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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: 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.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

Phenotypes of heterozygous (±) insertional mutants (SALK_045146) correlate with AtTCTP2 expression levels. (A) qRT-PCR was performed to determine transcript levels of AtTCTP2, AtTCTP1, and 18S rRNA (as endogenous control) using total RNA extracted from several AtTCTP2 mutants and comparing their expression against WT lines. (B) Severity of phenotype of heterozygous (±) AtTCTP2 mutants ordered by area/size correlates with AtTCTP2 expression levels, and are not associated to a decrease in AtTCTP1 transcript levels. Both qRT-PCR and analysis of phenotype were performed in plants 25 days after germination. Size bars = 2 cm.
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Figure 2: Phenotypes of heterozygous (±) insertional mutants (SALK_045146) correlate with AtTCTP2 expression levels. (A) qRT-PCR was performed to determine transcript levels of AtTCTP2, AtTCTP1, and 18S rRNA (as endogenous control) using total RNA extracted from several AtTCTP2 mutants and comparing their expression against WT lines. (B) Severity of phenotype of heterozygous (±) AtTCTP2 mutants ordered by area/size correlates with AtTCTP2 expression levels, and are not associated to a decrease in AtTCTP1 transcript levels. Both qRT-PCR and analysis of phenotype were performed in plants 25 days after germination. Size bars = 2 cm.

Mentions: In addition to AtTCTP1 (At3g16640), Arabidopsis harbors another TCTP gene, AtTCTP2 (At3g05540). RT-PCR assays failed to detect this mRNA, and a T-DNA insertion mutant shows a wild-type phenotype, reasons for which AtTCTP2 had been considered a pseudogene (Berkowitz et al., 2008). However, another insertional mutant, SALK_045146 (Alonso et al., 2003) shows a lethal phenotype during early stages of development (Figure 1A). Thus, a more thorough analysis was carried out in order to determine whether AtTCTP2 is a pseudogene. This insertion is located in the fourth exon of the gene; other T-DNA insertions of this gene fall within introns, which could explain their non-lethal phenotype. Two weeks after germination, homozygous SALK_045146 mutants (−/−) developed necrosis in leaves and eventually died (Figure 1A). Other defects were smaller size and fewer rosette leaves. No mutant survived past the early rosette stage, and no intact RNA could be recovered at this stage. Interestingly, heterozygous plants (+/−) displayed an intermediate phenotype. Plants showed a delay in bolting, were shorter and had fewer rosette leaves (Figures 1B,C). Only 15–20% of the heterozygous plants bolted, but only after more than 3 months. This mRNA was quantified to test whether AtTCTP1 was silenced in (+/−) plants and thus causing the observed phenotype. This could only be carried out in (+/−) plants, because the RNA of (−/−) mutants was quite degraded when the phenotype was evident and thus could not be isolated for RT-PCR. AtTCTP1 mRNA accumulated to even higher levels than in wild type plants; as expected, AtTCTP2 levels were lower than in wild type (Figure 2). Moreover, the severity of the phenotype correlated with decreased levels of AtTCTP2 mRNA in different heterozygous lines, while those for AtTCTP1 mRNA did not show such correlation, although in all cases were higher than in WT plants (Figure 2). Therefore, loss of one copy of the AtTCTP2 gene leads to a significant decrease in AtTCTP2 mRNA, and to a considerable increase in the AtTCTP1 mRNA. A digital droplet PCR assay was carried out to determine the number of T-DNA insertions in two SALK_045146 heterozygous mutant lines; in these cases only one insertion was found (Figure S1A). Photosynthesis rate, on the other hand, was not affected in heterozygous plants (Figure S1B).


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)

Phenotypes of heterozygous (±) insertional mutants (SALK_045146) correlate with AtTCTP2 expression levels. (A) qRT-PCR was performed to determine transcript levels of AtTCTP2, AtTCTP1, and 18S rRNA (as endogenous control) using total RNA extracted from several AtTCTP2 mutants and comparing their expression against WT lines. (B) Severity of phenotype of heterozygous (±) AtTCTP2 mutants ordered by area/size correlates with AtTCTP2 expression levels, and are not associated to a decrease in AtTCTP1 transcript levels. Both qRT-PCR and analysis of phenotype were performed in plants 25 days after germination. Size bars = 2 cm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Phenotypes of heterozygous (±) insertional mutants (SALK_045146) correlate with AtTCTP2 expression levels. (A) qRT-PCR was performed to determine transcript levels of AtTCTP2, AtTCTP1, and 18S rRNA (as endogenous control) using total RNA extracted from several AtTCTP2 mutants and comparing their expression against WT lines. (B) Severity of phenotype of heterozygous (±) AtTCTP2 mutants ordered by area/size correlates with AtTCTP2 expression levels, and are not associated to a decrease in AtTCTP1 transcript levels. Both qRT-PCR and analysis of phenotype were performed in plants 25 days after germination. Size bars = 2 cm.
Mentions: In addition to AtTCTP1 (At3g16640), Arabidopsis harbors another TCTP gene, AtTCTP2 (At3g05540). RT-PCR assays failed to detect this mRNA, and a T-DNA insertion mutant shows a wild-type phenotype, reasons for which AtTCTP2 had been considered a pseudogene (Berkowitz et al., 2008). However, another insertional mutant, SALK_045146 (Alonso et al., 2003) shows a lethal phenotype during early stages of development (Figure 1A). Thus, a more thorough analysis was carried out in order to determine whether AtTCTP2 is a pseudogene. This insertion is located in the fourth exon of the gene; other T-DNA insertions of this gene fall within introns, which could explain their non-lethal phenotype. Two weeks after germination, homozygous SALK_045146 mutants (−/−) developed necrosis in leaves and eventually died (Figure 1A). Other defects were smaller size and fewer rosette leaves. No mutant survived past the early rosette stage, and no intact RNA could be recovered at this stage. Interestingly, heterozygous plants (+/−) displayed an intermediate phenotype. Plants showed a delay in bolting, were shorter and had fewer rosette leaves (Figures 1B,C). Only 15–20% of the heterozygous plants bolted, but only after more than 3 months. This mRNA was quantified to test whether AtTCTP1 was silenced in (+/−) plants and thus causing the observed phenotype. This could only be carried out in (+/−) plants, because the RNA of (−/−) mutants was quite degraded when the phenotype was evident and thus could not be isolated for RT-PCR. AtTCTP1 mRNA accumulated to even higher levels than in wild type plants; as expected, AtTCTP2 levels were lower than in wild type (Figure 2). Moreover, the severity of the phenotype correlated with decreased levels of AtTCTP2 mRNA in different heterozygous lines, while those for AtTCTP1 mRNA did not show such correlation, although in all cases were higher than in WT plants (Figure 2). Therefore, loss of one copy of the AtTCTP2 gene leads to a significant decrease in AtTCTP2 mRNA, and to a considerable increase in the AtTCTP1 mRNA. A digital droplet PCR assay was carried out to determine the number of T-DNA insertions in two SALK_045146 heterozygous mutant lines; in these cases only one insertion was found (Figure S1A). Photosynthesis rate, on the other hand, was not affected in heterozygous plants (Figure S1B).

Bottom Line: 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.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