Combined Proteomic and Transcriptomic Analysis Identifies

Combined Proteomic and Transcriptomic Analysis Identifies Differentially Expressed Pathways Associated to Pinus radiata Needle Maturation Luis Valledor,*,† Jesu ´ s V. Jorrıń,‡ Jose Luis Rodrı´guez,† Christof Lenz,§ Mo ´ nica Meijo ´ n,† † † Roberto Rodrı´guez, and Maria Jesu ´ s Can ˜ al ´ rea de Fisiologıá Vegetal, Departamento B.O.S., Instituto Universitario de EPIPHYSAGE Research Group, A Biotecnologıá de Asturias (IUBA), Universidad de Oviedo, Oviedo, Spain, Plant Proteomics-Agricultural and Plant Biochemistry Research Group, Departamento de Bioquı´mica y Biologıá Molecular, Universidad de Co´rdoba, Co´rdoba, Spain, and AB Sciex, Central Europe, Frankfurter Strausse 129 B, Darmstadt, Germany Received February 24, 2010

Needle differentiation is a very complex process that leads to the formation of a mature photosynthetic organ from pluripotent needle primordia. The proteome and transcriptome of immature and fully developed needles of Pinus radiata D. Don were compared to described changes in mRNA and protein species that characterize the needle maturation developmental process. A total of 856 protein spots were analyzed, defining a total of 280 spots as differential between developmental stages, from which 127 were confidently identified. A suppressive subtractive library (2048 clones, 274 non redundant contigs) was built, and 176 genes showed to be differentially expressed. The Joint data analysis of proteomic and transcriptomic results provided a broad overview of differentially expressed pathways associated with needle maturation and stress-related pathways. Proteins and genes related to energy metabolism pathways, photosynthesis, and oxidative phosphorylation were overexpressed in mature needles. Amino acid metabolism, transcription, and translation pathways were overexpressed in immature needles. Interestingly, stress related proteins were characteristic of immature tissues, a fact that may be linked to defense mechanisms and the higher growth rate and morphogenetic competence exhibited by these needles. Thus, this work provides an overview of the molecular changes affecting proteomes and transcriptomes during P. radiata needle maturation, having an integrative vision of the functioning and physiology of this process. Keywords: Pinus radiata • proteomics • transcriptomics • needle development

Introduction Forests are ecosystems of great environmental and economic importance, providing food and raw materials for processing industries, and are the major CO2 traps that play an important role in climate change-prevention strategies.1 In recent decades, a significant increase in forest production has been achieved by employing fast growing species like Pinus radiata D. Don, but the production must still be improved by reforesting only with clonal elite individuals.2 To achieve this goal, ongoing upgrading and clonal multiplication programs, based on classical breeding and biotechnology, must overcome the challenge that tree maturation involves. On the one hand, tree maturation processes are responsible for the achievement of desirable traits that define an individual as an elite tree (growth capacity and straight shaft, among others), but on the other hand, these differentiation processes * To whom correspondence should be addressed. Luis Valledor. E-mail: [email protected]. Phone: +34 985 104 834. Fax: +34 985 104 777. † Universidad de Oviedo. ‡ Universidad de Co´rdoba. § AB Sciex.

3954 Journal of Proteome Research 2010, 9, 3954–3979 Published on Web 05/28/2010

are linked to the loss of cell plasticity, constituting a barrier to clonal multiplication and genetic manipulation in forest upgrading programs.2-4 Despite the importance of maturation on forest productivity and tree clonal upgrading programs, little is known about the molecular basis of the regulation of these developmental processes. Multidisciplinary efforts have been made to gain a better understanding of maturation, but only an incomplete understanding has been achieved, even in economically relevant species such as P. radiata.5-9 Since maturation of the complete tree system is a very complex process, and the actual knowledge of this process is very limited, the first step should be to study it in isolated organs. Needle fascicles do not only constitute the photosynthetic organ and the main external perception site but are also one of the main tissue sources employed for tree cloning and in vitro micropropagation.3 As such, they are ideally suited to study maturation. Needle development and maturation occur progressively and are the consequence of cell differentiation processes that define the future organ shape, tissue functions, and its competence.9 To achieve these changes, it is clear that specific genes must be regulated to be expressed only in certain 10.1021/pr1001669

 2010 American Chemical Society

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Analysis of Pinus Needle Maturation cells and situations, first to initiate the leaf growth and define its polarity, second to reach an adequate size and morphology, and finally to completely specialize the cells to gain tissue function and maturity.10-12 All of these changes should be reflected in variations in the needle transcriptome and proteome. The study of the variations in gene and protein expression after needle maturation is not only interesting from a basic biological perspective but also from an applied perspective because it is after these processes that photosynthesis reaches its maximum, defining the growth rate of the tree. However, the morphogenetic competence decreases, limiting clonal propagation and other biotechnological procedures. The feasibility of taking a direct classical transcriptomic or proteomic approach in P. radiata is hindered by the lack of protein and gene information in public databases. Despite the availability of high throughput proteomic platforms and the possibilities of combining these with transcriptomics or metabolomics to cross validate data and improve biological understanding, combined proteomic and transcriptomic analyses of leaf maturation are scarce, even in model species.13 The major efforts to characterize leaf maturation have been performed in Arabidopsis,12-14 Oryza,15-17 and Soybean18 using transcriptomic or proteomic approaches. Leaf primordia, in Arabidopsis, are initiated at the flanks of a group of undifferentiated and proliferative cells within the shoot apical meristem, which requires the acrivity of the class I KNOTTED1LIKE HOMEOBOX (KNOXI) genes for its establishment and maintenance.14 Leaves are produced as peg-like structures that initially elongate via rapid cell division.12-14 Unlike the shoot meristem, which exhibits radical symmetry, the leaf adquires bilateral symmetry concurrent with its emergence. The leaf primordium next transitions from growth by active cell division to growth exclusively through cell expansion in a wave of differentiation beginning ate the leaf tip and progressing to the leaf base. The mature leaf form is achieved as this cell expansion.12-14 In spite of the recent advances in sequencing the genome of poplar, conifers genomics is mainly based in EST analyses and focused into fiber and wood production19,20 with no reference to leaf development in any of them. None of these classes of studies have been performed to study leaf-needle maturation in tree species.21,22 Working with nonmodel species like Pinus radiata, for which most of the high throughput analyses tools (microarrays, easy protein MS/MS identification) are not available, make us develop custom EST libraries and 2-DE protein reference map of needle proteins prior to performing differential expression analyses. The leaf is a highly specialized organ for photosynthesis in higher plants. It has been shown in several reports that rates and activities of photosynthesis are highly dependent on the development and age of the leaf, and this is also correlated with the accumulation of proteins such as RIBULOSE-1,5BISPHOSPHATE CARBOXYLASE/OXYGENASE (RuBisCO) and other photosynthesis-related proteins.18 Physiological and biochemical analyses provide a better understanding of leaf development but unfortunately cannot provide information on the molecular mechanisms involved in gene and protein expression at the various developmental stages. In this work, the changes in the proteome and transcriptome, which occur during needle expansion, differentiation stages comparing immature growing needles, characterized by a low tissue differentiation and high morphogenetic capability, and mature needles, a specialized photosynthetic organ with no

Figure 1. (a) Mature and (b) immature growing needles.

morphogenetic capabilities,9 have been studied for first time. Proteomic analyses were performed by 2-DE (IEF-PAGE) focusing on experimental design and strong statistical analyses. Differential spots were identified by LC-ESI-MS/MS employing a substitution-tolerant database search algorithm. Transcriptomic expression analysis was only possible after construction of a P. radiata EST library and the employment of dot-blot macroarrays and RT-qPCR to test the expression levels of each EST. The protein reference map together with transcriptomic analyses provide clues about the nature of the proteins/transcripts and their abundance and may be used to determine the main gene functions expressed or the major metabolic pathways at work. Thus, this work gives a representative picture of the proteomic and transcriptomic structure of Pinus needles and an overview of the needle maturation biology.

Material and Methods Plant Material. Mature (12 month old) and immature (3-5 week old, active growth) needles (Figure 1) were taken from field Pinus radiata plantations at La Reigada (Asturias, Spain; 43°26′46′′N, 6°00′42′′W, altitude 500 m). Seven different adult trees present in the outermost tree-row of the plantation, approximately 15 years old, were sampled. Needles were collected from immature and mature needle fascicles taken from twigs of the last segment of auxiblasts. The auxiblasts were located at the same tree level and orientation and were sampled on the same day and hour, during the active growth season (spring). Needles were washed in situ with tap water, dried with filter paper, and then frozen in liquid nitrogen immediately. Samples were stored at -80 °C until RNA or protein extractions were done. Proteomics Experiments. Protein Sample Preparation and 2-DE. Protein isolation and subsequent gel electrophoresis were performed as described by Valledor et al. (2008),23 with minor modifications: DTT concentration was increased to 0.1% in TCA/Acetone extraction buffer; all precipitations were done for 1.5 h at -80 °C. In brief, for each developmental stage, 500 µg of protein were loaded onto precast IPG strips (pH 5-8 linear gradient, 17 cm; Bio-Rad, Hercules, USA), and seven biological replicates were done for each kind of needle. Due to the need of a large amount of material, needles from the same auxiblast in the same stage of development were pooled, aiming to reduce environmental variation inside each sample. Second dimension electrophoresis was performed in two batches in 13% polyacrylamide gels run in a DODECA-Cell (BioRad, Hercules, USA), striving to keep the variation between batches to a minimum. One analytical replicate for each of the seven biological replicates were done. Following 2-DE, gels were stained three times with colloidal CBB (G-250) and immediately imaged with a GS-800 (Bio-Rad, Hercules, USA) to avoid fading. 2-DE Gel Data Analysis. Digitalized gel images were analyzed with PDQuest 8 (Bio-Rad, Hercules, USA) using 3-fold over background as a minimum criterion for the presence or absence for the guided protein spot detection method. This criterion includes almost all spots of the gels and some noise, a spot by spot visual validation being required following Journal of Proteome Research • Vol. 9, No. 8, 2010 3955

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automated analysis to increase the reliability of the matching. Spot intensities were obtained for later quantitative analysis. Experimental pI was determined using a 5-8 linear scale over the total length of the IPG strip. Mr values were calculated by mobility comparisons with protein standard markers (SDS Molecular weight standards, Broad range, Bio-Rad, Hercules, USA) run in a separate lane on the gel. Missing spot volumes were estimated from the data set employing a sequential K-Nearest Neighbor algorithm25 using R 2.7.0 environment.26 This procedure was only performed if the spot was consistent (appeared in at least 5 out the 7 replicates). After missing values imputation, total spot intensity per gel was used to normalize spot intensities (% of individual spot intensity/Σ% spot intensity of each gel) to compensate for variations between gel replicates. These data sets were also cubic-root transformed to reduce the spot volume-spot deviation dependency. Selection of differentially abundant protein spots was performed after two-tailed t test analysis, employing a Bonferroni correction for an initial R ) 0.05 (power of test 0.9291 for detecting variations from 0.1% between normalized spot volumes). A multivariate analysis was performed over the whole set of spots and on those showing differences. Principal Component Analysis (PCA) was applied both to whole data sets and to differentially expressed spots (SPSS v. 15, SPSS Inc., Chicago, IL). Samples were clustered employing Cluster 3.0 employing Euclidean distance method over a complete linkage dissimilarity matrix and plotted employing Java Treeview 1.1.3 software. Protein Identification. Spots were automatically excised (Investigator ProPic, Genomic Solutions), transferred to multiwell 96 plates and digested with modified porcine trypsin (sequencing grade; Promega Corporation Madison, WI) using a ProGest (Genomics Solution) digestion station as previously described.23 Digested samples were loaded onto Tempo 1D nanoLC System (AB Sciex, Deutschland) coupled to a hybrid triple quadrupole/linear ion trap mass spectrometer (4000 Q TRAP LC-MS/MS System, AB Sciex, Deutschland). Briefly, samples were concentrated and desalted on a trapping column (PepMap C18 300 µm/5 mm, Dionex Europe, Deutschland; loading solvent aqueous 2% acetonitrile, 0.1% formic acid; 20 µL/min) and separated on a microbore column (PepMap C18 75 µm/ 15 cm, Dionex; 30 min linear gradient 5 to 40% acetonitrile vs 0.1% formic acid; 300 nL/min). The eluent was analyzed using a hybrid triple quadrupole/linear ion trap mass spectrometer (4000 Q TRAP LC-MS/MS System, AB Sciex, Deutschland) equipped with a heated Desolvation Chamber Interface set to 150 °C and operated under Analyst 1.4.1 software (AB Sciex, Deutschland). A fused silica tip (SilicaTip 360-20-10-N, New Objective, Woburn, USA) was used at an electrospray voltage of 2800 V to achieve ionization. The mass spectrometer’s scan cycle consisted of a Linear Ion Trap survey scan (Enhanced MS scan, scan range 400-1250 amu, scan speed 4000 amu/s, fixed fill time 20 ms), high resolution linear ion trap scans of up to five detected precursors (Enhanced Resolution scan, isolation width (5 amu, scan speed 250 amu/s, dynamic fill time) to determine the precursor molecular weight and charge state, and finally up to five product ion scans using Q2 collision cell fragmentation with Linear Ion Trap detection (Enhanced Product Ion scan, isolation width (1 amu, scan range 100-1400 amu, scan speed 4000 amu/s, dynamic fill time). The collision energy for each 3956

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precursor was determined dynamically depending on its molecular weight and charge state. Proteins were identified by searching the LC/MS/MS data using the Paragon algorithm27 present in ProteinPilot 2.0.1 Software (Applied Biosystems) against a combined plant protein sequence database containing sequence entries from SwissProt, trEMBL and Genbank (CDSplant_20050302.fasta, Applied Biosystems). Peaklist generation in ProteinPilot 2.0.1 Software is parameter-free and was used as such. The following settings were used for database searching: Cys alkylation, Iodacetamide; Digestion, Trypsin; Special Factors, Gel-Based ID; Species Filter, None; Search Effort, Thorough; ID focus, Amino acid substitutions. Afterward, the ProGroup algorithm present in Protein Pilot Software assembled the evidence reported by the Paragon Algorithm and assigned the Total Protein and Unused scores to each identification. The Total Protein Score is based on all peptides pointing to a protein, but peptide identifications can only contribute to the Unused Score of a protein to the extent that their spectra have not already been used to justify more confident proteins. In addition, Pro Group Algorithm also could distinguish protein isoforms, a specific protein isoform would be reported only if unique evidence (peptide) existed for this isoforms (AB Sciex, technical note). Only proteins with Unused Scores greater than 4.00, corresponding to a 99% or higher confident identification were included in the final protein list. Ccombining Pro Group and Paragon Algorithms greatly reduced the redundancy and suppressed the false positives; however, some protein isoforms were not able to be distinguished based on the peptides identified. In this case the proteins are referred as the generic protein family name. Transcriptomic Experiments. RNA Extraction and Amplification. RNA was isolated from 75 mg of frozen tissue according to Chang et al. (1993)28 Resulting pellets were purified with RNeasy Clean Up and treated with RNase-free DNase (both from Qiagen, Dusseldorf, Germany). RNA integrity was tested in agarose gel. For SSH approximately 1 µg of total RNA from immature and mature needles were transcribed into cDNA and amplified using the SuperSMART cDNA synthesis kit (Clontech Laboratories, Inc., Mountain View, USA). SSH Library Construction and Sequencing. cDNA suppressive subtractive hybridization (SSH) was performed using PCRSelect Subtraction Kit (Clontech Laboratories, Inc., Mountain View, CA) according to manufacturer’s instructions. The cDNA of immature needles was used as a tester and the cDNA of mature needles as a driver for mature library. For the mature needles library, cDNA of immature needles was used as driver. PCR products of subtracted cDNAs were directly inserted into pGEM T-Easy Vector (Promega Corporation, Madison, USA) and transformed into competent Escherichia coli DH5R cells employing TransformAID Kit (Fermentas International Inc., Ontario, Canada). Clone selection was performed employing IPTG, X-Gal and ampicillin. Clones were picked to establish each subtracted cDNA library constituted by 1024 clones each. Selected clones were single-pass sequenced (Macrogen Inc., Seoul, Korea). Sequences were trimmed using SeqTrim v0.098 - w0.1929 and Geneious 4.0.2 (Biomatters Inc. Auckland, New Zealand) and then assembled into 2 different batches (immature and mature) employing SeqMan Pro 7.1 (Lasergene, DNASTAR Inc., Madison, USA) to obtain sequence contigs. Contig sequence homology searches were performed using BLAST 2.2.18 software (NCBI) and custom local databases (described in Supporting Information). Homologies that showed

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Analysis of Pinus Needle Maturation Table 1. Number of Detected Spots in the Different Needle Extracts Used for Proteome Analysis number of spots

Immature Mature

consistent spots

Table 2. Mean Coefficient of Variation Determined after the Different Steps of Data Processing raw data

min/max

average

total

common

qualitative differences

718/735 695/724

726 ( 5 706 ( 12

715 692

551 551

164 141

e-values lower than 1 × 10-10 were considered significant. Functional classification was performed according to KEGG. Dot Blot Macroarray. Macroarrays were designed to test the differential expression of the identified contigs obtained after SSH. cDNA was PCR amplified directly from E. coli clones employing SP6 and T7 primers, and spotted into Hybond N+ membranes (GE Healthcare, Piscataway, USA). 96 probes were spotted in each membrane. In total 174 probes corresponding to identified genes were spotted on the arrays. RNA was reverse transcribed (Superscript III first strand synthesis kit, Invitrogen, Darmstadt, Germany) and tail-labeled with DIG-UTP alkalilabile employing a terminal transferase (Roche, Basel, Switzerland). The chemiluminiscence assay (CSPD ready to use, Roche, Basel, Switzerland) was done according to manufacturer and autoradiography was performed in a LAS-3000 mini system (Fujifilm Life Science, Tokyo, Japan). Images were analyzed with Quantity One V.4.6.3 (Bio-Rad, Hercules, USA). (A complete protocol description is available in Supporting Information.) qRT-PCR. Quantitative PCR was performed as follows: 10 ng of cDNA, 5pM of each primer and Perfecta SYBR Green (Quanta Biosciences, Gaithersburg, USA) was mixed and amplified using the ABI 7900HT system. Superscript III first strand synthesis kit (Invitrogen, Darmstadt, Germany) was used for labeling cDNA synthesis. Three measurements for each transcript and biological situation were analyzed using the Prism software (Applied Biosystems, Life technologies). Relative quantifications were performed for all genes and 18S, actin and GAPDH were used as loading controls. Primer sequences and Tm are indicated in Table S1, Supporting Information.

Results Proteome Analysis. Differential Expression Analysis. The protein yield of the employed protocol for protein isolation and solubilization was very similar in both mature (3.45 ( 0.84 µg of protein per mg of fresh tissue) and immature (3.90 ( 0.34 µg of protein per mg of fresh tissue) needles. Table 1 shows the number of total spots detected and the number of common and exclusive (those that were qualitatively different for each kind of sample) spots found after 2-DE PAGE of protein extracts from immature and mature needles (Figure 1). A total of 856 spots were considered for differential expression analysis: 551 spots were common to both kinds of needles while 164 and 141 spots were only detected, respectively, in immature or mature needles. Raw data was processed in three steps, missing value estimation, normalization and transformation, to reduce the variance between samples, increasing the significance of multivariate statistics and clustering algorithms. These methods were effective in reducing the coefficient of variation of each of the condition spots, from a mean Coefficient of Variation of 43 or 40% for raw data to a 14 or 13% for transformed data of immature and mature needles data sets, respectively (Table 2; Table S2; Figure S1, Supporting Information). A set of differentially abundant protein spots was selected from immature and mature needles. For that purpose, a very

imputation

normalization transformation

Immature 42.54 ( 23.18 41.13 ( 21.83 37.30 ( 22.71 Mature 39.74 ( 22.93 37.66 ( 21.15 33.57 ( 21.99

14.23 ( 7.97 13.23 ( 8.77

strict method, two-tailed t test with the Bonferroni correction, was used in differential analysis. Out of 856 studied spots, 280 showed a differential accumulation between immature and mature needles (Figure 2). Multivariate Analysis. The importance of studying the whole protein pattern and performing a multivariate analysis was also considered. Principal Component Analysis (PCA) was applied to all spots (856) and differentially expressed (280) spot data sets. For the data set containing all spots, out of the potential 856 principal components (PCs), the first 10 PCs accounted for 95% of the biological variability, a number that was reduced to the first component in the differentially expressed spot data set (Table 3). The employment of these components, plotting PC1 and PC2, allowed the effective separation of samples into their original groups (Figure 3), the plot structure not being greatly different between whole and differential spot data sets. Cluster analysis of the proteomics expression data was used to identify groups of similarly expressed proteins in the samples. Differential spots which were identified were clustered employing the Euclidean distance method over a complete linkage dissimilarity matrix (Figure 4). Spots were compared, employing a clustering method and a representation of quantitative variations between stages, determining two main groups based on the Euclidean distance coefficient. There was a short distance between biological replicates, but very large distances between the samples of the different needles indicating that 2-DE maps are very different, as the PCA had shown before. Protein Sequence Identification. Out of the 280 differentially abundant spots, a total 150 spots, selected based in their abundance, qualitative, and abundance-ratio differences between samples, were subjected to in gel tryptic digestion and later mass spectrometry analysis. Positive sequence hits, those that showed ProteinPilot scores greater than 4.00, corresponding to a 99% or higher confident identification, were obtained in 127 spots. For the remaining spots, no hits with enough confidence were found after querying the database. A total of 97 protein species were identified, some of them were present in more than one spot (Table 4). This comigration of proteins is possible if both present the same experimental pI and Mr. The presence of the same protein in more than one spot (i.e., RuBisCO LARGE SUBUNIT, ENOLASE, SAM SYNTHASE 2) has been reported. While for most of the identified proteins close theoretical and experimental Mr values were obtained, for others lower experimental than theoretical values were obtained, these probably being degradation products of the native protein (i.e., RuBisCO large subunit). In other cases, there were only small differences in pI described, suggesting the existence of post-translational modifications or isoforms (i.e., SAM SYNTHASE 2, 70 kDa HSP). There was another set of proteins in which the pI and Mr were not coincident with the heterologous sequence in the database. In most of the cases, this was because only a fragment of the protein sequence was included in the database. All information about this experiment following MIAPE standards is available at the ProteoRed public repository (see Supporting Information). The 97 non redundant protein species were classified into 17 categories according to the Kyoto Encyclopedia of Genes Journal of Proteome Research • Vol. 9, No. 8, 2010 3957

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Table 3. Principal Components (PCs) Calculated from All and Differentially Expressed Spots Data Sets total variance explained initial Eigenvalues all spots component

total

1 2 3 4 5 6 7 8 9 10 11 12 13

555.76 43.22 38.51 32.65 28.32 26.82 25.93 22.88 19.80 18.45 17.13 15.04 12.50

differentially expressed

% of cumulative variance %

64.85 5.04 4.49 3.81 3.30 3.13 3.03 2.67 2.31 2.15 2.00 1.76 1.46

64.85 69.89 74.39 78.20 81.50 84.63 87.66 90.32 92.63 94.79 96.79 98.54 100.00

total

265.14 2.92 2.18 1.77 1.41 1.33 1.01 0.95 0.78 0.75 0.67 0.58 0.51

% of cumulative variance %

94.69 1.04 0.78 0.63 0.50 0.48 0.36 0.34 0.28 0.27 0.24 0.21 0.18

94.69 95.73 96.51 97.15 97.65 98.12 98.49 98.83 99.11 99.37 99.61 99.82 100.00

and Genomes (KEGG), based on the putative functions of protein translation, folding, modification, and degradation (21 proteins), stress and defense (14 proteins), photosynthesis (13 proteins), and transcription and DNA replication (12 proteins), the principal groups found. The differences of protein accumulation levels between functional groups and type of needle are shown in Figure 5. Transcriptome Analysis. SSH was applied to RNA from both kinds of needles to normalize mRNA levels and to enrich for genes that are up-regulated at each of these developmental

stages. Clones (576) of each library were single-pass sequenced. After sequence trimming and contig building 196 and 143 sequences were obtained for immature and mature needle libraries, respectively. The sequence information of both ligenbraries is available in public repositories (GenBank Accessions GO096063 to GO096400). Sequence homology searches were performed locally using BLASTx against a custom database which identified 167 and 64 sequences respectively (Table 5). Some of the sequences showed significant homologies to the same database accession; a total of 176 sequences showed non redundant accessions. The differential expressions of these 176 non redundant sequences were validated by dot blotting (Supplementary Figure 2, Supporting Information) and quantitative PCR (Table S3, Supporting Information). Supplementary Table 4 summarizes the library construction and validation process (Supporting Information). The 176 identified sequences were classified into 17 categories based on their homologies and according to KEGG. The principal groups found were protein translation, folding, modification and degradation (31 sequences), transport (19 sequences), stress and defense and transcription and DNA replication (17 sequences each), with a very different distribution between the different maturation stages (Figure S3, Supporting Information). The comparison of transcriptomic and proteomic data, focusing on the analysis of the different functional groups, showed that the number of different protein species and genes is proportional in most of the categories. Secondary metabolism, transport, and cell cycle are represented only by a few protein species but a higher number of genes (Figure 6). The

Figure 2. (a) Master gel combining spots of immature (B1) and mature (B12) needles protein extracts. The relative Mr is given on the left, while the pI is given at the top of the figure. (b) Three sections of the 2-DE gels showing two representative B1 and B12 gels. Red and green arrows point to up and down accumulated spots, respectively, while black arrows point to spots only detected in one kind of protein extract. 3958


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Figure 3. 2-D representation of the main principal components obtained by PCA (a) over all spots data set or (b) over the differential spot data set.

number of gene-protein pairs was scarce with only 14 mRNAprotein identities found. These proteins were identified in 33 spots, representing 14.48 and 19.40% of the total protein amount from 2-DE gels of immature and mature needles, respectively. The differences of volume ratios of these proteins were transformed to fold difference between type of needles, as was also observed for the mRNA expression levels. As presented in Figure 7 and Table S3 (Supporting Information), the results from proteomic and real time PCR analysis demonstrated that some correlation trend exists between the differential expression profiles of the gene and the accumulation of the protein (RBCS, RBCL, RBCA, eEF2, RCC1), but in some cases (GSTU, APX, PPA) an opposite trend between protein and mRNA expression was described.

Discussion Needle maturation is a key developmental process for tree development and forest productivity since needles are the major photosynthetic organ of the coniferous trees. In addition, plant leaves are the best solar panels ever built, and they also perform well as air purifiers factory.12-14 An understanding of the processes underlying needle maturation thus provides an insight into a basic process in biology, modulation of which may have far-reaching significance strategies to improve forests yields and environment. The maturation-related loss of morphogenetic capacity that impedes in vitro plant regeneration is a phenomenon of great importance, both for applied (clonal breeding) and basic (factors limiting the cell totipotency of the mature needles) perspectives.9 Immature needles showed a higher number of spots (Table 1) and total RNA content (data not shown) than mature needles, a fact that may reflect the higher metabolic complexity with a higher number of active pathways that show developing leaves.15 But these metabolic changes are not known in depth, so this work is aimed to describe the changes that occur during the developmental transition of needle maturation, focusing on the joint analysis of the proteomic and transcriptomic data and on the biological interpretation of these changes. 2-DE Proteome Analysis. We have placed special emphasis on controlling technical variability to increase the power of the statistical analyses.30 Samples were taken from seven different individuals, trying to maintain the variation between needle developmental stages as low as possible, to reduce experimental noise. This number of biological replicates was proven to be adequate for proteomic analyses in P. radiata needles.23 After the manual revision of the matching of all of the 856 different spots (Figure 2) a K-Nearest-Neighbor (KNN) algorithm was employed to impute missing values. Although it has

been reported that there are more accurate methods to deal with missing values, such as BCPA, KNN is still a current method providing enough statistical power and its use is better than classical missing value estimations or performing multivariate analysis in the presence of missing data.25,30,31 After missing value imputation, spot volumes were normalized employing the mean intensity of corresponding gel as normalization factor. Variance of the whole data set was differentially distributed, with larger spots showing larger variance than small spots (Figure S1; Table S2, Supporting Information). This effect is related to image acquisition and processing steps32 and was reduced applying a cubic root transformation (Figure S1, Supporting Information; other transformations not shown). This workflow proved to be effective reducing the CV by almost 30% to a final 14% (Table 2), this value being significantly lower than that obtained for other tree species when not applying all of the processing steps, i.e., date palm33 or holm oak34 with 25% or 58% of mean CV respectively. Differentially abundant spots were defined after applying Student’s t test with a Bonferroni correction of the significance level (FWER procedure). Despite being a very conservative and strict approximation, mainly used in the discovery of strong biomarkers,35 280 spots were found differential between the studied maturation stages. The importance of multivariate analyses36,37 was also considered. A multivariate unsupervised PCA analysis was performed on the data sets. The first 13 principal components (PC) accounted for 100% of the variability of each whole or differential spot data set (Table 3) but with a different distribution. In the whole spot data set, a 95% variance was reached after the accumulation of the first 10 PCs, while in the differentially abundant data set only the first PC is needed. This fact reflects the strong selection force that was applied to the original data set, maybe caused by the strictness of the FWER procedure. Independent of this consideration, the plot structure for PC1 and PC2 did not greatly differ between data sets (Figure 3), the spots with high loading over these PCs (matrix not shown) being in concordance with those spots selected by the t test. Data clustering and heat map building (Figure 4) showed the same two group structure, coincidental with the PC1 correlation, which determined the accumulation level in immature (positive correlation) or mature (negative correlation) needles. These spots could be candidates for biomarkers of specific tissue activity at each developmental stage that would be of great interest for applied use (discussed later). This application of PCA is commonly employed in biomarker discovery38 and has been recently applied to plant developmental analyses.33 Journal of Proteome Research • Vol. 9, No. 8, 2010 3959

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Figure 4. Hierarchical clustering of the 127 functionally identified protein spots. Two main branches can be distinguished. The first one gathers “mature” spot cluster, which includes proteins overexpressed in that developmental stage, whereas the other one includes the proteins overexpressed in immature needles.

Protein identification was achieved employing LC-ESI-MS/ MS and database searching using the Paragon algorithm, resulting in the identification of 65% of the analyzed spots, a slightly lower percentage than previously described for iden3960


tifications dealing with this species.23 This can be explained by the fact that we only considered a valid identification when at least two peptides with unused scores above 2.00 were detected, representing a more critical and stringent evaluation

protein

reference organism

6.14 0.882 0.047 0.658 0.064 B1

3104 30.53

1.56 × 10-005 Ribulose 1,5-bisphosphate carboxylase-oxygenase large subunit (EC 4.1.1.39) [Fragment] 7.25 0.529 0.029 0.328 0.024 B1 3.19 × 10-006 Ribulose 1,5-bisphosphate carboxylase-oxygenase large subunit (EC 4.1.1.39) [Fragment] 5.42 0.000 0.000 0.441 0.062 B12 1.49 × 10-006 Ribulose 1,5-bisphosphate carboxylase-oxygenase large subunit (EC 4.1.1.39) [Fragment] 7.05 0.038 0.100 0.648 0.092 B12 3.94 × 10-006 Ribulose 1,5-bisphosphate carboxylase-oxygenase large subunit (EC 4.1.1.39) [Fragment])

7205 34.45

1001 22.77

Ribose-5-phosphate isomerase (EC 5.3.1.6)

16.1

Clermontia kakeana

trm|Q32015

11.0

26.9

trm|Q8WKJ1 22.8

Cajanus cajan

Coniogramme japonica trm|Q33311

trm|Q33420

18.9

trm|Q7XVP0 14.2

trm|Q8LPC3 19.7

spt|Q9M4S8 24.5

trm|O82428

Drypetes roxburghii

Oryza sativa

Physcomitrella patens

Fragaria x ananassa

1.98 × 10-006 Triosephosphate isomerase, chloroplast precursor (EC 5.3.1.1)

2.80 × 10-005 Plastidic aldolase (EC 4.1.2.13)

Datisca glomerata

1.35 × 10-004 Rubisco activase (EC 6.3.4.-) [Fragment]

7308 39.18

7509 44.76

4

6

5

9

6

12

5

2

7

2

7

15.1

27.6

Chloroplast: photosynthesis: carbohydrate pathways 1.47 × 10-007 Ribulose-5-phosphate-3-epimerase Arabidopsis thaliana trm|Q9SAU2 19.9 (EC 5.1.3.1)

spt|Q9S841

trm|Q6ZFJ3

1* 7

13

4

1*

6.5

7.24 0.558 0.056 0.400 0.040 B1

16.9

trm|Q7M1Y9 58.3 trm|O23254 18.7

spt|P12112

10.6

covf UniPepg

trm|Q7M1Y9 62.7

5015 18.97

1.002 0.064 0.559 0.081 B1

6.06 0.833 0.105 0.502 0.034 B1

Pinus monticola

6.61 0.533 0.111 1.318 0.076 B12 7.86 × 10-006 Photosystem II PsbP protein [Fragment]

5004 25.26

3405 42.21

Arabidopsis thaliana

5.59 0.000 0.000 0.596 0.062 B12 2.38 × 10-007 Photosystem II MSP protein [Precursor]

1208 33.27

5.85 1.234 0.057 0.724 0.066 B1

Oryza sativa

7.22 0.000 0.000 0.875 0.041 B12 2.16 × 10-009 Ferredoxin-NADP(H) oxidoreductase (EC 1.18.1.2)

7212 36.09

Pinus monticola Arabidopsis thaliana

2.28 × 10-006 Photosystem II PsbP protein 1.64 × 10-005 Serine hydroxymethyltransferase (EC 2.1.2.1)

7.31 0.531 0.041 0.282 0.014 B1 7.45 0.499 0.057 0.292 0.042 B1

Triticum aestivum

7.31 × 10-006 ATP synthase alpha chain (EC 3.6.3.14)

8803 76.54 8605 51.87

2108 30.24

accessione

Chloroplast: photosynthesis: electron transfer chain 5.11 × 10-007 Apocytochrome F [Precursor] Zea mays spt|P46617

p-valued

7.29 0.499 0.028 0.328 0.019 B1

mean SD (() mean SD (() OAc

8602 50.37

pI

B12b

7.19 0.375 0.044 0.000 0.000 B1

Mr

B1b

7109 28.84

SSP

experimental

Table 4. Simplified List of Identified Proteins that are Differentially Expressed between Immature (B1) and Mature (B12) Needlesa

LTYYTPEYQTKDTDILAAFR TFQGPPHGIQVER RLRLEDLR AVYECLR

LTYYTPEYKTKDTDILAAFR QRNHGIHFR

LTYYTPQYQTKDTDILAAFR TVWTDGLTSLDRYK

GVNPWIEVDGGVTPANAYK DYAEAIKGIK VPDFIK IGVCTGIFR SFQCELVFAK HVIGEDDQFIGK VIACIGEKLEER VASPQQAQEVHVAVR TFDVCYEQLK RHVIGEDDQFIGKK FFVGGNWK GILAIDESNATCGK LASIGLENNEVNR GSGGRFVVIVDESK PELNLVK TFQGPPHGIQVER TSIVGNVFGFKALR DDENVNSQPFMRWR

QVIDIIPPGPELLFSEGESIK IGPVPGKI VVNALAKPIDGK QTGKTAVATDTILNQK EAYPGDVFYLHSR AYGEAANVFGAPK VCDLCNITINKNAVYGDSSALSPGGVR ALDFRPKLLICGGSAYPR DFEDKINFAVFPALQGGPHNHQIGALAVALK DNTYVYMCGLKGMEK LVYTNDQGEIVKGVK GSSFLDPKGRGGSTGYDNAVALPAGGR VPFLFTVK AYGEAANVFGAPKK

high scoring peptidesh


research articles

Journal of Proteome Research • Vol. 9, No. 8, 2010 3961

3962

Mr

32.65

29.44

32.95

25.27

26.95

29.39

33.88

28.13

35.29

46.5

47.71

SSP

4106

4105

6104

2007

5007


5107

6204

5005

6313

2503

4603

6.28

5.68

6.84

6.63

6.93

6.65

6.71

5.85

6.76

6.27

6.28

pI

experimental

Table 4. Continued

0.000

0.597

0.000

0.000

0.000

0.000

0.043

0.501

0.000

0.000

0.000

mean

0.000

0.104

0.000

0.000

0.000

0.000

0.114

0.031

0.000

0.000

0.000

SD (()

B1b

0.342

1.253

0.333

0.406

0.880

0.400

1.174

1.776

0.837

0.359

0.528

mean

0.040

0.117

0.039

0.061

0.134

0.068

0.132

0.222

0.122

0.038

0.042

SD (()

B12b

B12

B12

B12

B12

B12

B12

B12

B12

B12

B12

B12

OAc

Ribulose 1,5-bisphosphate carboxylase-oxygenase large subunit (EC 4.1.1.39) [Fragment] Ribulose 1,5-bisphosphate carboxylase-oxygenase large subunit (EC 4.1.1.39) [Fragment] Ribulose 1,5-bisphosphate carboxylase-oxygenase small subunit (EC 4.1.1.39) [Fragment, putative] Plastidic aldolase (EC 4.1.2.13) Rubisco activase (EC 6.3.4.-)

Transketolase-like protein (EC 2.2.1.1)

4.32 × 10-006

2.16 × 10-006

3.83 × 10-005

5.01 × 10-007

4.71 × 10-007

2.31 × 10-006

5.86 × 10-007

Ribulose 1,5-bisphosphate carboxylase-oxygenase large subunit (EC 4.1.1.39) [Precursor] Ribulose 1,5-bisphosphate carboxylase-oxygenase large subunit (EC 4.1.1.39) [Fragment]

Ribulose 1,5-bisphosphate carboxylase-oxygenase large subunit (EC 4.1.1.39) [Fragment] Ribulose 1,5-bisphosphate carboxylase-oxygenase large subunit (EC 4.1.1.39) [Fragment] Ribulose 1,5-bisphosphate carboxylase-oxygenase large subunit (EC 4.1.1.39) [Fragment]

protein

6.09 × 10-006

1.80 × 10-006

2.71 × 10-007

5.01 × 10

-008

p-valued


Pinus halepensis

Physcomitrella patens

Pinus pinaster

Limonium cesium

Juniperus virginiana

Batis maritima

Pinus radiata

Declieuxia fruticosa

Ensete ventricosum

Rothmannia longifera

reference organism

trm|Q9LZY8

trm|Q9M431

trm|Q8LPC3

trm|Q8RVI7

trm|O47057

trm|Q9MVE8

trm|Q39288

spt|P24679

trm|Q9ZS74

trm|Q32225

trm|P94043

accessione

13.3

34.5

19.7

41.2

16.0

31.4

53.7

21.3

22.5

13.2

5.1

covf

9

6

2

4

5

10

10

1*

10

4

2

UniPepg

GILAIDESNATCGK LASIGLENNEVNR LIGINPIMMSAGELESGDAGEPAK MCVLFINDLDAGAGR SFQCELVFAK VPLILGIWGGK YREAADIVK FLAIDAVEK ILYDEVMR NGNTGYDEIR VTTTIGYGSPNK

LGLSARNYGR MSGGDHIHAGTVVGK GLLLHIHR GGLDFTKDDENVNSQPFMR FETLSYLPQLTEEQLVKEVEYLLR VIGFDNVR

PLLGCTIKPK ALRLEDLR LTYYTPEYQTK DTDILAAFR LEDLRIPPAYSK TFKGPPHGIQVER YGRPLLGCTIK VALEACVQAR EGNEVIREATKWSPELAAACEVWKGIK

LTYYTPEYKTKDTDILAAFR TFQGPPHGIQVER DDENVNSQPFMR DNGLLLHIHR MSGGDHIHAGTVVGK VALEACVKAR MAKNARRPPNLNVPK

DDENLNSQPFMRWRDRFLFCAEAIYK MSGGDHIHAGTVVGK VALEACVQAR

VALEACVKAR


research articles Valledor et al.

39.24

5409

13.29

39.24

5409

5003

29.63

7105

29.59

69.35

4804

0109

43.72 39.06

8501 6312

38.41

52.63

3609

8304

55.2

6711

37.43

13.41

3006

8302

Mr

SSP

6.56

5.28

7.41

7.3

6.57

6.57

7.21

6.28

7.32 6.97

6.18

6.96

6.05

pI

experimental

Table 4. Continued

0.630

0.000

0.842

0.264

0.536

0.536

0.311

0.606

0.626 1.012

0.932

0.527

0.000

mean

0.212

0.000

0.124

0.034

0.061

0.061

0.031

0.059

0.029 0.117

0.089

0.031

0.000

SD (()

B1b

2.144

0.342

0.413

0.693

0.000

0.000

0.838

0.462

0.349 0.504

0.654

0.319

0.947

mean

0.103

0.033

0.033

0.037

0.000

0.000

0.065

0.037

0.017 0.031

0.052

0.077

0.042

SD (()

B12b

B12

B12

B1

B12

B1

B1

B12

B1

B1 B1

B1

B1

B12

OAc

Triosephosphate isomerase (EC 5.3.1.1)

protein

Fragaria x ananassa

reference organism

Enolase (EC 4.2.1.11)

Enolase (EC 4.2.1.11) Fructose 1,6-bisphosphate aldolase (EC 4.1.2.13) Phosphoglycerate mutase (EC 5.4.2.1) Carbonic anhydrase (EC 4.2.1.1)

1.29 × 10-006

3.72 × 10-007 7.25 × 10-006 1.00 × 10-004 5.23 × 10-007

Oryza sativa

Solanum tuberosum

Arabidopsis thaliana Avena sativa


Spinacia oleracea

dihydrolipoamide succinyltransferase [Putative]

Malate dehydrogenase (EC 1.1.1.37) [Precursor]

4.27 × 10-007

9.35 × 10-007


Oryza sativa

Medicago sativa

NADH-ubiquinone oxidoreductase chain 1 (EC 1.6.5.3) [Fragment] Soluble inorganic pyrophosphatase (EC 3.6.1.1)

1.65 × 10-007 2.57 × 10-006

K+ channel protein

3.12 × 10-005

Solanum tuberosum

Glycine max


Mitochondria: electron transfer (oxidative phosphorilation)

Malate dehydrogenase (EC 1.1.1.37) [Precursor]

4.27 × 10-007

Mitochondria: piruvate pathways and tricarboxylic acic cycle

6-phosphogluconate dehydrogenase (EC 1.1.1.44)

5.09 × 10-005

Cytosol: glycolysis and other carbon metabolism enzymes

1.50 × 10-009

p-valued

spt|Q43187

trm|Q37475

trm|Q39151

trm|Q93ZA7

trm|Q7XVM2

trm|O48906

trm|Q69MC9

trm|Q9XE59

trm|Q9ZW34 trm|Q9LLD7

trm|Q8RWM8

trm|Q94KU1

spt|Q9M4S8

accessione

22.7

17.1

19.2

22.9

18.9

14.0

4.9

12.3

10.1 27.1

10.4

14.5

15.6

covf

6

2

7

3

13

6

2

3

7 2

6

6

5

UniPepg

IIAVCADDPEYR TGLIKVDR FFEDYK

AEEIMGQAIR IFWGGPGPNDK SPLASGVLTGK FALENYK SLVDDVLR YDQLMGLGRK

ALEALKPELKASIEK IQNAGTEVVEAK FVESSLR PMMYVALTYDHR LGLMSGFIK GLVVPVIR MNFAEIEK PIINP INAGIVKTLCEGVAK IQNGGTEVVEAK

VAAQVDSGPCVTYIGK ICSYAQGMNLIR ERLPANLVQAQR GFPISVYNR VIIMLVK VQIVGDDLLVTNPK IIGPALIGK SCNALLLK YNQLLR SCNALLLK GILAIDESNATCGK RLASIGLENNEVNR YAGMLQYDGELK MKALEIAER FMVVACADSR YMVFACSDSR

FFVGGNWK HVIGEDDQFIGKK VIACIGEK TFDVCYEQLK



research articles


3964 1.53 × 10

Alpha-1,4-glucan-protein synthase (EC 2.4.1.186)


ATLPSQMIEKGQNRVVEASLTLIR VLSDAGDVPIQEMR FGVEQYEMHSFSK DTVDGMTAMVAAK LNRPTRLNALSPDAFAEIPRAMALLDR

9

7.33 0.344 0.049 0.000 0.000 B1

6.06 0.833 0.105 0.502 0.034 B1

5.95 0.830 0.036 0.370 0.017 B1

6.07 0.450 0.040 0.000 0.000 B1

6.41 0.646 0.064 0.412 0.026 B1

6.74 0.479 0.033 0.000 0.000 B1

6.43 0.000 0.000 0.386 0.049 B12 7.70 × 10-007 Glutamine synthetase cytosolic isozyme (EC 6.3.1.2)

8810 62.23

3405 42.21

3601 48.81

3611 47.9

4610 51.18

6612 49.77

4204 35.28

Oryza sativa

2.08 × 10-008 S-adenosylmethionine synthetase (EC 2.5.1.6)

Pinus sylvestris

Pinus contorta

3.13 × 10-005 S-adenosylmethionine synthetase 2 (EC 2.5.1.6)

8.2

spt|P52783

19.3

trm|QA6VZ2 17.9

trm|Q9FVG7 20.4

Arabidopsis thaliana trm|Q9SJL8

9.83 × 10-008 S-adenosylmethionine synthetase (EC 2.5.1.6) [Putative]

trm|Q9FVG7 33.1

trm|Q9ZS52 24.5

Pinus sylvestris

Pinus contorta

trm|Q8RUL6 10.7

Oryza sativa

8.89 × 10-008 S-adenosylmethionine synthetase 2 (EC 2.5.1.6)

1.57 × 10-006 Enoyl CoA hydratase-like (EC 4.2.1.74) 1.35 × 10-004 Glutamine synthetase (EC 6.3.1.2)

6.57 0.536 0.061 0.000 0.000 B1

5409 39.24

7

5

6

3

9

3

EHIAAYGEGNER TLSHPVTDPKDLPK SMREEGGIKVIK YLDENTIFHLNPSGRFVIGGPHGDAGLTGR TNMVMVFGEITTKADVDYEQIVRK TQVTIEYRNEGGAMVPER EIGFISDDVGLDADHCK SIVAAGLAR FVIGGPHGDAGLTGR TQVTVEYK SVVAAGLAR NGTCPWLRPDGKTQVTIEYR FVIGGPHGDAGLTGR ADVDYEQIVRK SIVAAGLAR TNMVMVFGEITTKANVDYEKIVR FVIGGPHGDAGLTGR TQVTVEYR SLSGPVSSVK AGVVLSFDPK EGGFEVIK

GGFYGNVFR AAYDIVR

6

7.25 0.357 0.031 0.000 0.000 B1

8610 47.7

2

QIVGGTLKDNDDSLETNFVSK

6.87 0.648 0.078 0.290 0.021 B1

6307 37.57

Aminoacid and nitrogen metabolism 1*

GVAVEDSSSPHGVR TTLGGSAEYPYPR NGDNILTNR ELLVGKDDEILKTETK IGGAADVFVGDIR

Candida glabrata spt|Q6FPN5 8.0 1.12 × 10-005 1-(5-phosphoribosyl)-5[(5-phosphoribosylamino) methylideneamino] imidazole-4-carboxamide isomerase (EC 5.3.1.16) 7.89 × 10-008 Alanine--glyoxylate aminotransferase Arabidopsis thaliana trm|Q9SR86 7.5 2 homologue 3 (EC 2.6.1.44) [Precursor, mitochondrial] Pinus taeda trm|Q9AY33 26.4 4.27 × 10-007 Arginase (EC 3.5.3.1)

3

VICDHLGLGVK DINALEQHIK


2

Oryza sativa

Solanum tuberosum trm|Q9SAP1 10.4

2

covf UniPepg

trm|Q94HJ5 26.3

Lipoxygenase (EC 1.13.11.12)

accessione

Solanum tuberosum spt|Q8RU27 12.6

Lipid metabolism

6.68 0.638 0.039 0.907 0.062 B12 9.00 × 10-005 3-beta hydroxysteroid dehydrogenase/isomerase protein (EC 1.1.1.-) [Putative]

-004

1.35 × 10

reference organism

Cell wall biosynthesis

protein

5108 31.91

-004

p-valued

5.68 0.681 0.059 0.447 0.034 B1


2806 72.75

pI

B12b

6.06 0.833 0.105 0.502 0.034 B1

Mr

B1b

3405 42.21

SSP

experimental

Table 4. Continued


47.7

59.61 16.26

29.59

8610

7707 3005

0109

28.84

7109

76.54

36.71

4203

8803

41.66

8418

34.93

39.18

7308

3204

37.6

4308

45.3

11.38

4013

7501

33.54

1201

51.87

42.36

5406

8605

Mr

SSP

5.28

7.13 6.03

7.25

7.31

6.11

6.99

7.45

7.19

6.34

7.38

7.24

6.49

6.22

5.42

6.68

pI

experimental

0.000

0.421 1.185

0.357

0.531

0.408

0.627

0.499

0.375

0.555

0.628

0.558

0.644

0.545

0.000

0.617

0.000

0.051 0.100

0.031

0.041

0.049

0.050

0.057

0.044

0.071

0.079

0.056

0.044

0.128

0.000

0.041

SD (()

B1b

mean

Table 4. Continued

0.342

0.265 0.658

0.000

0.282

0.000

0.310

0.292

0.000

0.299

0.000

0.400

0.445

0.000

0.529

0.991

mean

0.033

0.032 0.054

0.000

0.014

0.000

0.022

0.042

0.000

0.028

0.000

0.040

0.022

0.000

0.081

0.123

SD (()

B12b

B12

B1 B1

B1

B1

B1

B1

B1

B1

B1

B1

B1

B1

B1

B12

B12

OAc

Nuclear transcription factor Y subunit C-1 Translation elongation factor 2 (eEF2) NGATHA3 transcription factor WRKY transcription factor 1 Regulator of chromosome condensation (RCC1) protein family

Ethylene-responsive transcription factor 7

5.39 × 10-007 2.28 × 10-006 7.89 × 10-008 3.76 × 10-005 1.45 × 10-005

1.65 × 10-007

1.64 × 10

5.11 × 10 -005

-007

Maturase K

Cytosine-5 methyltransferase (EC 2.1.1.37) DNA methyltransferase 101 (EC 2.1.1.37)

1.11 × 10-006

7.76 × 10

Vigna unguiculata

Oryza sativa


Arabidopsis thaliana Picea glauca


Guillardia theta


Stangeria eriopus

Peperomia caperata


Beta vulgaris

Zea mays


Transcription and DNA replication

Aminoimidazolecarboximide ribonucleotide transformylase/ inosine monophosphate cyclohydrolase (EC 2.1.2.3)

Trad-like protein

DNA-directed RNA polymerase (EC 2.7.7.6) DNA-directed RNA polymerase, subunit B (EC 2.7.7.6) FRUITFULL-like MADS-box (Fragment)

-007

1.56 × 10


Pinus sylvestris

reference organism

Nucleotide metabolism

Acetolactate synthase (EC 2.2.1.6)

Glutamine synthetase cytosolic isozyme (EC 6.3.1.2)

protein

3.39 × 10-005

-005

1.46 × 10-005

2.85 × 10

-005

2.42 × 10-006

3.47 × 10

-004

p-valued

spt|Q9LDE4

spt|Q9SI37 trm|Q9XEL4

trm|Q6DT80

trm|Q98S45

trm|Q9SMP0

trm|Q8MEX5

trm|Q7XBK9

trm|Q9LK40

trm|Q9MF58

trm|Q8LPU6

trm|Q9SEG3

trm|Q84XA4

trm|Q69KV4

trm|Q94B64

spt|P52783

accessione

3.3

3.7 67.1

15.2

5.2

12.8

4.4

10.5

7.1

6.0

2.6

2.4

8.5

5.5

3.1

48.7

covf

1*

3 6

4

2

2

5

3

6

3

8

4

4

2

2

5

UniPepg

SLLEQNSVMIK LILL ESSIIALR LSVK PPMGQPAGPGGMMIGRPAMDP NDIAAAITR IMEKALEGIKISK ILLK GQMGAASSSSALR LPPL TKTNGFEK ASADVEFRCFVGGLAWSTDDRSLQEAFS PYGEVVESK GFGFVTFNDEQSMR SITVNPAQSR GYRGGGGGGGYGGSR YGGGGSEGGSWR NLRGPKAK

IKVNVQVEVFKNTVVK

DTVGDLPK TSLKI MSVRV

ISLPR

SMMISVAMILK SPSR KAGDDVKGAALASDAFFPFAWK ILEAKKNEPGKLSLRQVGGGW

AGVVLSFDPK EHISAYGEGNER DIVDAHYK EGGFEVIKK SLSGPVSSVKELPK EGGFEVIKK SLSGPVSSVKELPK EGGFEVIKK SLSGPVSSVKELPK EGGFEVIKK SLSGPVSSVKELPK EGGFEVIK



research articles


3966

6.22

5.91

6.07

5.58

5.77 6.31

7.13

6.97

7.24

7.18

6.3

5.5

6.33

6.74

4001 25.29

3011 21.83

3611 47.9

1016 20.74

2006 22.44 4108 28.51

7707 59.61

6312 39.06

7308 39.18

7806 67.29

4107 30.13

1604 52.1

4605 50.9

6612 49.77

pI

7.33

Mr

8202 36.61

SSP

experimental

B1b

B12b


0.479

0.614

0.600

0.617

0.322

0.558

1.012

0.421

0.819 0.684

0.641

0.450

0.487

0.000

0.000

0.033

0.046

0.034

0.037

0.026

0.056

0.117

0.051

0.062 0.031

0.077

0.040

0.066

0.000

0.000

0.000

0.324

0.344

0.383

0.191

0.400

0.504

0.265

0.512 0.536

0.301

0.000

0.000

0.524

0.498

0.000

0.035

0.021

0.048

0.008

0.040

0.031

0.032

0.063 0.028

0.030

0.000

0.000

0.104

0.088

Maturase K

protein

B1

B1

B1

B1

B1

B1

B1

B1

B1 B1

B1

B1

B1

Arabidopsis thaliana Picea glauca

Capsella rubella

reference organism

Eukaryotic translation initiation factor 5A-2 30S ribosomal protein S14 [chloroplastic] 40S ribosomal protein S12 Type I protein geranylgeranyl transferase beta subunit (EC 2.5.1.59) 40S ribosomal protein S3 Chaperonin 21 [Chloroplastic] Chaperonin, delta-subunit [Cytosolic] spt|P06370

spt|Q9AXQ5

trm|Q9LJX4 trm|Q9XEL4

trm|Q9GF58

accessione

Brassica rapa


Oryza sativa

2.77 × 10-006 26S proteasome regulatory subunit 6A [Homologue]

2.25 × 10-006 26S proteasome regulatory subunit 6B [Homologue]

2.08 × 10-008 26S proteasome regulatory subunit 7

Oryza sativa


Oryza sativa

Saccharomyces cerevisiae

Glycine max

Neurospora crassa Vitis vinifera

trm|Q6K8W1

spt|Q9SEI4

spt|O23894

trm|Q6H852

23.2

30.6

22.6

28.1

14.6

8.0

trm|Q7 × 672 trm|Q9ZU75

9.3

15.9

11.0 13.5

42.0 12.1

17.0

13.8

12.7 25.2

20.6

7

9

8

3

4

6

3

10

1* 2

4 3

1*

2

8 4

6

covf UniPepg

spt|P40087

trm|Q9ZRX1

trm|Q7RV52 trm|Q6B4 V4

Hordeum vulgare spt|Q9XHS0 Schizosaccharomyces pombe spt|P32434

Marchantia polymorpha

Lycopersicon esculentum

Protein translation, folding, modification and degradation

7.25 × 10-006 DNA-damage inducible protein DDI1 (v-SNARE-master 1) 1.56 × 10-005 Calcium dependent protein kinase [Putative] 6.25 × 10-006 RUB1-conjugating enzyme [putative ] 2.05 × 10-005 20S proteasome, R2 subunit

3.76 × 10-005

1.98 × 10-006 2.06 × 10-004

1.88 × 10-005

9.83 × 10

-008

1.17 × 10

-006

RNA binding protein-like B12 1.08 × 10-005 Cold, Circadian Rhythm and RNA binding 2

-006

p-valued

B12 5.53 × 10


Table 4. Continued

AANGVVIATEKKLPSILVDETSVQK QAQQYYRLYKETIPVTQLVRI STDDFNGAQLKAVCVEAGMLALR RFDSEVSGDREVQR GVLLYGPPGTGK ACAAQTNATFLK ADILDPALMR LLENEIR AVANHTTAAFIRVVGSEFVQK ILSTINRELLKPSASVALHR QIGIDPPRGVLLYGPPGTGK FDAQTGADREVQR LVFQVCTSK LCPNSTGADIRSVCTEAGMYAIR AVANRTDACFIRVIGSELVQK FDDGVGGDNEVQR TYGLGPYSTSIK GVLCYGPPGTGK VSPTDIEEGMR

EESQSNNGRGASTVK

RRMTAAQALSHPWIR

MLVELSK ATGCNVLLIQK GSNQLVLDEAER FLIAGGGAPEIELSR IDDIVTVR TGMGPTPTGRSTAGATTATGRTFPEQTIK

AAQVQAAAEAARQEEQAGEEEAAAPAAEE TAGGLLLTEASK

VVGCSCIVVK DLIKNFVELCKTSQGHFR

HLLREMAHACLLPGVTK

TFPQQAGTIRKNGYIVIK

SSHFRSTSYQVLFER VNINQLSKDNLEFLGYLSSL LELSDIAGR ASADVEFR SITVNPAQSR



2108 30.24

6010 17.52 5021 13.45

5.85 1.234 0.057 0.724 0.066 B1

1.47 × 10

-007

Oryza sativa Thermomyces lanuginosus

Zea mays

Ascorbate peroxidase (EC 1.11.1.11)

Pinus pinaster

8.4

23.5 42.2

trm|Q6RY58 59.4

trm|Q75M06 4.6 spt|P10365 25.6

trm|Q93XF4

Schizosaccharomyces pombe spt|P10989 Picea rubens trm|Q9SPI7

Cytoskeleton

Stress and defense related proteins

1.11 × 10-008 Kinesin heavy chain [Fragment] 6.89 0.480 0.049 0.204 0.016 B1 1.35 × 10-005 Tubulin gamma-2 chain 6.48 0.447 0.200 0.947 0.082 B12 1.20 × 10-003 Actin

6.03 0.758 0.047 0.000 0.000 B1

3009 23.38

3.84 × 10 Actin 3.40 × 10-007 Actin

5.47 0.499 0.033 0.317 0.013 B1 5.65 0.429 0.035 0.275 0.029 B1

-006

trm|Q9M524 25.1

Tsuga heterophylla

6.41 × 10-006 Phenylcoumaran benzylic ether reductase TP5 (EC 1.1.1.-) [Homologue]

9.7

12.4

8.5

trm|Q9LL41

Secondary metabolism


spt|O48551

trm|Q6R1M9 11.7

3.39 × 10-005 Phenylcoumaran benzylic ether Pinus taeda reductase PT1 (EC 1.1.1.-)

1603 52.4 2501 44.56

7.63 0.696 0.055 0.411 0.034 B1

6.84 0.067 0.116 0.741 0.032 B12 1.34 × 10-006 ATP-dependent Clp protease proteolytic subunit ClpR4 [Putative]

6107 29.84

8207 37.27

5.72 0.327 0.035 0.458 0.035 B12 3.47 × 10-004 Cell division protease Arabidopsis thaliana ftsH homologue 2 (EC 3.4.24.-) [Cholorplastic precursor]

2810 68.68

6.34 0.555 0.071 0.299 0.028 B1

trm|Q8LB10 11.1

Glycine max

6.27 0.000 0.000 0.359 0.038 B12 2.71 × 10-007 Proteasome subunit R6 (EC 3.4.25.1)

4105 29.44

4203 36.71

trm|O80860

Brassica napus

trm|Q8RXD6 22.6

trm|Q9FXA4 25.4

6.46 0.000 0.000 0.442 0.066 B12 2.08 × 10-006 20S proteasome R1 subunit [Fragment]


4110 30.35

1.64 × 10-005 Plant U-Box 26

6

2 5

5

8 12

4

4

2

5

2

2

12

7

4

covf UniPepg

trm|Q8LD27 19.7

accessione



reference organism

5.58 0.356 0.028 0.669 0.021 B12 3.87 × 10-007 E3 ubiquitin-protein ligase BRE1-Like 1(EC 2.6.2.-)

PBA1 Endopeptidase 20S proteasome subunit R (EC 3.4.25.1) [Precursor]

protein

1406 42.27

6.23 × 10

-006

p-valued

7.45 0.499 0.057 0.292 0.042 B1


8605 51.87

pI

B12b

6.53 0.636 0.023 0.464 0.034 B1

Mr

B1b

5002 28.26

SSP

experimental

Table 4. Continued

EGLLQLPSDKALLADPSFAVYVQKYAQDEDA FFADYAEAHLKLSELGFADA TGGPFGTMRYGAELAHGANSGLDIAVRLLEPIK NCAPIMVRIAWHSAGTYDVK VKAYPTVSEEYKAAIDK EIVALSGAHTLGR LPDATKGPDHLR

NIMVSSYARNKEASQAK AGFAGDDAPR

DLYGNVVMSGGTTMYPGIADR AGFAGDDAPRAVFPSIVGR AEYDESGPSIVHR DAYVGDEAQSKR EITALAPSSMK GYSFTTTAER KPMNTNDNQTENVNLDK

YTTVDEYLSN AQLLESFK FLPSEFGNDCDR KVDVVISAVK TYLSEEEVLKK VDVVISAVK

SGSAADSQVVSDYVR TVIINSEGVTR TSTGMYVANR AVSLAIAR LAADFDR LLLL RHFSSISPSEAAAAVK ADIGNGLEQARSR ADVQSLSGVLCRK QRDLQDMETVLK MLGKMSNLQNK SGSAADSQVVSDYVR TVIINSEGVTR AAGITSIGVR ATEIEVGVVRK IVAGMEGTVMTDGK QVSVDVPDVK FLEYLDKDR GQATDVEIAR



research articles


3968

Mr

50.37

76.94

72.75

61.91

70.15

51.69 16.26

28.34

17.45

30.31

17.64

17.64

17.64

17.64

17.64

22.44

41.43

SSP

8602

1810

2806

3809

5808


8607 3005

6001

0009

1103

1012

1012

1012

1012

1012

2006

2405

5.76

5.77

5.42

5.42

5.42

5.42

5.42

5.47

5.28

6.8

7.69 6.03

6.62

6.19

5.68

5.57

7.29

pI

experimental

Table 4. Continued

0.336

0.819

0.000

0.000

0.000

0.000

0.000

0.359

0.000

0.555

0.714 1.185

0.664

0.942

0.681

0.878

0.499

mean

0.036

0.062

0.000

0.000

0.000

0.000

0.000

0.050

0.000

0.040

0.067 0.100

0.048

0.099

0.059

0.074

0.028

SD (()

B1b

0.627

0.512

0.453

0.453

0.453

0.453

0.453

0.446

0.843

0.353

0.332 0.658

0.365

0.430

0.447

0.515

0.328

mean

0.028

0.063

0.108

0.108

0.108

0.108

0.108

0.031

0.141

0.036

0.040 0.054

0.028

0.044

0.034

0.035

0.019

SD (()

B12b

B12

B1

B12

B12

B12

B12

B12

B12

B12

B1

B1 B1

B1

B1

B1

B1

B1

OAc

70 kDa Heat shock protein 70 like

60 kDa Heat shock protein 60 [Fragment]

Stress-induced protein sti1-like

LEA protein Tau class glutathione S-transferase (EC 2.5.1.18) Pathogenesis related protein [Putative] Ascorbate peroxidase (EC 1.11.1.11) Dehydroascorbate reductase (EC 1.8.5.1) Dehydroascorbate reductase (EC 1.8.5.1) [Putative] Dehydroascorbate reductase (EC 1.8.5.1) [Putative] Dehydroascorbate reductase (EC 1.8.5.1) [Putative] Dehydroascorbate reductase (EC 1.8.5.1) [Putative] Dehydroascorbate reductase (EC 1.8.5.1) [Putative] Universal Stress Protein Family Putative oxidoreductase, zinc-binding

1.53 × 10-004

8.94 × 10-006

8.75 × 10-006

6.40 × 10-006 1.45 × 10-005

4.41 × 10-005

5.19 × 10-003 3.19 × 10-005 3.19 × 10-005 3.19 × 10-005 3.19 × 10-005 3.19 × 10-005 1.98 × 10-006 1.24 × 10-006

3.98 × 10-006

70 kDa Heat shock cognate 70 kDa

F5M15.5

protein

2.35 × 10-005

7.31 × 10

-006

p-valued

Oryza sativa


Oryza sativa

Oryza sativa

Oryza sativa

Oryza sativa

Oryza sativa

Brassica juncea

Pinus pinaster

Oryza sativa

Bromus inermis Pinus tabuliformis


Prunus dulcis


Petunia x hybrida


reference organism

trm|Q7EYM8

trm|Q9M328

trm|Q67UK9

trm|Q67UK9

trm|Q67UK9

trm|Q67UK9

trm|Q67UK9

trm|Q8LJP9

trm|Q6RY58

trm|Q7XW56

trm|Q6L5L9 trm|Q6DNI8

trm|Q9STH1

trm|Q8H6U4

trm|Q9SZJ3

spt|P09189

gb|AAF79625

accessione

23.3

15.0

16.9

16.9

16.9

16.9

16.9

17.1

28.5

9.9

34.1 13.2

12.5

25.1

29.6

17.1

14.6

covf

5

3

4

4

4

4

4

2

5

1*

4 4

5

14

9

8

22

UniPepg

EGLLQLPSDKALLADPSFAVYVQK TGGPFGTMR ISAADLSLAPK YPEPPLATPPEK ISAADLSLAPK VLLTIEEK ISAADLSLAPK VLLTIEEK ISAADLSLAPK VLLTIEEK ISAADLSLAPK VLLTIEEK ISAADLSLAPK VLLTIEEK LDSIVMGSR LYWGDAR ATGKVVISPIP FVVTSDGSVLEK VAAAALNPVDAKR

GPGVQTPVIVR APGVQTPVIVRF DAGVISGLNVMRIINEPTAAAIAYGLDK NQVAMNPTNTVFDAKR ATAGDTHLGGEDFDNR NAVVTVPAYFNDSQR TTPSYVAFTDSER IINEPTAAALSYGMNNK EVDEVLLVGGMTR DKATGKEQNITIR NSADTTIYSVEK VQEIVSEIFGK NVVLEQSFGAPK SVAAGMNAMDLR VGKEGVITIADGK ISGGVAVLK VTDALNATK AGIIDPLK NPNNLNLYMKDKR DFEPAIETFQK LVSAGIVQVR CIELDPSFTK AGQTTEATK VLVGLEEK IPLETQFPR VAEFAMQIR RAVGVAPLAWSAGIARYAK



6.46

7.21

5.41

7.38

5.98 6.56

7.21

7.29

5.89

6.88 7.13

6.5

5.88

pI

B12b

0.000

0.390

0.503

0.628

0.459 0.630

0.390

0.499

0.512

0.515 0.421

1.002

0.368

0.000

0.045

0.058

0.079

0.086 0.212

0.045

0.028

0.084

0.031 0.051

0.064

0.036

0.442

0.833

0.320

0.000

0.000 2.144

0.833

0.328

0.000

0.307 0.265

0.559

0.431

0.066

0.064

0.043

0.000

0.000 0.103

0.064

0.019

0.000

0.022 0.032

0.081

0.028

Plantago major Arabidopsis thaliana

-006

Oryza sativa

B12 2.08 × 10-006 Nodulin MtN21 [Putative]

B1

Oryza sativa


B12 3.05 × 10-007 ATB2 oxidoreductase

Hypothetical protein At2g27280

Oryza sativa

7.76 × 10

Unknown biological process

1.99 × 10-005 Hypothetical protein B1097D05.32

B1

-007

Ustilago maydis Zea mays

Cell cycle B1 7.76 × 10 Cell cycle regulatory protein B12 2.57 × 10-006 Retinoblastoma-related p rotein

B1

trm|Q6Z7C3

trm|Q7XT99

trm|Q8LJ71

trm|Q8S8I5

10.9

28.5

4.7

10.6

6.6 10.6

30.3

spt|P40691

trm|Q8J214 trm|Q8H0J6

9.2

8.5

8.4 13.7

trm|Q6C775

trm|Q6C775

trm|Q5ZF60 trm|O64842

2

4

3

6

7 6

3

10

10

1* 2

17

5

covf UniPepg

trm|Q8W593 20.3

accessione

Aspergillus fumigatus trm|Q6MYL7 10.8

3.60 × 10-006 Histidine Kinase protein Yarrowia lipolytica family, wooden Leg, WOL. [Putative] B1 7.31 × 10-006 Histidine Kinase protein Yarrowia lipolytica family, wooden Leg, WOL. [Putative] B12 3.05 × 10-007 Auxin-induced protein PCNT115 Nicotiana tabacum

B1 B1

reference organism


Signaling GTPase activator protein, putative

Lactoylglutathione lyase (EC 4.4.1.5)

protein

5.33 × 10-006 Lectin-like protein 1 3.76 × 10-005 Root Specific Kinase 1, ARSK1 (EC 2.7.1.-)

2.80 × 10

B1

-005

B12 2.77 × 10

p-valued -002


B1b

SLEPLEAEQAVSEKEMGSDGTEERKSSIKEAA KEVPK IPYVEM VGDLRRVAVDAFAR LATGEPLR IKYIGLSEASASTIR DVEEDIIPTCR VPIEVTIGELKK KAAASGGGAR

EANAFNPFAK QTKMDLLLSPSSR

RLDIDCIDLYYQHR DVEEEIIPTCR

LNLNAWVVDSLEEAPLPEDR AVAR

RRKKKAGVPTSK SSGIV QGQAGGNYCVTAR RLSLSDISDPSSPMSVMDDLSHSFTSQK GFIDDKVKPGIEAQPVAVKALDLHGHQGHR LNLNAWVVDSLEEAPLPEDR

GPTPEPLCQVMLR ITREPGPLPGISTK ITACLDPDGWK


a Proteins were classified according to Kyoto Encyclopedia of Genes and Genomes. b Mean and SD of normalized spot volumes (% of total spot intensity) of B1 and B12 data sets. c Overaccumulated (OA) in B1 or B12 needles. d P-value resulted of applying two tailed t test to compare normalized each spot volumes of B1 and B12 data sets. e Accession number according to: Swiss-Prot (spt); trmEMBL (trm); GenBank (gb). f Percentage of sequence covered by identified unique peptides. g Number of unique peptides identified. h Description of the amino sequences of unique peptides with Protein Pilot scores above 5.00. i For single-peptide identification of proteins, the precursor charge and m/z, peptide scores and spectra are available as Supporting Information (Table S5 and Figures S4-S12).

30.35

30.06 13.29

3208 5003

4110

39.77

7307

39.77

50.37

8602

7307

21.4

3010

59.52

62.33 59.61

6804 7707

0704

18.97

5015

41.66

33.16

2208

8418

Mr

SSP

experimental

Table 4. Continued


research articles


research articles

Valledor et al.

Figure 5. Expression levels (% of total spot volumes in gels) of the different functional groups established after protein identification. Identified B1 and B12 proteins represented 59.93 and 58.36% of total spot volumes, respectively.

of the database searching results than those originally suggested by the software manufacturer. In this way, we increased the number of rejected identifications (decreasing the final number of identified proteins) but ensured the correctness of the identified ones. These proteins belong to a wide set of metabolic pathways (Table 4; Figure 4), being the main represented functional groups transcription, stress, energy (photosynthesis and oxidative phosphorylation), and protein metabolism pathways. The Paragon algorithm has been proven as effective in other non model plant systems.39,40 Transcriptomic Analysis. As it has been known, the dot blotting has not the sensibility of microarray or qPCR based analysis. The current macroarray has been effectively used to validate the SSH-based selection of genes, which drastically changes their expression during needle maturation, a purpose for which dot-blotting is a current methodology.41,42 The macroarray analyses disclosed 176 differentially expressed genes between immature and mature needles (Table 5). The largest number of sequences belonged to the functional groups of photosynthesis, protein metabolism and transport. But, for instance, by SSH and macroarray validation, transcription factors and cell fate proteins, which in general are rather difficult to detect by 2-DE, were also identified, complementing the results obtained by 2-DE. This combination of SSH-based transcriptomic and 2-DE proteomic analysis was revealed as a useful approach to obtain a broad range of proteins and transcripts in species with a very low database coverage like Pinus radiata with a total of 304 differentially expressed genes and proteins described (Figure 6). Combining Proteomics and Transcriptomics. Performing the expression analysis both, the mRNA and protein level provided an insight into the relationship between the timing of gene expression and protein function during development. 3970


The sequences of the cDNAs of the SSH libraries were BLASTed against the peptidic tags employed to identify the different protein species, finding 14 identities. On the one hand, this matching rate is similar to that observed in comparative proteomic and microarray-based studies comparing embryo development in rapeseed43 or wood formation in maritime pine,44 a fact that states the adequateness of the techniques employed in this study, but on the other hand, this rate is significantly lower than that obtained in fully sequenced species like rice.45 This difference can be explained by taking into account the sequence database limitations for P. radiata with only a small number of sequences available for the comparison with the obtained peptide tags. Furthermore, and due to the limitations of the employed proteomic approach, only a small fraction of protein species were detected, while SSH libraries are designed to enrich the sample with low abundance transcripts making it more difficult to find protein-transcript identities. The correlation between the changes of protein species accumulation to the corresponding mRNA expression during needle maturation was studied on the 14 protein-gene identities that were found (Figure 6). A positive correlation between the corresponding mRNA and protein levels was found for eEF, RCBL, RCBS, RCC1, HSP70, and RNA Pol II, demonstrating that a substantial proportion of changes in protein species accumulation are a consequence of variations in mRNA levels. Gygi et al.46 have proposed that for high abundance proteins the correlation between mRNA and protein accumulation level is stronger than for low abundance proteins. At this point it must be said that the presence of these proteins in multiple spots indicates a posttranscriptional effect that must also be considered because it may define the biological activities of these species.

research articles


Table 5. List of the 232 Contigs Isolated by SSH in which Significant Homologies with Sequences Available in Public Databases Were Founda clone PRAb

accessionc

Bp

homology accesiond

description

B1206_G9

Chloroplast: photosynthesis: electron transfer chain GO096109 286 Photosystem II Oxygen-evolving enhancer protein 3 GO096120 351 Photosystem II Oxygen-evolving enhancer protein 3 GO096140 771 Photosystem II core complex psbY protein GO096251 827 Photosystem II core complex psbY protein GO096272 405 Photosystem I reaction center, subunit XI GO096273 249 Photosystem I subunit A GO096274 1455 Photosystem II 44 kDa reaction center GO096276 258 Photosystem II 44 kDa reaction center protein (P6 protein) GO096278 646 Photosystem II 44 kDa reaction center protein (P6 protein) GO096279 120 Photosystem II 44 kDa reaction center protein (P6 protein) GO096290 831 Photosystem II D2 (psbD) and photosystem II CP43 (psbC) proteins GO096282 439 Photosystem II CP43 protein (psbC) GO096280 654 Photosystem II D2 (psbD) and photosystem II CP43 (psbC) proteins GO096281 659 Photosystem II D2 (psbD) and photosystem II CP43 (psbC) proteins GO096283 681 Photosystem II D2 (psbD) and photosystem II CP43 (psbC) proteins GO096284 685 Photosystem II D2 (psbD) and photosystem II CP43 (psbC) proteins GO096285 742 Photosystem II D2 (psbD) and photosystem II CP43 (psbC) proteins GO096286 776 Photosystem II D2 (psbD) and photosystem II CP43 (psbC) proteins GO096287 720 Photosystem II D2 (psbD) and photosystem II CP43 (psbC) proteins GO096288 585 Photosystem II D2 (psbD) and photosystem II CP43 (psbC) proteins GO096289 798 Photosystem II D2 (psbD) and photosystem II CP43 (psbC) proteins GO096291 749 Photosystem II D2 (psbD) and photosystem II CP43 (psbC) proteins GO096292 797 Photosystem II D2 (psbD) and photosystem II CP43 (psbC) proteins GO096293 755 Photosystem II D2 (psbD) and photosystem II CP43 (psbC) proteins GO096294 781 Photosystem II D2 (psbD) and photosystem II CP43 (psbC) proteins GO096295 618 Photosystem II D2 (psbD) and photosystem II CP43 (psbC) proteins GO096297 657 Photosystem II Oxygen-evolving enhancer protein 3-1, precursor GO096298 286 Photosystem II Oxygen-evolving enhancer protein 3-1, precursor GO096299 391 Photosystem II Oxygen-evolving enhancer protein 3-1, precursor GO096300 366 Photosystem II Oxygen-evolving enhancer protein 3-2, precursor GO096301 662 Photosystem II Oxygen-evolving enhancer protein 3-2, precursor GO096311 398 Type II Chlorophyll A-B binding protein precursor

B01DG_03 B1205_D10 B1206_H11 B1204_F9 B12DG_01

GO270978 GO096266 GO096268 GO096305 GO270973

B0102_H2 B0103_D6 B0104_D10 B0106_H6 B1205_F1 B1206_G4 B1205_D1 B1205_B1 B1204_D2 B1205_B7 B1202_D12 B1201_E3 B1203_C7 B1205_G8 B1205_E4 B1206_A12 B1203_E2 B1201_F3 B1201_A12 B1203_E6 B1201_D10 B1206_B10 B1202_D11 B1201_F11 B1202_B10 B1203_B7 B1204_C9 B1206_D5 B1202_H7 B1201_E6 B1203_D5

CABA02_F3

B0102_G2 B0104_B2

GO096106 GO096131

Chloroplast: photosynthesis: carbohydrate pathways Serine hydroxymethyltransferase 4 (EC 2.1.2.1) Glyceraldehyde 3-phosphate dehydrogenase A (EC 1.2.1.12) NADP-dependent malate dehydrogenase (EC 1.1.1.37) Ribulose-1,5-bisphosphate carboxylase/oxygenase activase Ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (EC 4.1.1.39) 460 Ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (EC 4.1.1.39)

590 655 684 448 350

Cytosol: glycolysis and other carbon metabolism enzymes 436 Alpha-amylase precursor (EC 3.2.1.1) 767 Alpha-L-fucosidase 1 (EC 3.2.1.51)

E-value

diffe

AT4G21280.11 AT4G21280.21 AT1G67740.11 AT1G67740.11 TC1011572 ATC95561 TC805522 TC805522 TC447572 TC447572 TC163152

8 × 10-18 4 × 10-28 1 × 10-16 6 × 10-23 2 × 10-87 3 × 10-43 0.0 2 × 10-54 8 × 10-91 4 × 10-23 7 × 10-71

B1 B1 B1 NT B12 B12 B12 NT NT NT B12

TC805522 TC805522

3 × 10-93 1 × 10-16

NT NT

CF444953

7 × 10-46

NT

TC805522

3 × 10-39

NT

TC805522

1 × 10-60

NT

TC805522

7 × 10-28

NT

TC805522

3 × 10-73

NT

TC805522

4 × 10-21

NT

TC805522

4 × 10-67

NT

TC447572

1 × 10-54

NT

TC447572

9 × 10-86

NT

TC936172

6 × 10-57

NT

TC805522

4 × 10-36

NT

TC447572

3 × 10-31

NT

TC805522

1 × 10-41

NT

TC1041952

1 × 10-27

B12

TC963472

2 × 10-43

NT

TC815562

2 × 10-74

NT

TC815562

3 × 10-50

NT

TC104192

5 × 10-46

B12

TC936172

1 × 10-85

B12

AT4G13930.11 TC56822 TC493552 TC879712 ABD041983

5 × 10-93 8 × 10-95 1 × 10-142 4 × 10-88 5 × 10-063

B1f B12 B12 B12f B12f

TC922282

0.0

B12f

TC441772 AT2G28100.11

4 × 10-77 3 × 10-080

B1 B1


research articles

Valledor et al.

Table 5. Continued clone PRAb

accessionc

Bp

description

723 804 746 767 656 811

homology accesiond

E-value

diffe

AT2G28100.11 AT2G36970.11 AT5G55180.11 TC965652 AW7548533 TC933392

1 × 10-037 8 × 10-041 3 × 10-081 1 × 10-172 1 × 10-010 8 × 10-088

B1 B1 B1 B1 B1 B1

TC933392

1 × 10-128

B1

AT5G583301 TC884792

1 × 10-110 5 × 10-027

B12f B12f

B0104_A7 B0106_C3 B0104_D1 B0101_E5 B0102_B4 B0106_E6

GO096130 GO096212 GO096139 GO096073 GO096084 GO096232

B0101_F10

GO096075

B12DG_02 B1201_G7

GO270974 GO096263

Alpha-L-fucosidase 1 (EC 3.2.1.51) UDP-glucoronosyl/UDP-glucosyl transferase (EC 2.4.1.-) Beta-1,3-glucanase-like protein (EC 3.2.1.58) Granule-bound starch synthase 1 (EC 2.4.1.21) Phosphotransferase EII (EC 2.7.1.-) Pyrophosphate-dependent 6-phosphofructose-1-kinase beta subunit (EC 2.7.1.90) 584 Pyrophosphate-dependent 6-phosphofructose-1-kinase beta subunit (EC 2.7.1.90) 684 Malate dehydrogenase 685 Carbonic anhydrase protein family (EC 4.2.1.1)

B0104_F10 BA02_F4

GO096148 GO270980

Mitochondria: electron transfer (oxidative phosphorylation) 395 Putative quinone oxidoreductase (EC 1.6.5.3) AT1G23740.11 720 Inorganic pyrophosphatase (EC 3.6.1.1) TC622742

2 × 10-27 0.0

B1 B12f

B0106_D3 B0106_E12

GO096221 GO096228

Cell wall 864 Adheshin related protein, similar to UniRef100_A9TWU0 753 Cell wall-plasma membrane linker protein

TC633492 Q393534

4 × 10-98 1 × 10-10

B1 B1

B0103_C9 B0103_F4 B0102_G11

GO096117 GO096125 GO096104

Lipid metabolism 606 Lantoside 15-O-Acetylesterase (EC 3.1.1.-) 509 Beta-ketoacyl-CoA synthase (EC 2.3.1.119) 766 CXE carboxylesterase (EC 3.1.1.-)

TC854922 TC523902 DV9962843

1 × 10-135 1 × 10-100 1 × 10-122

B1 B1 B1

TC713752

1 × 10-160

B1

TC784332 TC664842 TC849982 AT1G23820.11 AT1G70310.11

1 × 10-179 1 × 10-105 2 × 10-086 3 × 10-83 1 × 10-83

B1 B1 B1 B1 B1

TC1016492 TC845132 TC845132 TC119712 AT3G522601 AT4G01850.11

9 × 10-055 1 × 10-112 3 × 10-098 4 × 10-116 5 × 10-037 4 × 10-036

B1 B1 B1 NT B1 NSf

TC946752 AT5G186201 CF6676263 CF6676263 TC811532 TC886152 TC886152 TC886152 TC993502

1 × 10-017 1 × 10-124 1 × 10-101 2 × 10-088 8 × 10-065 2 × 10-054 3 × 10-019 9 × 10-057 1 × 10-160

B1f B1 B1 B1 B1 B1 B1 B1 B1

TC1015552 TC1124182 TC987572 TC975712 TC848372 CF3883563 CF3883563 EX417423 TC941282 TC855162 TC954782

3 × 10-020 5 × 10-067 1 × 10-126 1 × 10-105 5 × 10-012 1 × 10-131 1 × 10-123 3 × 10-040 1 × 10-043 6 × 10-091 9 × 10-051

B1 B1 B1 B1 B1 B1 B1 NSf B12 B12 B12

TC914072 TC1027322 AT5G61790.11 TC990112

1 × 10-166 0.0 3 × 10-017 1 × 10-125

B1 B1 B1 B1

B0105_B12 B0104_C6 B0101_A7 B0104_E11 B0105_B10

Aminoacid and nitrogen metabolism GO096113 1408 3-hydroxyisobutyryl-coenzyme A hydrolase-like protein (EC 3.1.2.4) GO096160 1183 Anthranilate phosphoribosyltransferase-like protein (EC 2.4.2.18) GO096137 765 Tryptophan synthase beta subunit (EC 4.2.1.20) GO096064 498 Cyanate lyase (EC 4.2.1.104) GO096144 739 Spermidine synthase 1 (EC 2.5.1.16) GO096159 842 Spermidine synthase 2 (EC 2.5.1.16)

B0102_F9 B0103_F11 B0106_D1 B0104_E1 B0101_G7 B01DG_02

GO096103 GO096122 GO096217 GO096142 GO096077 GO270977

737 765 769 910 945 231

B0101_B9 B0101_C5 B0102_C11 B0106_A3 B0106_E4 B0105_B7 B0105_D12 B0106_E3 B0106_F10

GO096066 GO096068 GO096090 GO096200 GO096230 GO096165 GO096174 GO096229 GO096234

764 764 776 745 408 642 799 652 739

B0105_C1 B0104_C10 B0104_E10 B0106_F12 B0105_C11 B0101_B12 B0106_G8 B1206_D11 B1204_D6 B1206_H10 B1201_F2

GO096166 GO096133 GO096143 GO096235 GO096167 GO096065 GO096245 GO096265 GO096307 GO096308 GO096312

802 597 779 785 744 765 840 410 712 703 912

B0106_F5 B0106_C10 B0105_G12 B0104_E6

Protein translation, folding, modification, and degradation GO096236 890 40S ribosomal protein S10 GO096211 1178 Ribosomal L1 domain containing 1 GO096189 814 Calnexin-like protein GO096147 614 Calnexin-like protein

B0103_B8

3972

Nucleotide metabolism Adenine phosphoribosyltransferase (EC 2.4.2.7) Cytidine deaminase 1 like (EC 3.5.4.5) Cytidine deaminase protein family (EC 3.5.4.5) Guanine nucleotide-binding beta subunit-like protein Pseudo tRNA uridine synthase protein family (EC 5.4.99.-) S-Adenosylmethionine synthetase 2 (EC 2.5.1.6) Transcription and DNA replication Regulator of chromosome condensation (RCC1) protein family Chromatin-remodeling protein 11 bHLH transcription activator (MYC4 protein family) bHLH transcription activator (MYC4 protein family) DNA binding helicase/hydrolase (EC 3.6.1.-) DNA binding protein MYC2-like DNA binding protein MYC2-like DNA binding protein MYC2-like Mediator of RNA polymerase II transcription subunit 31-like protein Putative transcription factor Transcription elongation factor 1-alpha Transcription elongation factor 1-alpha Transcription elongation factor 1-gamma Transcription regulator protein family Transcriptional corepressor Leunig Transcriptional corepressor Leunig DNA-directed RNA polymerase (EC 2.7.7.6) Surfeit gene 6-like Transcriptional regulator ArsR family like U2 snRNP auxiliary splicing factor


research articles

Analysis of Pinus Needle Maturation Table 5. Continued clone PRAb

accessionc

Bp

homology accesiond

E-value

diffe

B0105_E8 B0105_F1 B0105_H6 B0104_E5 B0105_H11 B0106_G5 B0103_G6 B0105_C5 B0105_H5 B0106_B2 B0106_D7 B0105_D3 B0104_B7 B0102_H6 B0102_D3 B0105_G10 B0105_F4 B01DG_04 B0106_G3 B0104_G8 B0105_H7 B01SC_E9 B0104_F12 B0105_F12 B0102_D12 B0106_D2 B1202_C11 B1201_A1

GO096182 GO096183 GO096194 GO096146 GO096192 GO096244 GO096126 GO096169 GO096193 GO096206 GO096224 GO096175 GO096132 GO096110 GO096094 GO096188 GO096186 GO270979 GO096243 GO096152 GO096195 GO096256 GO096149 GO096184 GO096093 GO096220 GO096309 GO096316

722 253 813 421 840 835 632 835 731 1027 817 1019 767 1046 724 860 654 649 546 669 862 318 745 818 725 859 662 690

Calnexin-like protein Citoplasmic protein chaperone family Cysteine proteinase RD19a precursor (EC 3.4.22.-) Putative valyl-tRNA synthetase (EC 6.1.1.9) Putative valyl-tRNA synthetase (EC 6.1.1.9) RNA binding helicase protein family (EC 3.6.1.-) RNA binding protein family RNA binding protein family T-complex polypeptide protein 1-like T-complex polypeptide protein 1-like Eukaryotic translation initiation factor 4-like protein Eukaryotic translation initiation factor 4-like protein Eukaryotic translation initiation factor 4G Translation elongation factor 1-alpha Translation elongation factor 1-alpha Translation elongation factor 1-alpha Translation elongation factor 1-gamma 3 elongation factor Tu family protein (eeF2) Ubiquitin DegP protease 9 (EC 3.4.21.107) putative DegP protease (EC 3.4.21.-) Serine endopeptidase family (EC 3.4.21.-) Protease Do-like 9 (EC 3.4.21.-) Protease Do-like 9 (EC 3.4.21.-) Protease Do-like 9 (EC 3.4.21.-) Proteasome subunit alpha type Translation elongation factor 2 Ubiquitin-protein ligase. F-box protein family (EC 6.3.2.19)

TC941302 TC911552 TC809992 TC970412 TC919052 TC918662 TC982662 AT3G08620.11 TC983402 TC891732 TC2697152 TC1202392 AT3G60240.41 TC1124182 BQ6556723 TC1124182 TC975712 AT3G22980.11 TC1069702 TC89532 TC2984752 TC8605822 AT5G40200.11 TC2984752 TC961822 TC72317 TC662012 DR177843

1 × 10-085 3 × 10-051 1 × 10-139 2 × 10-061 2 × 10-033 0.0 1 × 10-036 2 × 10-057 4 × 10-075 0.0 1 × 10-037 3 × 10-075 1 × 10-040 0.0 1 × 10-036 1 × 10-164 1 × 10-142 2 × 10-063 2 × 10-095 1 × 10-128 2 × 10-088 1 × 10-051 1 × 10-068 1 × 10-104 1 × 10-101 1 × 10-151 1 × 10-077 7 × 10-120

NT B1 NT B1 B1 B1 B1 B1 B1 B1 B1 B1 B1 B1 NT NT B1 NT B1 B1 B1 B1 B1 B1 B1 B1 B12f NT

B0102_G12 B0103_C4 B0106_D4 B0102_E11 B0101_H3 B0102_D9 B0103_D11 B0105_B2 B0101_C6 B0103_D4 B0106_C9 B0101_G11 B0104_C4 B0105_D7 B0106_E5 B1205_E2

GO096105 GO096115 GO096222 GO096097 GO096079 GO096095 GO096118 GO096161 GO096069 GO096119 GO096216 GO096076 GO096136 GO096176 GO096231 GO096303

715 736 846 768 657 659 741 284 763 765 784 703 769 854 865 298

Secondary metabolism 4-coumarate:CoA ligase protein family (EC 6.2.1.12) 4-coumarate:CoA ligase protein family (EC 6.2.1.12) 4-coumarate:CoA ligase protein family (EC 6.2.1.12) Caffeic acid O-methyltransferase (EC 2.1.1.68) Carbon-sulfur lyase-like protein (EC 2.8.1.7) Carbon-sulfur lyase-like protein (EC 2.8.1.7) Chalcone synthase (EC 2.3.1.74) Dienelactone hydrolase-like protein family (EC 3.1.1.45) Leucoanthocyanidin dioxygenase (EC 1.14.11.19) Mevalonate disphosphate decarboxylase (EC 4.1.1.33) Mevalonate disphosphate decarboxylase (EC 4.1.1.33) Serine/threonine protein kinase (EC 2.7.11.-) Trans-cinnamate 4-monooxygenase like (EC 1.14.13.11) Trans-cinnamate 4-monooxygenase like (EC 1.14.13.11) Trans-cinnamate 4-monooxygenase like (EC 1.14.13.11) Pyridoxal biosynthesis protein PDX1 (Sor-Like protein) (EC 4.-.-.-)

TC1110582 TC1110582 TC1110582 TC973652 AT5G16940.11 AT5G16940.11 TC8603342 AT3G23600.21 AT4G22880.21 TC817852 TC1080962 AT1G14000.11 TC1090032 TC1090032 TC1090032 TC107712

4 × 10-083 1 × 10-127 0.0 1 × 10-151 1 × 10-041 3 × 10-024 1 × 10-047 3 × 10-023 5 × 10-087 1 × 10-178 4 × 10-040 5 × 10-040 1 × 10-128 1 × 10-161 0.0 58 × 10-042

B1 B1 B1 B1 B1 B1 B1 B1 B1 B1 B1 B1 B1 B1 B1 B12

B0103_G8 B0105_B6

GO096127 GO096164

725 794

Actin depolymerizing factor Actin depolymerizing factor

TC824152 TC824152

1 × 10-153 1 × 10-127

B1 B1

B0103_H1 B0105_E1 B0102_E7 B0106_D9 B0101_H5 B0102_B6 B0105_D1 CABA04_H8 B0101_E3 B0102_B10 B0102_E10 B01DG_01 B0103_C8 B1227_H5

GO096128 GO096179 GO096100 GO096226 GO096080 GO096085 GO096172 GO270981 GO096072 GO096083 GO096096 GO270976 GO096116 GO096261

748 991 684 776 653 798 799 199 538 738 583 82 759 684

Stress and defense related proteins Dehydration-induced protein 19 (Similar to fiber protein Fb2) Dehydration-responsive protein RD22 Dehydrin-like protein (LEA) Dehydrin-like protein (LEA) LEA protein Mitochondrial small heat shock protein Mitochondrial small heat shock protein Heat shock protein 70 Thaumatin-like protein Disease resistance protein, putative Wound-induced protein WIN1 Ascorbate peroxidase Dirigent protein pDIR18 (Similar to UniRef100_Q27J94) Avirulence-responsive protein AIG1-like

TC921732 AT5G25610.11 TC104422 TC104422 TC427282 TC812612 AT5G51440.11 AT2G32120.21 TC809732 AT3G04220.11 TC934362 AT1G07890.11 TC1065182 AT1G33970.41

1 × 10-104 4 × 10-017 6 × 10-114 63 × 10-048 1 × 10-12 1 × 10-136 2 × 10-020 6 × 10-22 1 × 10-108 7 × 10-025 1 × 10-116 1 × 10-008 8 × 10-043 2 × 10-025

B1 B1 B1 B1 B1 B1 B1 NSf B1 B1 B1 B1f B1 NT

description

Cytoskeleton


research articles

Valledor et al.

Table 5. Continued clone PRAb

accessionc

B1206_B9 B1202_F6 B1203_E7 B12DG_03

GO096302 GO096310 GO096318 GO270975

735 579 672 93

B0102_G7 B0106_H8 B0104_G10 B0102_B9 B01SC_H9 B0101_C2 B0102_B8 B0104_D11 B0102_E3 B0105_E2 B1206_F6

GO096107 GO096253 GO096151 GO096088 GO096258 GO096067 GO096087 GO096141 GO096099 GO096181 GO096262

763 841 723 752 446 414 774 807 752 623 435

B0102_A7

GO096082

B0106_H4 B0106_F6 B0103_B2 B0103_E11 B0106_B12 B0106_A2 B0103_F12 B0106_B4 B0106_D6 B0102_F12 B0106_E8 B0104_C3 B0105_B1 B0106_H3 B0101_H6 B0101_H12 B0106_H9 B0106_C5 B0102_D11 B0104_C7 B0106_D8 B0104_C12

GO096250 GO096237 GO096112 GO096121 GO096205 GO096199 GO096123 GO096208 GO096223 GO096102 GO096233 GO096135 GO096158 GO096249 GO096081 GO096078 GO096254 GO096213 GO096092 GO096138 GO096225 GO096134

B0105_E10

GO096180

B1202_H4

GO096259

B1202_B4

GO096271

B0105_F3 B0102_E2 B0101_D11 B0106_H12 B0105_G1 B0105_C6 B0104_E2 B0105_G8 B0103_B11 B0106_A1 B0106_G10 B01SC_D9 B01SC_G9 3974

GO096185 GO096098 GO096071 GO096248 GO096187

homology accesiond

E-value

diffe

Puroindoline B protein, putative Agglutinin alpha chain Early light inductable protein 1 (ELIP1) Tau class glutathione S-transferase (EC 2.5.1.13)

CAQ43070.23 TC964022 AT3G22840.11 AT1G78370.11

1 × 10-010 1 × 10-108 6 × 10-033 3 × 10-004

B12 B12 NT B12f

Signaling Methyl-accepting chemotaxis sensory transducer Peptidyl-prolyl cis-trans isomerase (EC 5.2.1.8) Plasma membrane proton ATPase (EC 3.6.3-) Protein Kinase 5 (EC 2.7.1.-) Protein Kinase 5 (EC 2.7.1.-) Sensor protein like Sterol carrier protein family (Similar to SCP-2) (EC 2.3.1.176) Sterol carrier protein family (Similar to SCP-2) (EC 2.3.1.176) Receptor like Serine/threonine protein kinase (Similar to RFK3) COP9 signalosome complex subunit 3 (Signalosome subunit 3) Calmoduline domain protein kinase 9(EC 2.7.1.-)

CT5750213 TC622432 TC118862 TC571822 TC571822 TC973762 TC869462 TC869462 NP6521493 DV9924993 AT3G20410.11

7 × 10-063 1 × 10-109 7 × 10-119 1 × 10-112 2 × 10-073 1 × 10-056 5 × 10-070 0.0 7 × 10-044 2 × 10-091 1 × 10-012

B1 B1 B1 B1 B1 B1 B1 B1 B1 NT B1

TC6476232

1 × 10-073

B1

AT5G64940.21 AT5G64940.21 AT5G64940.21 TC862942 TC1045462 TC885762 TC808032 TC808032 TC808032 TC808032 TC808032 BF6095943 AT1G68570.11 AT1G68570.11 TC1063522 2 TC93498 TC862942 TC8142922 TC9451712 TC8325912 TC945662 C8566423

1 × 10-035 3 × 10-061 5 × 10-010 1 × 10-123 2 × 10-009 1 × 10-122 1 × 10-125 0.0 5 × 10-027 1 × 10-032 1 × 10-161 3 × 10-022 2 × 10-071 4 × 10-088 1 × 10-102 1 × 10-159 2 × 10-087 1 × 10-077 1 × 10-053 1 × 10-030 1 × 10-104 1 × 10-092

NT B1 B1 B1 B1 B1 B1 B1 B1 B1 B1 B1 B1 B1 B1 B1 B1 B1 B1 B1 B1 NT

AT5G43960.11

9 × 10-056

NT

TC647622

1 × 10-082

B12

Q393534

3 × 10-16

B12

AT1G72880.11 TC934002 TC504732 TC504732 AT1G26355.11

1 × 10-069 1 × 10-12 1 × 10-105 1 × 10-112 2 × 10-019

B1 B1 B1 B1 B1

TC919192 TC1052692 DV9846813 AT3G19000.21

9 × 10-038 1 × 10-136 6 × 10-056 2 × 10-046

B1 B1 B1 B1

AT3G19000.11

6 × 10-095

B1

AT3G19000.11

6 × 10-025

B1

TC867972 TC1112072

2 × 10-090 3 × 10-033

B1 B1

Bp

description

Transport 729 ABC-type Fe3+-hydroxamate transport system periplasmic component precursor 678 ATP-binding cassette (ABC) protein-like, putative 754 ATP-binding cassette (ABC) protein-like, putative 730 ATP-binding cassette (ABC) protein-like, putative 699 Aquaporin TIP1-1 606 Porin 591 Porin MIP1-4 729 Gamma-tonoplast intrinsic protein family 1149 Gamma-tonoplast intrinsic protein family 794 Gamma-tonoplast intrinsic protein family 728 Gamma-tonoplast intrinsic protein family 821 Gamma-tonoplast intrinsic protein family 427 Myosin motor protein 814 Oligopeptide membrane transporter 839 Peptide transporter, putative 530 Nonspecific lipid-transfer protein 4 738 Hexose carrier protein 768 Putative aquaporin family membrane transport protein 876 Intracellular vesicle-mediated protein transport protein, putative 711 Transport membrane protein, putative 750 SNF7-like protein family 501 Voltage-gated chloride channel protein family 729 Nuclear transport factor 2 (NTF2) family protein/RNA recognition motif (RRM)-containing protein 1240 Nuclear transport factor 2 (NTF2) family protein/RNA recognition motif (RRM)-containing protein 1157 ABC-type Fe3+-hydroxamate transport system periplasmic component precursor 717 Cell wall-plasma membrane linker protein 862 580 751 834 802

Cell cycle Acid pyrophosphatase survival protein SurE (EC 3.1.3.2) Translationally controlled tumor protein (TCTP) Pleiade (PLE) like Pleiade (PLE) like SPIRAL1-LIKE2 like protein (EC 3.-.-.-)

Other biological process GO096170 838 Heavy-metal-associated domain-containing protein family GO096145 628 Cytochrome P450 78A4 GO096191 1160 Histidine-rich glycoprotein GO096111 760 Iron/ascorbate-dependent oxidoreductase protein family (EC 1.-.-.-) GO096196 1266 Iron/ascorbate-dependent oxidoreductase protein family (EC 1.-.-.-) GO096239 807 Iron/ascorbate-dependent oxidoreductase protein family (EC 1.-.-.-) GO096255 436 Oxidoredcutase protein family (EC 1.-.-.-) GO096257 307 Oxidoreductase protein family (EC 1.-.-.-)


research articles

Analysis of Pinus Needle Maturation Table 5. Continued clone PRAb

accessionc

Bp

description

homology accesiond

E-value

diffe

B0102_B7 B0104_F4 B0106_B3 B1203_A6 B1202_A6 B1201_E9 B1204_F7 B1202_H9

GO096086 GO096150 GO096207 GO096264 GO096260 GO096267 GO096270 GO096306

765 861 833 666 362 390 601 696

Protein with nucleotide-diphospho-sugar transferase domain ECT4 like protein Short-chain dehydrogenase/reductase (SDR) family protein Cytochrome C biogenesis protein Alpha/beta hydrolase family protein (EC 3.1.-.-) Haloacid dehalogenase-like hydrolase family protein (EC 3.6.3.1) Oxidoreductase, zinc-binding dehydrogenase family (EC 1.-.-.-) Selenium-binding pentatricopeptide repeat protein family

TC878092 TC736252 TC1065182 CT583462 CF3882793 AT2G41250.11 TC103802 TC110802

1 × 10-179 1 × 10-177 2 × 10-090 2 × 10-010 2 × 10-021 1 × 10-061 16 × 10-08 51 × 10-041

B1 B1 B1 B12 B12 B12 B12 B12

B0106_G2 B0105_A6 B0101_E8 B0105_C3 B0101_C9 B0106_F7 B0102_D1 B0105_G5 B0105_B3 B1202_G10 B1206_E8 B1206_B11 B1202_F4 B1205_D5

GO096242 GO096156 GO096074 GO096168 GO096070 GO096238 GO096091 GO096190 GO096162 GO096313 GO096315 GO096317 GO096319 GO096399

905 804 322 712 735 862 716 859 880 799 402 395 422 720

TC595322 TC815902 TC8115312 TC840172 TC902212 TC902212 CT5793433 TC1127602 TC50473 TC961112 AT4G28100.11 CO362953 TC672232 A9NK114

1 × 10-136 7 × 10-054 1 × 10-016 1 × 10-148 8 × 10-064 1 × 10-176 4 × 10-045 1 × 10-084 1 × 10-144 2 × 10-055 8 × 10-006 1 × 10-087 9 × 10-036 8 × 10-20

NT NT NT NT NT NT NT NT NT NT NT NT NT NT

B1205_D3

GO096314

715

TC968642

1 × 10-112

NT

B1202_B9

GO096400

741

TC968642

5 × 10-058

NT

Unclassified Uncharacterized protein (Similar to UniRef100_A7QLT5 Cluster) Uncharacterized protein (Similar to UniRef100_A9NU27) Uncharacterized protein (Similar to UniRef100_A9S9 × 0) Uncharacterized protein (Similar to UniRef100_A9U166) Uncharacterized protein (Similar to UniRef100_Q10DM3) Uncharacterized protein (Similar to UniRef100_Q10DM3) Uncharacterized protein (Similar to UniRef100_Q5C027) Uncharacterized protein (Weakly similar to UniRef100_Q9LJU8) Unknown protein (Similar to UniRef100_A7QGT7) Uncharacterized protein (homologue to UniRef100_A7Q919) Uncharacterized protein (Similar to At4g28100 precursor) Uncharacterized protein (Similar to At3g09180) Uncharacterized protein (Weakly similar to UniRef100_Q9AU17) Uncharacterized protein (Similar to Picea sitchensis WS02718_E07) Uncharacterized protein (Similar to Picea sitchensis WS0287_C08) Uncharacterized protein (Similar to Picea sitchensis WS0287_C08)

a Functional classification was based according to Kyoto Encyclopedia of Genes and Genomes. b Clone reference in PRA library. c dbEST accession number. d Homology accession number according to public databases: (1) TAIR8pep, (2) TGI, (3) Genbank, and (4) Uniprot. e Differential expression analysis. B1, overexpressed in B1 needles; B12, overexpressed in B12 needles; NS, not significant difference of expression between samples; NT, not tested. f Expression analized by qPCR.

However, some discrepancies have been found: GSTU, APX and PPi showed an opposite trend between the changes in protein accumulation and mRNA expression. SAMS2, RCA, SHM4, CA and MDH protein and mRNA followed the same trend, but without any significant correlation. These observations are in concordance with other protein-mRNA comparative analyses reported in plant systems.43,47,48 All of these discrepancies indicate that some post-transcriptional and posttranslational events might occur at the mRNA and/or protein expression levels during needle development process, although some of the above-described variations could be artifacts due to the technical limitation of current methods of measurement. Fournier et al.49 recently described that mRNA expression and protein accumulation exhibited a delayed correlation in Saccharomyces cerevisiae; furthermore, the turnovers of proteins and mRNAs are uncoupled.50,51 These phenomena may add more variability to this analysis, because the plant material was sampled in the early morning, and the effect of cold and the absence of light at night alter the ratios mRNA-protein accumulation. Therefore, for future studies dealing with similar experimental systems, we propose quantifying translation and transcription processes, instead of proteins and mRNAs, to obtain more accurate results. Biological Interpretation of Needle Developmental Transition. The formation of leaf is a basic aspect of plant development; however, except in model species such as Arabidopsis,12-14 the control of leaf development is an unknown process. The actors involved in compound leaf development have been divided into two functional modules that regulate and allow the two events required for compound leaf development. The

first module allows the maintenance of an undifferentiated state that accounts for the higher morphogenetic potential of compound leaves compared to simple leaves. Class I KNOX expression is re-established during the development of most species with compound leaves and antagonizes gibberrellin signaling that promotes cell growth and differentiation.14 Needle maturation implies processes of cell division and programmed death, related to the formation of organ structure and morphogenesis determination,10,12 and cell differentiation, associated to tissue functional competence and maturation.9 Mature needles showed an overexpression of photosynthesis related pathways (chloroplast electron transfer chain, carbohydrate biosynthesis) compared with immature needles (Figure 4). These results are supported by the observations that mature needles exhibited a higher number of photosynthetically active chloroplasts and higher rates of energy production.52 Also, Ahsan and Komatsu18 observed in soybean that the net photosynthesis rate and chlorophyll content present an agedependent correlation in leaves. These results suggest that proteins involved in carbon assimilation, folding and assembly, and energy may work synchronously and show a linear correlation to photosynthesis at developmental stages of leaves. The key enzymes for these processes are the RuBisCO complex and its activator the RuBisCO ACTIVASE.53 Since the protein content of mature and immature needles is similar, and the photosyntesis-related proteins represent 20% of the total protein of mature needles, it can explain the reduced number of spots and the lower number of differential genes detected of mature compared to immature needles. Journal of Proteome Research • Vol. 9, No. 8, 2010 3975

research articles

Valledor et al.

Figure 6. Number of nonredundant proteins and cDNA sequences that were differentially expressed in immature (B1) and mature (B12) needles gathered by functional groups.

RuBisCO subunits correlates, being more expressed in mature needles (Figure 6). HSP70, a protein related to the cytoplasmatic prefolding of RuBisCO SMALL SUBUNIT,54 is overexpressed and overabundant in immature needles probably because in these needles, with higher biosynthetic activity, the participation of this chaperone is required for the correct folding of the newly synthesized proteins. In the case of the ionic potential modulators of RuBisCO such as CARBONIC ANHYDRASE, which is implied in CO2 capture, liberation, and pH regulation55,56 and RuBisCO ACTIVASE, an enzyme that facilitates the release of sugar phosphate inhibitors from RuBisCO catalytic sites,57 there was higher expression and accumulation levels in mature needles.

Figure 7. Heatmap comparing the differential expression levels of the 14 mRNA-protein identities that were found. Abundance values were adjusted and centered (by using Cluster 3.0 software) considering the expression levels in B1 needles. Red and green values indicate overexpression/accumulation in immature and mature needles, respectively.

The fold-change of mRNA expression and protein accumulation levels between mature and immature needles of the two 3976


Related to energy production, INORGANIC PYROPHOSPHATASE, which stimulates photosynthesis by hydrolyzing inorganic pyrophosphate (PPi) being an alternative to ATP as energy donor for various energy-requiring reactions,58 and MALATE DEHYDROGENASE, which is implied in a NADPH photoactivation,59 are also overexpressed in mature needles. Electron transfer chain transcripts like P6, psbC, psbD, OXYGEN EVOLVING ENHANCERS, and CHLOROPHYLL BINDING PROTEIN are overexpressed in mature needles, while psbY is expressed at higher levels in immature needles (Tables 4 and 5). It is important to note that in immature needles the photosynthetic machinery is present but may not be correctly regulated and tuned. This hypothesis is supported by the detection of higher mRNA and protein accumulation levels of SHM4 in immature needles, and enzymes related to photores-

research articles

Analysis of Pinus Needle Maturation piratory production of serine being involved in a GS/GOGAT strategy to survive photorespiratory impairment.60 The high expression levels of these regulatory and buffering proteins give them an important role to maintain homeostasis and protect the photosynthetic machinery against crucial photosynthetic impairment challenges. In immature needles compared to mature needles is observed an overexpression of stress/defense-related pathway. LATE EMBRYOGENESIS ABUNDANT (LEA) and HEAT SHOCK PROTEIN 70 (HSP70) represent two proteins and mRNA overaccumulated and overexpressed in immature needles. During the leave maturation higher photosynthetic pigment content and the maximum photosynthetic capacity is gradually reached,18 so that during early leaf development stages the plant is very fragile against any stress (biotic or abiotic) being these proteins closely related to these mechanisms. Glycolysis and carbohydrate metabolism related proteins, like ENOLASE, FRUCTOSE ALDOLASE, MITOCHONDRIAL MALATE DEHYDROGENASE, or PHOSPHOGLICERATE MUTASE and its transcripts are overexpressed in immature needles, indicating that growing needles, a biosynthetically active tissue, are a strong energy sink. An increase of fructokinase activity was also reported in young leaves of soybean.18 The higher morphogenetic competence that is exhibited by the growing needles should be reflected in changes of the cell cycle regulatory proteins or related transcription factors.11,61 But, in general, the detection of these proteins through 2-DE is rather difficult because they are often present at very low abundances, may have basic pIs, and may be present in various modified forms that alter their mobility on 2-DE gels.62 Only 3 nonredundant cell cycle and 10 transcription related proteins were found by 2-DE PAGE whereas in transcriptomic analysis we described 5 cell cycle regulatory and 19 transcription related mRNAs showing differential expression in their levels. Although some Class-III homeodomain/leu zipper transcription factors, which regulate organ adaxial-abaxial polarity,63 and cyclins and cyclin-dependent kinases, which regulate cell division and leaf expansion,64 have been described, none of them have been found in the obtained set of genes and proteins. CELL CYCLE REGULATORY PROTEIN and PLEIADE, SPIRAL1, TCTP, and SurE transcripts, which are also associated with morphogenesis-related cell division,65-67 were found to be upregulated in immature needles, whereas RETINOBLASTOMA RELATED PROTEIN2, which recruits a histone deacetylase to control gene transcription playing a negative role in cell proliferation68 and members of the E3-UBIQUITIN-PROTEIN protein family, which was related to the control of the growth period in Arabidopsis leaves,69 were present in abundance in mature needles. Polyamines are also implied in cell growth, division, and death70 in this way S-ADENOSYL-METHIONINE SYNTHASE2 and SPERMIDINE SYNTASE 1 and 2 are over abundant/overexpressed in immature needles. These results are in concordance with previous studies in Arabidopsis which correlate changes in these transcripts with the development of mature leaves.10,64 The transcription regulation related proteins can be grouped into transcription factors or chromatin regulating proteins. The identified transcription factors, which are up-regulated in immature needles, can also be grouped into 3 different families which have been identified as being correlated with plant development. For example, transcription factors bHLH, and MYC2 have been found to associate with a WD40 repeat protein

which initiates multiple cellular differentiation pathways in a range of plants,71 and the WRKY family to abiotic and biotic stress response.72 Chromatin remodeling-related proteins, which are related to gene regulation by compaction or decompaction of the chromatin, such as RCC1,73 DNA METHYLTRANSFERASES,74 and SAMS2, which are also related to DNA and Histone methylation,75 were found mainly in immature needles. When using the available reference maps of Pinus radiata, mature needles (presented in this work and in Valledor et al.23) are characterized for presenting similar amount of glycolysis and other carbon metabolism enzymes as exhibited in Arabidopsis76 and rapeseed leaves.77 But the lower abundance of carbon-fixation related proteins (about a half that is present in rapeseed leaves) and the high proportion of transcription, protein biosynthesis, and stress-related proteins compared to these species is striking. Quantitative accurate comparisons between angiosperm and gymnosperm leaves cannot be made with available data sets, which showed different methodologies and protein classification schemes. In conclusion, this work provides an overview of the molecular changes affecting proteomes and transcriptomes during P. radiata needle maturation processes having an integrative vision of the functioning and physiology of this process. Furthermore, the development of 2-DE map and gene library allows different levels and complexities of analysis, from large studies including the global and functional classification of genes and proteins to the metabolic level analyzing pathways and networks. Mature needles are characterized by an overexpression of energy-related and photosynthetic pathways. Conversely, immature needles showed biosynthesis, stressrelated, and defense mechanisms, a fact that may be linked to the higher growth rate and the fragility against biotic and abiotic stresses characteristic of these needles. The developed tools will be of special interest for applied and basic investigations regarding Pinus radiata ranging from the selection of elite trees for clonal breeding propagation and the study of biotic and abiotic stresses to plant development.

Acknowledgment. The financial support needed to guarantee our work has come from Spanish National Projects AGL2004-00810/FOR and AGL2007-62907/FOR. Spanish Ministry of Science and Innovation supported the fellowships of all young researchers. We also thank Dr. Kevin Dalton for his linguistic revision of the text. Supporting Information Available: Supplementary tables and figures. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Bonan, G. B. Forests and Climate Change: Feedbacks, and the Climate Benefits of Forests. Science 2008, 320, 1444–1449. (2) Walter, C.; Carson, M.; Carson, S. Conifers. In Biotechnology in Agriculture and Forestry (Transgenic Crops V); Pua, E. C., Davey, M. R., Eds.; Springer-Verlag: Berlin Heidelberg, 2007; pp 447-471. (3) Valledor, L.; Rodrı´guez, R.; Sańchez, P.; Fraga, M. F.; et al. Propagation of Pinus genotypes regardless of age. In Protocols of micropropagation for Woody Trees and Fruits; Jain, M., Ha¨ggman, H., Eds.; Springer: Dordrecht, 2007; pp 137-146. (4) Kumar, S.; Burdon, R. D.; Stovold, G. T.; Gea, L. D. Implications of selection history on genetic architecture of growth, form, and wood-quality traits in Pinus radiata. Can. J. For. Res. 2008, 38, 2372–2381. (5) Fraga, M. F.; Rodriguez, R.; Can ˜ al, M. J. Reinvigoration of Pinus radiata is associated with partial recovery of juvenile-like polyamine concentrations. Tree Physiol. 2003, 23, 205–209.


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Combined Proteomic and Transcriptomic Analysis Identifies

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