Proteomics of Thlaspi caerulescens accessions and an inter-accession cross segregating for zinc accumulation

Abstract

Metal hyperaccumulator plants have previously been characterized by transcriptomics, but reports on other profiling techniques are scarce. Protein profiles of Thlaspi caerulescens accessions La Calamine (LC) and Lellingen (LE) and lines derived from an LCxLE cross were examined here to determine the co-segregation of protein expression with the level of zinc (Zn) hyperaccumulation. Although hydrophobic proteins such as membrane transporters are not disclosed, this approach has the potential to reveal other proteins important for the Zn hyperaccumulation trait. Plants were exposed to metals. Proteins were separated using two-dimensional electrophoresis and those showing differences among accessions, lines or metal exposures were subjected to mass-spectrometric analysis for identification. Crossing decreased the number of different proteins in the lines compared with the parents, more so in the shoots than in the roots, but the frequencies of Zn-responsive proteins were about the same in the accessions and the selection lines. This supports the finding that the Zn accumulation traits are mainly determined by the root and that Zn accumulation itself is not the reason for the co-segregation. This study demonstrates that crossing accessions with contrasting Zn accumulation traits is a potent tool to investigate the mechanisms behind metal hyperaccumulation. Four tentatively identified root proteins showed co-segregation with high or low Zn accumulation: manganese superoxide dismutase, glutathione S-transferase, S-formyl glutathione hydrolase, and translation elongation factor 5A-2. However, these proteins may not be the direct determinants of Zn accumulation. The role of these and other tentatively identified proteins in Zn accumulation and tolerance is discussed.

Fig. 1.

Protein spots identified in shoots (A) and roots (C) of LC×LE inter-accession cross lines and shoots (B) and roots (D) of T. caerulescens LC/LE parental accessions. (Tables 2–4; see Supplementary Figs S1, S3, S4, and Supplementary Tables S1–S4 at JXB online).

Fig. 2.

Examples of root spots with different intensity among the T. caerulescens parental accessions LC and LE and lines from inter-accession cross (49L, 77L, 6L; low-Zn-accumulating lines, 19H, 70H; high-Zn-accumulating lines). (A, B) Eukaryotic elongation factor 5A-2; (C, D) manganese superoxide dismutase 1; (E, F) S-formylglutathione hydrolase (Table 2; Fig. 1). Vertical bars represent mean values ±SD of the spot in three gels. 0 Zn, 0 μM ZnSO₄; 2 Zn, 2 μM ZnSO₄; 10 Zn, 10 μM ZnSO₄; 100 Zn, 100 μM ZnSO₄.