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Arabidopsis thaliana DOF6 negatively affects germination in non-after-ripened seeds and interacts with TCP14.

Rueda-Romero P, Barrero-Sicilia C, Gómez-Cadenas A, Carbonero P, Oñate-Sánchez L - J. Exp. Bot. (2011)

Bottom Line: While constitutive over-expression of DOF6 produced aberrant growth and sterility in the plant, its over-expression induced upon seed imbibition triggered delayed germination, abscisic acid (ABA)-hypersensitive phenotypes and increased expression of the ABA biosynthetic gene ABA1 and ABA-related stress genes.DOF6 was found to interact in a yeast two-hybrid system and in planta with TCP14, a previously described positive regulator of seed germination.The expression of ABA1 and ABA-related stress genes was also enhanced in tcp14 knock-out mutants.

View Article: PubMed Central - PubMed

Affiliation: Universidad Politécnica de Madrid, Campus de Montegancedo, Pozuelo de Alarcón, Madrid, Spain.

ABSTRACT
Seed dormancy prevents seeds from germinating under environmental conditions unfavourable for plant growth and development and constitutes an evolutionary advantage. Dry storage, also known as after-ripening, gradually decreases seed dormancy by mechanisms not well understood. An Arabidopsis thaliana DOF transcription factor gene (DOF6) affecting seed germination has been characterized. The transcript levels of this gene accumulate in dry seeds and decay gradually during after-ripening and also upon seed imbibition. While constitutive over-expression of DOF6 produced aberrant growth and sterility in the plant, its over-expression induced upon seed imbibition triggered delayed germination, abscisic acid (ABA)-hypersensitive phenotypes and increased expression of the ABA biosynthetic gene ABA1 and ABA-related stress genes. Wild-type germination and gene expression were gradually restored during seed after-ripening, despite of DOF6-induced over-expression. DOF6 was found to interact in a yeast two-hybrid system and in planta with TCP14, a previously described positive regulator of seed germination. The expression of ABA1 and ABA-related stress genes was also enhanced in tcp14 knock-out mutants. Taken together, these results indicate that DOF6 negatively affects seed germination and opposes TCP14 function in the regulation of a specific set of ABA-related genes.

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Related in: MedlinePlus

mRNA levels of ABA-related genes in germinating freshly harvested (FH) ioexDOF6 seeds. Expression of genes involved in ABA metabolism and signalling (A) and ABA-inducible genes (B) was compared by quantitative RT-PCR between wild-type (WT) and ioexDOF6 FH seeds 48 hours after imbibition in 50 μM estradiol. Gene-specific mRNA levels are relative to UBC21. All the UBC21 normalized expression values are relative to the values obtained for each gene in the WT sample.
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fig5: mRNA levels of ABA-related genes in germinating freshly harvested (FH) ioexDOF6 seeds. Expression of genes involved in ABA metabolism and signalling (A) and ABA-inducible genes (B) was compared by quantitative RT-PCR between wild-type (WT) and ioexDOF6 FH seeds 48 hours after imbibition in 50 μM estradiol. Gene-specific mRNA levels are relative to UBC21. All the UBC21 normalized expression values are relative to the values obtained for each gene in the WT sample.

Mentions: Total RNA was isolated from seeds and other tissues, as described by Oñate-Sánchez and Vicente-Carbajosa (2008). Seeds (15 mg) from each genotype were germinated on moistened filter paper and samples were taken at different hours after imbibition (hai). The Transcriptor First Strand cDNA Synthesis kit (Roche Diagnostics, Mannheim, Germany) was used to synthesize cDNA. For quantitative reverse transcription (RT)-PCR, 8–16 ng cDNA was used as template together with 0.5 μM of forward and reverse specific oligonucleotides (Supplementary Table S1) and Power SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA). Cycling conditions (ABI Prism 7300; Applied Biosystems) were as follows: 10 min at 95 °C and 50 cycles of 15 s at 95 °C and 60 s at 60 °C, linked to a default dissociation stage programme to detect non-specific amplification. Three technical and at least two biological replicates were included in every experiment and the ubiquitin gene UBC21 (At5g25760) was used to normalize expression levels. Therefore, gene-specific mRNA levels shown in all figures except Fig. 5 are relative to UBC21.


Arabidopsis thaliana DOF6 negatively affects germination in non-after-ripened seeds and interacts with TCP14.

Rueda-Romero P, Barrero-Sicilia C, Gómez-Cadenas A, Carbonero P, Oñate-Sánchez L - J. Exp. Bot. (2011)

mRNA levels of ABA-related genes in germinating freshly harvested (FH) ioexDOF6 seeds. Expression of genes involved in ABA metabolism and signalling (A) and ABA-inducible genes (B) was compared by quantitative RT-PCR between wild-type (WT) and ioexDOF6 FH seeds 48 hours after imbibition in 50 μM estradiol. Gene-specific mRNA levels are relative to UBC21. All the UBC21 normalized expression values are relative to the values obtained for each gene in the WT sample.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3295388&req=5

fig5: mRNA levels of ABA-related genes in germinating freshly harvested (FH) ioexDOF6 seeds. Expression of genes involved in ABA metabolism and signalling (A) and ABA-inducible genes (B) was compared by quantitative RT-PCR between wild-type (WT) and ioexDOF6 FH seeds 48 hours after imbibition in 50 μM estradiol. Gene-specific mRNA levels are relative to UBC21. All the UBC21 normalized expression values are relative to the values obtained for each gene in the WT sample.
Mentions: Total RNA was isolated from seeds and other tissues, as described by Oñate-Sánchez and Vicente-Carbajosa (2008). Seeds (15 mg) from each genotype were germinated on moistened filter paper and samples were taken at different hours after imbibition (hai). The Transcriptor First Strand cDNA Synthesis kit (Roche Diagnostics, Mannheim, Germany) was used to synthesize cDNA. For quantitative reverse transcription (RT)-PCR, 8–16 ng cDNA was used as template together with 0.5 μM of forward and reverse specific oligonucleotides (Supplementary Table S1) and Power SYBR Green Master Mix (Applied Biosystems, Foster City, CA, USA). Cycling conditions (ABI Prism 7300; Applied Biosystems) were as follows: 10 min at 95 °C and 50 cycles of 15 s at 95 °C and 60 s at 60 °C, linked to a default dissociation stage programme to detect non-specific amplification. Three technical and at least two biological replicates were included in every experiment and the ubiquitin gene UBC21 (At5g25760) was used to normalize expression levels. Therefore, gene-specific mRNA levels shown in all figures except Fig. 5 are relative to UBC21.

Bottom Line: While constitutive over-expression of DOF6 produced aberrant growth and sterility in the plant, its over-expression induced upon seed imbibition triggered delayed germination, abscisic acid (ABA)-hypersensitive phenotypes and increased expression of the ABA biosynthetic gene ABA1 and ABA-related stress genes.DOF6 was found to interact in a yeast two-hybrid system and in planta with TCP14, a previously described positive regulator of seed germination.The expression of ABA1 and ABA-related stress genes was also enhanced in tcp14 knock-out mutants.

View Article: PubMed Central - PubMed

Affiliation: Universidad Politécnica de Madrid, Campus de Montegancedo, Pozuelo de Alarcón, Madrid, Spain.

ABSTRACT
Seed dormancy prevents seeds from germinating under environmental conditions unfavourable for plant growth and development and constitutes an evolutionary advantage. Dry storage, also known as after-ripening, gradually decreases seed dormancy by mechanisms not well understood. An Arabidopsis thaliana DOF transcription factor gene (DOF6) affecting seed germination has been characterized. The transcript levels of this gene accumulate in dry seeds and decay gradually during after-ripening and also upon seed imbibition. While constitutive over-expression of DOF6 produced aberrant growth and sterility in the plant, its over-expression induced upon seed imbibition triggered delayed germination, abscisic acid (ABA)-hypersensitive phenotypes and increased expression of the ABA biosynthetic gene ABA1 and ABA-related stress genes. Wild-type germination and gene expression were gradually restored during seed after-ripening, despite of DOF6-induced over-expression. DOF6 was found to interact in a yeast two-hybrid system and in planta with TCP14, a previously described positive regulator of seed germination. The expression of ABA1 and ABA-related stress genes was also enhanced in tcp14 knock-out mutants. Taken together, these results indicate that DOF6 negatively affects seed germination and opposes TCP14 function in the regulation of a specific set of ABA-related genes.

Show MeSH
Related in: MedlinePlus