Maize (Zea mays L.) seedling leaf nuclear proteome and differentially expressed proteins between a hybrid and its parental lines

To better understand the underlying molecular basis of leaf development in maize, a reference map of nuclear proteins in basal region of seedling leaf was established using a combination of 2DE and MALDI‐TOF‐MS. In total, 441 reproducible protein spots in nuclear proteome of maize leaf basal region were detected with silver staining in a pH range of 3–10, among which 203 spots corresponding to 163 different proteins were identified. As expected, proteins implicated in RNA and protein‐associated functions were overrepresented in nuclear proteome. Remarkably, a high percentage (10%) of proteins was identified to be involved in cell division and growth. In addition, comparative nuclear proteomic analysis in leaf basal region of highly heterotic hybrid Mo17/B73 and its parental lines was also performed and 52 of 445 (11.69%) detected protein spots were differentially expressed between the hybrid and its parental lines, among which 16 protein spots displayed nonadditively expressed pattern. These results indicated that hybridization between two parental lines can cause changes in the expression of a variety of nuclear proteins, which may be responsible for the observed leaf size heterosis.     

Identification of differentially expressed proteins between hybrid and parents in wheat (Triticum aestivum L.) seedling leaves

In spite of commercial use of heterosis in agriculture, the molecular basis of heterosis is poorly understood. To gain a better understanding of the molecular basis of wheat heterosis, we carried out a comparative proteomic analysis in seedling leaves between wheat hybrid and parents. Common wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD) Line 3338 and spelt wheat (Triticum spelta L., 2n = 6x = 42, AABBDD) Line 2463 were used to produce a heterotic F₁ hybrid. The expression patterns of the total proteins were compared in seedling leaves between hybrid and its parents by using two-dimensional gel electrophoresis with two pH ranges for the first dimension separation. Among ~900 protein spots reproducibly detected, 49 protein spots were identified as being differentially expressed between hybrid and its parental lines (P < 0.05) for more than 1.5-folds. Six possible modes of differential expression were observed, including high- and low-parent dominance, underdominance, and overdominance, uniparent silencing and uniparent dominance. Moreover, 30 of the 49 differentially expressed protein spots were identified, which were involved in metabolism, signal transduction, energy, cell growth and division, disease and defense, secondary metabolism. These results indicated that wheat hybridization can cause protein expression differences between hybrid and its parents; these proteins were involved in diverse physiological process pathways, which might be responsible for the observed heterosis. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. X. Song and Z. Ni have equally contributed to this work.     

Nonadditive protein accumulation patterns in Maize (Zea mays L.) hybrids during embryo development

Heterosis describes the superior performance of heterozygous F(1)-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 F(1)-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.     

Analysis of nonadditive protein accumulation in young primary roots of a maize (Zea mays L.) F(1)-hybrid compared to its parental inbred lines

Heterosis describes the superior performance of heterozygous F(1)-hybrids compared to their homozygous parental inbred lines. Heterosis is already manifested during early maize (Zea mays L.) primary root development. In this study, the most abundant soluble proteins have been investigated before the phenotypic manifestation of heterosis in 3.5-day-old primary roots in the flint inbred line UH002, the dent inbred line UH301 and the corresponding hybrid UH301 x UH002. In CBB-stained 2-DE gels, 150 of 304 detected proteins (49%) were accumulated in a nonadditive fashion in the hybrid compared to the average of their parental inbred lines (Student's t-test: p < 0.05). Remarkably, expression of 51% (76/150) of the nonadditively accumulated proteins exceeded the high parent or was below the low parent. ESI-MS/MS identified 75 of the 76 proteins that belonged to these expression classes. The most abundant functional classes among the 75 proteins that were encoded by 60 different genes were metabolism (58%) and disease and defense (19%). Nonadditive protein accumulation in primary roots of maize hybrids might be associated with heterosis manifestation. Identification of these proteins could therefore contribute to the better understanding of the molecular basis of heterosis.     

Relationship between gene expression and hybrid vigor in primary root tips of young maize (Zea mays L.) plantlets

Summary To provide an insight into the molecular basis of heterosis, we investigated gene expression in primary root tips of a heterotic maize hybrid (B73 × Mo17) and its parental lines (B73 and Mo17). This analysis was carried out (i) by differential plaque hybridization of a recombinant cDNA library made to poly(A) RNA isolated from B73 × Mo17 primary root tips, and (ii) by comparing with two-dimensional gel electrophoresis proteins synthesized in vitro in the rabbit reticulocyte system by poly(A) RNA isolated, at different stages of development, from the three genotypes. The results showed that there are sets of proteins and mRNAs that are differentially synthesized and expressed in the F₁ primary root tips in comparison to the parental lines. Moreover, results from the survey of 21 major in-vitrosynthesized polypeptide variants, from mRNAs of primary root tips of the parental lines and their F₁ hybrid, indicated that in seven instances hybrid proteins translated in vitro were more abundant or possibly new. In most of the remaining cases, hybrid spots were similar in intensity to the same protein produced by one of the two parental lines.     

Proteomic analysis of muscle between hybrid abalone and parental lines Haliotis gigantea Reeve and Haliotis discus hannai Ino

To understand the potential molecular mechanism of heterosis, protein expression patterns were compared from hybrids of Haliotis gigantea (G) and Haliotis discus hannai (D) using two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight analyses. Expression differences were observed in muscle samples from the four groups with 673±21.0 stained spots for H. discus hannai ♀ × H. discus hannai ♂ (DD), 692±25.6 for H. gigantea ♀ × H. gigantea ♂ (GG), 679±16.2 for H. discus hannai ♀ × H. gigantea ♂ (DG) (F₁ hybrid) and 700±19 for H. gigantea ♀ × H. discus hannai ♂ (GD) (F₁ hybrid). Different 2-DE image muscle protein spots had a mirrored relationship between purebreds and the F₁ hybrid, suggesting that all stained spots in F₁ hybrid muscle were on 2-DEs from parents. DD and DG clustered together first, and then clustered with GD, whereas the distance of DD and GG was maximal according to hierarchical cluster analysis. We identified 136 differentially expressed protein spots involved in major biological processes, including energy metabolism and stress response. Most energy metabolism proteins were additive, and stress-induced proteins displayed additivity or over-dominance. In these 136 identified protein spots, hybrid offspring with additivity or over-dominance accounted for 68.38%. Data show that a proteomic approach can provide functional prediction of abalone interspecific hybridization.     

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

The phenomenon of heterosis describes the increased agronomic performance of heterozygous F(1) plants compared to their homozygous parental inbred plants. Heterosis is manifested during the early stages of root development in maize. The goal of this study was to identify nonadditive gene expression in primary roots of maize hybrids compared to the average expression levels of their parental inbred lines. To achieve this goal a two-step strategy was used. First, a microarray preselection of nonadditively expressed candidate genes was performed. Subsequently, gene expression levels in a subset of genes were determined via high-throughput quantitative real-time (qRT)-PCR experiments. Initial microarray experiments identified 1941 distinct microarray features that displayed nonadditive gene expression in at least 1 of the 12 analyzed hybrids compared to the midparent value of their parental inbred lines. Most nonadditively expressed genes were expressed between the parental values (>89%). Comparison of these 1941 genes with nonadditively expressed genes identified in maize shoot apical meristems via the same experimental procedure in the same genotypes revealed significantly less overlap than expected by pure chance. This finding suggests organ-specific patterns of nonadditively expressed genes. qRT-PCR analyses of 64 of the 1941 genes in four different hybrids revealed conserved patterns of nonadditively expressed genes in different hybrids. Subsequently, 22 of the 64 genes that displayed nonadditive expression in all four hybrids were analyzed in 12 hybrids that were generated from four inbred lines. Among those genes a superoxide dismutase 2 was expressed significantly above the midparent value in all 12 hybrids and might thus play a protective role in heterosis-related antioxidative defense in the primary root of maize hybrids. The findings of this study are consistent with the hypothesis that both global expression trends and the consistent differential expression of specific genes contribute to the organ-specific manifestation of heterosis.     

Comparative proteome analysis of drought-sensitive and drought-tolerant rapeseed roots and their hybrid F1 line under drought stress

Rapeseed (Brassica napus L.), which is the third leading source of vegetable oil, is sensitive to drought stress during the early vegetative growth stage. To investigate the initial response of rapeseed to drought stress, changes in the protein expression profiles of drought-sensitive (RGS-003) and drought-tolerant lines (SLM-003), and their F1 hybrid, were analyzed using a proteomics approach. Seven-day-old rapeseed seedlings were treated with drought stress by restricting water for 7 days, and proteins were extracted from roots and separated by two-dimensional polyacrylamide gel electrophoresis. In the sensitive rapeseed line, 35 protein spots were differentially expressed under drought stress, and proteins related to metabolism, energy, disease/defense, and transport were decreased. In the tolerant line, 32 protein spots were differentially expressed under drought stress, and proteins involved in metabolism, disease/defense, and transport were increased, while energy-related proteins were decreased. Six protein spots in F1 hybrid were common among expressed proteins in the drought-sensitive and -tolerant lines. Notably, tubulin beta-2 and heat shock protein 70 were decreased in the drought-sensitive line and hybrid F1 plants, while jasmonate-inducible protein and 20S proteasome subunit PAF1 were increased in the F1 hybrids and drought-tolerant line. These results indicate that (1) V-type H⁺ ATPase, plasma-membrane associated cation-binding protein, HSP 90, and elongation factor EF-2 have a role in the drought tolerance of rapeseed; (2) The decreased levels of heat shock protein 70 and tubulin beta-2 in the drought-sensitive and hybrid F1 lines might explain the reduced growth of these lines in drought conditions.     

Vacuolar H+-ATPase Is Expressed in Response to Gibberellin during Tomato Seed Germination

Completion of germination (radicle emergence) by gibberellin (GA)-deficient (gib-1) mutant tomato (Lycopersicon esculentum Mill.) seeds is dependent upon exogenous GA, because weakening of the endosperm tissue enclosing the radicle tip requires GA. To investigate genes that may be involved in endosperm weakening or embryo growth, differential cDNA display was used to identify mRNAs differentially expressed in gib-1 seeds imbibed in the presence or absence of GA₄₊₇. Among these was a GA-responsive mRNA encoding the 16-kD hydrophobic subunit c of the V₀ membrane sector of vacuolar H⁺-translocating ATPases (V-ATPase), which we termed LVA-P1. LVA-P1 mRNA expression in gib-1 seeds was dependent on GA and was particularly abundant in the micropylar region prior to radicle emergence. Both GA dependence and tissue localization of LVA-P1 mRNA expression were confirmed directly in individual gib-1 seeds using tissue printing. LVA-P1 mRNA was also expressed in wild-type seeds during development and germination, independent of exogenous GA. Specific antisera detected protein subunits A and B of the cytoplasmic V₁ sector of the V-ATPase holoenzyme complex in gib-1 seeds only in the presence of GA, and expression was localized to the micropylar region. The results suggest that V-ATPase plays a role in GA-regulated germination of tomato seeds.     

Comparative proteomic study reveals dynamic proteome changes between superhybrid rice LYP9 and its parents at different developmental stages

Heterosis is a common phenomenon in which the hybrids exhibit superior agronomic performance than either inbred parental lines. Although hybrid rice is one of the most successful apotheoses in crops utilizing heterosis, the molecular mechanisms underlying rice heterosis remain elusive. To gain a better understanding of the molecular mechanisms of rice heterosis, comparative leaf proteomic analysis between a superhybrid rice LYP9 and its parental cultivars 9311 and PA64s at tillering, flowering and grain-filling stages were carried out. A total of 384 differentially expressed proteins (DP) were detected and 297 DP were identified, corresponding to 222 unique proteins. As DP were divided into those between the parents (DP_PP) and between the hybrid and its parents (DP_HP), the comparative results demonstrate that proteins in the categories of photosynthesis, glycolysis, and disease/defense were mainly enriched in DP. Moreover, the number of identified DP_HP involved in photosynthesis, glycolysis, and disease/defense increased at flowering and grain-filling stages as compared to that at the tillering stage. Most of the up-regulated DP_HP involved in the three categories showed greater expression in LYP9 at flowering and grain-filling stages than at the tillering stage. In addition, CO₂ assimilation rate and apparent quantum yield of photosynthesis also showed a greater increase in LYP9 at flowering and grain-filling stages than at the tillering stage. These results suggest that the proteins involved in photosynthesis, glycolysis, and disease/defense as well as their dynamic regulation at different developmental stages may be responsible for heterosis in rice.     

Genome Properties and Prospects of Genomic Prediction of Hybrid Performance in a Breeding Program of Maize

Maize (Zea mays L.) serves as model plant for heterosis research and is the crop where hybrid breeding was pioneered. We analyzed genomic and phenotypic data of 1254 hybrids of a typical maize hybrid breeding program based on the important Dent × Flint heterotic pattern. Our main objectives were to investigate genome properties of the parental lines (e.g., allele frequencies, linkage disequilibrium, and phases) and examine the prospects of genomic prediction of hybrid performance. We found high consistency of linkage phases and large differences in allele frequencies between the Dent and Flint heterotic groups in pericentromeric regions. These results can be explained by the Hill–Robertson effect and support the hypothesis of differential fixation of alleles due to pseudo-overdominance in these regions. In pericentromeric regions we also found indications for consistent marker–QTL linkage between heterotic groups. With prediction methods GBLUP and BayesB, the cross-validation prediction accuracy ranged from 0.75 to 0.92 for grain yield and from 0.59 to 0.95 for grain moisture. The prediction accuracy of untested hybrids was highest, if both parents were parents of other hybrids in the training set, and lowest, if none of them were involved in any training set hybrid. Optimizing the composition of the training set in terms of number of lines and hybrids per line could further increase prediction accuracy. We conclude that genomic prediction facilitates a paradigm shift in hybrid breeding by focusing on the performance of experimental hybrids rather than the performance of parental lines in testcrosses.     

Gene Expression Analysis in Sorghum Hybrids and Their Parental Lines at Critical Developmental Stages in Relation to Grain Yield Heterosis by Exploiting Heterosis-Related Genes from Major Cereals

Relative expression levels of selected genes from the Heterosis-Related Gene Database exhibiting more than 90% homology with sorghum were studied in hybrids and their respective parental lines for a better understanding on the molecular basis of heterosis. A high (27A × RS 673) and a low heterotic hybrid (7A × CB 26) of sorghum along with their parental lines were used for this purpose. Twenty (15 maize and 5 rice) genes exhibiting more than 90% homology with that of sorghum were identified. The maize genes ZmHG13, ZmHG16, and ZmhG19 exhibited more than fourfold increase over the male parent (RS 673) of high heterotic hybrid during booting stage, which started decreasing during flowering stage. Similarly, the rice genes OsHG1 and OsHG12 recorded >?2.5-fold increase. However, these genes recorded less than twofold increase during the same stage of the plant in the low heterotic hybrid. Notably, among the genes that exhibited higher expression in the highly heterotic hybrid were those coding for proteins, which were known to play crucial roles in the manifestation of heterosis in plants.     

Cellular Responses during Morphological Transformation in Azospirillum brasilense and Its flcA Knockout Mutant

FlcA is a response regulator controlling flocculation and the morphological transformation of Azospirillum cells from vegetative to cyst-like forms. To understand the cellular responses of Azospirillum to conditions that cause morphological transformation, proteins differentially expressed under flocculation conditions in A. brasilense Sp7 and its flcA knockout mutant were investigated. Comparison of 2-DE protein profiles of wild-type (Sp7) and a flcA deletion mutant (Sp7-flcAΔ) revealed a total of 33 differentially expressed 2-DE gel spots, with 22 of these spots confidently separated to allow protein identification. Analysis of these spots by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and MASCOT database searching identified 48 proteins (≥10% emPAI in each spot). The functional characteristics of these proteins included carbon metabolism (beta-ketothiolase and citrate synthase), nitrogen metabolism (Glutamine synthetase and nitric oxide synthase), stress tolerance (superoxide dismutase, Alkyl hydroperoxidase and ATP-dependent Clp protease proteolytic subunit) and morphological transformation (transducer coupling protein). The observed differences between Sp7 wild-type and flcA ⁻ strains enhance our understanding of the morphological transformation process and help to explain previous phenotypical observations. This work is a step forward in connecting the Azospirillum phenome and genome.     

Proteomic profiling of eggs from a hybrid abalone and its parental lines: Haliotis discus hannai Ino and Haliotis gigantea

Proteomic analysis was performed on the eggs of hybrid abalone and their corresponding parental lines. A total of 915 ± 19 stained protein spots were detected from Haliotis discus hannai♀ × H. discus hannai♂ (DD), 935 ± 16 from H. gigantea♀ × H. gigantea♂ (GG) and 923 ± 13 from H. gigantea♀ × H. discus hannai♂ (GD). The spots from DD and GD were clustered together. The distance between DD and GG was maximal by hierarchical cluster analysis. A total of 112 protein gel spots were identified; of these, 59 were abalone proteins. The proteins were involved in major biological processes including energy metabolism, proliferation, apoptosis, signal transduction, immunity, lipid metabolism, electron carrier proteins, protein biosynthesis and decomposition, and cytoskeletal structure. Three of 20 differential expression protein spots involved in energy metabolism exhibited as upregulated in GD, 13 spots exhibited additivity, and four spots exhibited as downregulated in the offspring. Eleven protein spots were expressed at the highest level in DD. The proteins involved in stress responses included superoxide dismutase, peroxiredoxin 6, thioredoxin peroxidase and glutathione‐S‐transferase. Two of seven differential expression protein spots involved in response to stress exhibited as upregulated in GD, three exhibited additivity, and two exhibited as downregulated. These results might suggest that proteomic approaches are suitable for the analysis of hybrids and the functional prediction of abalone hybridization.     

DNA Methylation Alterations at 5′-CCGG Sites in the Interspecific and Intraspecific Hybridizations Derived from Brassica rapa and B. napus

DNA methylation is an important regulatory mechanism for gene expression that involved in the biological processes of development and differentiation in plants. To investigate the association of DNA methylation with heterosis in Brassica, a set of intraspecific hybrids in Brassica rapa and B. napus and interspecific hybrids between B. rapa and B. napus, together with parental lines, were used to monitor alterations in cytosine methylation at 5′-CCGG sites in seedlings and buds by methylation-sensitive amplification polymorphism analysis. The methylation status of approximately a quarter of the methylation sites changed between seedlings and buds. These alterations were related closely to the genomic structure and heterozygous status among accessions. The methylation status in the majority of DNA methylation sites detected in hybrids was the same as that in at least one of the parental lines in both seedlings and buds. However, the association between patterns of cytosine methylation and heterosis varied among different traits and between tissues in hybrids of Brassica, although a few methylation loci were associated with heterosis. Our data suggest that changes in DNA methylation at 5′-CCGG sites are not associated simply with heterosis in the interspecific and intraspecific hybridizations derived from B. rapa and B. napus.