Acquisition, conservation, and loss of dual-targeted proteins in land plants

Abstract

The dual-targeting ability of a variety of proteins from Physcomitrella patens, rice (Oryza sativa), and Arabidopsis (Arabidopsis thaliana) was tested to determine when dual targeting arose and to what extent it was conserved in land plants. Overall, the targeting ability of over 80 different proteins from rice and P. patens, representing 42 dual-targeted proteins in Arabidopsis, was tested. We found that dual targeting arose early in land plant evolution, as it was evident in many cases with P. patens proteins that were conserved in rice and Arabidopsis. Furthermore, we found that the acquisition of dual-targeting ability is still occurring, evident in P. patens as well as rice and Arabidopsis. The loss of dual-targeting ability appears to be rare, but does occur. Ascorbate peroxidase represents such an example. After gene duplication in rice, individual genes encode proteins that are targeted to a single organelle. Although we found that dual targeting was generally conserved, the ability to detect dual-targeted proteins differed depending on the cell types used. Furthermore, it appears that small changes in the targeting signal can result in a loss (or gain) of dual-targeting ability. Overall, examination of the targeting signals within this study did not reveal any clear patterns that would predict dual-targeting ability. The acquisition of dual-targeting ability also appears to be coordinated between proteins. Mitochondrial intermembrane space import and assembly protein40, a protein involved in oxidative folding in mitochondria and peroxisomes, provides an example where acquisition of dual targeting is accompanied by the dual targeting of substrate proteins.

Figure 1.

Experimental design to investigate dual targeting of proteins in land plants. A, Tree diagram showing the approximate time (in millions of years ago) that the major group of plants diverged. The four species of land plants used in this study, Arabidopsis, rice, P. glauca, and P. patens, are shown. Additionally, C. reinhardtii and C. variabilis were included in the phylogenetic analysis. B, Verifying the P. patens-derived organelle-specific markers for mitochondria, plastids (rbcS [for small subunit of Rubisco]), and peroxisomes (thiolase and malate synthase) fused to GFP. Accession numbers are indicated. The biolistic transformation of each P. patens-derived organelle marker was carried out with previously published organelle marker sets, with the mitochondrial, plastid, and peroxisomal (SRL [for Cucurbita spp. malate synthase]) targeting signal fused to RFP (Carrie et al., 2009b). In addition, cytochrome oxidase IV-mCherry (Nelson et al., 2007) and P. patens AOX-mCherry were used as mitochondrial markers in onion epidermal cells and P. patens protonemal tissues, respectively. Targeting was tested in Arabidopsis cell suspensions, onion epidermal cells, and P. patens protonemal tissues. Scale bar indicates 20 µm. Mitochondria (M), plastids (Pl), and peroxisomes (Px) are indicated, respectively.

Figure 2.

Dual targeting of DNA topoisomerase to mitochondria and plastids. A, Phylogenetic analysis of genes encoding DNA topoisomerases (Top) from Arabidopsis (At), rice (Os), P. patens (Pp), P. glauca (Pg), C. reinhardtii (Cr), and C. variabilis (Cv) using MEGA 5 (see “Materials and Methods”). B, Table of Top proteins from Arabidopsis, rice, P. patens, P. glauca, C. reinhardtii, and C. variabilis with genomic loci numbers, targeting predictions (see “Materials and Methods”), and experimental localization based on GFP targeting. C, GFP images of the targeting ability of tested Top proteins. Dual targeting of AtTopIA1 and OsTopI was evident in Arabidopsis cell suspensions and onion epidermal cells. In contrast, dual targeting of PpTop was only evident in onion epidermal cells and P. patens protonemal tissues. Mitochondria (M) and plastids (Pl) are indicated, respectively. Scale bar indicates 20 µm. Cyto, Cytosol; N, nuclear; S, secretory pathway.

Figure 3.

Dual targeting of DNA helicase to mitochondria and plastids. A, Phylogenetic analysis of genes encoding DNA helicase (Hel) from Arabidopsis (At), rice (Os), P. patens (Pp), C. reinhardtii (Cr), and C. variabilis (Cv) using MEGA 5 (see “Materials and Methods”). There was no ortholog found in P. glauca. B, Table of Hel proteins from Arabidopsis, rice, P. patens, C. reinhardtii, and C. variabilis with genomic loci numbers, targeting predictions, and experimental localization based on GFP tagging. C, GFP images of the targeting ability of Hel proteins tested. Dual targeting of OsHel and PpHel1 was evident in Arabidopsis cell suspensions, onion epidermal cells, and P. patens protonemal tissues. Mitochondria (M) and plastids (Pl) are indicated, respectively. Scale bar indicates 20 µm. Cyto, Cytosol; ER, endoplasmic reticulum; N, nuclear.

Figure 4.

Dual targeting of DNA polymerase to mitochondria and plastids. A, Phylogenetic analysis of genes encoding DNA polymerases (Pol) from Arabidopsis (At), rice (Os), P. patens (Pp), P. glauca (Pg), and C. reinhardtii (Cr) using MEGA 5 (see “Materials and Methods”). There was no ortholog found in C. variabilis. B, Table of the Pol proteins from Arabidopsis, rice, P. patens, P. glauca, and C. reinhardtii with genomic loci numbers, predictions using a variety of prediction programs, and experimental localization based on GFP tagging. C, GFP images of targeting ability of tested Pol proteins. Dual targeting of OsPol1 was evident in all three tested cells types. Only plastid targeting could be detected for PpPol1 in all three tissues, while plastid and mitochondrial targeting was detected for PpPol2 in onion epidermal cells and P. patens protonemal tissues. Mitochondria (M) and plastids (Pl) are indicated, respectively. Scale bar indicates 20 µm. Cyto, Cytosol; N, nuclear.

Figure 5.

Dual targeting of the enzymes of the ascorbate-glutathione cycle. A, Overview of the ascorbate-glutathione cycle. B, Phylogenetic analysis of the genes encoding GR from Arabidopsis (At), rice (Os), P. glauca (Pg), P. patens (Pp), C. reinhardtii (Cr), and C. variabilis (Cv) using MEGA 5. C, Table of GR proteins from Arabidopsis, rice, P. patens, P. glauca, C. reinhardtii, and C. variabilis with genomic loci numbers, targeting predictions, and experimental localization based on GFP tagging. D, GFP images of targeting ability of tested GR proteins. Dual targeting of GR from Arabidopsis, rice, and P. patens was detected in all tested tissues. Mitochondria (M) and plastids (Pl) are indicated, respectively. Scale bar indicates 20 µm. DHAR, Dehydroascorbate reductase; ASC, ascorbate; DHA, dehydroascorbate; GSH, glutathione; GSSG, glutathione disulfide; M, mitochondria; Pl, plastids; Cyto, cytosol.

Figure 6.

Dual targeting of APX to mitochondria and plastids. A, Phylogenetic analysis of genes encoding APX from Arabidopsis (At), rice (Os), P. glauca (Pg), P. patens (Pp), and C. reinhardtii (Cr) using MEGA 5 (see methods). There was no ortholog found in C. variabilis. B, GFP images of targeting ability of tested APX proteins. Dual targeting of AtSAPX was evident in Arabidopsis cell suspensions and onion epidermal cells. Four rice APX proteins were tested. OsAPX5 and OsAPX6 showed targeting to mitochondria, while OsAPX7 and OsAPX8 showed targeting to plastids. PgAPX1 showed targeting to both mitochondria and plastids in onion epidermal cells, while PpAPX1 showed targeting to plastids only in all tested tissues. C, Table of the APX proteins from Arabidopsis, rice, and P. patens with genomic loci numbers, targeting predictions, and experimental localization based on GFP targeting. Mitochondria (M) and plastids (Pl) are indicated, respectively. Scale bar indicates 20 µm. Cyto, Cytosol; ER, endoplasmic reticulum.

Figure 6.

Dual targeting of APX to mitochondria and plastids. A, Phylogenetic analysis of genes encoding APX from Arabidopsis (At), rice (Os), P. glauca (Pg), P. patens (Pp), and C. reinhardtii (Cr) using MEGA 5 (see methods). There was no ortholog found in C. variabilis. B, GFP images of targeting ability of tested APX proteins. Dual targeting of AtSAPX was evident in Arabidopsis cell suspensions and onion epidermal cells. Four rice APX proteins were tested. OsAPX5 and OsAPX6 showed targeting to mitochondria, while OsAPX7 and OsAPX8 showed targeting to plastids. PgAPX1 showed targeting to both mitochondria and plastids in onion epidermal cells, while PpAPX1 showed targeting to plastids only in all tested tissues. C, Table of the APX proteins from Arabidopsis, rice, and P. patens with genomic loci numbers, targeting predictions, and experimental localization based on GFP targeting. Mitochondria (M) and plastids (Pl) are indicated, respectively. Scale bar indicates 20 µm. Cyto, Cytosol; ER, endoplasmic reticulum.

Figure 7.

Dual targeting of MDHAR to mitochondria and plastids. A, Phylogenetic analysis of genes encoding MDHAR from Arabidopsis (At), rice (Os), P. glauca (Pg), P. patens (Pp), and C. reinhardtii (Cr) using MEGA 5 (see “Materials and Methods”). There is no ortholog found in C. variabilis. B, GFP images of targeting ability of the tested MDHAR proteins. Dual targeting of AtMDHAR6 was evident in Arabidopsis cell suspensions and onion epidermal cells. While OsMDHAR1.1 displayed dual targeting to mitochondria and plastids, OsMDAHR1.2 only showed mitochondrial targeting ability. Likewise for both P. patens and P. glauca, of the two proteins tested for each species, only one (PgMDHAR2 and PpMDHAR2 for P. glauca and P. patens, respectively) displayed dual-targeting ability. C, Table of the MDHAR proteins from Arabidopsis, rice, P. glauca, P. patens, and C. reinhardtii with genomic loci numbers, targeting predictions, and experimental localization based on GFP tagging. Mitochondria (M) and plastids (Pl) are indicated respectively. Scale bar indicates 20 µm. Cyto, Cytosol; ER, endoplasmic reticulum; EX, extracellular; S, secretory pathway.

Figure 7.

Dual targeting of MDHAR to mitochondria and plastids. A, Phylogenetic analysis of genes encoding MDHAR from Arabidopsis (At), rice (Os), P. glauca (Pg), P. patens (Pp), and C. reinhardtii (Cr) using MEGA 5 (see “Materials and Methods”). There is no ortholog found in C. variabilis. B, GFP images of targeting ability of the tested MDHAR proteins. Dual targeting of AtMDHAR6 was evident in Arabidopsis cell suspensions and onion epidermal cells. While OsMDHAR1.1 displayed dual targeting to mitochondria and plastids, OsMDAHR1.2 only showed mitochondrial targeting ability. Likewise for both P. patens and P. glauca, of the two proteins tested for each species, only one (PgMDHAR2 and PpMDHAR2 for P. glauca and P. patens, respectively) displayed dual-targeting ability. C, Table of the MDHAR proteins from Arabidopsis, rice, P. glauca, P. patens, and C. reinhardtii with genomic loci numbers, targeting predictions, and experimental localization based on GFP tagging. Mitochondria (M) and plastids (Pl) are indicated respectively. Scale bar indicates 20 µm. Cyto, Cytosol; ER, endoplasmic reticulum; EX, extracellular; S, secretory pathway.

Figure 8.

Dual targeting of HXK to mitochondria and plastids. A, Phylogenetic analysis of genes encoding HXK from Arabidopsis (At), Rice (Os), P. glauca (Pg), P. patens (Pp), C. reinhardtii (Cr), and C. variabilis (Cv) using MEGA 5 (see “Materials and Methods”). B, Table of the HXK proteins from Arabidopsis, rice, P. glauca, P. patens, C. reinhardtii, and C. variabilis with genomic loci numbers, targeting predictions, and experimental localization based on GFP tagging. C, GFP tagging of several HXK proteins from Arabidopsis showed that no dual targeting was evident for any HXK protein in Arabidopsis. D and E, GFP tagging of several HXK proteins from P. patens revealed that PpHXK2, PpHXK3, PpHXK5, PpHXK7, PpHXK9, and PpHXK11 displayed dual-targeting ability. Mitochondria (M) and plastids (Pl) are indicated respectively. Scale bar indicates 20 µm. Cyto, Cytosol; ER, endoplasmic reticulum; EX, extracellular; S, secretory pathway.

Figure 8.

Dual targeting of HXK to mitochondria and plastids. A, Phylogenetic analysis of genes encoding HXK from Arabidopsis (At), Rice (Os), P. glauca (Pg), P. patens (Pp), C. reinhardtii (Cr), and C. variabilis (Cv) using MEGA 5 (see “Materials and Methods”). B, Table of the HXK proteins from Arabidopsis, rice, P. glauca, P. patens, C. reinhardtii, and C. variabilis with genomic loci numbers, targeting predictions, and experimental localization based on GFP tagging. C, GFP tagging of several HXK proteins from Arabidopsis showed that no dual targeting was evident for any HXK protein in Arabidopsis. D and E, GFP tagging of several HXK proteins from P. patens revealed that PpHXK2, PpHXK3, PpHXK5, PpHXK7, PpHXK9, and PpHXK11 displayed dual-targeting ability. Mitochondria (M) and plastids (Pl) are indicated respectively. Scale bar indicates 20 µm. Cyto, Cytosol; ER, endoplasmic reticulum; EX, extracellular; S, secretory pathway.

Figure 8.

Dual targeting of HXK to mitochondria and plastids. A, Phylogenetic analysis of genes encoding HXK from Arabidopsis (At), Rice (Os), P. glauca (Pg), P. patens (Pp), C. reinhardtii (Cr), and C. variabilis (Cv) using MEGA 5 (see “Materials and Methods”). B, Table of the HXK proteins from Arabidopsis, rice, P. glauca, P. patens, C. reinhardtii, and C. variabilis with genomic loci numbers, targeting predictions, and experimental localization based on GFP tagging. C, GFP tagging of several HXK proteins from Arabidopsis showed that no dual targeting was evident for any HXK protein in Arabidopsis. D and E, GFP tagging of several HXK proteins from P. patens revealed that PpHXK2, PpHXK3, PpHXK5, PpHXK7, PpHXK9, and PpHXK11 displayed dual-targeting ability. Mitochondria (M) and plastids (Pl) are indicated respectively. Scale bar indicates 20 µm. Cyto, Cytosol; ER, endoplasmic reticulum; EX, extracellular; S, secretory pathway.

Figure 8.

Dual targeting of HXK to mitochondria and plastids. A, Phylogenetic analysis of genes encoding HXK from Arabidopsis (At), Rice (Os), P. glauca (Pg), P. patens (Pp), C. reinhardtii (Cr), and C. variabilis (Cv) using MEGA 5 (see “Materials and Methods”). B, Table of the HXK proteins from Arabidopsis, rice, P. glauca, P. patens, C. reinhardtii, and C. variabilis with genomic loci numbers, targeting predictions, and experimental localization based on GFP tagging. C, GFP tagging of several HXK proteins from Arabidopsis showed that no dual targeting was evident for any HXK protein in Arabidopsis. D and E, GFP tagging of several HXK proteins from P. patens revealed that PpHXK2, PpHXK3, PpHXK5, PpHXK7, PpHXK9, and PpHXK11 displayed dual-targeting ability. Mitochondria (M) and plastids (Pl) are indicated respectively. Scale bar indicates 20 µm. Cyto, Cytosol; ER, endoplasmic reticulum; EX, extracellular; S, secretory pathway.

Figure 9.

Targeting ability of Mia40 in P. patens. A, Phylogenetic analysis of genes encoding Mia40 in Arabidopsis (At), rice (Os), P. glauca (Pg), P. patens (Pp), C. reinhardtii (Cr), and C. variabilis (Cv) using MEGA 5 (see “Materials and Methods”). B, Table of the Mia40 proteins from Arabidopsis, rice, P. patens, P. glauca, C. reinhardtii, and C. variabilis with genomic loci numbers, targeting prediction, and experimental localization based on GFP tagging. C, GFP tagging of P. patens Mia40, displaying cytosolic targeting in all tested tissues. Scale bar indicates 20 µm. M, Mitochondria; Px, peroxisomes; Pl, plastids; Cyto, cytosol; EX, extracellular; N, nuclear; Cyto, cytosol.

Figure 10.

The evolution of targeting of Mia40 and putative substrate proteins. In plants such as Arabidopsis, G. max, and P. trichocarpa, Mia40 is dual targeted to mitochondria and peroxisomes. This dual targeting is also accompanied by the targeting of Mia40 substrates to the same organelles. However, in plants such as C. reinhardtii, V. carteri, C. variabillis, P. patens, and P. glauca, Mia40 is not predicted to be targeted to any organelle and was confirmed for P. patens in the current study (Fig. 9). This finding is accompanied by either the absence of substrate proteins or the absence of targeting of substrate proteins to peroxisomes. Monocot species such as rice, Brachypodium distachyon, and maize appear to be an intermediate, as they contain a dual-targeted Mia40 but lack substrate proteins in the peroxisome. This suggests how dual targeting of one protein may facilitate the acquisition of whole metabolic pathways between different organelles. See Supplemental Figure S3 for sequence alignment of Mia40 from various plants and the predicted targeting signals highlighted.