Interaction of the Response Regulator ARR4 with Phytochrome B in Modulating Red Light Signaling Uta Sweere, Klaus Eichenberg, Jens Lohrmann, Virtudes Mira-Rodado, Isabel Bäurle, Jörg Kudla, Ferenc Nagy, Eberhard Schäfer, and Klaus Harter

Supplementary Material

Reference (16). Supplemental Figure 1. Overexpression of ARR4 modulates the responsiveness of Arabidopsis plants to red light. (A) Root elongation response of ARR4-6 and ARR4-9 compared with wild type (WT). Seedlings were grown for 72 hours either under continuous red light (white bars) or in darkness (black bars). Root length was measured manually. (B) Flowering of wild-type (WT), ARR4-6, and ARR4-9 plants under long day conditions (16 hours light/8 hours dark). The number of leaves developed at flowering was determined as described previously (26). The mean and standard deviation of leaves from 10 plants per line are shown.

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Reference (24).

Experimental conditions for Fig. 1.

Arabidopsis plants were grown on soil under short-day conditions (16 hours light:8 hours dark). Wild-type, phyA- (phyA-201) and phyB- (phyB-5) mutant seedlings were grown for 4 days on paper in darkness or (pulse-) irradiated with white (2.8 × 10^-6 mol/m²s), red (3.2 × 10^-6 mol/m²s), or far-red (0.5 × 10^-6 mol/m²s ) light. Plant tissue and seedlings were extracted in SDS-sample buffer, and extracts were subjected to SDS polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblots as described in (15).

ARR2 and ARR4 were detected with antisera generated in mice. As recombinant, (His)₆-tagged antigens a protein fragment of the output domain of ARR2 (amino acid 204 to 644) and a protein fragment of ARR4 (amino acid 9 to 259) were expressed in the Escherichia coli strain BL21(DE3). Recombinant proteins were purified on Ni-NTA-agarose (Qiagen) followed by in-gel purification as described previously (23). The antisera were diluted 1:500 (-ARR2) or 1:1000 (-ARR4) in TBST (15) before use for immunoblots.

Experimental conditions for Fig. 2.

ARR4, PHYA, PHYB and PHYB-101 cDNAs were cloned into pASK-IBA2 (ARR4), pAA7 (PHYA) and pYES (PHYB, PHYB-101). Strep-tagged ARR4 was expressed from the pASK-IBA2 construct in E. coli strain XL1-blue and purified on StrepTactin beads (IBA, Germany). Expression of PHYA, PHYB and PHYB-101 apoproteins in yeast and self-assembly to the holoproteins with phycocyanobilin in crude protein extracts was done as described (12). 200 ng of Strep-tagged ARR4 were added to an aliquot of yeast extract containing 15 l of StrepTactin beads and 2 g of photoreversible phy irradiated for 10 min with red or with far-red light to obtain Pfr or Pr, respectively. After 60 min of incubation at 4°C, the beads were washed 4 times with RIPA buffer (12). The proteins were eluted in 35 l RIPA buffer (12) containing 10 mM desthiobiotin. For in planta interaction assay 5-day-old, dark-grown ARR4-overexpressing seedlings were extracted in extraction buffer [EB; 50 mM Tris/HCl pH 8.0, 150 mM NaCl, 1 mM dithiothreitol (DTT), 0.05 % Tween-20, Complete^TM protease inhibitor cocktail]. The extract was centrifuged (100.000g, 10 min, 4°C). Protein (2 g ) was supplemented with 20 l ARR4 antiserum and 20 l of protein A/G beads and incubated at 4°C for 2 hours. The beads were washed 4 times with EB. The bound proteins were eluted in 30 l of 10 mM glycine pH 3.0. Samples were subjected to SDS-PAGE and immunoblot. Phytochromes were detected by using the monoclonal antibodies mAP5 for phyA and mAT1 for phyBs (22). ARR4 was probed with the specific antiserum described above.

For yeast two-hybrid studies ARR4, PHYA and PHYB cDNAs and cDNA fragments were cloned into pGAD424 (ARR4, PHYB^FL, PHYB^179-1171) and pGBT9 (ARR4, PHYB1-173, PHYA1-137) and the assays for protein-protein interaction (growth on interaction-selective media CSM-L,W,H and CSM-L,W,A; -galactosidase activity) were performed as reported (14). For the analysis of the expression of selected GAL4-AD and GAL4-BD fusion proteins, yeast was extracted in EB and subjected to immunoblot analysis. AD-phyB^FL and AD-phyB^179-1171 were detected with mAT1 (22), whereas BD-phyA^1-137 and BD-phyB^1-173 were detected with the GAL4-BD-specific monoclonal antibody RK5C1 (Santa Cruz Biotechnology).

For the analysis of the intracellular partitioning of ARR4 the corresponding ARR4 cDNA was cloned into the GFP vector pMAV4 (23). Transient transformation of parsley protoplasts and microscopic techniques were carried out as described (23).

Experimental conditions for Figs. 3 and 4

For the generation of transgenic Arabidopsis lines, the ARR4 cDNA was cloned into the binary vector pPCV812 (15). Introduction of the construct into Agrobacterium tumefaciens strain GV3101, transformation of Arabidopsis (Columbia and ABO/A^-) and selection for transgenic plants were carried out as described in (15). Growth of seedlings on paper and determination of hypocotyl length were carried out as reported (15).

In vivo Pfr-to-Pr dark reversion in yeast and dark-grown Arabidopsis seedlings was measured in a dual wavelength ratio spectrophotometer as described in (12) and (17), respectively. The fluence-rate of the blue light source was 1.8 × 10^-6 mol/m²s and of the red light source used for the irradiation of ABO/A^- and ARR4-overexpressing ABO/A^- lines, respectively, was 0.4 × 10^-8 mol/m²s. The HSC70 antiserum (27) served as a loading control.

References

12. T. Kunkel et al., Eur. J. Biochem. 215, 587 (1993).

14. J. Lohrmann et al., Mol. Genet. Genomics 265, 2 (2001).

15. S. Kircher et al., Plant Cell 11, 1445 (1999).

17. K. Eichenberg, L. Hennig, A. Martin, E. Schäfer, Plant Cell Environ. 23, 311 (2000).

22. A. Nagatani et al., Cell Physiol. 25, 1059 (1985).

23. S. Kircher et al., J. Cell Biol. 144, 201 (1999).

26. L. Krall, J. W. Reed, Proc. Natl. Acad. Sci. U.S.A. 97, 8169 (2000).

27. D. Neumann, L. Nover, Biol. Zentbl. 108, 1 (1989).