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

Abstract

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.

Figure 1.

2-DE gels of B. oleracea, B. rapa, their hybrids, and synthetic B. napus stem proteome. Spot 1992 is a maternal spot (B. oleracea HDEM specific) and is absent in all amphiploid 2-DE gels. Spot 1992 was identified by mass spectrometry as a pollen allergen-like protein [At1g24020] and is absent in root proteome.

Figure 2.

2-DE gels of B. oleracea, B. rapa, their hybrids, and synthetic B. napus stem (A) and root (B) proteomes. In the stem, spot 753 deviates from additivity in amphiploids and displays the B. rapa pattern (Z1 dominance) while in the root, spot 753 displays an additive pattern. Spot 753 was identified by mass spectrometry as an enolase [At2g36530].

Figure 3.

2-DE gels of B. oleracea, B. rapa, their hybrids, and synthetic B. napus stem proteome. Spot 2041 displays a nonadditive overdominant pattern. Spot 2041 was identified by mass spectrometry as the small subunit of Rubisco [At1g67090] and is absent in root proteome.

Figure 4.

2-DE gels of B. oleracea, B. rapa, their hybrids, and synthetic B. napus stem (A) and root (B) proteomes. Polypeptide 530 (arrow) displays a nonadditive pattern in amphiploids with an opposite behavior in the stem (downregulation) and in the root (upregulation): in the stem, spot 530 exhibits a quantification value between the midparent prediction and the B. rapa pattern (“intermediary” pattern) while in the root, spot 530 shows positive overdominance.

Figure 5.

2-DE gels of B. oleracea, B. rapa, F₁ hybrids, and synthetic B. napus stem proteomes. Polypeptide 1239 shows a progressive decrease of their abundance in hybrids, S0 amphidiploids, and S1 amphidiploids and globally displays an “intermediary” pattern, while spot 1230 exhibits an additive profile. In root, spot 1239 displays the same progressive intermediary pattern as in stem (not shown). Spot 1239 was identified by mass spectrometry as a phosphoglycerate kinase-like protein [At1g79550].

Figure 6.

2-DE gels of B. oleracea, B. rapa, their hybrids, and synthetic B. napus stem (A) and root (B) proteomes. Spots 1445, 1441, 1458, and 1473 are Z1 specific (absent in HDEM). In both organs, spots 1445 and 1458 deviate from additivity in amphiploids and display an “intermediary” pattern between midparent prediction and the HDEM pattern (downregulation). On the contrary, spot 1441 is upregulated (intermediary pattern) and spot 1473 is additive. Downregulation of spots 1445 and 1458 is partially compensated by spot 1441 upregulation. All these spots were identified using mass spectrometry as similar to mitochondrial NAD-dependent malate dehydrogenase [At1g53240].

Figure 7.

Functional classification of nonadditively expressed genes in stem (73 genes) and root (75 genes) of synthetic B. napus. Stem and root proteome reference maps from Pisum sativum and Medicago truncatula were used as controls (Mathesius et al. 2001; Schiltz et al. 2004), on the basis of 106 and 138 identifications, respectively. Only categories representing ≥2% of the proteins were represented: 01, metabolism; 02, energy; 10, cell cycle and DNA processing; 12, protein synthesis; 14, protein fate (folding, modification, destination); 20, cellular transport, transport facilitation, and transport routes; 32, cell rescue, defense, and virulence; 34, interaction with the cellular environment; 40, cell fate; 41, development (systemic); and 42, biogenesis of cellular components. Other categories (<2%) as well as unclassified proteins and proteins without clear-cut classification were grouped in the “Other” category. The distributions between nonadditively expressed genes and the reference map were not significantly different (χ², P = 0.602 in stem and P = 0.153 in root).