anal. Biochem. 2001, 292, 125–129. (23) Laemmli, U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970, 227, 680–685. (24) Candiano, G.; Bruschi, M.; Musante, L.; Santucci, L.; Ghiggeri, G. M.; Carnemolla, B.; Orecchia, P.; Zardi, L.; Righetti, P. G. Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteomic analysis. Electrophoresis 2004, 25, 1327–1333. (25) Gallardo, K.; Le Signor, C.; Vandekerckhove, J.; Thompson, R. D.; Burstin, J. Proteomics of Medicago truncatula seed development establishes the time frame of diverse metabolic processed related to reserve accumulation. Plant Physiol. 2003, 133, 664–682. (26) Lara-Nun ˜ ez, A.; Romero-Romero, T.; Ventura, J. L.; Blancas, V.; Anaya, A. L.; Cruz-Ortega, R. Allelochemical stress causes inhibition of growth and oxidative damage in Lycopersicon esculentum Mill. Plant Cell Environ. 2006, 29, 2009–2016. (27) Okuda, T.; Matsuda, Y.; Yamanaka, A.; Sagisaka, S. Abrupt increase in the level of hydrogen peroxide in leaves of winter wheat is caused by cold treatment. Plant Physiol. 1991, 97, 1265–1267. (28) Basra, A. S. Cotton Fibers: Developmental Biology, Quality Improvement, and Textile Processing; Food Products Press: NY, 1999; pp 16-20. (29) Calvert, C. M.; Gant, S. J.; Bowles, D. J. Tomato annexins p34 and p35 bind to F-Actin and display nucleotide phosphodiesterase activity inhibited by phospholipid binding. Plant Cell 1996, 8, 333– 342. (30) Vojtek, A. B.; Der, C. J. Increasing complexity of the Ras signaling pathway. J. Biol. Chem. 1998, 273, 19925–19928. (31) Ueda, T.; Yoshizumi, T.; Anai, T.; Matsui, M.; Uchimiya, H.; Nakano, A. AtGDI2, a novel Arabidopsis gene encoding a Rab GDP dissociation inhibitor1. Gene 1998, 206, 137–143. (32) Davletova, S.; Rizhsky, L.; Liang, H.; Shengqiang, Z.; Oliver, D. J.; Coutu, J.; Shulaev, V.; Schlauch, K.; Mittler, R. Cytosolic ascorbate peroxidase 1 is a central component of the reactive oxygen gene network of Arabidopsis. Plant Cell 2005, 17, 268–281. (33) Kwon, S. I.; Lee, H.; An, C. S. Differential expression of three catalase genes in the small radish (Rhaphanus sativus L. var. sativus). Mol. Cell 2007, 24, 37–44. (34) Potikha, T. S.; Collins, C. C.; Johnson, D. I.; Delmer, D. P.; Levine, A. The involvement of hydrogen peroxide in the differentiation of secondary walls in cotton fibers. Plant Physiol. 1999, 119, 849– 858. (35) Kurek, I.; Kawagoe, Y.; Jacob-Wilk, D.; Doblin, M.; Delmer, D. Dimerization of cotton fiber cellulose synthase catalytic subunits occurs via oxidation of the zinc-binding domains. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 11109–11114. (36) Amino, S.; Takeuchi, Y.; Komamine, A. Changes in enzyme activities involved in formation and interconversion of UDP-sugars during the cell cycle in a synchronous culture of Catka- rantkus roseus. Physiol. Plant 1985, 64, 111–117. (37) Delmer, D. P. Cellulose biosynthesis. Annu. Rev. Plant Physiol. 1987, 38, 259–290. (38) Ruan, Y. L.; Llewellyn, D. J.; Furbank, R. T. Suppression of sucrose synthase gene expression represses cotton fiber cell initiation, elongation, and seed development. Plant Cell 2003, 15, 952–964. (39) Ruan, Y. L.; Llewellyn, D. J.; Furbank, R. T.; Chourey, P. S. The delayed initiation and slow elongation of fuzz-like short fibre cells in relation to altered patterns of sucrose synthase expression and plasmodesmata gating in a lintless mutant of cotton. J. Exp. Bot. 2005, 56, 977–984. (40) Leterrier, M.; Del Rı´o, L. A.; Corpas, F. J. Cytosolic NADP-isocitrate dehydrogenase of pea plants: genomic clone characterization and functional analysis under abiotic stress conditions. Free Radical Res. 2007, 41, 191–199. (41) Hodges, M.; Flesch, V.; Ga´lvez, S.; Bismuto, E. Higher plant NADP+-dependent isocitrate dehydrogenases, ammonium assimilation and NADPH production. Plant Physiol. Biochem. 2003, 41, 577–585. (42) Kichey, T.; Heumez, E.; Pocholle, D.; Pageau, K.; Vanacker, H.; Dubois, F.; Le Gouis, J.; Hirel, B. Combined agronomic and

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physiological aspects of nitrogen management in wheat highlight a central role for glutamine synthetase. New Phytol. 2006, 169, 265–278. Henkes, S.; Sonnewald, U.; Badur, R.; Flachmann, R.; Stitt, M. A small decrease of plastid transketolase activity in antisense tobacco transformants has dramatic effects on photosynthesis and phenylpropanoid metabolism. Plant Cell 2001, 13, 535–551. Freshour, G.; Bonin, C. P.; Reiter, W. D.; Albersheim, P.; Darvill, A. G.; Hahn, M. G. Distribution of fucose-containing xyloglucans in cell walls of the mur1 mutant of Arabidopsis. Plant Physiol. 2003, 131, 1602–1612. Perrin, R. M.; Jia, Z.; Wagner, T. A.; O’Neill, M. A.; Sarria, R.; York, W. S.; Raikhel, N. V.; Keegstra, K. Analysis of xyloglucan fucosylation in Arabidopsis. Plant Physiol. 2003, 132, 768–778. Hori, H.; Kaushal, G. P.; Elbein, A. D. Fucosylation of membrane proteins in soybean cultured cells: effects of tunicamycin and swainsonine. Plant Physiol. 1985, 77, 687–694. Wang, S.; Lee, D. P.; Gong, N.; Schwerbrock, N. M.; Mashek, D. G.; Gonzalez-Baro´, M. R.; Stapleton, C.; Li, L. O.; Lewin, T. M.; Coleman, R. A. Cloning and functional characterization of a novel mitochondrial N-ethylmaleimide-sensitive glycerol-3-phosphate acyltransferase (GPAT2). Arch. Biochem. Biophys. 2007, 465, 347– 358. Zanelli, C. F.; Valentini, S. R. Is there a role for eIF5A in translation. Amino Acids 2007, 33, 351–358. Zanelli, C. F.; Maragno, A. L.; Gregio, A. P.; Komili, S.; Pandolfi, J. R.; Mestriner, C. A.; Lustri, W. R.; Valentini, S. R. eIF5A binds to translational machinery components and affects translation in yeast. Biochem. Biophys. Res. Commun. 2006, 348, 1358–1366. Malave´, T. M.; Forney, J. D. Identification of a developmentally regulated translation elongation factor 2 in Tetrahymena thermophila. Gene 2004, 326, 97–105. Ivanova, E. V.; Kolosov, P. M.; Birdsall, B.; Kelly, G.; Pastore, A.; Kisselev, L. L.; Polshakov, V. I. Eukaryotic class 1 translation termination factor eRF1) the NMR structure and dynamics of the middle domain involved in triggering ribosome-dependent peptidyl-tRNA hydrolysis. FEBS J. 2007, 274, 4223–4237. Eckermann, C.; Eichel, J.; Schro¨der, J. Plant methionine synthase: new insights into properties and expression. Biol Chem. 2000, 381, 695–703. Radchuk, V. V.; Sreenivasulu, N.; Radchuk, R. I.; Wobus, U.; Weschke, W. The methylation cycle and its possible functions in barley endosperm development. Plant Mol. Biol. 2005, 59, 289– 307. Young, L. S.; Harrison, B. R.; Narayana Murthy, U. M.; Moffatt, B. A.; Gilroy, S.; Masson, P. H. Adenosine kinase modulates root gravitropism and cap morphogenesis in Arabidopsis. Plant Physiol. 2006, 142, 564–573.

(55) Moffatt, B. A.; Stevens, Y. Y.; Allen, M. S.; Snider, J. D.; Pereira, L. A.; Todorova, M. I.; Summers, P. S.; Weretilnyk, E. A.; MartinMcCaffrey, L.; Wagner, C. Adenosine kinase deficiency is associated with developmental abnormalities and reduced transmethylation. Plant Physiol. 2002, 128, 812–821. (56) Lee, P. T.; Hsu, A. Y.; Ha, H. T.; Clarke, C. F. A C-methyltransferase involved in both ubiquinone and menaquinone biosynthesis: isolation and identification of the Escherichia coli ubiE gene. J. Bacteriol. 1997, 179, 1748–1754. (57) Shibata, M.; Tsuyama, M.; Takami, T.; Shimizu, H.; Kobayashi, Y. Accumulation of menaquinones with incompletely reduced side chains and loss of a-tocopherol in rice mutants with alternations in the chlorophyll moiety. J. Exp. Bot. 2004, 55, 1989–1996. (58) Mittler, R.; Vanderauwera, S.; Gollery, M.; Breusegem, F. V. Reactive oxygen gene network of plants. Trends Plant Sci. 2004, 9, 490– 498. (59) Jime´nez, A.; Herna´ndez, J. A.; Pastori, G.; del Rı´o, L. A.; Sevilla, F. Role of the ascorbate-glutathione cycle of mitochondria and peroxisomes in the senescence of pea leaves. Plant Physiol. 1998, 118, 1327–1335. (60) Green, M. A.; Fry, S. C. Apoplastic degradation of ascorbate: novel enzymes and metabolites permeating the plant cell wall. Plant Biosystems 2005, 139, 2–7. (61) Green, M. A.; Fry, S. C. Vitamin C degradation in plant cells via enzymatic hydrolysis of 4-O-oxalyl-L-threonate. Nature 2005, 433, 83–87. (62) Gonza´lez-Reyes, J. A.; Hidalgo, A.; Caler, J. A.; Palos, R.; Navas, P. Nutrient uptake changes in ascorbate free radical-stimulated onion roots. Plant Physiol. 1994, 104, 271–276. (63) Alcaı´n, F. J.; Caler, J. A.; Serrano, A.; Co´rdoba, F.; Navas, P. Stimulation of onion root elongation by ascorbate and ascorbate free radical in Allium cepa L. Protoplasma 1995, 184, 31–35. (64) Wang, X. Regulatory functions of phospholipase D and phosphatidic acid in plant growth, development, and stress responses. Plant Physiol. 2005, 139, 566–573. (65) Liscovitch, M.; Czarny, M.; Fiucci, G.; Tang, X. Phospholipase D: molecular and cell biology of a novel gene family. Biochem. J. 2000, 345, 401–415. (66) Sang, Y.; Cui, D.; Wang, X. Phospholipase D and phosphatidic acidmediated generation of superoxide in Arabidopsis. Plant Physiol. 2001, 126, 1449–1458. (67) Cosgrove, D. J. Loosening of plant cell walls by expansins. Nature 2000, 407, 321–326. (68) Foreman, J.; Demidchik, V.; Bothwell, J. H.; Mylona, P.; Miedema, H.; Torres, M. A.; Linstead, P.; Costa, S.; Brownlee, C.; Jones, J. D.; Davies, J. M.; Dolan, L. Reactive oxygen species produced by NADPH oxidase regulate plant cell growth. Nature 2003, 422, 442–446.

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Journal of Proteome Research • Vol. 7, No. 11, 2008 4637