Analysis of gene expression in resynthesized Brassica napus allotetraploids: transcriptional changes do not explain differential protein regulation.

Polyploidy, or whole genome duplication, is a major evolutionary process that has shaped eukaryotic genomes, notably those of flowering plants. The mechanisms underlying the regulation of, and sharing of functions between, the duplicated genes originating from polyploidy events, which lead to novel phenotypes, remain to be elucidated. A previous comparative proteomic study identified 360 proteins that were differentially regulated between the diploid Brassica progenitors and their synthetic allotetraploid derivatives. For 102 of these proteins, using the same resynthesized Brassica napus allotetraploids, we assayed the accumulation of the transcripts of the corresponding genes. We compared transcript levels quantified in the synthetic allotetraploids with the mid-parent expression values. Although all of the genes surveyed encoded nonadditive proteins, we found that two-thirds of them had additive transcript levels, indicating that most of the differential protein regulation is not explained by transcriptional changes. Our data suggest that differential protein regulation is mainly governed by post-transcriptional modifications. Summarizing available data from transcriptomic studies of other synthetic allopolyploid models, we describe the general trends of transcript regulation in an allopolyploid genome and discuss putative underlying molecular mechanisms, with particular emphasis on the small RNA pathway for the post-transcriptional control of gene expression.

Comparative proteomics of leaf, stem, and root tissues of synthetic Brassica napus.

Comparative proteomics was applied to three vegetative organs of Brassica napus, the leaf, stem, and root using 2-DE. Among the >1600 analyzed spots, 43% were found to be common to all three organs, suggesting the existence of a "basal" or ubiquitous proteome composed of housekeeping proteins. The green organs, leaf, and stem, were closely related (approximately 80% common spots) while the root displayed more organ-specific polypeptides (approximately 10%). Reference maps were established using MS, allowing the identification of 93, 385, and 266 proteins in leaf, stem, and root proteomes, respectively. Bioinformatic analyses were also performed; in silico functional categorization and cellular localization allow obtaining a precise picture of the cell molecular network within vegetative organs. These proteome maps can be explored using the PROTICdb software at the following address: http://bioinformatique.moulon.inra.fr/proticdb/web_view/.

Differential regulation of gene products in newly synthesized Brassica napus allotetraploids is not related to protein function nor subcellular localization.

BACKGROUND: Allopolyploidy is a preeminent process in plant evolution that results from the merger of distinct genomes in a common nucleus via inter-specific hybridization. Allopolyploid formation is usually related to genome-wide structural and functional changes though the underlying mechanisms operating during this "genomic shock" still remain poorly known. The aim of the present study was to investigate the modifications occurring at the proteomic level following an allopolyploidization event and to determine whether these changes are related to functional properties of the proteins. In a previous report, we applied comparative proteomics to synthetic amphiploids of Brassica napus and to its diploid progenitors B. rapa and B. oleracea. Although several hundred polypeptides displayed additivity (i.e. mid-parent values) in the amphiploids, many of them showed non-additivity. Here, we report the in silico functional characterization of the "non-additive" proteins (the ones with a non-additive pattern of regulation) in synthetic B. napus. RESULTS: The complete set of non-additive proteins (335 in the stem and 205 in the root), as well as a subset of additive polypeptides (200 per organ), was identified by mass spectrometry. Several protein isoforms were found, and most of them (approximately 55%) displayed "different" or "opposite" patterns of regulation in the amphiploids, i.e. isoforms of the same protein showing both up-regulation and down-regulation in the synthetic B. napus compared to the mid-parent value. Components of protein complexes were identified of which approximately 50% also displayed "different" or "opposite" patterns of regulation in the allotetraploids. In silico functional categorization of the identified proteins was carried out, and showed that neither functional category nor metabolic pathway were systematically affected by non-additivity in the synthetic amphiploids. In addition, no subcellular compartment was found to be over- or under-represented among the proteins displaying non-additive values in the allopolyploids. CONCLUSION: Protein identification showed that functionally related polypeptides (isoforms and complex subunits) could be differentially regulated in synthetic B. napus in comparison to its diploid progenitors while such proteins are usually expected to display co-regulation. The genetic redundancy within an allopolyploid could explain why functionally related proteins could display imbalanced levels of expression. No functional category, no metabolic pathway and no subcellular localization was found to be over- or under-represented within non-additive polypeptides, suggesting that the differential regulation of gene products was not related to functional properties of the proteins. Thus, at the protein level, there is no evidence for the "genomic shock" expected in neo-polyploids and the overall topology of protein networks and metabolic pathways is conserved in synthetic allotetraploids of B. napus in comparison to its diploid progenitors B. rapa and B. oleracea.

Numerous and rapid nonstochastic modifications of gene products in newly synthesized Brassica napus allotetraploids.

Polyploidization is a widespread process that results in the merger of two or more genomes in a common nucleus. To investigate modifications of gene expression occurring during allopolyploid formation, the Brassica napus allotetraploid model was chosen. Large-scale analyses of the proteome were conducted on two organs, the stem and root, so that >1600 polypeptides were screened. Comparative proteomics of synthetic B. napus and its homozygous diploid progenitors B. rapa and B. oleracea showed that very few proteins disappeared or appeared in the amphiploids (<1%), but a strikingly high number (25-38%) of polypeptides displayed quantitative nonadditive pattern. Nonstochastic gene expression repatterning was found since 99% of the detected variations were reproducible in four independently created amphiploids. More than 60% of proteins displayed a nonadditive pattern closer to the paternal parent B. rapa. Interspecific hybridization triggered the majority of the deviations (89%), whereas very few variations (approximately 3%) were associated with genome doubling and more significant alterations arose from selfing (approximately 9%). Some nonadditive proteins behaved similarly in both organs, while others exhibited contrasted behavior, showing rapid organ-specific regulation. B. napus formation was therefore correlated with immediate and directed nonadditive changes in gene expression, suggesting that the early steps of allopolyploidization repatterning are controlled by nonstochastic mechanisms.

Autopolyploidy in cabbage (Brassica oleracea L.) does not alter significantly the proteomes of green tissues.

Polyploidization is a major evolutionary process in eukaryotes. In plants, genetic and epigenetic changes occur rapidly after formation of allopolyploids. Hybridization, rather than genome doubling itself, is considered as the main cause for the resulting differential gene expression. We studied the consequences of genome doubling alone in an autopolyploid model, by comparing two-dimensional gel electrophoresis (2-DE) gels of haploid, diploid, and tetraploid Brassica oleracea cabbages. Two fully homozygous lines, HDEM and RC, as well as two organs, leaf and stem, were studied. For the 558 common spots found present in all the 29 2-DE gels of the experiment, inter-organ and -genotype differences were the major sources of the variation in protein amounts: 41 and 10-13%, respectively. HDEM leaf and stem proteomes were not significantly affected by the ploidy level, since no qualitative variation was detected and since the number of quantitative variations could be due to chance. For RC, no qualitative variations were observed, but a few spots were significantly variable in protein amount. However, the number of inter-ploidy variations was of the same range as the number of intra-ploidy variations. In conclusion, whatever the ploidy level, leaf and stem proteomes remained globally unchanged in both cabbage lines.

Combining proteomic and genetic studies in plants.

Plant proteomics is still in its infancy, although numerous experiments have been undertaken since the end of the 1970s. In this review we focus on the interactions between proteomics and genetics. A given genome can express various proteomes according to differentiation, development, tissues, cells and subcellular compartments, and proteomes are modified in function of biotic and abiotic environment. These different proteomes and the way they respond to environment can be compared between genotypes, allowing the characterization of mutants or lines, the study of mutation pleiotropic effects, the genetic mapping of expressed genes. These comparisons also permit to hypothesize for "candidate proteins" that might be involved in the genetic variation of traits of economic or agronomic interest.