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Eukaryotic release factor 1-2 affects Arabidopsis responses to glucose and phytohormones during germination and early seedling development.

Zhou X, Cooke P, Li L - J. Exp. Bot. (2009)

Bottom Line: The eRF1-2 gene was found to be specifically induced by glucose.By contrast, the loss-of-function erf1-2 mutant exhibited resistance to paclobutrazol, suggesting that eRF1-2 may exert a negative effect on the GA signalling pathway.Collectively, these data provide evidence in support of a novel role of eRF1-2 in affecting glucose and phytohormone responses in modulating plant growth and development.

View Article: PubMed Central - PubMed

Affiliation: Robert W Holley Center for Agriculture and Health, U.S. Department of Agriculture-Agricultural Research Service, Cornell University, Ithaca, NY 14853, USA.

ABSTRACT
Germination and early seedling development are coordinately regulated by glucose and phytohormones such as ABA, GA, and ethylene. However, the molecules that affect plant responses to glucose and phytohormones remain to be fully elucidated. Eukaryotic release factor 1 (eRF1) is responsible for the recognition of the stop codons in mRNAs during protein synthesis. Accumulating evidence indicates that eRF1 functions in other processes in addition to translation termination. The physiological role of eRF1-2, a member of the eRF1 family, in Arabidopsis was examined here. The eRF1-2 gene was found to be specifically induced by glucose. Arabidopsis plants overexpressing eRF1-2 were hypersensitive to glucose during germination and early seedling development. Such hypersensitivity to glucose was accompanied by a dramatic reduction of the expression of glucose-regulated genes, chlorophyll a/b binding protein and plastocyanin. The hypersensitive response was not due to the enhanced accumulation of ABA. In addition, the eRF1-2 overexpressing plants showed increased sensitivity to paclobutrazol, an inhibitor of GA biosynthesis, and exogenous GA restored their normal growth. By contrast, the loss-of-function erf1-2 mutant exhibited resistance to paclobutrazol, suggesting that eRF1-2 may exert a negative effect on the GA signalling pathway. Collectively, these data provide evidence in support of a novel role of eRF1-2 in affecting glucose and phytohormone responses in modulating plant growth and development.

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Analysis of eRF1-2:GFP transgenic lines, subcellular localization of eRF1-2, and qRT-PCR analysis of the induction of eRF1-2 expression by sugar. (A) RT-PCR analysis revealed that eRF1-2 and GFP were highly expressed in the homozygous OV1-4 and OV13-11 overexpressing lines in comparison with WT plants (Col). (B) Immunoblot analysis of the eRF1-2 overexpressing plants. Ten microgram of proteins extracted from leaf tissues were separated by SDS-PAGE and immunoblotted with a GFP antibody. Arrow points to the fusion protein at approximately 85 kDa. Molecular sizes are indicated on the left. (C) Subcellular localization of eRF1-2 protein. GFP signal in the root elongation zone of a 3-d-old transgenic seedling expressing 35S:eRF1-2:GFP (1) and 35S:GFP (2). GFP signal in the guard cells of a 3-d-old transgenic seedling expressing 35S:eRF1-2:GFP (3) and 35S:GFP (4). (D) Expression of eRF1-2 was specifically induced by glucose. Arabidopsis seedlings (10-d-old) of WT plants were treated with 6% glucose, 6% mannitol, and water for 0, 2, 4, 6, 8, and 24 h. (This figure is available in colour at JXB online.)
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fig2: Analysis of eRF1-2:GFP transgenic lines, subcellular localization of eRF1-2, and qRT-PCR analysis of the induction of eRF1-2 expression by sugar. (A) RT-PCR analysis revealed that eRF1-2 and GFP were highly expressed in the homozygous OV1-4 and OV13-11 overexpressing lines in comparison with WT plants (Col). (B) Immunoblot analysis of the eRF1-2 overexpressing plants. Ten microgram of proteins extracted from leaf tissues were separated by SDS-PAGE and immunoblotted with a GFP antibody. Arrow points to the fusion protein at approximately 85 kDa. Molecular sizes are indicated on the left. (C) Subcellular localization of eRF1-2 protein. GFP signal in the root elongation zone of a 3-d-old transgenic seedling expressing 35S:eRF1-2:GFP (1) and 35S:GFP (2). GFP signal in the guard cells of a 3-d-old transgenic seedling expressing 35S:eRF1-2:GFP (3) and 35S:GFP (4). (D) Expression of eRF1-2 was specifically induced by glucose. Arabidopsis seedlings (10-d-old) of WT plants were treated with 6% glucose, 6% mannitol, and water for 0, 2, 4, 6, 8, and 24 h. (This figure is available in colour at JXB online.)

Mentions: The eRF1-2 protein contains no obvious predicted localization signal sequence. To experimentally determine the subcellular localization of eRF1-2 in plants, the eRF1-2:GFP chimeric gene under the control of the CaMV 35S promoter was introduced into Arabidopsis plants. Fifteen independent transformants were obtained and two homozygous lines (OV1-4 and OV13-11) of T3 plants were selected. RT-PCR analysis showed that eRF1-2 was highly expressed in these two overexpressing lines, which were further verified by PCR using GFP specific primers (Fig. 2A). Immunoblot analysis with the GFP antibody revealed that, the eRF1-2 transgenic lines contained a specific band close to the expected molecular weight of the fusion protein at approximately 85 kDa and an extra band at higher molecular weight (Fig. 2B), confirming that eRF1-2 was expressed in the transgenic Arabidopsis.


Eukaryotic release factor 1-2 affects Arabidopsis responses to glucose and phytohormones during germination and early seedling development.

Zhou X, Cooke P, Li L - J. Exp. Bot. (2009)

Analysis of eRF1-2:GFP transgenic lines, subcellular localization of eRF1-2, and qRT-PCR analysis of the induction of eRF1-2 expression by sugar. (A) RT-PCR analysis revealed that eRF1-2 and GFP were highly expressed in the homozygous OV1-4 and OV13-11 overexpressing lines in comparison with WT plants (Col). (B) Immunoblot analysis of the eRF1-2 overexpressing plants. Ten microgram of proteins extracted from leaf tissues were separated by SDS-PAGE and immunoblotted with a GFP antibody. Arrow points to the fusion protein at approximately 85 kDa. Molecular sizes are indicated on the left. (C) Subcellular localization of eRF1-2 protein. GFP signal in the root elongation zone of a 3-d-old transgenic seedling expressing 35S:eRF1-2:GFP (1) and 35S:GFP (2). GFP signal in the guard cells of a 3-d-old transgenic seedling expressing 35S:eRF1-2:GFP (3) and 35S:GFP (4). (D) Expression of eRF1-2 was specifically induced by glucose. Arabidopsis seedlings (10-d-old) of WT plants were treated with 6% glucose, 6% mannitol, and water for 0, 2, 4, 6, 8, and 24 h. (This figure is available in colour at JXB online.)
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2803205&req=5

fig2: Analysis of eRF1-2:GFP transgenic lines, subcellular localization of eRF1-2, and qRT-PCR analysis of the induction of eRF1-2 expression by sugar. (A) RT-PCR analysis revealed that eRF1-2 and GFP were highly expressed in the homozygous OV1-4 and OV13-11 overexpressing lines in comparison with WT plants (Col). (B) Immunoblot analysis of the eRF1-2 overexpressing plants. Ten microgram of proteins extracted from leaf tissues were separated by SDS-PAGE and immunoblotted with a GFP antibody. Arrow points to the fusion protein at approximately 85 kDa. Molecular sizes are indicated on the left. (C) Subcellular localization of eRF1-2 protein. GFP signal in the root elongation zone of a 3-d-old transgenic seedling expressing 35S:eRF1-2:GFP (1) and 35S:GFP (2). GFP signal in the guard cells of a 3-d-old transgenic seedling expressing 35S:eRF1-2:GFP (3) and 35S:GFP (4). (D) Expression of eRF1-2 was specifically induced by glucose. Arabidopsis seedlings (10-d-old) of WT plants were treated with 6% glucose, 6% mannitol, and water for 0, 2, 4, 6, 8, and 24 h. (This figure is available in colour at JXB online.)
Mentions: The eRF1-2 protein contains no obvious predicted localization signal sequence. To experimentally determine the subcellular localization of eRF1-2 in plants, the eRF1-2:GFP chimeric gene under the control of the CaMV 35S promoter was introduced into Arabidopsis plants. Fifteen independent transformants were obtained and two homozygous lines (OV1-4 and OV13-11) of T3 plants were selected. RT-PCR analysis showed that eRF1-2 was highly expressed in these two overexpressing lines, which were further verified by PCR using GFP specific primers (Fig. 2A). Immunoblot analysis with the GFP antibody revealed that, the eRF1-2 transgenic lines contained a specific band close to the expected molecular weight of the fusion protein at approximately 85 kDa and an extra band at higher molecular weight (Fig. 2B), confirming that eRF1-2 was expressed in the transgenic Arabidopsis.

Bottom Line: The eRF1-2 gene was found to be specifically induced by glucose.By contrast, the loss-of-function erf1-2 mutant exhibited resistance to paclobutrazol, suggesting that eRF1-2 may exert a negative effect on the GA signalling pathway.Collectively, these data provide evidence in support of a novel role of eRF1-2 in affecting glucose and phytohormone responses in modulating plant growth and development.

View Article: PubMed Central - PubMed

Affiliation: Robert W Holley Center for Agriculture and Health, U.S. Department of Agriculture-Agricultural Research Service, Cornell University, Ithaca, NY 14853, USA.

ABSTRACT
Germination and early seedling development are coordinately regulated by glucose and phytohormones such as ABA, GA, and ethylene. However, the molecules that affect plant responses to glucose and phytohormones remain to be fully elucidated. Eukaryotic release factor 1 (eRF1) is responsible for the recognition of the stop codons in mRNAs during protein synthesis. Accumulating evidence indicates that eRF1 functions in other processes in addition to translation termination. The physiological role of eRF1-2, a member of the eRF1 family, in Arabidopsis was examined here. The eRF1-2 gene was found to be specifically induced by glucose. Arabidopsis plants overexpressing eRF1-2 were hypersensitive to glucose during germination and early seedling development. Such hypersensitivity to glucose was accompanied by a dramatic reduction of the expression of glucose-regulated genes, chlorophyll a/b binding protein and plastocyanin. The hypersensitive response was not due to the enhanced accumulation of ABA. In addition, the eRF1-2 overexpressing plants showed increased sensitivity to paclobutrazol, an inhibitor of GA biosynthesis, and exogenous GA restored their normal growth. By contrast, the loss-of-function erf1-2 mutant exhibited resistance to paclobutrazol, suggesting that eRF1-2 may exert a negative effect on the GA signalling pathway. Collectively, these data provide evidence in support of a novel role of eRF1-2 in affecting glucose and phytohormone responses in modulating plant growth and development.

Show MeSH
Related in: MedlinePlus