Protein identification from two-dimensional electrophoresis gels usually allows the detection of several isoforms 2021. Protein isoforms may be i- the results of post-translational modifications (PTMs) of the same gene product, ii- gene products of paralogous genes, iii- gene products of homeologous genes (i.e. orthologous genes from the two distinct parental species merged in a common nucleus via hybridization) and iv- a combination of these different origins. Since the expression of isoforms may be controlled by partially shared or conserved regulatory pathways, it can be hypothesized that they should display an identical regulation within the same organ (for example, a proportional increase in their quantity). This hypothesis was tested in synthetic Brassica napus: the identification of spots (displaying additivity and non-additivity) allowed the detection of groups of isoforms in both organs: 104 groups of isoforms in the stem and 82 in the root (Table 2). Among these groups, 42 to 48% comprised spots that all displayed an identical regulation within one organ (all isoforms up-regulated, or all isoforms down-regulated in the synthetic amphiploids compared to the mid parent value), in accordance with the above hypothesis. Interestingly, 15 to 24% of the groups included isoforms with "opposite" regulation patterns, resulting in partial or complete compensation: for example, in the stem, spots #1352 and #1353 were respectively B. oleracea- and B. rapa-specific and were both identified as putative fructokinases (Figure 1). These proteins were likely encoded by homeologous genes, and both displayed non-additive patterns in synthetic Brassica napus: spot #1352 was up-regulated compared to the mid parent value, and such an increase was partially balanced by the down-regulation of spot #1353. The differential regulation of homeologous gene products in allopolyploids appeared as a relatively frequent mechanism, since 7 out of 14 of the putative pairs of homeologous proteins displayed contrasting patterns of regulation.

One of the great advantages of comparative proteomics is the possibility to observe post-translational modifications (PTMs) of the same gene product. Although it is not obvious to demonstrate formally whether multiple isoforms correspond to PTMs or to products of homologous/homeologous genes, proteins with post-translational modifications such as phosphorylation usually appear on the 2-DE gels as horizontal rows of spots, resulting in characteristic "trains of spots". Figure 2 shows a train of four spots (#370, #374, #382 and #383) for which analysis by mass spectrometry led to the identification of one single accession, namely an Hsp70-like protein. Moreover, previous studies have described post-translational modifications of HSP 70-related proteins 2223 so that the four detected spots most likely corresponded to different PTMs of HSP 70. Quantitative proteomic analysis revealed that spot #374 was additive in the synthetic B. napus, while isoform #370 was less abundant in amphiploids than expected under the additivity hypothesis whereas spots #382 and #383 were more abundant. Other examples of trains of spots with contrasting patterns of regulation were detected (26/44 for both organs), suggesting that the differential regulation of PTMs was not a rare event in the synthetic amphiploids of B. napus.

In order to know whether the distribution of the isoforms was similar between polypeptides displaying additivity and non-additivity, we counted the number of isoforms (Table 3). In both organs, significantly more isoforms were found among non-additive spots than among spots displaying additivity (χ² tests, P-values = 3.76^e-6 and 1.42^e-10 in the stem and the root respectively, both inferior to the p-value = 0.05 limit). Such a difference might reflect the experimental bias due to the differential sampling of additive and non-additive spots. Since we analyzed by mass spectrometry all the spots with non-additive values, we detected all the groups within which at least two isoforms displayed non-additive patterns. On the other hand, we identified only a random subset of the additive spots (two hundred spots per organ while we found 519 and 583 additive spots in the stem and root respectively), so we may have underestimated the number of groups of isoforms within the additive group.

Most metabolic pathways are driven by protein complexes. Thus, the stoichiometry of the different subunits might be a critical issue for cell processing, and the components of a protein complex are expected to display parallel variations of their respective amounts. These supposed co-regulation and coordinate expressions are known as "the balance hypothesis", and it has been suggested that both under-expression and over-expression (imbalance) of subunits shall be deleterious 2425. Hence, the "balance hypothesis" was tested in the synthetic amphiploids of Brassica napus: protein complexes with at least two identified distinct subunits were studied. Eleven and nine complexes were found in the stem and root respectively, and the pattern of regulation of their components was investigated (Table 4). For example, analysis of the stem proteome led to the identification of the alpha, beta, delta and gamma subunits of the chloroplastic ATP synthase, and all of these four subunits displayed non-additive up-regulation in B. napus synthetic amphiploids compared to the mid parent value. On the contrary, two subunits of the ribosomal complex, the 40S ribosomal protein SA (RPSaA) and the 60S acidic ribosomal protein P0 (RPP0A) 26, were found to be differentially expressed in the root, but with opposite patterns of regulation: two isoforms of RPSaA subunit were down-regulated while RPP0A was up-regulated in the amphiploids (Figure 3). Finally, only 5/11 complexes in stem and 2/9 in root encompassed subunits with identical patterns of regulation, demonstrating that the different subcomponents of a complex can be differentially regulated in neo-synthetized B. napus. These results suggest that the balance hypothesis might not be a general rule for the regulation of different complex subunits in synthetic allopolyploids.

One of the major goals of protein identification of both additive and non-additive spots was to find whether some functions or metabolic pathways were preferentially affected by allopolyploidization. Thus, the functional categorization of the identified polypeptides was carried out using the MIPS FunCat database available on line 27. Since the proportion of isoforms was significantly imbalanced between additive and non-additive spots, we did not consider the isoforms individually but we counted each group of isoforms as a single protein. Figure 4 shows the distribution of additive and non-additive spots into functional categories. In both organs, the identified proteins were mainly involved in metabolism (26% to 37%) and energy (14% to 21%), which was congruent with previous proteomic data 2128. Protein fate, transport, defense and biogenesis of cellular components were other well represented functional categories (5% to 10%), while an important proportion of the identified polypeptides were not assigned to any known function (30% to 42%). No functional category was found over- or under-represented for spots displaying non-additivity compared to "additive" spots (χ² tests, p-values > 0.05), suggesting that differential regulation of gene products in synthetic Brassica napus was not related to the function of the proteins.

In addition, we used the KEGG PATHWAY database 29 to identify the proteins involved in the same metabolic pathways (Table 5). No pathway was particularly altered by differential gene expression, since no proteins involved in the same pathway were found all up-regulated, or down-regulated in the synthetic amphiploids compared to the mid parent value. Moreover, the proteins involved in the same pathway could display different patterns of regulation: for example, most of the glycolytic enzymes were identified in stem proteome (Figure 5). Some of these proteins were up-regulated in the amphiploids (like the phosphoglucoisomerase), others were down-regulated (enolase) or displayed an additive pattern (phosphoglyceromutase). Taken together, these results indicated that neither functional category nor metabolic pathway were particularly targeted by differential regulation of gene expression in the amphiploids.

The subcellular localization of the proteins was assessed using the Gene Ontology GO annotations 30. As for the functional categorization of the proteins, each group of isoforms was taken into account only once in order to correct for the bias between additive and non-additive spots. Figure 6 shows the distribution of the identified proteins within cellular compartments: in both organs, most of the polypeptides were located in the organelles (chloroplast and mitochondria), in accordance with the main functional categories previously found, namely Energy and Metabolism. The fact that ~10% of the root proteins were located in the "chloroplast" actually indicate that these proteins were located in the plastids, and particularly in the amyloplasts, which are very abundant in root tissues 31. Other cellular localizations, such as cytosol, membrane and nucleus, were well represented in both organs, indicating that comparative proteomics allows the analysis of all the compartments of the cell. The comparison between non-additive and additive spots showed no specific subcellular localization (χ² tests, p-values > 0.05), which suggests that the regulation of gene products in the amphiploids was not related to subcellular localization.

Protein identification in synthetic amphiploids of B. napus allowed the discovery of several isoforms that could display different patterns of regulation. In particular, probable homeoallelic products were found with "opposite" regulation (up- and down-regulation in the synthetic allotetraploids compared to the mid parent value), suggesting that differential expression of homeologous genes could occur. Unequal contributions of homeoalleles to the transcriptome have already been evidenced in synthetic allotetraploids of Gossypium 1232. Here, we have shown that such a phenomenon was detectable at the proteomic level and was not a rare event. Moreover, our proteomic data suggest that the post-translational modifications of the same gene product could also be differentially regulated in the amphiploids. Differential regulation of PTMs has been described in a few proteomic analyses 33, but no data was available in the context of allopolyploidy. PTMs play a key role in the cell as the same gene product may have different activities, modified turn-overs, distinct subcellular locations, or altered interactions with proteins or nucleic acids depending on its PTMs. Thus, variation of amounts among protein isoforms (PTMs, but also homeoalleles or paralogous genes) may establish a novel metabolic equilibrium in synthetic Brassica napus in contrast to its diploid progenitors.

Complex subunits are usually supposed to present co-ordinate expressions 2425. Such a statement was supported by some experimental data showing that genes encoding subcomponents of a complex displayed the same pattern of regulation of their expressions 3435. In contradiction with this assumption, we have shown that the different subunits of a protein complex could be differentially regulated in newly synthesized B. napus. This result may reflect the instability of protein regulation in the very first step of allopolyploidization, and it will be of particular interest to investigate the evolution of such imbalance in the further generations of allotetraploids. Moreover, although the synthetic B. napus plants we analyzed were viable and morphologically "normal", neither phenotypic analyses nor relative fitness studies were undertaken. Thus, the imbalanced levels of isoforms and subunit complexes may have an impact on the fitness and/or the phenotype of the neo-allopolyploids that remains to be quantified. One can also suppose that, due to genetic and proteomic redundancies, expression constraints are relaxed within an amphiploid, allowing previously constrained genes to be differentially regulated and to evolve in a different way.

During the last few years, several synthetic allopolyploids have been investigated at the transcriptional level using various approaches (cDNA-SSCP in synthetic Gossypium allotetraploids 12, AFLP- cDNA in synthetic wheat allopolyploids 15, microarray in synthetic Senecio allopolyploids 13 and synthetic Gossypium allotetraploids 32, etc.). Non-additive transcription in hybrids and allopolyploids is now well documented, yet the genes concerned by non-additive expression are not well characterized. Recently, a transcriptional analysis of Arabidopsis allotetraploids showed that genes involved in "hormonal regulation" and in "cell defense and aging" were more susceptible to expression changes 11. In addition, the ethylene biosynthesis pathway was found globally repressed in two distinct lineages of synthetic A. suecica 11. In this report, we have described a thorough in silico functional analysis of proteins in synthetic B. napus, and we have shown that neither specific functional category nor metabolic pathway were over- or under-represented among non-additive proteins. This discrepancy between Arabidopsis transcriptional results and oilseed rape proteomic data may be explained by the fact that mRNA abundance and protein amounts are poorly related 19) or by the reference used (we compared the functional categorizations of non-additive proteins to additive ones, while Wang et al. compared the distribution among functional categories of non-additive genes to the whole set of 26,000 annotated genes in Arabidopsis 11). Moreover, differential regulation of gene expression may vary from one biological model to an other (Arabidopsis vs. Brassica).

Finally, this is the first in silico analysis of proteins in synthetic allotetraploids. Although in silico prediction is not a perfect process and may give erroneous information for some genes considered individually 36, bioinformatic tools are particularly relevant for genome-wide analysis and global consideration of the data. We have shown that non-additive and additive polypeptides could not be differentiated on the basis of their function nor subcellular localization, indicating that the regulation of gene products following allopolyploidization is not related to any functional feature of the proteins.

Polyploidy is often described as a genomic shock 3738, that may induce stress and defense responses. Our study showed that, in synthetic B. napus, the "cell rescue, defense and virulence" category was unchanged in the stem and root. In addition, Wang et al. showed that the "defense" class was under-represented when analyzing differential gene transcription in leaves and flower buds of synthetic Arabidopsis allotetraploids 11. These results suggest that neither stress nor defense responses were particularly enhanced following a recent polyploidy event for these two Brassicaceae species. Moreover, although stem and root proteomes were extensively modified in synthetic B. napus, the patterns were reproducible in independent amphiploid lineages 14 and our data indicated that, globally, the distribution of the proteins into metabolic pathways, functional categories and subcellular localizations was not disturbed. Thus, at the protein level, there is no evidence for a chaos or a large disorder following the merging of two genomes. Instead, a novel order establishes quickly and may evolve in further generations of synthetic B. napus. Hence, allopolyploidy can be seen as a mechanism making new from old: new expression patterns from old genomes, and perhaps this feature explains in part why polyploidy is such an undeniable evolutionary success.

The thorough in silico analysis of the non-additive proteins in synthetic B. napus indicated that the differential regulation of gene products was not related to functional properties of the proteins. The molecular mechanisms however remain to be investigated, such as the possible implication of transposable elements: more and more data suggest that transposable elements have played a key role in polyploid evolution such as Gossypium or Arabidopsis 939, and that their transcriptional activation could alter the expression of adjacent genes (like in wheat allopolyploids 40). Epigenetic mechanisms (changes in chromatin structure, histone modification, DNA methylation, etc.) are described in synthetic allopolyploids 4142 and may be responsible for the modification of gene expression in polyploids 4344. Furthermore, RNA interference, action of siRNAs and miRNAs and their implication in gene regulation, are thus good candidates to explain nonadditive gene expression 45. Finally, allopolyploidy not only induces the merger of homeologous genomes in a common nucleus, but also the juxtaposition of divergent regulatory networks. The interaction and the coordination of such homeologous networks in a newly synthesized allopolyploid may also explain the genome-wide modification of gene expression, as well as the maintenance at a viable level, in successful individual plants, of the equilibrium between the different functions, metabolic pathways and cellular localizations.

In a previous report, we applied comparative proteomics to synthetic Brassica napus (four independent lineages) and its diploid progenitors B. rapa and B. oleracea (two to five plants analyzed per genotype) 14. Our proteomic analysis showed that several spots (519 in stem and 583 in root) displayed a mid-parent value in synthetic Brassica napus compared to its diploid progenitors while many others (335 in stem, 205 in root) displayed non-additive amounts 14. About 150 of these non-additive polypeptides were identified by mass spectrometry and submitted to in silico analyses 14. However, the subset of non-additive polypeptides analyzed was too small to obtain accurate and relevant data on the functional categorization and subcellular localization of the non-additive proteins 14. Here, the remaining non-additive spots (~260 in stem and ~130 in root) were excised from 2-DE gels and submitted to mass spectrometry, so that the whole set of non-additive proteins in synthetic B. napus was identified.

In our previous study, no accurate "control group" was available (no additive spot was identified), so that we used reference proteomic maps of other organisms (Pisum sativum, Medicago truncatula) 14. Here, two hundred spots displaying additivity were also cut out for both organs (random excision among the 519 and 583 spots found additive in stem and root 14). These additive polypeptides were used as controls for subsequent in silico analyses. Mass spectrometry analyses were conducted as described in 14.

Distinct spots were considered as isoforms when they met the following criteria: 1 – the corresponding identified protein sequences presented more than 70% identity all along the sequences; 2 – the corresponding proteins had the same function (see below) 3 – the polypeptides were assessed in silico to the same cellular compartment (see cellular localization below) and 4 – the spots displayed similar molecular weight on 2-DE gels (to discard degradation or catabolic products).

Functional categorization and cellular localization of the proteins were conducted using the MIPS FunCat database (Version 2.0) available on line 27 and the TAIR's Gene Ontology (GO) annotations (release of February 2006) 4647 as described in 14. For both organs, the main molecular networks were identified using the KEGG PATHWAY database (release of February 2006) 29. χ² tests were carried out to test statistical differences between proteins fitting additivity or displaying non-additivity.