Protein subcellular relocalization of duplicated genes in Arabidopsis

Abstract

Gene duplications during eukaroytic evolution, by successive rounds of polyploidy and by smaller scale duplications, have provided an enormous reservoir of new genes for the evolution of new functions. Preservation of many duplicated genes can be ascribed to changes in sequences, expression patterns, and functions. Protein subcellular relocalization (protein targeting to a new location within the cell) is another way that duplicated genes can diverge. We studied subcellular relocalization of gene pairs duplicated during the evolution of the Brassicaceae including gene pairs from the alpha whole genome duplication that occurred at the base of the family. We analyzed experimental localization data from green fluorescent protein experiments for 128 duplicate pairs in Arabidopsis thaliana, revealing 19 pairs with subcellular relocalization. Many more of the duplicate pairs with relocalization than with the same localization showed an accelerated rate of amino acid sequence evolution in one duplicate, and one gene showed evidence for positive selection. We studied six duplicate gene pairs in more detail. We used gene family analysis with several pairs to infer which gene shows relocalization. We identified potential sequence mutations through comparative analysis that likely result in relocalization of two duplicated gene products. We show that four cases of relocalization have new expression patterns, compared with orthologs in outgroup species, including two with novel expression in pollen. This study provides insights into subcellular relocalization of evolutionarily recent gene duplicates and features of genes whose products have been relocalized.

Keywords: gene duplication; subcellular localization; whole genome duplication.

Fig. 1.—

Subcellular relocalization, asymmetric sequence evolution, and gene expression divergence in a pair of peroxidases. (A) GFP subcellular localization of PRX36. (B) GFP subcellular localization of PRX72. (C) RT-PCR expression assays of PRX36 and PRX72. Plus signs (+) indicate reactions with reverse transcriptase and minus signs (−) indicate reactions without reverse transcriptase. ACT2 and UBQ10 were positive controls. (D) PAML analysis of PRX36, PRX72, and their orthologs in other species. Numbers above the branches indicate the dN/dS ratios. dN analysis is shown in supplementary figure S4, Supplementary Material online. Species include: At, A. thaliana; Cr, Capsella rubella; Es, Eutrema salsugineum; Br, Brassica rapa; Cp, C. papaya; Gr, G. raimondii; Tc, T. cacao; Pt, P. trichocarpa; and Me, M. esculenta. See supplementary figures S2 and S4, Supplementary Material online, for locus numbers of each gene.

Fig. 2.—

Asymmetric sequence evolution in duplicated gene pairs. (A) Diagram showing the frequency of asymmetrically evolved duplicated pairs in duplicated pairs with different subcellular localization (SCL) and duplicated pairs with the same SCL. (B) Histogram showing the distribution of the difference in frequency of asymmetric sequence evolution between duplicated pairs with different SCL and those with the same SCL (DIF_sim) from 10,000 Monte Carlo randomization tests. Red line indicates the observed value (DIF_obs).

Fig. 3.—

Analysis of the pI difference (ΔpI) between duplicated pairs. Box plots of ΔpI in gene pairs with different subcellular localization (SCL) and those with the same subcellular localization. Higher ΔpI values indicate a greater difference in pI between the duplicates.

Fig. 4.—

Neolocalization, asymmetric sequence evolution, and gene expression divergence in a pair of VAMP proteins. (A) PAML analysis of VAMP genes. Numbers above the branches indicate the dN/dS ratios. dN analysis is shown in supplementary figure S5, Supplementary Material online. Subcellular localization for the proteins in A. thaliana is highlighted in red. VAMP722 is relocalized from PM/endosome to ER. Species include: At, A. thaliana; Al, Arabidopsis lyrata; Cr, Capsella rubella; Es, Eutrema salsugineum; Br, Brassica rapa; Cp, C. papaya; Gr, G. raimondii; Tc, T. cacao; Pt, P. trichocarpa; Me, M. esculenta; and Vv, V. vinifera. See supplementary figure S5, Supplementary Material online, for locus numbers of each gene. (B) Amino acid alignment showing the position of positively selected sites in the SNARE domain of VAMP723 inferred using the empirical Bayes approach.

Fig. 5.—

Neolocalization, asymmetric sequence evolution, and gene expression divergence in a pair of VTI proteins. (A) PAML analysis of VTI genes. Numbers above the branches indicate the dN/dS ratios. dN analysis is shown in supplementary figure S6, Supplementary Material online. Subcellular localization for the proteins in A. thaliana is highlighted in red. VTI14 was relocalized from TGN/PVC (trans-Golgi network/prevacuolar compartments) to endosome. Species include: At, A. thaliana; Al, Arabidopsis lyrata; Cr, Capsella rubella; Es, Eutrema salsugineum; Br, Brassica rapa; Cp, C. papaya; Gr, G. raimondii; Tc, T. cacao; Pt, P. trichocarpa; Me, M. esculenta; and Vv, V. vinifera. See supplementary figure S6, Supplementary Material online, for locus numbers of each gene. (B) RT-PCR expression assays of VTI14, VTI11, and VTI 13 in A. thaliana, and their orthologs in C. papaya and G. hirsutum. Plus signs (+) indicate reactions with reverse transcriptase and minus signs (−) indicate reactions without reverse transcriptase. ACT2 was a positive control in A. thaliana, whereas ACT1 was a positive control in C. papaya and G. hirsutum.

Fig. 6.—

Asymmetric sequence evolution, neolocalization, and gene expression divergence in a pair of K⁺ channel protein proteins. (A) PAML analysis of TPK genes. dN analysis is shown in supplementary figure S7, Supplementary Material online. Numbers above the branches indicate the dN/dS ratios. Species include: At, A. thaliana; Al, Arabidopsis lyrata; Cr, Capsella rubella; Es, Eutrema salsugineum; Br, Brassica rapa; Cp, C. papaya; Gr, G. raimondii; Tc, T. cacao; Pt, P. trichocarpa; and Me, M. esculenta. See supplementary figure S7, Supplementary Material online, for locus numbers of each gene. (B) RT-PCR expression assays of TPK4 and TPK5 in A. thaliana, and their orthologs in C. papaya and G. hirsutum. Plus signs (+) indicate reactions with reverse transcriptase and minus signs (−) indicate reactions without reverse transcriptase. ACT2 was a positive control in A. thaliana, whereas ACT1 was a positive control in C. papaya and G. hirsutum.

Fig. 7.—

Neolocalization and gene expression divergence in CPK2. (A) Alignment showing the N-terminal targeting region. Arrowheads indicate CPK2-specific amino acid changes in the CPK2 orthologs. Gene locus numbers are in supplementary figure S8, Supplementary Material online. (B) RT-PCR expression assays of CPK1 and CPK2 in A. thaliana, and their orthologs in C. papaya and G. hirsutum. Plus signs (+) indicate reactions with reverse transcriptase and minus signs (−) indicate reactions without reverse transcriptase. ACT2 was a positive control in A. thaliana, whereas ACT1 was a positive control in C. papaya and G. hirsutum.

Fig. 8.—

Alignment of subcellular targeting signal regions in PP2A B'. The C-terminal region of a pair of protein phosphatase 2A proteins, PP2A B′θ and PP2A B′η, and their orthologs in the outgroup species are shown. Signal peptides for peroxisome localization are indicated by inverted triangles. Gene locus numbers are in supplementary figure S10, Supplementary Material online.