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Peroxidases identified in a subtractive cDNA library approach show tissue-specific transcript abundance and enzyme activity during seed germination of Lepidium sativum.

Linkies A, Schuster-Sherpa U, Tintelnot S, Leubner-Metzger G, Müller K - J. Exp. Bot. (2009)

Bottom Line: These peroxidases were identified as orthologues of Arabidopsis AtAPX01, AtPrx16, and AtPrxIIE.The corresponding SALK lines displayed significant germination phenotypes.Possible implications of this tissue-specificity are discussed.

View Article: PubMed Central - PubMed

Affiliation: University of Freiburg, Faculty of Biology, Institute for Biology II, Botany/Plant Physiology, Schänzlestrasse 1, Freiburg, Germany.

ABSTRACT
The micropylar endosperm is a major regulator of seed germination in endospermic species, to which the close Brassicaceae relatives Arabidopsis thaliana and Lepidium sativum (cress) belong. Cress seeds are about 20 times larger than the seeds of Arabidopsis. This advantage was used to construct a tissue-specific subtractive cDNA library of transcripts that are up-regulated late in the germination process specifically in the micropylar endosperm of cress seeds. The library showed that a number of transcripts known to be up-regulated late during germination are up-regulated in the micropylar endosperm cap. Detailed germination kinetics of SALK lines carrying insertions in genes present in our library showed that the identified transcripts do indeed play roles during germination. Three peroxidases were present in the library. These peroxidases were identified as orthologues of Arabidopsis AtAPX01, AtPrx16, and AtPrxIIE. The corresponding SALK lines displayed significant germination phenotypes. Their transcripts were quantified in specific cress seed tissues during germination in the presence and absence of ABA and they were found to be regulated in a tissue-specific manner. Peroxidase activity, and particularly its regulation by ABA, also differed between radicles and micropylar endosperm caps. Possible implications of this tissue-specificity are discussed.

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Transcript levels of the three peroxidases identified in the subtractive cDNA library in different cress seed tissues and in dry micropylar seed ends (radical + cap) at different points during germination (in hours) in the absence (CON, control) and presence of 10 μM ABA. Transcript abundance was determined by qPCR and normalized against actin abundance. Note the different scales on the y-axes. Means of three biological replicates ±SE are shown. Cap, micropylar endosperm cap; Nme, non-micropylar endosperm; Cot, cotyledons.
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fig3: Transcript levels of the three peroxidases identified in the subtractive cDNA library in different cress seed tissues and in dry micropylar seed ends (radical + cap) at different points during germination (in hours) in the absence (CON, control) and presence of 10 μM ABA. Transcript abundance was determined by qPCR and normalized against actin abundance. Note the different scales on the y-axes. Means of three biological replicates ±SE are shown. Cap, micropylar endosperm cap; Nme, non-micropylar endosperm; Cot, cotyledons.

Mentions: Quantitative PCR was used to quantify the expression of the three peroxidases found in our subtractive library to be up-regulated during cress endosperm weakening (Fig. 3). The three cDNAs showed the highest similarities to AtAPx01, AtPrx16, and AtPrxIIE. In addition to expression in the endosperm cap, transcript levels in the non-micropylar endosperm, the radicle, and the cotyledons were investigated since peroxidase activity histostains, as well as activity assays, showed different activity in all tissues. Expression was investigated at the two points used for the subtractive library, i.e. at 8 h and 18 h after imbibition for all four tissues. In addition, micropylar endosperm caps and radicles were sampled from seeds imbibed in medium containing ABA at four different time points. Transcript levels in the dry micropylar end were also investigated. In the dry state, radicles and endosperm caps cannot be separated. RNA was thus extracted from radicles with the surrounding micropylar endosperm cap and the attached testa. Primers were designed based on the partial cress cDNAs obtained from the library.


Peroxidases identified in a subtractive cDNA library approach show tissue-specific transcript abundance and enzyme activity during seed germination of Lepidium sativum.

Linkies A, Schuster-Sherpa U, Tintelnot S, Leubner-Metzger G, Müller K - J. Exp. Bot. (2009)

Transcript levels of the three peroxidases identified in the subtractive cDNA library in different cress seed tissues and in dry micropylar seed ends (radical + cap) at different points during germination (in hours) in the absence (CON, control) and presence of 10 μM ABA. Transcript abundance was determined by qPCR and normalized against actin abundance. Note the different scales on the y-axes. Means of three biological replicates ±SE are shown. Cap, micropylar endosperm cap; Nme, non-micropylar endosperm; Cot, cotyledons.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2803213&req=5

fig3: Transcript levels of the three peroxidases identified in the subtractive cDNA library in different cress seed tissues and in dry micropylar seed ends (radical + cap) at different points during germination (in hours) in the absence (CON, control) and presence of 10 μM ABA. Transcript abundance was determined by qPCR and normalized against actin abundance. Note the different scales on the y-axes. Means of three biological replicates ±SE are shown. Cap, micropylar endosperm cap; Nme, non-micropylar endosperm; Cot, cotyledons.
Mentions: Quantitative PCR was used to quantify the expression of the three peroxidases found in our subtractive library to be up-regulated during cress endosperm weakening (Fig. 3). The three cDNAs showed the highest similarities to AtAPx01, AtPrx16, and AtPrxIIE. In addition to expression in the endosperm cap, transcript levels in the non-micropylar endosperm, the radicle, and the cotyledons were investigated since peroxidase activity histostains, as well as activity assays, showed different activity in all tissues. Expression was investigated at the two points used for the subtractive library, i.e. at 8 h and 18 h after imbibition for all four tissues. In addition, micropylar endosperm caps and radicles were sampled from seeds imbibed in medium containing ABA at four different time points. Transcript levels in the dry micropylar end were also investigated. In the dry state, radicles and endosperm caps cannot be separated. RNA was thus extracted from radicles with the surrounding micropylar endosperm cap and the attached testa. Primers were designed based on the partial cress cDNAs obtained from the library.

Bottom Line: These peroxidases were identified as orthologues of Arabidopsis AtAPX01, AtPrx16, and AtPrxIIE.The corresponding SALK lines displayed significant germination phenotypes.Possible implications of this tissue-specificity are discussed.

View Article: PubMed Central - PubMed

Affiliation: University of Freiburg, Faculty of Biology, Institute for Biology II, Botany/Plant Physiology, Schänzlestrasse 1, Freiburg, Germany.

ABSTRACT
The micropylar endosperm is a major regulator of seed germination in endospermic species, to which the close Brassicaceae relatives Arabidopsis thaliana and Lepidium sativum (cress) belong. Cress seeds are about 20 times larger than the seeds of Arabidopsis. This advantage was used to construct a tissue-specific subtractive cDNA library of transcripts that are up-regulated late in the germination process specifically in the micropylar endosperm of cress seeds. The library showed that a number of transcripts known to be up-regulated late during germination are up-regulated in the micropylar endosperm cap. Detailed germination kinetics of SALK lines carrying insertions in genes present in our library showed that the identified transcripts do indeed play roles during germination. Three peroxidases were present in the library. These peroxidases were identified as orthologues of Arabidopsis AtAPX01, AtPrx16, and AtPrxIIE. The corresponding SALK lines displayed significant germination phenotypes. Their transcripts were quantified in specific cress seed tissues during germination in the presence and absence of ABA and they were found to be regulated in a tissue-specific manner. Peroxidase activity, and particularly its regulation by ABA, also differed between radicles and micropylar endosperm caps. Possible implications of this tissue-specificity are discussed.

Show MeSH
Related in: MedlinePlus