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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

Plant regeneration occurs in leaves from regenerated tobacco plants harboring AtTCTP2. (A–D) Representative images of regeneration from four leaf explants 21 days after transformation with the AtTCTP2-overexpressing construct harbored by A. rhizogenes. Selected leaf explants (dashed) were cut and transferred to fresh solid MS basal medium (without hormone supplementation) and incubated under controlled conditions (16:8 photoperiod). (E–G) New tissue arose 21 days after incubation in three samples, while in one of four samples tested (H) no self-regeneration capacity was observed. Original explants are highlighted in arrowheads. (I) GFP amplification by final point PCR using total DNA extracted from each original explant (arrowheads) as template to test transgene presence. [Lane 1 corresponds to (A,E), lane 2 to (B,F), lane 3 to (C,G), and lane 4 to (D,H)]. Self-regeneration correlated with presence of the AtTCTP2-GFP transgene (lanes 1–3); in a plant where no regeneration occurred AtTCTP2 was not detected (lane 4). 18S rRNA was used as control for RNA integrity. Size bars = 1 cm.
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Figure 8: Plant regeneration occurs in leaves from regenerated tobacco plants harboring AtTCTP2. (A–D) Representative images of regeneration from four leaf explants 21 days after transformation with the AtTCTP2-overexpressing construct harbored by A. rhizogenes. Selected leaf explants (dashed) were cut and transferred to fresh solid MS basal medium (without hormone supplementation) and incubated under controlled conditions (16:8 photoperiod). (E–G) New tissue arose 21 days after incubation in three samples, while in one of four samples tested (H) no self-regeneration capacity was observed. Original explants are highlighted in arrowheads. (I) GFP amplification by final point PCR using total DNA extracted from each original explant (arrowheads) as template to test transgene presence. [Lane 1 corresponds to (A,E), lane 2 to (B,F), lane 3 to (C,G), and lane 4 to (D,H)]. Self-regeneration correlated with presence of the AtTCTP2-GFP transgene (lanes 1–3); in a plant where no regeneration occurred AtTCTP2 was not detected (lane 4). 18S rRNA was used as control for RNA integrity. Size bars = 1 cm.

Mentions: Remarkably, inoculation of A. rhizogenes harboring AtTCTP2 induced whole plant regeneration in tobacco (Figures 7A–E); this regeneration activity was quantitatively similar to CmTCTP (Hinojosa-Moya et al., 2013; Table 1). Interestingly, AtTCTP1 was unable to induce regeneration to levels higher than background (Figure 7F; Table 1). In those cases in which there was plant regeneration, no AtTCTP1-GFP transgene was detected (not shown), while GFP and 35S transgenes, and thus AtTCTP2, were amplified in all regenerated plants, indicating that regeneration requires the latter (Figure 7G). On the other hand, a construct in which the AtTCTP2 start codon was replaced by a stop codon failed to induce regeneration above background levels, indicating that the protein is required for this phenomenon to occur (Table 1). Leaves from regenerated plants were also capable of regenerating plants themselves in the absence of exogenously applied plant hormones. Randomly selected leaves were excised from regenerated plants (four from each plant; five plants were analyzed) and placed on minimal MS medium. All these leaves gave rise to whole plants in three out of four cases; the exception corresponded to a plant that resulted negative for GFP and thus for AtTCTP2 (Figure 8).


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)

Plant regeneration occurs in leaves from regenerated tobacco plants harboring AtTCTP2. (A–D) Representative images of regeneration from four leaf explants 21 days after transformation with the AtTCTP2-overexpressing construct harbored by A. rhizogenes. Selected leaf explants (dashed) were cut and transferred to fresh solid MS basal medium (without hormone supplementation) and incubated under controlled conditions (16:8 photoperiod). (E–G) New tissue arose 21 days after incubation in three samples, while in one of four samples tested (H) no self-regeneration capacity was observed. Original explants are highlighted in arrowheads. (I) GFP amplification by final point PCR using total DNA extracted from each original explant (arrowheads) as template to test transgene presence. [Lane 1 corresponds to (A,E), lane 2 to (B,F), lane 3 to (C,G), and lane 4 to (D,H)]. Self-regeneration correlated with presence of the AtTCTP2-GFP transgene (lanes 1–3); in a plant where no regeneration occurred AtTCTP2 was not detected (lane 4). 18S rRNA was used as control for RNA integrity. Size bars = 1 cm.
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Figure 8: Plant regeneration occurs in leaves from regenerated tobacco plants harboring AtTCTP2. (A–D) Representative images of regeneration from four leaf explants 21 days after transformation with the AtTCTP2-overexpressing construct harbored by A. rhizogenes. Selected leaf explants (dashed) were cut and transferred to fresh solid MS basal medium (without hormone supplementation) and incubated under controlled conditions (16:8 photoperiod). (E–G) New tissue arose 21 days after incubation in three samples, while in one of four samples tested (H) no self-regeneration capacity was observed. Original explants are highlighted in arrowheads. (I) GFP amplification by final point PCR using total DNA extracted from each original explant (arrowheads) as template to test transgene presence. [Lane 1 corresponds to (A,E), lane 2 to (B,F), lane 3 to (C,G), and lane 4 to (D,H)]. Self-regeneration correlated with presence of the AtTCTP2-GFP transgene (lanes 1–3); in a plant where no regeneration occurred AtTCTP2 was not detected (lane 4). 18S rRNA was used as control for RNA integrity. Size bars = 1 cm.
Mentions: Remarkably, inoculation of A. rhizogenes harboring AtTCTP2 induced whole plant regeneration in tobacco (Figures 7A–E); this regeneration activity was quantitatively similar to CmTCTP (Hinojosa-Moya et al., 2013; Table 1). Interestingly, AtTCTP1 was unable to induce regeneration to levels higher than background (Figure 7F; Table 1). In those cases in which there was plant regeneration, no AtTCTP1-GFP transgene was detected (not shown), while GFP and 35S transgenes, and thus AtTCTP2, were amplified in all regenerated plants, indicating that regeneration requires the latter (Figure 7G). On the other hand, a construct in which the AtTCTP2 start codon was replaced by a stop codon failed to induce regeneration above background levels, indicating that the protein is required for this phenomenon to occur (Table 1). Leaves from regenerated plants were also capable of regenerating plants themselves in the absence of exogenously applied plant hormones. Randomly selected leaves were excised from regenerated plants (four from each plant; five plants were analyzed) and placed on minimal MS medium. All these leaves gave rise to whole plants in three out of four cases; the exception corresponded to a plant that resulted negative for GFP and thus for AtTCTP2 (Figure 8).

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