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First published online 19 July 2006
doi: 10.1242/dev.02481

Development 133, 3213-3222 (2006)
Published by The Company of Biologists 2006
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Arabidopsis SPA proteins regulate photoperiodic flowering and interact with the floral inducer CONSTANS to regulate its stability

Sascha Laubinger1,*, Virginie Marchal2, José Gentilhomme2,{dagger}, Stephan Wenkel2,{ddagger}, Jessika Adrian1,§, Seonghoe Jang2, Carmen Kulajta1,, Helen Braun1,**, George Coupland2 and Ute Hoecker1,{dagger}{dagger},{ddagger}{ddagger}

1 Department of Plant Developmental and Molecular Biology, Geb. 26.03.02, University of Düsseldorf, D-40225 Düsseldorf, Germany.
2 Max Planck Institute for Plant Breeding, Carl-von-Linné Weg 10, D-50829 Cologne, Germany.


Figure 1
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  		Fig. 1. Visual phenotype of 78-day-old wild-type, spa single and spa triple mutants grown in SD. (A) spa1 mutants, but not spa2, spa3 and spa4 single mutants, flower early in SD. (B) A functional SPA1 gene, but not SPA2, is sufficient for normal flowering in SD.

 

Figure 2
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  		Fig. 2. FT transcript levels are drastically increased in SD-grown, early flowering spa mutants. (A) RT-PCR analysis of CO, FT, SOC1, FLC and ubiquitin (UBQ10) mRNA abundance in wild-type (WT) and spa mutant plants. (B-E) Quantification of mRNA abundance of the blots shown in A. The transcript levels of flowering time genes were normalized by the transcript levels of UBQ10. A representative experiment of two or three independent experiments is shown. Notice the difference in scale of FT/UBQ10 in the two graphs in C. Wild-type (Col), spa1-7, spa1-7 spa3 spa4 and spa2 spa3 spa4 (Col) plants were grown in SD for 25 days. RNA was isolated from these plants and used for RT-PCR analysis.

 

Figure 3
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  		Fig. 3. Transcript analysis of SPA1, SPA2, SPA3 and SPA4 in SD-grown plants. (A) RT-PCR analysis of all four SPA genes in 25-day-old SD-grown wild-type (Col) plants. (B-E) Quantification of the SPA transcript abundance of the blots shown in A. The transcript levels of SPA genes were normalized by the transcript levels of UBQ10. A representative experiment of two independent experiments is shown.

 

Figure 4
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  		Fig. 4. Early flowering and FT expression in SD-grown spa1 mutants depends on CO. (A) Visual phenotype of 83-day-old wild-type (WT), spa1-7, co, and spa1-7 co mutant plants grown in SD. (B) Flowering time in SD of genotypes shown in A. (C) RT-PCR analysis of FT and UBQ10 transcript levels in 25-day-old plants grown in SD. (D) Quantification of the blots shown in C. The transcript levels of FT were normalized by the transcript levels of UBQ10.

 

Figure 5
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  		Fig. 5. SPA proteins physically interact with CO. (A) SPA proteins interact with CO in vitro. Recombinant 35S-labelled CO was incubated with partially 35S-labelled GAD-SPA proteins or GAD, respectively, and co-immunoprecipitated with anti-GAD antibodies. Supernatant fractions (1.6%) and 33.3% of the pellet fractions were resolved by SDS-PAGE and visualized by autoradiography using a phosphorimager. (B) Co-localization of CFP-SPA1 and YFP-CO in transiently transfected Arabidopsis leaf epidermal cells. Images were taken by confocal microscopy. Scale bar: 4 µm. (C) FRET microscopy by acceptor photobleaching. Fluorescence intensities of CFP-SPA1 and of YFP-CO or YFP constructs before and after acceptor photobleaching. Scale bar: 5 µm. (D) Comparison of FRET efficiency after acceptor photobleaching measured in nuclei. Data are mean±s.d. of 10-20 cells from three separate experiments.

 

Figure 6
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  		Fig. 6. The CCT domain of CO is necessary for the in vitro interaction with SPA1. (A) Schematic representation of the deletion-derivatives of CO or His6-CO used in the in vitro binding assay. (B) Mapping of the SPA1-interacting domain of CO. Recombinant 35S-labelled CO or indicated CO-deletion proteins were incubated with partially 35S-labelled GAD-SPA1 or GAD, respectively, and co-immunoprecipitated with anti-GAD antibodies. Supernatant fractions (1.6%) and 33.3% of the pellet fractions were resolved by SDS-PAGE and visualized by autoradiography using a phosphorimager. (C) Quantification of the fractions of prey proteins that were co-immunoprecipitated by the indicated bait proteins GAD-SPA1 or GAD. Error bars depict the s.e.m. from two replicate experiments.

 

Figure 7
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  		Fig. 7. CO protein levels are strongly increased in spa1 spa3 spa4 triple mutants. (A) RT-PCR analysis of CO and UBQ10 transcript levels in wild-type plants, spa1 spa3 spa4 mutants and plants of a transgenic 35S-CO overexpressing line. (B) CO protein levels in nuclear extracts of wild-type plants, spa1 spa3 spa4 mutants and plants of a transgenic 35S-CO overexpressing line. As a loading control, blots were reprobed with an antibody against histone H3A. Plants were grown in LD for 12 days and harvested at ZT16.

 




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