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Plant and Cell Physiology Advance Access originally published online on June 20, 2009
Plant and Cell Physiology 2009 50(8):1463-1478; doi:10.1093/pcp/pcp092
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© The Author 2009. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org

Regulation of HSD1 in Seeds of Arabidopsis thaliana

Sébastien Baud1,4, Neel Reinhard Dichow1,4, Zsolt Kelemen1,5, Sabine d’Andréa2, Alexandra To1, Nathalie Berger1, Michel Canonge2, Jocelyne Kronenberger1,3, David Viterbo1, Bertrand Dubreucq1, Loïc Lepiniec1, Thierry Chardot2 and Martine Miquel1,*

1Institut Jean-Pierre Bourgin, UMR Biologie des semences, INRA/AgroParisTech, F-78000 Versailles, France
2CBAI, UMR Chimie Biologique, INRA/AgroParisTech, F-78850 Thiverval-Grignon, France
3Institut Jean-Pierre Bourgin, Plateforme de cytologie et imagerie végétale INRA-IJPB, F-78000 Versailles, France

*Corresponding author: E-mail, Martine.Miquel{at}versailles.inra.fr; Fax, +33-1-30-83-31-11.


  Abstract

The hydroxysteroid dehydrogenase HSD1, identified in the proteome of oil bodies from mature Arabidopsis seeds, is encoded by At5g50600 and At5g50700, two gene copies anchored on a duplicated region of chromosome 5. Using a real-time quantitative reverse transcriptase–polymerase chain reaction (RT–PCR) approach, the accumulation of HSD1 mRNA was shown to be specifically and highly induced in oil-accumulating tissues of maturing seeds. HSD1 mRNA disappeared during germination. The activity of HSD1 promoter and the localization of HSD1 transcripts by in situ hybridization were consistent with this pattern. A complementary set of molecular and genetic analyses showed that HSD1 is a target of LEAFY COTYLEDON2, a transcriptional regulator able to bind the promoter of HSD1. Immunoblot analyses and immunolocalization experiments using anti-AtHSD1 antibodies established that the pattern of HSD1 deposition faithfully reflected mRNA accumulation. At the subcellular level, the study of HSD1:GFP fusion proteins showed the targeting of HSD1 to the surface of oil bodies. Transgenic lines overexpressing HSD1 were then obtained to test the importance of proper transcriptional regulation of HSD1 in seeds. Whereas no impact on oil accumulation could be detected, transgenic seeds exhibited lower cold and light requirements to break dormancy, germinate and mobilize storage lipids. Interestingly, overexpressors of HSD1 over-accumulated HSD1 protein in seeds but not in vegetative organs, suggesting that post-transcriptional regulations exist that prevent HSD1 accumulation in tissues deprived of oil bodies.

Keywords: Arabidopsis thaliana - hydroxysteroid dehydrogenase 1 - LEAFY COTYLEDON2 - oil bodies - seed - triacylglycerol

Abbreviations: DAA, days after anthesis; DW, dry weight; HSD, hydroxysteroid dehydrogenase; SDR, short-chain dehydrogenase reductase.

4These authors contributed equally to this work.

5Present address: Laboratoire de Virologie Moléculaire et Structurale, UMR CNRS 2472, Bât. 14B, 1 Avenue de la Terrasse, F-91198 Gif-sur-Yvette Cedex, France.

(Received May 14, 2009; Accepted June 18, 2009)
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