New Phytologist 184: 885-897 (2009)

The NADPH-Oxidase AtrbohB plays a role in Arabidopsis seed after-ripening ^[W]

Abstract
• Seeds can enter a state of dormancy, in which they do not germinate under optimal environmental conditions. Dormancy can be broken during seed after-ripening in the low-hydrated state.
• By screening enhancer trap lines of Arabidopsis, we identified a role for the NADPH-oxidase AtrbohB in after-ripening. Semiquantitative PCR was used to investigate AtrbohB transcripts in seeds. These methods were complemented with a pharmacological approach using the inhibitor diphenylene iodonium chloride (DPI) and biomechanical measurements in the Brassicaceae seed model system cress (Lepidium sativum) as well as protein carbonylation assays.
• atrbohB mutants fail to after-ripen and show reduced protein oxidation. AtrbohB pre-mRNA is alternatively spliced in seeds in a hormonally and developmentally regulated manner. AtrbohB is a major producer of superoxide in germinating Arabidopsis seeds, and inhibition of superoxide production by diphenylene iodonium (DPI) leads to a delay in Arabidopsis and cress seed germination and cress endosperm weakening.
• Reactive oxygen species produced by AtrbohB during after-ripening could act via abscisic acid (ABA) signalling or post-translational protein modifications. Alternative splicing could be a general

mechanism in after-ripening: by altered processing of stored pre-mRNAs seeds could react quickly to environmental changes.

Key words: abscisic acid (ABA) • after-ripening • alternative splicing • Arabidopsis thaliana • cress (Lepidium sativum) • seed dormancy • seed germination • NADPH-oxidase (Rboh)

Financial support: This work was supported by grants from the Deutsche Forschungsgemeinschaft (grant no. DFG LE720/6) to G.L.-M. and by grants to M.A.T., i.e. BIO 2007-66806 (Plant Nacional de I+D, Spain) and IRG RTD REG/T.2 (2007) D/562971 (European Community).