Supplementary Data

Nuclear Phytochrome B regulates leaf flattening through Phytochrome Interacting Factors.

Henrik Johansson and Jon Hughes1

Department of Plant Physiology, Justus Liebig University, D35390 Giessen, Germany

1 To whom correspondence should be addressed.

E-mail:

TEL: +49/0 641 99 35430

FAX: +49/0 641 99 35429


Material and Methods

Plant material and growth conditions

All lines used in this study are in the Columbia WT background. phyB-9, phyB-9 BFL, phyB-9 B651-NLS, phyB-9 B651-NES, pif4-101pif5-3, phyB-9pif4-101pif5-3 and PIF5-OX have been described previously (Lorrain et al., 2008; Palagyi et al., 2010; Reed et al., 1993). To generate phot1-5phot2-1 and phyB-9phot1-5phot2-1, phyB-9 was crossed with phot1-5phot2-1gl1 (Kinoshita et al., 2001). Seedlings homozygous for phot1-5phot2-1 were selected in the F2 generation based on their lack of phototropic bending towards blue light (and later confirmed by the loss of chloroplast relocation in response to both low and high fluence rates of blue light). Seedlings from the F3 generation were then grown in red light to identify F2 plants homozygous for PHYB and GL1 (phot1-5phot2-1) or for phyB-9 and GL1 (phyB-9phot1-5phot2-1) respectively.

Seeds were sown directly on soil containing 25% (v/v) vermiculite and kept in darkness at 4°C for 3 days to synchronise germination. Plants were then moved to white light (100 μmol m-2 s-1 PAR) long day conditions (16/8, L/D) at 23°C for 4 weeks. For the temperature experiment, plants were treated similarly but after 1 week half were moved to 28°C for 3 weeks.

Leaf flatness

Leaf flatness was measured essentially as previously described (Kozuka et al., 2011). Briefly, a 2 mm cross section at the middle of the 5th (unless stated otherwise) rosette leaf was excised using a scalpel and then photographed. The total width of the leaf section and the distance between the two edges was measured using ImageJ (http://rsb.info.nih.gov/ij/) and the flatness parameter X calculated from the quotient. As 0 < X < 1, the data was not normally distributed, especially in the case of treatments in which leaves were nearly flat (X→1). Gaussian distributions were obtained with -log(1-X) transformation, however. The means and standard errors were thus calculated following this transformation and plotted accordingly. Significant differences were then estimated using Student's t-tests on the transformed data. Non-parametric Mann-Whitney U tests of the raw data (using SPSS 17.0.1 software) gave comparable results.


Supplementary Figures

Supplementary Figure 1. phyB restores the curled leaf phenotype of the phot1phot2 mutant.

Leaf flatness measurement of WT, phyB, phot1phot2 and phyBphot1phot2. Error bars represent SE (n≥14). (***) and (**) represent a significance of p<0.001 and p<0.01 compared to the WT respectively.

Supplementary Figure 2. Leaf flatness of phot1phot2 but not phyB is increased by warm temperatures.

(A) Measurements of leaf flatness of WT (data also appear in Fig. 1D) and phot1phot2 in plants grown constantly at 23°C for 4 weeks, or moved to 28°C after one week at 23°C. Error bars represent SE (n≥19). (B) Measurements of the 4th rosette leaf flatness of phyB and phyBphot1phot2 mutants grown as in (A). Error bars represent SE (n=18). (**) and (*) represent a significance of p<0.01 and p<0.05 respectively.

Supplementary Figure 3. B651-NLS suppress leaf flattening in response to warm temperatures.

Measurements of leaf flatness of WT, phyB BFL and phyB B651-NLS in plants grown constantly at 23°C for 4 weeks, or moved to 28°C after one week at 23°C. Error bars represent SE (n=18). (***) represents a significance of p<0.001.

Supplementary References

Kinoshita, T., Doi, M., Suetsugu, N., Kagawa, T., Wada, M., and Shimazaki, K. (2001). phot1 and phot2 mediate blue light regulation of stomatal opening. Nature 414:656-660.

Lorrain, S., Allen, T., Duek, P.D., Whitelam, G.C., and Fankhauser, C. (2008). Phytochrome-mediated inhibition of shade avoidance involves degradation of growth-promoting bHLH transcription factors. Plant J 53:312-323.

Kozuka, T., Kong, S.G., Doi, M., Shimazaki, K., and Nagatani, A. (2011). Tissue-autonomous promotion of palisade cell development by phototropin 2 in Arabidopsis. Plant Cell 23:3684-3695.

Palagyi, A., Terecskei, K., Adam, E., Kevei, E., Kircher, S., Merai, Z., Schafer, E., Nagy, F., and Kozma-Bognar, L. (2010). Functional analysis of amino-terminal domains of the photoreceptor phytochrome B. Plant Physiol 153:1834-1845.

Reed, J.W., Nagpal, P., Poole, D.S., Furuya, M., and Chory, J. (1993). Mutations in the gene for the red/far-red light receptor phytochrome B alter cell elongation and physiological responses throughout Arabidopsis development. Plant Cell 5:147-157.