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Plant Molecular Biology

, Volume 83, Issue 4–5, pp 351–363 | Cite as

Isolation and characterization of rice (Oryza sativa L.) E3-ubiquitin ligase OsHOS1 gene in the modulation of cold stress response

  • Tiago Lourenço
  • Helena Sapeta
  • Duarte D. Figueiredo
  • Mafalda Rodrigues
  • André Cordeiro
  • Isabel A. Abreu
  • Nelson J. M. Saibo
  • M. Margarida OliveiraEmail author
Article

Abstract

Plants can cope with adverse environmental conditions through the activation of stress response signalling pathways, in which the proteasome seems to play an important role. However, the mechanisms underlying the proteasome-mediated stress response in rice are still not fully understood. To address this issue, we have identified a rice E3-ubiquitin ligase, OsHOS1, and characterized its role in the modulation of the cold stress response. Using a RNA interference (RNAi) transgenic approach we found that, under cold conditions, the RNAi::OsHOS1 plants showed a higher expression level of OsDREB1A. This was correlated with an increased amount of OsICE1, a master transcription factor of the cold stress signalling. However, the up-regulation of OsDREB1A was transient and the transgenic plants did not show increased cold tolerance. Nevertheless, we could confirm the interaction of OsHOS1 with OsICE1 by Yeast-Two hybrid and bi-molecular fluorescence complementation in Arabidopsis protoplasts. Moreover, we could also determine through an in vitro degradation assay that the higher amount of OsICE1 in the transgenic plants was correlated with a lower amount of OsHOS1. Hence, we could confirm the involvement of the proteasome in this response mechanism. Taken together our results confirm the importance of OsHOS1, and thus of the proteasome, in the modulation of the cold stress signalling in rice.

Keywords

Abiotic stress Proteasome Cold stress signalling Transcription factors 

Notes

Acknowledgments

This work was supported by the Fundação para a Ciência e Tecnologia (FCT) through the projects POCI/BIA-BCM/56063/2004, PTDC/BIA_BCM/099836/2008, Pest-OE/EBQ/LA0004/2011. We would like to thank Dr. Ko Shimamoto (Nara Institute of Science and Technology, Japan) for the Gateway-based vector, pANDA, used in rice transformation for the production of the RNAi::OsHOS1 plants and Dr. Alejandro Ferrando for providing the BiFC vectors (www.ibmcp.upv.es/FerrandoLabVectors.php). TL (SFRH/BPD/34943/2007), DDF (SFRH/BD/29258/2006), AC (SFRH/BD/74946/2010) and IAA (SFRH/BPD/78314/2011) are grateful to Fundação para a Ciência e a Tecnologia (FCT) for their fellowships. NS was supported by Programa Ciência 2007, financed by POPH (QREN).

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11103_2013_92_MOESM1_ESM.docx (14 kb)
Supplementary material 1 List of primers used in this work. (DOCX 13 kb)
11103_2013_92_MOESM2_ESM.tif (3.9 mb)
Supplementary material 2 A. Agarose gel showing the amplification products of the different primer combination used to identify OsHOS1. B. Deduced amino-acid sequence of the longest ORF from the identified OsHOS1 gene. C. Sequence alignment of the identified OsHOS1 sequence and the sequence present in TIGR database (http://rice.plantbiology.msu.edu). (TIFF 3947 kb)
11103_2013_92_MOESM3_ESM.tif (227 kb)
Supplementary material 3 Northern blot analysis for the detection of small interference RNAs (siRNAs) in WT and RNAi::OsHOS1 plants, using the RNAi::OsHOS1 fragment as probe. As loading control, 5S rRNA was used as probe. (TIFF 226 kb)
11103_2013_92_MOESM4_ESM.tif (3.1 mb)
Supplementary material 4 A. Expression profile analysis of OsDREB1A through northern blot in- WT and OsHOS1 silencing plants under cold stress. Specific primers for the OsDREB1A fragment used as probe are available on Online Resource 1. Total RNA ethidium bromide staining was used as loading control. B. Agarose gel from the sqRT-PCR analysis of OsDREB1A expression in transformed rice protoplasts under control (0 h) or cold stress (3 h). The expression of Ubiquitin-Conjugating Enzyme E2 (Ubc) was used as internal control. C. Relative expression analysis of OsHOS1 in the transformed protoplasts as compared to control at 0 h and 3 h of cold stress. The relative OsHOS1 expression values of control samples were normalized to 1. (TIFF 3221 kb)

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Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Tiago Lourenço
    • 1
    • 2
  • Helena Sapeta
    • 1
    • 2
  • Duarte D. Figueiredo
    • 1
    • 2
  • Mafalda Rodrigues
    • 1
    • 2
  • André Cordeiro
    • 1
    • 2
  • Isabel A. Abreu
    • 1
    • 2
  • Nelson J. M. Saibo
    • 1
    • 2
  • M. Margarida Oliveira
    • 1
    • 2
    Email author
  1. 1.Genomics of Plant Stress laboratory, Instituto de Tecnologia Química e BiológicaUniversidade Nova de LisboaOeirasPortugal
  2. 2.iBETOeirasPortugal

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