Results. Seeds of Arabidopsis thaliana ecotype Cape Verde Islands (Cvi) were deeply dormant upon harvest, and slowly lost this primary dormancy during dry.
Expression of germination- and dormancy-associated genes is controlled by light and nitrate
Cassandra Patemanb, Fay Christodouloua, Peter E. Tooropa, Henk W.M. Hilhorstc, Bill E. Finch-Savageb
a Millennium
Seedbank Project, RBG Kew, Wakehurst Place, Ardingly, United Kingdom
HRI, University of Warwick, Wellesbourne, United Kingdom
c Laboratory of Plant Physiology, Wageningen University, Wageningen, The Netherlands b Warwick
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Seeds of Arabidopsis thaliana ecotype Cape Verde Islands (Cvi) were deeply dormant upon harvest, and slowly lost this primary dormancy during dry afterripening (Fig. 1). Potassium nitrate broke dormancy efficiently, and even enhanced germination of non-dormant seeds (not shown). All seeds required white light to complete germination.
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Results
Microarray studies revealed two genes that were associated with dormancy, a sinapoyltransferase (SNG), involved in hydrolysis of peptides, and a small heat shock protein (sHSP) that putatively acts as a chaperonin. Two genes were associated with germination, encoding a GA-stimulated (GAST-like) protein and an expansin (EXP) involved in cell growth. GAST-like
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Imbibition time (h) Fig.1 Germination of Cvi seeds in white light at 20°C after 2, 3 and 5 months dry afterripening, in water or in 10mM KNO3.
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Quantitative RT-PCR showed that expression of the GAST-like and EXP was higher in non-dormant than in primary dormant seed (NDD vs. PDD), and that white light and nitrate strongly enhanced expression in both dormant and non-dormant seed (Fig. 2). This was not necessarily related to germination, as in the case of PDL.
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Expression of the SNG and sHSP was higher in primary dormant seeds than in non-dormant seeds. Light and nitrate did not change expression in non-dormant seed. The anticipated decreased transcription in the light in primary dormant seed was found for the SNG but not for the sHSP. Apart from the unexpected result for the sHSP, which might play a role in protein refolding as a protection against light-induced damage, expression of these four genes generally reflects the state of dormancy. Furthermore, a distinction can be made between seeds that are dormant (PDD), non-dormant (NDD) and those induced to germinate (PDLN, NDL).
Fig.2 Quantitative RT-PCR data of the GAST-like and EXP (top) and the SNG and sHSP (bottom) genes in primary dormant seeds imbibed for 24h in the dark in water (PDD), in the light in water (PDL), or in the light in 10mM KNO3 (PDLN), and in non-dormant seeds imbibed for 24h in the dark in water (NDD), in the light in water (NDL), or in the light in 10mM KNO3 (NDLN). Values and error bars represent mean and standard deviation of four independent RNA samples after 40 cycles, normalised for rpS18.
Nitrate not only enhanced expression of EXP in the light, but also in the dark (Fig. 3). The synergistic effect of light and nitrate on the induction of EXP concur with the promotion of germination.
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PCR cycles (n) Fig.3 Expression of the EXP gene using RT-PCR in non-dormant seeds imbibed for 24h in the dark with or without 10mM KNO3, and in the light with or without 10mM KNO3. Three independent RNA samples were used. Left: expression levels after 30, 35, 40 and 45 cycles. Right: gel with PCR samples after 35 cycles.
Conclusions • The dormancy-breaking and germination-promoting effect of nitrate is reflected in expression of the GAST-like, EXP, SNG and sHSP genes; light induces an inconsistent pattern. • Non-dormant seeds are distinguishable from those induced to germinate.
