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Open Access Research article

In Medicago truncatula, water deficit modulates the transcript accumulation of components of small RNA pathways

Cláudio Capitão1*, Jorge AP Paiva2, Dulce M Santos3 and Pedro Fevereiro14

Author Affiliations

1 Laboratório de Biotecnologia de Células Vegetais, Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Apartado 127, 2781-901 Oeiras, Portugal

2 Instituto de Investigação Científica e Tropical, Centro das Florestas e Produtos Florestais, Tapada da Ajuda, 1349-017 Lisboa, Portugal

3 Instituto de Investigação Científica e Tropical, Centro de Veterinária e Zootecnia, Av. Universidade Técnica 1300-477 Lisboa, Portugal

4 Departamento de Biologia Vegetal, Faculdade de Ciências, Universidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal

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BMC Plant Biology 2011, 11:79  doi:10.1186/1471-2229-11-79

Published: 10 May 2011

Additional files

Additional file 1:

Scheme showing the water regime imposed to M. truncatula plants. The average of the relative water content (RWC) of each experimental group is shown.

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Open Data

Additional file 2:

A. thaliana Dicer-like and Argonaute sequences used for identification of DCLs and AGOs in M. truncatula. The Arabidopsis Information Resource (TAIR) accession number of the genes and their mRNA and protein accession numbers in NCBI database are shown.

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Additional file 3:

The Minimum Information for Publication of Quantitative Real Time PCR Experiments (MIQE) check list. A complete list of all the procedures used in the qPCR experiment.

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Additional file 4:

Primers used for the quantification of transcript accumulation by qPCR in M. truncatula. Indication of the amplification product size (Amplicon size) and the PCR efficiency used for each pair of primers obtained from real-time PCR Miner software (version 2.2). The efficiency for each gene was calculated doing the arithmetic mean of all efficiencies given by PCR Miner.

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Additional file 5:

Annotation of MtAGO12b gene in M. truncatula genome using IMGAG (Mt3.0) and Fgenesh software. IMGAG gives three independent annotated sequences (Medtr2g074590.1, Medtr2g074600.1 and Medtr2g074610.1) on the other hand Fgenesh annotates them as only one sequence. The image was obtained in the Medicago GBrowse from J. Craig Venture Institute [39].

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Additional file 6:

Annotation of MtAGO11 gene in M. truncatula genome using IMGAG (Mt3.0) and Fgenesh software. IMGAG annotates three independent annotated sequences (Medtr3g016400.1, Medtr3g016410.1 and Medtr3g016420) while Fgenesh annotates them as only one sequence. The image was obtained in the Medicago GBrowse from J. Craig Venture Institute [39].

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Open Data

Additional file 7:

Amino acid alignment of the PIWI domains of M. truncatula (Mt) and A. thaliana (At) Argonaute proteins. The protein sequences were aligned using T-Coffee software [46-48]. The amino acids residues corresponding to the conserved aspartate, aspartate and histidine (DDH) catalytic triad residues are marked in black, while the A. thaliana Argonaute 1 histidine in the position 800 (H800) is in yellow. Amino acid positions corresponding to the beginning and end of the PIWI Domains in each protein are mentioned. TtAGO, Thermus thermophilus-0026 Argonaute (gi:46255097); PfAGO, Pyrococcus furiosus-0537 Argonaute (gi:18976909); AaAGO Aquifex aeolicus-1447 Argonaute (gi:15606619); HsPIWI, human PIWI (gi:24431985); HsAGO1, human Argonaute1 (gi:6912352); HsAGO2, human Argonaute2 (gi:29171734).

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Additional file 8:

Relative accumulation of MtDCL3, MtAGO4b, MtAGO4c, MtAGO7 mRNAs in M. truncatula. The shoots of M. truncatula plants were analyzed in the different water treatment conditions imposed to the plants. Values are the mean of two technical replicates of three independent cDNAs for each treatment and bars represent standard errors. The relative mRNA accumulation was calculated using L2 as the reference gene and normalized against the shoot control treatment. A One Way ANOVA Test of significance was used to compare the four conditions in each organ followed by the Tukey Test (p-value <0.05).. Ct, Control; MWD, Moderate Water Deficit; SWD, Severe Water Deficit, Rec, Recovery.

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Additional file 9:

Expression of miR162 and miR168 in various organs and seedling phase of M. truncatula plants. Northern-blot analysis of shoots (S), roots (Rt), 8-day-old seedlings (Sd), young immature seed pods (Sp) and flowers (F) of M. truncatula plants in control conditions. The small nuclear RNA U6 was used as internal loading control for quantification of RNA gel blot signals which were normalized against the shoot samples (numbers indicated under each lane). The membrane was first hybridized with miR168 probe and then striped and rehybridized with miR162 probe. The molecular marker (M) is shown in the left and present three different sizes: 17 nt, 21 nt and 24nt.

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