Limits...
AtTCTP2, an Arabidopsis thaliana homolog of Translationally Controlled Tumor Protein, enhances in vitro plant regeneration.

Toscano-Morales R, Xoconostle-Cázares B, Cabrera-Ponce JL, Hinojosa-Moya J, Ruiz-Salas JL, Galván-Gordillo SV, Guevara-González RG, Ruiz-Medrano R - Front Plant Sci (2015)

Bottom Line: Leaf explants transformed with Agrobacterium rhizogenes harboring AtTCTP2, but not AtTCTP1, led to whole plant regeneration with a high frequency.Insertion of a sequence present in AtTCTP1 but absent in AtTCTP2 demonstrates that it suppresses the capacity for plant regeneration; also, this phenomenon is enhanced by the presence of TCTP (AtTCTP1 or 2) in the nuclei of root cells.This confirms that AtTCTP2 is not a pseudogene and suggests the involvement of certain TCTP isoforms in vegetative reproduction in some plant species.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Plant Molecular Biology, Department of Biotechnology and Bioengineering, CINVESTAV Mexico City, Mexico.

ABSTRACT
The Translationally Controlled Tumor Protein (TCTP) is a central regulator of cell proliferation and differentiation in animals, and probably also in plants. Arabidopsis harbors two TCTP genes, AtTCTP1 (At3g16640), which is an important mitotic regulator, and AtTCTP2 (At3g05540), which is considered a pseudogene. Nevertheless, we have obtained evidence suggesting that this gene is functional. Indeed, a T-DNA insertion mutant, SALK_045146, displays a lethal phenotype during early rosette stage. Also, both the AtTCTP2 promoter and structural gene are functional, and heterozygous plants show delayed development. AtTCTP1 cannot compensate for the loss of AtTCTP2, since the accumulation levels of the AtTCTP1 transcript are even higher in heterozygous plants than in wild-type plants. Leaf explants transformed with Agrobacterium rhizogenes harboring AtTCTP2, but not AtTCTP1, led to whole plant regeneration with a high frequency. Insertion of a sequence present in AtTCTP1 but absent in AtTCTP2 demonstrates that it suppresses the capacity for plant regeneration; also, this phenomenon is enhanced by the presence of TCTP (AtTCTP1 or 2) in the nuclei of root cells. This confirms that AtTCTP2 is not a pseudogene and suggests the involvement of certain TCTP isoforms in vegetative reproduction in some plant species.

No MeSH data available.


Related in: MedlinePlus

Domain swapping between AtTCTP2 and AtTCTP1 alters their regeneration capacity. (A) Amino acid sequence alignment of AtTCTP2 and AtTCTP1 versions with ClustalX. The main difference between unmodified (normal/endogenous) protein versions is located in the 13 amino acid region shown by arrowheads and red-dashed rectangle; this region was swapped between AtTCTP1 and AtTCTP2 in order to generate the modified versions for both proteins. (B) Predictive three-dimensional structures of AtTCTP2 and AtTCTP1 (unmodified and modified versions) were modeled with the SWISS-MODEL automated protein structure homology-modeling server and processed using Deep View program (Swiss-Pdb-Viewer); dashed circles underline the region with more structural differences in all four versions. (C) Rate of plant regeneration is altered when both AtTCTP2 and AtTCTP1 are modified. Transformation of tobacco explants was performed with the four-overexpression versions (fused to GFP) previously mentioned. Three transformation events were performed with 30 explants per biological replicate (n ≈ 90), given as mean ± SE. Plant regeneration rate of AtTCTP2 was reduced significantly while that of AtTCTP1 increased in the modified versions (mAtTCTP2 and mAtTCTP1) relative to the unmodified ones (AtTCTP2 and AtTCTP1).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4489097&req=5

Figure 10: Domain swapping between AtTCTP2 and AtTCTP1 alters their regeneration capacity. (A) Amino acid sequence alignment of AtTCTP2 and AtTCTP1 versions with ClustalX. The main difference between unmodified (normal/endogenous) protein versions is located in the 13 amino acid region shown by arrowheads and red-dashed rectangle; this region was swapped between AtTCTP1 and AtTCTP2 in order to generate the modified versions for both proteins. (B) Predictive three-dimensional structures of AtTCTP2 and AtTCTP1 (unmodified and modified versions) were modeled with the SWISS-MODEL automated protein structure homology-modeling server and processed using Deep View program (Swiss-Pdb-Viewer); dashed circles underline the region with more structural differences in all four versions. (C) Rate of plant regeneration is altered when both AtTCTP2 and AtTCTP1 are modified. Transformation of tobacco explants was performed with the four-overexpression versions (fused to GFP) previously mentioned. Three transformation events were performed with 30 explants per biological replicate (n ≈ 90), given as mean ± SE. Plant regeneration rate of AtTCTP2 was reduced significantly while that of AtTCTP1 increased in the modified versions (mAtTCTP2 and mAtTCTP1) relative to the unmodified ones (AtTCTP2 and AtTCTP1).

Mentions: AtTCTP1 and AtTCTP2 share a ~80% similarity at the amino acid level, the most striking difference being a deletion of 13 amino acids in AtTCTP2 relative to AtTCTP1 (Figure 10A). Modified versions of these proteins were obtained in which this domain was deleted in AtTCTP1, and added in AtTCTP2. The predicted structure of mAtTCTP1 resembled AtTCTP2, while mAtTCTP2 resembled AtTCTP1 (Figure 10B). Regeneration assays in tobacco showed that, indeed, mAtTCTP1 behaved as AtTCTP2 and mAtTCTP2 as AtTCTP1 in terms of regeneration frequency (Figure 10C). Thus, while mATCTP1 regeneration activity increased from ~3 to ~13%, this decreased from ~35 to ~16% in mATCTP2 (Table S2). Furthermore, localization of these proteins was modified. Indeed, mAtTCTP1 was found in nuclei of roots of regenerated plants, while mAtTCTP2 localized to cytoplasm and, in a very small proportion, nuclei (Figure 11 and Figure S8). Thus, nuclear localization of AtTCTP1 and 2 in root cortical cells correlates with their capacity to induce plant regeneration. This was observed consistently in samples from independent transformants (Figure S8). It must be mentioned that the predicted structure of CmTCTP and AtTCTP2 are more similar between them than to AtTCTP1 (Figure S9).


AtTCTP2, an Arabidopsis thaliana homolog of Translationally Controlled Tumor Protein, enhances in vitro plant regeneration.

Toscano-Morales R, Xoconostle-Cázares B, Cabrera-Ponce JL, Hinojosa-Moya J, Ruiz-Salas JL, Galván-Gordillo SV, Guevara-González RG, Ruiz-Medrano R - Front Plant Sci (2015)

Domain swapping between AtTCTP2 and AtTCTP1 alters their regeneration capacity. (A) Amino acid sequence alignment of AtTCTP2 and AtTCTP1 versions with ClustalX. The main difference between unmodified (normal/endogenous) protein versions is located in the 13 amino acid region shown by arrowheads and red-dashed rectangle; this region was swapped between AtTCTP1 and AtTCTP2 in order to generate the modified versions for both proteins. (B) Predictive three-dimensional structures of AtTCTP2 and AtTCTP1 (unmodified and modified versions) were modeled with the SWISS-MODEL automated protein structure homology-modeling server and processed using Deep View program (Swiss-Pdb-Viewer); dashed circles underline the region with more structural differences in all four versions. (C) Rate of plant regeneration is altered when both AtTCTP2 and AtTCTP1 are modified. Transformation of tobacco explants was performed with the four-overexpression versions (fused to GFP) previously mentioned. Three transformation events were performed with 30 explants per biological replicate (n ≈ 90), given as mean ± SE. Plant regeneration rate of AtTCTP2 was reduced significantly while that of AtTCTP1 increased in the modified versions (mAtTCTP2 and mAtTCTP1) relative to the unmodified ones (AtTCTP2 and AtTCTP1).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4489097&req=5

Figure 10: Domain swapping between AtTCTP2 and AtTCTP1 alters their regeneration capacity. (A) Amino acid sequence alignment of AtTCTP2 and AtTCTP1 versions with ClustalX. The main difference between unmodified (normal/endogenous) protein versions is located in the 13 amino acid region shown by arrowheads and red-dashed rectangle; this region was swapped between AtTCTP1 and AtTCTP2 in order to generate the modified versions for both proteins. (B) Predictive three-dimensional structures of AtTCTP2 and AtTCTP1 (unmodified and modified versions) were modeled with the SWISS-MODEL automated protein structure homology-modeling server and processed using Deep View program (Swiss-Pdb-Viewer); dashed circles underline the region with more structural differences in all four versions. (C) Rate of plant regeneration is altered when both AtTCTP2 and AtTCTP1 are modified. Transformation of tobacco explants was performed with the four-overexpression versions (fused to GFP) previously mentioned. Three transformation events were performed with 30 explants per biological replicate (n ≈ 90), given as mean ± SE. Plant regeneration rate of AtTCTP2 was reduced significantly while that of AtTCTP1 increased in the modified versions (mAtTCTP2 and mAtTCTP1) relative to the unmodified ones (AtTCTP2 and AtTCTP1).
Mentions: AtTCTP1 and AtTCTP2 share a ~80% similarity at the amino acid level, the most striking difference being a deletion of 13 amino acids in AtTCTP2 relative to AtTCTP1 (Figure 10A). Modified versions of these proteins were obtained in which this domain was deleted in AtTCTP1, and added in AtTCTP2. The predicted structure of mAtTCTP1 resembled AtTCTP2, while mAtTCTP2 resembled AtTCTP1 (Figure 10B). Regeneration assays in tobacco showed that, indeed, mAtTCTP1 behaved as AtTCTP2 and mAtTCTP2 as AtTCTP1 in terms of regeneration frequency (Figure 10C). Thus, while mATCTP1 regeneration activity increased from ~3 to ~13%, this decreased from ~35 to ~16% in mATCTP2 (Table S2). Furthermore, localization of these proteins was modified. Indeed, mAtTCTP1 was found in nuclei of roots of regenerated plants, while mAtTCTP2 localized to cytoplasm and, in a very small proportion, nuclei (Figure 11 and Figure S8). Thus, nuclear localization of AtTCTP1 and 2 in root cortical cells correlates with their capacity to induce plant regeneration. This was observed consistently in samples from independent transformants (Figure S8). It must be mentioned that the predicted structure of CmTCTP and AtTCTP2 are more similar between them than to AtTCTP1 (Figure S9).

Bottom Line: Leaf explants transformed with Agrobacterium rhizogenes harboring AtTCTP2, but not AtTCTP1, led to whole plant regeneration with a high frequency.Insertion of a sequence present in AtTCTP1 but absent in AtTCTP2 demonstrates that it suppresses the capacity for plant regeneration; also, this phenomenon is enhanced by the presence of TCTP (AtTCTP1 or 2) in the nuclei of root cells.This confirms that AtTCTP2 is not a pseudogene and suggests the involvement of certain TCTP isoforms in vegetative reproduction in some plant species.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Plant Molecular Biology, Department of Biotechnology and Bioengineering, CINVESTAV Mexico City, Mexico.

ABSTRACT
The Translationally Controlled Tumor Protein (TCTP) is a central regulator of cell proliferation and differentiation in animals, and probably also in plants. Arabidopsis harbors two TCTP genes, AtTCTP1 (At3g16640), which is an important mitotic regulator, and AtTCTP2 (At3g05540), which is considered a pseudogene. Nevertheless, we have obtained evidence suggesting that this gene is functional. Indeed, a T-DNA insertion mutant, SALK_045146, displays a lethal phenotype during early rosette stage. Also, both the AtTCTP2 promoter and structural gene are functional, and heterozygous plants show delayed development. AtTCTP1 cannot compensate for the loss of AtTCTP2, since the accumulation levels of the AtTCTP1 transcript are even higher in heterozygous plants than in wild-type plants. Leaf explants transformed with Agrobacterium rhizogenes harboring AtTCTP2, but not AtTCTP1, led to whole plant regeneration with a high frequency. Insertion of a sequence present in AtTCTP1 but absent in AtTCTP2 demonstrates that it suppresses the capacity for plant regeneration; also, this phenomenon is enhanced by the presence of TCTP (AtTCTP1 or 2) in the nuclei of root cells. This confirms that AtTCTP2 is not a pseudogene and suggests the involvement of certain TCTP isoforms in vegetative reproduction in some plant species.

No MeSH data available.


Related in: MedlinePlus