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EST sequencing and time course microarray hybridizations identify more than 700 Medicago truncatula genes with developmental expression regulation in flowers and pods

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Abstract

To evaluate the molecular mechanisms during pod and seed formation in legumes, starting with the development of reproductive organs, we constructed two cDNA libraries from developing flowers (MtFLOW) and pods including seeds (MtPOSE) of the model plant Medicago truncatula Gaertner. A total of 2,516 expressed sequence tags (ESTs) clustered into 1,776 nonredundant sequences (2k-set), which were annotated and assigned to functional classes. While about 30% of the ESTs encoded proteins of yet unknown function, typical annotations pointed to seed storage proteins, LTPs and lipoxygenases. The 2k-set was used to upgrade Mt6k-RIT microarrays (Küster et al. in J Biotechnol 108: 95, 2004) to Mt8k versions representing approximately 6,300 nonredundant M. truncatula genes. These were used to perform time course expression profiling studies based on hybridizations of samples that covered eight different developmental stages from flower buds to almost mature pods versus leaves as a common reference. About 180 up- and 70 downregulated genes were typically found for each stage and in total, 782 genes were either twofold up- or downregulated in at least one of the eight stages investigated. Based on this set, a combination of self-organizing map and hierarchical clustering revealed genes displaying expression regulation during characteristic stages of M. truncatula flower and pod development. Amongst those, several genes encoded proteins related to seed metabolism and development including novel regulators and proteins involved in signaling.

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Abbreviations

EST:

Expressed sequence tag

LTP:

Nonspecific lipid transfer protein

MENS:

Medicago EST navigation system

SOM:

Self-organizing map

TC:

Tentative consensus sequence

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Acknowledgements

We thank Anke Becker (Institute of Genome Research, CeBiTec, Bielefeld University) for printing Mt8k microarrays. We are grateful to Thomas Bekel, Daniela Bartels, Michael Dondrup and Folker Meyer (Bioinformatics Resource Facility, CeBiTec, Bielefeld University) as well as Jérôme Gouzy (INRA-CNRS Toulouse, France) for providing indispensable bioinformatics tools. We also thank Victoria Bartelsmeier, Tanja Thias and Eva Schulte–Berndt (CeBiTec, Bielefeld University) for excellent technical support. We are grateful to Etienne-Pascal Journet, Pascal Gamas, Diederik van Tuinen and Vivienne Gianinazzi-Pearson (INRA-CNRS Toulouse, France; INRA Dijon, France) for sharing probes from the Mt6k-RIT collection. This work was supported by the European Union projects MEDICAGO (QLG-CT2000-00676) and GRAIN LEGUMES (FOOD-CT-2004-506223). Helge Küster acknowledges financial support by the “International NRW Graduate School in Bioinformatics and Genome Research”.

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  1. Lehrstuhl für Genetik, Fakultät für Biologie, Universität Bielefeld, Postfach 100131, 33501, Bielefeld, Germany

    Christian Firnhaber, Alfred Pühler & Helge Küster

  2. International NRW Graduate School in Bioinformatics and Genome Research, Center for Biotechnology (CeBiTec), Universität Bielefeld, 33594, Bielefeld, Germany

    Helge Küster

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  1. Christian Firnhaber
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  2. Alfred Pühler
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Correspondence to Helge Küster.

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Firnhaber, C., Pühler, A. & Küster, H. EST sequencing and time course microarray hybridizations identify more than 700 Medicago truncatula genes with developmental expression regulation in flowers and pods. Planta 222, 269–283 (2005). https://doi.org/10.1007/s00425-005-1543-3

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