Nonadditive Protein Accumulation Patterns in Maize (Zea mays L

Sep 14, 2010 - Received July 12, 2010. Heterosis describes the superior performance of heterozygous F1-hybrid plants compared to their homozygous ...
0 downloads 0 Views 4MB Size
Download PDF
Nonadditive Protein Accumulation Patterns in Maize (Zea mays L.) Hybrids during Embryo Development Caroline Marcon,†,‡ Andre´ Schu ¨ tzenmeister,§ Wolfgang Schu ¨ tz,| Johannes Madlung,| § Hans-Peter Piepho, and Frank Hochholdinger*,†,‡ ZMBP, Center for Plant Molecular Biology, Department of General Genetics, University of Tuebingen, 72076 Tuebingen, Germany, INRES, Institute of Crop Science and Resource Conservation, Chair for Crop Functional Genomics, University of Bonn, 53115 Bonn, Germany, Institute for Crop Production and Grassland Research, Bioinformatics Unit, University of Hohenheim, Fruwirthstrasse 23, 70599 Stuttgart, Germany, and Proteome Center Tuebingen, Interfaculty Institute for Cell Biology, University of Tuebingen, 72076 Tuebingen, Germany Received July 12, 2010

Heterosis describes the superior performance of heterozygous F1-hybrid plants compared to their homozygous parental inbred lines. In the present study, heterosis was detected for length, weight, and the time point of seminal root primordia initiation in maize (Zea mays L.) embryos of the reciprocal F1-hybrids UH005xUH250 and UH250xUH005. A two-dimensional gel electrophoresis (2-DE) proteome survey of the most abundant proteins of the reciprocal hybrids and their parental inbred lines 25 and 35 days after pollination revealed that 141 of 597 detected proteins (24%) exhibited nonadditive accumulation in at least one hybrid. Approximately 44% of all nonadditively accumulated proteins displayed an expression pattern that was not distinguishable from the low parent value. Electrospray ionization-tandem mass spectrometry (ESI-MS/MS) analyses and subsequent functional classification of the 141 proteins revealed that development, protein metabolism, redox-regulation, glycolysis, and amino acid metabolism were the most prominent functional classes among nonadditively accumulated proteins. In 35-day-old embryos of the hybrid UH250xUH005, a significant up-regulation of enzymes related to glucose metabolism which often exceeded the best parent values was observed. A comparison of nonadditive protein accumulation between rice and maize embryo data sets revealed a significant overlap of nonadditively accumulated proteins suggesting conserved organ- or tissue-specific regulatory mechanisms in monocots related to heterosis. Keywords: maize • heterosis • embryo • proteome • two-dimensional gel electrophoresis (2-DE)

Introduction Heterosis, or hybrid vigor, describes the superior performance of heterozygous F1-hybrids compared to their corresponding homozygous parental inbred lines.1 Heterosis is of outstanding agronomic importance in allogamous species as for instance maize and is typically quantified in adult plants in terms of increased yield, biomass, or speed of development.2 However, increased vigor can already be observed shortly after germination in terms of seedling biomass,3 root development,4 and even during the very early stages of embryo development,5,6 hence allowing for a molecular dissection of the early stages of heterosis manifestation. Although different quantitative models have been proposed to explain the genetic basis of hybrid vigor, little consensus * Corresponding author. Address: University of Bonn, INRES, Institute of Crop Science and Resource Conservation, Chair for Crop Functional Genomics, Katzenburgweg 1a, 53115 Bonn, Germany. Email: frank.hochholdinger@ uni-bonn.de. Tel: +49 228 73 60334. Fax: +49 228 73 60333. † University of Tuebingen, ZMBP. ‡ University of Bonn, INRES. § University of Hohenheim. | University of Tuebingen, Proteome Center. 10.1021/pr100718d

 2010 American Chemical Society

has emerged on their contribution to the manifestation of heterosis.7-10 The dominance (or complementation) hypothesis suggests that deleterious alleles at different loci in the homozygous parental lines can be complemented in highly heterozygous F1-hybrid plants.11 Alternatively, the overdominance hypothesis attributes heterosis to allelic interactions at one or multiple loci in hybrids that result in superior traits compared to the homozygous parental inbred lines.1 Finally, the epistasis hypothesis suggests that heterosis is caused by the multiplicative coaction of nonallelic genes leading to epistatic interactions. A drawback of these quantitative genetic models is that they cannot be directly associated with gene expression or protein accumulation patterns in order to explain the molecular basis of heterosis. Recent studies in maize, rice, tomato, and Arabidopsis provided first insights into the phenomenon of heterosis on the molecular level.1,5,12,13 In maize, it has been demonstrated that distinct inbred lines differed significantly in their gene content due to the loss of individual genes at multiple loci.14-16 Such noncollinear haplotypes can complement the gene loss of individual inbred lines in the resulting heterozygous hybrid plants by hemizygous complementation, which is in support Journal of Proteome Research 2010, 9, 6511–6522 6511 Published on Web 09/14/2010

research articles

Marcon et al. 17

of the dominance hypothesis. Since in maize many genes are present in functionally redundant gene families, gene deletions in maize inbred lines might have only minor quantitative effects on plant performance. However, hemizygous complementation of multiple genes with minor quantitative effects might explain an increased performance of hybrids.17 Moreover, heterosis-associated gene expression has been analyzed in several studies by comparing expression profiles in hybrids and their parental inbred lines. For instance, heterosis-associated gene expression was assayed during heterosis manifestation in 3.5-day-old maize primary roots via cDNA microarray experiments.18 In this study, about 18% of the transcripts were nonadditively expressed in hybrid roots in at least 1 of 12 tested hybrids. Moreover, suppression subtractive hybridization (SSH) in combination with microarray hybridization experiments was applied to identify differentially expressed genes in maize embryos and endosperm 6 days after pollination (dap). Between 2 and 13% of these preselected genes displayed an expression level different from additivity.5 In contrast to these studies which revealed a relatively low number of genes being expressed differently from the parental average in hybrids, in other transcriptome surveys nonadditive gene expression prevailed as for instance in meristems19 and immature ears.20 The observed differences in these transcriptome studies might be caused by the use of different genotypes, plant tissues, and experimental designs. Intriguingly, when transcriptomes of maize primary roots and maize shoot apical meristems from identical genotypes were analyzed under the same experimental conditions, less overlap of nonadditively expressed genes was discovered than expected by chance.18 This suggests tissue-specific expression of nonadditive genes at least in primary roots and shoot apical meristems. This notion is also supported by the observation that maize displays significant differences in the degree of heterosis in distinct tissues or developmental stages.21 Proteomics combines the resolution of two-dimensional gel electrophoresis (2-DE) with protein identification via mass spectrometry.22 In recent years, proteomics was successfully applied to determine different aspects of root and embryo development in maize and rice.13,23-29 Thus far, little is known about heterosis-related protein accumulation patterns. A comparison of nonadditive protein accumulation patterns in mature hybrid rice embryos compared to embryos of their parental inbred lines revealed 23% nonadditively accumulated proteins.13 Similarly, in a 2-DE proteome analysis of 3.5-dayold maize primary roots 49% of the detected proteins were accumulated in a nonadditive manner in the hybrids compared to their parental inbred lines. Among those, 51% displayed an expression above the high or below the low parent expression value.30 In addition to transcriptomic and proteomic analyses of heterosis manifestation metabolomic profiling31,32 and integrated network analyses33,34 were performed. Moreover, cis and trans-regulatory effects on allelic expression were studied via high-throughput sequencing in order to dissect heterosis.35 The present study focused on the reciprocal hybrids UH005xUH250 and UH250xUH005 for which heterosis has been demonstrated with respect to several root traits compared to their parental inbred lines UH005 and UH250 in a previous study.4 Two developmental stages of immature maize embryo have been selected for the analyses: before (25 days after pollination, dap) and after (35 dap) the initiation of seminal roots. These stages are crucial during early root development, 6512

Journal of Proteome Research • Vol. 9, No. 12, 2010

and the molecular insights provided by this study might help to better understand the molecular networks involved in heterosis manifestation.

Materials and Methods Plant Material. The maize inbred lines UH005 (German national listing of plant varieties (NLPV) AC M9379) from the flint pool and UH250 (German NLPV AC M9005, Iowa Stiff Stalk) from the dent pool were propagated for phenotypic and proteomic analyses in the summer season of 2008 in the nursery of the University of Tuebingen. Plants of the inbred lines UH005 and UH250 were self-pollinated and crossed to generate the reciprocal hybrid genotypes UH005xUH250 and UH250xUH005. Prior to protein extraction embryos were manually isolated from 25 and 35 dap kernels of the selfed homozygous inbred lines UH005 and UH250 and the hybrids UH005xUH250 and UH250xUH005. Embryos were immediately frozen in liquid nitrogen after harvest. Each biological replicate was obtained from a pool of 20-30 embryos collected from three individual ears resulting in approximately 500 mg of fresh embryo material. Determination of Embryo Length and Weight. At least 60 embryos per genotype and time-point harvested from three independent ears were transferred into ethanol prior to phenotypic analyses. For each genotype/stage combination length and weight were determined in 30 embryos. Embryo length was analyzed using a binocular Stemi SV8 (Zeiss, Oberkochen, Germany) and documented with a Canon Powershot G2 digital camera (Canon, Krefeld, Germany). Data analysis was performed with Image-Pro Express software (version 4.5.1.3., MediaCybernetics, Silver Spring, MD, USA). Embryo weight was measured using the Acculab special accuracy balance ALC 110.4 (Acculab, Sartorius, Go¨ttingen, Germany). Histological Analyses of Embryos. Embryos were fixed in 4% (w/v) paraformaldehyde for 12 h at 4 °C and subsequently embedded in paraffin as previously described.36 Sections of 12 µm were prepared with a Leica 2035 Biocut-microtome (Leica, Nussloch, Germany). Deparaffinized embryo sections were stained with Safranin O (AppliChem, Darmstadt, Germany) and Fast Green (Sigma-Aldrich, Taufkirchen, Germany) as previously described.37 Stained embryo sections were analyzed under a Zeiss-Axioskop HBO 100W/2 microscope (Zeiss, Jena, Germany) and documented with a Canon Powershot G2 digital camera (Canon, Krefeld, Germany). Protein Isolation. For each biological replicate, total soluble protein extracts were isolated from approximately 500 mg of frozen embryos by an acetone-methanol precipitation protocol.38 Dried protein pellets were then resuspended in 400 µL of a solution containing 7 M urea (Sigma-Aldrich), 2 M thiourea (Sigma-Aldrich), 2% CHAPS (AppliChem), 1.25% (v/v) Bio-Lyte 3/10 (Biorad, Hercules, USA), 50 mM DTT (AppliChem), a trace of bromephenol blue (AppliChem), and one tablet of protease inhibitor complete (Roche, Mannheim, Germany) per 10 mL solution. Subsequently, the samples were ultrasonicated for 15 min, vortexed for 15 s, and ultrasonicated again for 15 min. The insoluble fraction was precipitated by centrifugation at 12000g for 60 min. The soluble protein extracts were frozen at -80 °C O/N and subjected to 2-DE protein separation. 2-DE Separation of Embryo Proteins, Gel Staining, and Quantification of Protein Spots. Per biological replicate 300 µg of total protein extract were subjected to isoelectric focusing using a PROTEAN IEF Cell (Biorad) and 24 cm immobilized, linear pH 4-7 gradients (ReadyStrip IPG strips, Biorad) followed

Proteomics of Maize Embryo Heterosis by rehydration at 50 V O/N. The following parameters were used for the isoelectric focusing (IEF): 250 V for 1 h, 500 V for 2 h, 1000 V for 1 h, voltage increase to 4000 V in 1 h, 4000 V for 2 h, voltage increase to 10 000 V in 2 h to a final setting of 60 000 Vh. The strips were equilibrated in buffer 1 (6 M urea, 4% (w/ v) SDS, 50 mM Tris, 1% (w/v) DTT, 30% glycerol) and subsequently in buffer 2 (6 M urea, 0.8% (w/v) SDS, 50 mM Tris pH 8.8, 4% (w/v) iodacetamid, 30% glycerol) for 15 min each. After equilibration, the proteins were separated based on their Mr’s in 12% SDS-PAGE gels using the PROTEAN Plus Dodeca Cell electrophoresis system (Biorad). After electrophoresis, the proteins were stained according to a modified colloidal Coomassie Blue staining protocol39 containing 10% (w/v) ammonium sulfate, 8.5% (v/v) phosphoric acid, 20% (v/v) methanol, and 0.12% (w/v) Coomassie Blue G250. The protein staining was performed for 72 h on an orbital shaker and was subsequently documented with an ImageMaster high-resolution flatbed scanner (Amersham Biosciences, Uppsala, Sweden). The gel image-files were exported and the detected proteins were normalized using PDQuest software (Biorad). During normalization 1 000 000 pixels were assigned to all visible spots of each 2-DE gel proportional to each spot’s intensity. The resulting relative spot intensities were used for statistical analyses. Statisitical Analyses. The statistical analyses for embryo length and weight, as well as for the embryo proteins, were done by a one-way analysis of variance (ANOVA) model. This approach enables great flexibility in estimating specific contrasts (heterosis and pairwise contrasts). The general linear model can be written as yij ) µ + βi + eij where yij is the jth observation of genotype i, µ is the general mean, present in every observation, βi is the fixed effect of genotype i, and eij is the independent, normally distributed measurement error of the jth observation for genotype i, that 2 is, eij i.i.d. ∼ N(0,σ ). In case of the embryo proteins, this model was fitted to each protein separately. The embryo length and weight were analyzed at different developmental stages using this model. Mid-parent heterosis (MPH) was evaluated using the appropriate linear contrast. If βˆ represents the vector of genotype estimates, and L is a row vector of coefficients specifying the appropriate linear contrast, the null hypothesis is H0:Lβ ) 0 which is tested using a t test.40 If A and B represent the means for the parental genotypes, and AB corresponds to the mean of the hybrid, the MPH can be written as: Lβ ) MPH ) AB -

1 1 A- B 2 2

To apply pairwise t tests, another linear contrast L could be used which is equivalent to performing separate two-sample t tests. If C and D represent the respective means of two genotypes, this contrast can be written as Lβ ) C - D

research articles To account for multiple testing, the false discovery rate (FDR) was controlled at the 5% level41 for the resulting p-values obtained for the embryo length and weight. For the embryonic proteins FDR-adjustment according to Benjamini-Yekutieli42 was used, which is a step-up procedure valid for general dependency structures. Dependencies can be assumed among proteins, which led to this choice of multiplicity adjustment. Relative MPH was calculated as the increase of the parental mean in percent by the formula MPH% ) MPH/MPV × 100, where the midparental value (MPV) given by MPV ) A/2 + B/2. Similarly, best-parent-heterosis (BPH) was calculated by BPH% ) (AB - BP)/BP × 100, where AB represents the mean of the hybrid and BP ) max(A,B), BP corresponds to the mean of the better parent. Classification of Nonadditively Accumulated Protein. The nonadditively accumulated proteins were classified in different categories as previously described.43 Briefly, proteins that show significantly higher accumulation in the hybrid than in both parental inbred lines were designated as “above high parent” (Figure 5: ++). Accordingly, hybrid accumulation values which were significantly lower than the lowest parental inbred value were classified as “below low parent” (Figure 5: --). Proteins which were significantly different from the parent with the lower expression value but not different from the higher parental expression value are described as “high parent” (Figure 5: +). Similarly, if the protein accumulation in the hybrid differed significantly from the high parental value but not from the low parent the category is called “low parent” (Figure 5: -). Finally, if the accumulation in the hybrid was significantly higher than in the lower parent but significantly lower than in the better parent, the hybrid displayed partial dominance (Figure 5: (). NanoHPLC-ESI-MS/MS Mass Spectrometry. All 141 protein spots identified as nonadditively accumulated between the parental inbred lines and the hybrids were excised from representative gels and digested in-gel using trypsin (sequencing grade, Promega, Mannheim, Germany) and subsequently eluted from the gel. Reversed-phase nano-LC-MS/MS was either performed as described elsewhere26 using an Ultimate nanoflow LC system (Dionex/LC Packings, Idstein, Germany) coupled to a QSTAR Pulsar i hybrid QqTOF mass spectrometer (Applied Biosystems/MDS Sciex, Darmstadt, Germany), equipped with a nanoelectrospray ion source (New Objective, Woburn, USA) or using a Proxeon Easy-LC coupled to a 4000QTRAP mass spectrometer (Applied Biosystems) with comparable parameters for the LC gradient and the data acquisition in the MS. As the Proxeon LC was operated in a setup without a precolumn the samples were cleaned up prior to LC-MS using StageTips.44 Analysis of LC-MS Data. Proteins were identified by correlating the ESI-MS/MS spectra with the protein sequence database of Zea mays at PlantGDB (http://www.plantgdb.org as of 02/05/09) using the MOWSE-algorithm as conducted in the MS search engine MASCOT (Matrix Science Ltd. London, UK).45 The parameter settings of the mass tolerances were fixed to 0.5 Da (QSTAR) or 1.5 Da (QTRAP) for the precursor ion and 0.5 Da for the fragment ions for both instruments, respectively. One missed cleavage was permitted and as modifications carbamidomethyl (fixed) and oxidation of methionine (variable) were included. The Mascot result file was analyzed using MSQuant (http:// msquant.sourceforge.net). All identified peptides were sorted and separated in three categories according to their Mascot Journal of Proteome Research • Vol. 9, No. 12, 2010 6513

research articles

Marcon et al.

Figure 1. Phenotypic analyses of embryos at two time-points (25 dap and 35 dap). Embryo length (A, C) and weight (B, D) of the inbred lines UH005, UH250, and the reciprocal hybrids UH005xUH250, UH250xUH005 were measured. Each data point represents 30 replicate measurements. Asterisks indicate that the hybrid differs significantly from the midparent value, MPV (*: p e 0.01; **: p e 0.001; ***: p e 0.0001; MPV: bold line). Error bars represent standard deviations per group.

ion scores based on significance thresholds (p e 0.01, p e 0.05, p e 0.1). The protein was successfully validated if the sum of all associated peptide scores exceeded the sum of the threshold scores of the lower peptide groups (p e 0.05 and p e 0.1; sum >89 and >71 for QSTAR and QTRAP data, respectively). Peptide sequences of all identified proteins are summarized in Table S1. The functional assignment of the proteins was implemented according to the MapMan Bin classification.46 Comparison of Different Nonadditively Accumulated Protein Data Sets. Homology comparisons of mature rice embryo and 3.5-day-old maize primary root proteins with the present data set were performed by downloading the protein data sets to a local stand alone blast server. Blast analyses of all these data sets were performed against the 107 unique maize proteins identified in this study (cut off value: E e 1 × 10-10). 6514

Journal of Proteome Research • Vol. 9, No. 12, 2010

Results Morphological and Histological Dissection of 25- and 35-day-old Immature Maize Embryos of Reciprocal Hybrids and Their Parental Inbred Lines. In order to study the early stages of heterosis manifestation in immature maize embryos, length and weight of embryos harvested from the inbred lines UH005 and UH250 and the reciprocal hybrids generated from these parents were analyzed 25 and 35 dap (Figure 1). For each genotype/stage combination, 30 embryos were analyzed. The 25 dap UH250 embryos were significantly longer and heavier than those of UH005 at the same developmental stage (Figure 1A,B). For both measurements, the midparent value (MPV) was calculated by the formula MPV ) A/2 + B/2. The embryos of the hybrid UH005xUH250 (25 dap) displayed values similar to the MPV. In contrast, length and weight of UH250xUH005 embryos significantly exceeded the MPV (Figure 1A,B). Length and weight of the 35-day-old embryos showed similar values for the homozygous inbred

Proteomics of Maize Embryo Heterosis

research articles

Figure 2. Safranin/fastgreen stained maize embryos 25 dap (A-D) and 35 dap (E-H) shown as transverse sections. A and E represent the inbred line UH005, B and F the hybrid UH005xUH250, C and G hybrid UH250xUH005, and D and H display the inbred genotype UH250 at the two time-points. While all 25-day-old embryos do not initiate seminal roots at this developmental stage, both hybrids of the 35-day-old embryos show seminal root initiation (arrows) in contrast to the parental lines (UH005 and UH250). Vascular bundles are indicated by an asterisk. Size bars: 1 mm.

lines UH005 and UH250 (Figure 1C,D). However, at this developmental stage both reciprocal hybrids significantly exceeded the MPV for length and weight. In summary, at both developmental stages the hybrid UH250xUH005 showed higher degrees of midparent (MPH%) and best-parent heterosis (BPH%) than UH005xUH250 indicating that the hybrid UH250xUH005 performs significantly better than the better of the two homozygous parental inbred lines. Transverse histological microsections were generated for 25 and 35 dap embryos of the reciprocal hybrids and their parental inbred lines. The 25 dap embryos of all four genotypes did not display any seminal root primordia (Figure 2A-D). In contrast, at 35 dap the reciprocal hybrids displayed seminal root primordia (Figure 2F,G), while their parental inbred lines (Figure 2E,H) did not. Isolation and 2-DE Separation of Embryo Soluble Proteins. On the basis of the histological results that distinguished 25and 35-day-old hybrid embryos by the absence and presence of seminal root primordia, a comparative proteome analysis was performed. The goal of this study was to identify proteins that are nonadditively accumulated in the hybrids UH005xUH250 and UH250xUH005 before (25 dap) and after seminal root initiation in hybrids (35 dap). Total soluble proteins were extracted in three biological replicates for each of the four genotypes at both developmental stages. Proteins were first separated by isoelectric focusing (IEF) on a linear gradient ranging from pH 4 to 7. Subsequently, proteins were separated according to their molecular masses using SDS-PAGE and then stained with colloidal CBB. All protein spots that were consistently detected in each of the three biological replicates of a

particular genotype were recorded. In total, 597 different protein spots were identified. Representative 2-DE gels of each genotype/stage combination are shown in Figures 3 and 4. The protein spot intensities on each replicate gel were normalized using PDQuest software (BioRad) to compensate for variations in spot intensity that are not related to protein accumulation (Table S2, Supporting Information). Identification of Nonadditively Accumulated Proteins in the Hybrids UH005xUH250 and UH250xUH005 at Two Developmental Stages of the Embryo. In order to identify nonadditively expressed proteins a two-step strategy was applied. First, hybrid proteins which accumulated at levels significantly different from the MPV of the inbred lines were determined by a one-way-ANOVA analysis and subsequent pairwise t tests. Among the 597 different protein spots identified on 2-DE gels, 141 protein spots (24%) displayed accumulation levels that deviated significantly from the MPV, in at least one hybrid. Significant reciprocal effects (p e 0.1) were identified in only two (1.4%) of the 141 nonadditively expressed proteins (spots 24, 26; Table S3 and S4, Supporting Information). All nonadditively accumulated proteins were then categorized according to a model previously suggested43 (Figure 5). Several of the 141 nonadditively expressed proteins were nonadditively accumulated in more than one genotype/stage combination. Hence, a total of 193 nonadditive expression patterns are summarized in Figure 5. Collectively, 8% of the proteins (15 out of 193) showed “below low parent” (--) expression in the hybrid genotypes. The largest proportion of nonadditively expressed proteins (86/193 or 44%) could be classified as “low parent” Journal of Proteome Research • Vol. 9, No. 12, 2010 6515

research articles

Marcon et al.

Figure 3. Representative 2-DE protein maps of soluble proteins extracted from 25 dap (A-D) embryos. Isoelectric focusing of the protein extracts in the first dimension was performed on linear IPG strips pH 4-7. In the second dimension, proteins were separated according to their molecular masses on 12% SDS-PAGE gels. Proteins were stained with a colloidal Coomassie dye. Proteins that were differentially accumulated in the hybrid genotypes are numbered on the gels. The mass spectrometric identification of these proteins is summarized in Tables S3 and S4, Supporting Information. The identified peptide fragments are summarized in Table S1, Supporting Information.

(-), whereas only 7 of 193 of the nonadditively accumulated proteins (4%) displayed partial dominance ((). In addition, 34 of the 193 proteins (18%) showed “high parent” (+) expression in the hybrids. Finally, 51 of the 193 nonadditively expressed proteins (26%) displayed an expression level that significantly exceeded that of the better parent (++). In summary, most proteins (120/193 or 62%) being nonadditively expressed in the hybrid lines resembled the expression level of the lower (-) or the better parent (+). The individual hybrids revealed a variable number of nonadditively expressed proteins. Furthermore, the hybrids exhibited differences in the distribution of the nonadditively accumulated proteins in the distinct categories, independently of the hybrid genotype or developmental stage (Figure 5). In UH005xUH250 (25 dap), 43 proteins were identified that deviated from the MPV, whereas the reciprocal hybrid UH250xUH005 (25 dap) showed only nine nonadditively expressed proteins. In summary, the hybrid genotypes of this developmental stage displayed 52 nonadditively accumulated proteins and most of these proteins showed “low” (-) or even 6516

Journal of Proteome Research • Vol. 9, No. 12, 2010

“below low parent” (--) expression. On the other hand, the 35 dap embryos of the hybrid UH005xUH250 displayed 34 proteins that were accumulated differently from additivity. They were mainly attributed to the class “low” (-). Interestingly, the reciprocal hybrid UH250xUH005 (35 dap) showed the largest number of nonadditively expressed proteins (107), which were mainly distributed between the classes -, +, and ++. Remarkably, the major part of the nonadditively accumulated proteins (100 out of 141) was exclusively detected in only one of the hybrid lines and developmental stages (Tables S3 and S4, Supporting Information). Only five protein spots were differently expressed from the MPV in two hybrids of the same developmental stage and additionally in a hybrid of the second developmental stage. Remarkably, among the five proteins detected in three of the hybrids four displayed the same accumulation tendency (Tables S3 and S4, Supporting Information). Mass Spectrometric Identification and Functional Annotation of Nonadditively Expressed Proteins. All 141 nonadditively accumulated protein spots were eluted from representative

Proteomics of Maize Embryo Heterosis

research articles

Figure 4. Representative 2-DE protein maps of soluble proteins extracted from 35 dap (A-D) embryos. Separation, staining, and protein identification were performed as described in Figure 3.

2-DE gels (Figures 3 and 4), digested with trypsin, and analyzed in a nano-HPLC-ESI-MS/MS spectrometer. Proteins not yet annotated in PlantGDB were annotated via blastp database searches (NCBI.nr database, cutoff value: E e 1× 10-10). The 141 proteins identified in this study were represented by 107 unique GenBank accessions. Among those, 19 accessions were represented by multiple protein spots. The storage proteins globulin 2 (spots 6, 54-55, 70-74, 99, 113, 121) and the globulin 1 S allele precursor (spots 1, 13, 15, 46-47, 76, 106, 110-111) were identified in 11 and 9 spots, respectively. Moreover, three proteins involved in glycolysis and TCA were represented by three spots of the same GenBank accession, respectively: an enolase 1 (spots 45, 50, 57), a triosephosphate isomerase (spots 7, 23, 95), and a succinate dehydrogenase flavoprotein subunit (spots 28, 129, 132). Other protein accessions that were represented by more than one protein spot are summarized in Tables S3 and S4, Supporting Information. All proteins differentially accumulated in 25- and 35-day-old embryos were functionally classified according to the MapMan Bin classification system46 (Table 1). The most prominent functional class was development (17%; 25 of 141 proteins) followed by protein

metabolism (11%; 16/141) and redox-regulation (8%; 11/141). Other proteins were classified in the categories amino acid metabolism (7%; 10/141), glycolysis (7%; 10/141), and tricarboxylic acid cycle (TCA)/organic acid transformation (6%; 9/141). The remaining proteins were distributed in 19 other functional classes listed in Table 1. Three of the annotated proteins were classified in two MapMan Bins: a precursor of dehydrogenase dihydrolipoamide dehydrogenase 4 (spot 35) mapped to redox-regulation and TCA/organic acid transformation. Moreover, the pyridoxin biosynthesis protein ER1 (spot 31) was classified in the MapMan Bins hormone metabolism and cofactor and vitamine metabolism. Finally, an ankyrin repeat domain-containing protein 2 (spots 114 and 115) was classified in the categories RNA and cell organization (Tables S3 and S4, Supporting Information). Up-regulation of Glucose Metabolism in Hybrid Embryos. Among the protein spots that are nonadditively expressed in at least one of the hybrid embryo genotype/stage combinations, 23 proteins (16%), represented by 17 Genbank accessions, were involved in carbohydrate metabolism (Figure 6, Tables S3 and S4, Supporting Information). According to the MapMan Bin Journal of Proteome Research • Vol. 9, No. 12, 2010 6517

research articles

Marcon et al. Table 1. Functional Classification and Abundance of All Differentially Accumulated Proteins in Hybrid Embryos (25 dap, 35 dap) MapMan Binsa

33.1, 33.3 29.2, 29.3, 29.5, 29.6 21, 21.2, 21.5 4.1, 4.5, 4.7, 4.8, 4.11, 4.12 13.1, 13.2 8.1, 8.2

Figure 5. Classification of accumulation trends of 141 differentially accumulated proteins in the hybrid UH005xUH250 and UH250xUH005 at two developmental stages (25 dap and 35 dap) as previously described.43 The numbers above the columns reflect the absolute numbers of proteins in each category.

classification system,46 four of these proteins are functioning upstream of glycolysis supplying intermediates for the glycolytic pathway (spots 58, 63, 65, 107) and 10 are directly involved in glycolysis (spots 7, 23, 34, 45, 50, 57, 66, 95, 140, 141). Furthermore, nine of the nonadditively accumulated enzymes are functioning within the TCA (spots 20, 25, 35, 28, 80, 86, 129, 132, 136). Among those, three catalyze the conversion of pyruvate to acetyl-CoA (spots 25, 35, 80). Remarkably, 10 of these 23 protein spots displayed “above high parent” expression in the 35-day-old UH250xUH005 (Figure 6, Tables S3 and S4, Supporting Information). The other genotype/stage combinations revealed a low number of nonadditively accumulated proteins related to carbohydrate metabolism showing a more heterogeneous distribution across the different expression classes. Homology Comparison of Different Nonadditively Accumulated Protein Data Sets. All 107 distinct protein accessions of the immature maize hybrid embryos were compared to nonadditively expressed proteins of mature embryos of rice hybrids13 and such proteins identified in maize primary roots.30 Homology was identified for 17/42 proteins (41%) of rice hybrids compared to nonadditively accumulated proteins in immature maize hybrid embryos (Figure 7 and Table S5, Supporting Information). In total 24/58 proteins (41%) of 3.5day-old maize roots displayed high similarity to nonadditively expressed embryonic maize proteins. Protein homology comparison of maize roots with rice embryos displayed only one protein (acidic ribosomal protein P0, T04309) to be similar in both surveys. One protein, a heat shock cognate 70 kDa protein 2 (spot 12), was identified throughout the three proteome studies (Figure 7 and Table S5, Supporting Information).

Discussion Although adult traits of maize display the highest degree of heterosis,2 hybrid vigor can already be detected during embryo 6518

Journal of Proteome Research • Vol. 9, No. 12, 2010

35.2 20, 20.2 17.2, 17.5 1.3 5.10 23.2, 23.3, 23.4 27.3, 27.4 16.2, 16.4 18, 18.2 19.3 24.1, 24.2 26.2, 26.8 31.1 2.1, 2.2 11.1 3.5 7.1 9.9 25

functional classification

no. of proteins

development protein metabolism

25 16

redox-regulationb glycolysis

11 11

amino acid metabolism tricarboxylic acid cycle/organic acid transformationb not assigned stress hormone metabolismc photosynthesis, Calvin cycle fermentation nucleotide metabolism RNAd secondary metabolism cofactor and vitamine metabolismc tetrapyrrole synthesis biodegradation of xenobiotics misc cell organizationd major carbohydrate metabolism lipid metabolism minor CHO metabolism oxidative pentose phosphate pathway mitochondrial electron transport/ATP synthesis C1-metabolism

10 9 9 7 6 4 4 4 4 3 3 2 3 3 3 2 2 1 1 1 1 145

a Functional assignment according to the MapMan Bin classification.46 gi 224111924 Precursor of dehydrogenase dihydrolipoamide dehydrogenase 4 [P. trichocarpa] (spot 35) mapped to two bins: Redox-regulation and TCA/organic acid transformation. c gi 226528529 Pyridoxin biosynthesis protein ER1 [Z. mays] (spot 31) mapped to two bins: Hormone metabolism and Cofactor and vitamine metabolism. d gi 195652911 Ankyrin repeat domain-containing protein 2 [Z. mays] (spots 114, 115) mapped to two bins: RNA and Cell organization. b

development as early as six days after pollination.6 In the present study, phenotypic parameters of immature embryos were compared between reciprocal hybrids and their parental inbred lines 25 and 35 days after pollination. These experiments demonstrated that size and weight of the hybrid UH250xUH005 significantly exceeded the midparent values at both developmental stages (Figure 1). MPH% for embryo length was between 15-34% for UH250xUH005 and 1-5% for UH005xUH250 (Figure 1). These results are consistent with a previous survey in which six dap embryos of the hybrid UH250xUH005 (MPH 51%) displayed a higher degree of heterosis concerning embryo length than the reciprocal hybrid UH005xUH250 (MPH -11%) at the same developmental stage,5 suggesting a faster growth of this hybrid during embryo development. Analysis of transverse sections of inbred and hybrid embryos (Figure 2) revealed that the initiation of seminal root primordia during embryogenesis was dependent on the developmental stage and the genotype. In previous studies, seminal root primordia were detectable between 22 and 40 dap in different maize inbred lines depending on their genetic background.27,47,48 In the present study, seminal roots were present in 35 dap hybrid embryos but absent in their parental inbred lines, suggesting

Proteomics of Maize Embryo Heterosis

research articles

Figure 6. Seventeen enzymes (in bold) involved in carbohydrate metabolism were differentially accumulated in distinct hybrids (in brackets). --: below low parent expression; -: low parent expression; (: partial dominance; +: high parent expression; ++: above high parent expression.

that the hybrids UH005xUH250 and UH250xUH005 develop faster than their parental inbred lines.

Figure 7. Overlap of unique maize proteins identified in this study that display homology with nonadditively expressed protein identified in rice mature embryos13 and 3.5-day-old maize primary roots.30

In this survey, 24% (141 of 597) of the unique soluble proteins were accumulated differently from additivity in at least one of the reciprocal hybrids. In previous heterosis-related proteome studies, the fractions of nonadditively proteins in hybrid plants varied. In 3.5-day-old hybrid maize primary roots 49% of all proteins were accumulated differently from additivity,30 whereas in mature hybrid rice embryos 23% of the soluble proteins were nonadditively accumulated.13 In total, 193 nonadditive expression patterns were detected in the four genotype/stage combinations analyzed in this study. Among those, 27% (52 proteins) were identified in 25-day-old hybrid embryos, while the majority of nonadditively accumulated proteins (73%; 141 proteins) were associated with 35-day-old hybrids (Figure 5, Tables S3 and S4, Supporting Information). This observation suggests that the increasing morphological and histological differences manifested between embryos of inbred lines and hybrids during the two developmental stages are also reflected on the proteome level. Journal of Proteome Research • Vol. 9, No. 12, 2010 6519

research articles Nonadditively accumulated proteins identified in the 25 and 35 dap hybrids UH005xUH250 and UH250xUH005 were categorized based on their relative expression levels with reference to the parental inbred lines according to the classification of Stupar and Springer.43 Most of the nonadditively expressed proteins (66%) showed accumulation levels either similar to or between the parental values (classes: +, -, (, Figure 5). The remaining proteins were expressed at levels below those of the lower parent (8%) or above of those of the better parent (26%). Consistent with this data, other expression studies including maize embryos, young seedlings, and immature ears3,5,20,43 also described the majority of nonadditively expressed genes to be expressed at levels between the values of the parental inbred lines on the transcriptome level. In contrast, one heterosis related transcriptome study of maize shoot apical meristems19 as well as the proteome analysis of 3.5-day-old maize primary roots30 demonstrated that about 50% of the analyzed genes/ proteins displayed expression levels above the high or below the low parental value. Different tissues and organs within a hybrid plant display significant differences in their degree of heterosis which might partly explain the high variation between the results of these studies.9,49 In contrast to the phenotypic reciprocal effects observed between the hybrids only two (1.4%) of 141 nonadditively expressed proteins in this study showed significant reciprocal effects (p e 0.1; spots 24, 26; Tables S3 and S4, Supporting Information), suggesting that phenotypic reciprocal differences are not necessarily correlated with reciprocal effects of the most abundant soluble proteins in maize hybrids. In total, 19 unique protein accessions that were expressed in a nonadditive fashion were represented by between two and 11 protein spots in the present study (Tables S3 and S4, Supporting Information). In a previous study,30 seven unique accessions were nonadditively accumulated and were also represented several times. Such proteins might represent different posttranslational modifications which not only influence their function but also change the chemical and physical character (Mr’s, pI) of the protein leading to individual positions on 2-DE gels.50 The resulting pool of proteins can have different functions, or specificities, and may expand the properties of the cellular proteome. Four of the nonadditively accumulated proteins showed the same accumulation pattern in three of the four analyzed hybrids (Tables S3 and S4, Supporting Information). Among those, GrpE1 (spot 9; ++) belongs to the functional class of protein metabolism. GrpE1 functions as a cochaperone of Hsp70 chaperones which are involved in many cellular processes such as preventing aggregation of proteins, assisting refolding, protein import and translocation.51 Remarkably, a member of the Hsp70 family (spot 12) was also up-regulated (++) in 25 dap UH005xUH250 and 35 dap UH250xUH005. Such heat shock cognate 70 kDa proteins (Hsc70) are mainly involved in the correct folding of de novo synthesized polypeptides and in the import/translocation of proteins precursors in cell organelles.51 The up-regulation of GrpE1 and Hsc70 in maize hybrids during embryo development could be related to an increased capacity or effectiveness of protein folding and translocation in hybrid maize embryos. Remarkably, Hsp70 was the only nonadditively accumulated protein that could be identified in rice embryos, 3.5-day-old maize roots and in maize embryos13,30 (Figure 7) which might imply a general role of the processes controlled by this class of proteins during the manifestation of heterosis. 6520

Journal of Proteome Research • Vol. 9, No. 12, 2010

Marcon et al. All four hybrid combinations showed “above high parent” (++) expression for an ankyrin repeat domain-containing protein 2 (AKR2; spots 114, 115; Tables S3 and S4, Supporting Information). Ankyrin repeats are degenerate 33-amino-acidrepeats serving as domains for protein-protein interaction.52 In Arabidopsis, AKR2 interacts with ascorbate peroxidase 3 (APX3) which detoxifies hydrogen peroxides (H2O2). AKR2 may be involved in the regulation of APX3 activity in plants indicating the importance of AKR2 for the antioxidation metabolism and disease resistance in plants.53 A better resistance to disease or to reactive oxygen species could be beneficial for hybrids and might thus contribute to heterosis. Carbohydrates are the most abundant organic metabolites in most living organisms and are a primary source of chemical energy.54 In the present study, proteins involved in glucose metabolism were found to be the largest up-regulated class of proteins in the 35-day-old hybrid embryos UH250xUH005 (Figure 6). An up-regulation of the glucose metabolism could be associated with the increased speed of embryo development detected for the hybrids studied in this survey (Figure 2). Remarkably, similar glucose metabolizing proteins, especially enzymes of the gycolysis and the TCA, were detected to be significantly up- or down-regulated in several 2-DE proteome studies.13,27,28,30 Moreover, a metabolome study detected several metabolites with nonadditive accumulation patterns in hybrid maize kernels (8-30 dap).31 Here, the hybrid kernels revealed higher values compared to the midparent values for the metabolites glucose-6-phosphate, starch, and sucrose. Among the most abundant nonadditively accumulated proteins identified in mature embryos of rice hybrids,13 41% (17/ 42) were similar to nonadditively accumulated proteins in immature maize hybrid embryos (Figure 7). This suggests conserved organ- or tissue-specific regulatory mechanisms in monocots related to heterosis. In contrast, only ∼5% of the nonadditively accumulated embryonic rice proteins13 displayed similarity to such proteins identified in maize roots,30 while for 41% of the nonadditively accumulated maize primary root proteins30 nonadditively expressed homologues were identified in maize embryos. Hence, while heterosis associated nonadditive protein patterns are apparently conserved to a certain degree between different organs of a species such as maize, little conservation of such patterns is observed between these organs between different closely related monocot species such as maize and rice. In summary, the presented data suggest that reciprocal maize hybrids exhibit heterosis already during embryo development for traits such as length, weight, or seminal root primordia formation. Moreover, these heterotic traits are associated with nonadditive protein accumulation and in particular with an up-regulation of sugar metabolism during later stages of embryo development. Finally, comparison of proteome data sets representing data of maize and rice hybrid embryos suggested conserved organ- or tissue-specific regulatory mechanisms in monocots related to heterosis.

Acknowledgment. This project was supported by grants of the DFG (Deutsche Forschungsgemeinschaft) priority program SPP (Schwerpunktprogramm) 1149 “heterosis in plants” to F.H. and H.P.P. We thank Inga Buchen for excellent technical help with 2-DE experiments, Dr. Anja Paschold (University of Tuebingen) for her helpful comments on the manuscript, and Prof. Albrecht

research articles

Proteomics of Maize Embryo Heterosis Melchinger and his co-workers (University of Hohenheim) for providing seeds of the inbred lines and hybrids used in this study.

Supporting Information Available: Table S1. Peptide fragments identified via ESI-MS/MS. Table S2. Mean protein spot intensities after normalization and division by the standard deviation of the 597 proteins detected in 25- and 35-day-old embryos. Table S3. Mass spectrometric identification (ESI-MS/ MS) and annotation of proteins extracted from 25 dap maize embryos that revealed nonadditive accumulation in the hybrids UH005xUH250 and UH250xUH005 compared to its parental lines UH005 and UH250 after separation on 2-DE gels. Table S4. Mass spectrometric identification (ESI-MS/MS) and annotation of proteins extracted from 35 dap maize embryos that revealed nonadditive accumulation in the hybrids UH005xUH250 and UH250xUH005 compared to its parental lines UH005 and UH250 after separation on 2-DE gels. Table S5. Maize embryo proteins for which similar proteins were identified in hybrids of 3.5-day-old maize primary roots or mature rice embryos. This material is available free of charge via the Internet at http://pubs.acs.org.

(17) (18)

(19)

(20)

(21) (22)

(23)

References (1) Shull, G. H. Beginning of the heterosis concept. In Heterosis; Gowen, J. W., Ed.; Iowa State College Press: Ames, IA, 1952; pp 14-48. (2) Falconer, M. S., Mackay, T. F. C. Introduction to Quantitative Genetics, 4th ed.; Longman: Harlow, 1996. (3) Swanson-Wagner, R. A.; Jia, Y.; DeCook, R.; Borsuk, L. A.; Nettleton, D.; Schnable, P. S. All possible modes of gene action are observed in a global comparison of gene expression in a maize F1 hybrid and its inbred parents. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 6805–6810. (4) Hoecker, N.; Keller, B.; Piepho, H. P.; Hochholdinger, F. Manifestation of heterosis during early maize (Zea mays L.) root development. Theor. Appl. Genet. 2006, 112, 421–429. (5) Jahnke, S.; Sarholz, B.; Thiemann, A.; Kuhr, V.; Gutierrez-Marcos, J. F.; Geiger, H. H.; Piepho, H. P.; Scholten, S. Heterosis in early seed development: a comparative study of F1 embryo and endosperm tissues 6 days after fertilization. Theor. Appl. Genet. 2010, 120, 389–400. (6) Meyer, S.; Pospisil, H.; Scholten, S. Heterosis associated gene expression in maize embryos 6 days after fertilization exhibits additive, dominant and overdominant pattern. Plant Mol. Biol. 2007, 63, 381–391. (7) Birchler, J. A.; Auger, D. L.; Riddle, N. C. In search of the molecular basis of heterosis. Plant Cell 2003, 15, 2236–2239. (8) Birchler, J. A.; Yao, H.; Chudalayandi, S. Unraveling the genetic basis of hybrid vigor. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 12957–12958. (9) Hochholdinger, F.; Hoecker, N. Towards the molecular basis of heterosis. Trends Plant Sci. 2007, 12, 427–432. (10) Springer, N. M.; Stupar, R. M. Allelic variation and heterosis in maize: how do two halves make more than a whole. Genome Res. 2007, 17, 264–275. (11) Jones, D. F. Dominance of linked factors as a means of accounting for heterosis. Genetics 1917, 2, 466–479. (12) Vuylsteke, M.; van Eeuwijk, F.; Van Hummelen, P.; Kuiper, M.; Zabeau, M. Genetic analysis of variation in gene expression in Arabidopsis thaliana. Genetics 2005, 171, 1267–1275. (13) Wang, W.; Meng, B.; Ge, X.; Song, S.; Yang, Y.; Yu, X.; Wang, L.; Hu, S.; Liu, S.; Yu, J. Proteomic profiling of rice embryos from a hybrid rice cultivar and its parental lines. Proteomics 2008, 8, 4808– 4821. (14) Brunner, S.; Fengler, K.; Morgante, M.; Tingey, S.; Rafalski, A. Evolution of DNA sequence nonhomologies among maize inbreds. Plant Cell 2005, 17, 343–360. (15) Song, R.; Messing, J. Gene expression of a gene family in maize based on noncollinear haplotypes. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 9055–9060. (16) Springer, N. M.; Ying, K.; Fu, Y.; Ji, T.; Yeh, C. T.; Jia, Y.; Wu, W.; Richmond, T.; Kitzman, J.; Rosenbaum, H.; Iniguez, A. L.; Barbazuk, W. B.; Jeddeloh, J. A.; Nettleton, D.; Schnable, P. S. Maize inbreds

(24) (25)

(26)

(27)

(28)

(29) (30)

(31)

(32)

(33)

(34)

(35)

exhibit high levels of copy number variation (CNV) and presence/ absence variation (PAV) in genome content. PLoS Genet. 2009, 5, e1000734. Fu, H.; Dooner, H. K. Intraspecific violation of genetic colinearity and its implications in maize. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 9573–9578. Hoecker, N.; Keller, B.; Muthreich, N.; Chollet, D.; Descombes, P.; Piepho, H. P.; Hochholdinger, F. Comparison of maize (Zea mays L.) F1-hybrid and parental inbred line primary root transcriptomes suggests organ-specific patterns of nonadditive gene expression and conserved expression trends. Genetics 2008, 179, 1275–1283. Uzarowska, A.; Keller, B.; Piepho, H. P.; Schwarz, G.; Ingvardsen, C.; Wenzel, G.; Lu ¨ bberstedt, T. Comparative expression profiling in meristems of inbred-hybrid triplets of maize based on morphological investigations of heterosis for plant height. Plant Mol. Biol. 2007, 63, 21–34. Guo, M.; Rupe, M. A.; Yang, X.; Crasta, O.; Zinselmeier, C.; Smith, O. S.; Bowen, B. Genome-wide transcript analysis of maize hybrids: allelic additive gene expression and yield heterosis. Theor. Appl. Genet. 2006, 113, 831–845. Springer, N. M.; Stupar, R. M. Allele-specific expression patterns reveal biases and embryo-specific parent-of-origin effects in hybrid maize. Plant Cell 2007, 19, 2391–2402. Go¨rg, A.; Obermaier, C.; Boguth, G.; Harder, A.; Scheibe, B.; Wildgruber, R.; Weiss, W. The current state of two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 2000, 21, 1037–1053. Dembinsky, D.; Woll, K.; Saleem, M.; Liu, Y.; Fu, Y.; Borsuk, L. A.; Lamkemeyer, T.; Fladerer, C.; Madlung, J.; Barbazuk, B.; Nordheim, A.; Nettleton, D.; Schnable, P. S.; Hochholdinger, F. Transcriptomic and proteomic analyses of pericycle cells of the maize primary root. Plant Physiol. 2007, 145, 575–588. Hochholdinger, F.; Guo, L.; Schnable, P. S. Lateral roots affect the proteome of the primary root of maize (Zea mays L.). Plant Mol. Biol. 2004, 56, 397–412. Hochholdinger, F.; Woll, K.; Guo, L.; Schnable, P. S. The accumulation of abundant soluble proteins changes early in the development of the primary roots of maize (Zea mays L.). Proteomics 2005, 5, 4885–4893. Liu, Y.; Lamkemeyer, T.; Jakob, A.; Mi, G.; Zhang, F.; Nordheim, A.; Hochholdinger, F. Comparative proteome analyses of maize (Zea mays L.) primary roots prior to lateral root initiation reveal differential protein expression in the lateral root initiation mutant rum1. Proteomics 2006, 6, 4300–4308. Muthreich, N.; Schu ¨ tzenmeister, A.; Schu ¨ tz, W.; Madlung, J.; Krug, K.; Nordheim, A.; Piepho, H. P.; Hochholdinger, F. Regulation of the maize (Zea mays L.) embryo proteome by RTCS which controls seminal root initiation. Eur. J. Cell Biol. 2010, 89, 242–249. Saleem, M.; Lamkemeyer, T.; Schu ¨ tzenmeister, A.; Fladerer, C.; Piepho, H. P.; Nordheim, A.; Hochholdinger, F. Tissue specific control of the maize (Zea mays L.) embryo, cortical parenchyma, and stele proteomes by RUM1 which regulates seminal and lateral root initiation. J. Proteome Res. 2009, 8, 2285–2297. Sauer, M.; Jakob, A.; Nordheim, A.; Hochholdinger, F. Proteomic analysis of shoot-borne root initiation in maize (Zea mays L.). Proteomics 2006, 6, 2530–2541. Hoecker, N.; Lamkemeyer, T.; Sarholz, B.; Paschold, A.; Fladerer, C.; Madlung, J.; Wurster, K.; Stahl, M.; Piepho, H. P.; Nordheim, A.; Hochholdinger, F. Analysis of nonadditive protein accumulation in young primary roots of a maize (Zea mays L.) F1-hybrid compared to its parental inbred lines. Proteomics 2008, 8, 3882– 3894. Ro¨misch-Margl, L.; Spielbauer, G.; Schu ¨ tzenmeister, A.; Schwab, W.; Piepho, H. P.; Genschel, U.; Gierl, A. Heterotic patterns of sugar and amino acid components in developing maize kernels. Theor. Appl. Genet. 2010, 120, 369–381. Spielbauer, G.; Margl, L.; Hannah, L. C.; Ro¨misch, W.; Ettenhuber, C.; Bacher, A.; Gierl, A.; Eisenreich, W.; Genschel, U. Robustness of central carbohydrate metabolism in developing maize kernels. Phytochemistry 2006, 67, 1460–1475. Andorf, S.; Selbig, J.; Altmann, T.; Poos, K.; Witucka-Wall, H.; Repsilber, D. Enriched partial correlations in genome-wide gene expression profiles of hybrids (A. thaliana): a systems biological approach towards the molecular basis of heterosis. Theor. Appl. Genet. 2010, 120, 249–259. Steinfath, M.; Ga¨rtner, T.; Lisec, J.; Meyer, R. C.; Altmann, T.; Willmitzer, L.; Selbig, J. Prediction of hybrid biomass in Arabidopsis thaliana by selected parental SNP and metabolic markers. Theor. Appl. Genet. 2010, 120, 239–247. Guo, M.; Yang, S.; Rupe, M.; Hu, B.; Bickel, D. R.; Arthur, L.; Smith, O. Genome-wide allele-specific expression analysis using massively

Journal of Proteome Research • Vol. 9, No. 12, 2010 6521

research articles

(36)

(37) (38)

(39)

(40) (41) (42) (43) (44)

6522

parallel signature sequencing (MPSS) reveals cis- and trans-effects on gene expression in maize hybrid meristem tissue. Plant Mol. Biol. 2008, 66, 551–563. Lim, J.; Helariutta, Y.; Specht, C. D.; Jung, J.; Sims, L.; Bruce, W. B.; Diehn, S.; Benfey, P. N. Molecular analysis of the SCARECROW gene in maize reveals a common basis for radial patterning in diverse meristems. Plant Cell 2000, 12, 1307–1318. Hetz, W.; Hochholdinger, F.; Schwall, M.; Feix, G. Isolation and characterization of rtcs a mutant deficiant in the formation of nodal roots. Plant J. 1996, 10, 845–857. Damerval, C.; Vienne, D.; Zivy, M.; Thiellement, H. Technical improvements in two-dimensional electrophoresis increase the level of genetic variation detected in wheat seedling proteins. Electrophoresis 1986, 7, 52–54. Neuhoff, V.; Arold, N.; Taube, D.; Ehrhardt, W. Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250. Electrophoresis 1988, 9, 255– 262. Searle, S. R. Linear Models; John Wiley & Sons, Inc.: New York, 1971. Benjamini, Y.; Hochberg, Y. Controlling the false discovery rate: A practical and powerful approach to multiple testing. J. R. Stat. Soc. Ser. B 1995, 57, 289–300. Benjamini, Y.; Yekutieli, D. The control of the false discovery rate in multiple testing under dependency. Ann. Stat. 2001, 29, 1165– 1188. Stupar, R. M.; Springer, N. M. Cis-transcriptional variation in maize inbred lines B73 and Mo17 leads to additive expression patterns in the F1 hybrid. Genetics 2006, 173, 2199–2210. Rappsilber, J.; Mann, M.; Ishihama, Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat. Protoc. 2007, 2, 1896–1906.

Journal of Proteome Research • Vol. 9, No. 12, 2010

Marcon et al. (45) Perkins, D. N.; Pappin, D. J.; Creasy, D. M.; Cottrell, J. S. Probabilitybased protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 1999, 20, 3551–3567. (46) Thimm, O.; Bla¨sing, O.; Gibon, Y.; Nagel, A.; Meyer, S.; Kru ¨ ger, P.; Selbig, J.; Mu ¨ ller, L. A.; Rhee, S. Y.; Stitt, M. MAPMAN: a userdriven tool to display genomics data sets onto diagrams of metabolic pathways and other biological processes. Plant J. 2004, 37, 914–939. (47) Feldman, L. The maize root. In The Maize Handbook; Freeling, M., Walbot, V., Eds.; Springer: New York, 1994, 29-37. (48) Sass, J. L. Morphology. In The Maize Handbook; Springer: New York, 1977; pp 2937. (49) Paschold, A.; Marcon, C.; Hoecker, N.; Hochholdinger, F. Molecular dissection of heterosis manifestation during early maize root development. Theor. Appl. Genet. 2010, 120, 383–388. (50) Kumar, Y.; Khachane, A.; Belwal, M.; Das, S.; Somsundaram, K.; Tatu, U. ProteoMod: A new tool to quantitate protein posttranslational modifications. Proteomics 2004, 4, 1672–1683. (51) Wang, W.; Vinocur, B.; Shoseyov, O.; Altman, A. Role of plant heatshock proteins and molecular chaperones in the abiotic stress response. Trends Plant Sci. 2004, 9, 244–252. (52) Michaely, P.; Bennett, V. The ANK repeat: a ubiquitous motif involved in macromolecular recognition. Trends Cell Biol. 1992, 2, 127–129. (53) Yan, J.; Wang, J.; Zhang, H. An ankyrin repeat-containing protein plays a role in both disease resistance and antioxidation metabolism. Plant J. 2002, 29, 193–202. (54) Plaxton, W. C. The organization and regulation of plant glycolysis. Annu. Rev. Plant Physiol. Plant Mol. Biol. 1996, 47, 185–214.

PR100718D