SUPPLEMENTAL MATERIALS AND METHODS

[Ca2+]cyt Measurements in Response to Cold

Each seedling containing reconstituted coelenterazine in a 1.5 mL tube was set on the plate holder of the Lumicounter in complete darkness. The basal luminescence level was measured for at least 20 s (5 times /s). Then 0.6 mL ice water was added to the tube with pipettor for cold treatment and the aequorin luminescence was recorded for 60 s. Room temperature water was added to the tube for control. During the whole process, perturbations should be avoided, which maybe elicit mechanical signaling. In this way, mechanical stimulation and the time required to put the tube back to the photomultiplier tube were minimal. To estimate the Ca2+ concentrations from the luminescence values, 0.1 mL solution (including 2 M CaCl2 and 20% ethanol) was quickly injected into the tube to discharge all of the remaining aequorin after the experiment. In vivo Ca2+ concentrations were estimated according to the method of Knight et al. (1996).

GST Pull-down Assays

For GST pull-down assays, the coding regions of PKS1 were amplified by PCR with primers that contained appropriate restriction sites, and the amplified fragments were inserted into the plasmid pGEX-6P1 (Amersham Biosciences). The recombinant PKS1-GST fusion proteins were expressed using the pGEX-6P1-PKS1 vectors. The coding regions of PIN1 and CAM4 were amplified by PCR with primers that contained appropriate restriction sites, and the amplified fragments were inserted into the plasmids pCITE-4a-c(+) (Novagen). PIN1 and CAM4 proteins that were biotinylated on Lys residues were produced from pCITE-PIN1 and pCITE-CAM4 using an in vitro transcription and translation assay kit (TNT Quick Coupled Transcription/ Translation system; Promega) with Transcend Biotinylated Lys tRNA incorporation and detection, according to the manufacturer’s instructions. Phot1 and phot2 proteins were acquired from Arabidopsis etiolated seedling. Polyclonal antibodies raised against the plasma membrane phot1 and phot2 were provided by Shimazaki and were described previously (Doi et al. 2004, Ueno et al. 2005). Immunodetection was performed according to Kinoshita and Shimazaki (1999).

RT-PCR Analysis for PKS1, PIN1 and CAM4

The primers used for gene expression analysis by semi-quantitative RT-PCR are as follows: PIN1 (Forward, 5'-CGCTTACGGCTCTGTCAAATGG-3'; Reverse, 5'- ATCACCTTAGCCTGCGTCGTTT-3'); PKS1 (Forward, 5'-ACAAAGAGTGAAGGGAGTG-3'; Reverse, 5'-CATTGGGAATCTAGGAACG-3'); CAM4 (Forward, 5'-CTCTGATTCAGGTGATTCGTG-3'; Reverse, 5'-AGTCAATGGTTCCGTTTCC-3'). ACTIN2 (Forward, 5'- TTCCTCATGCCATCCTCCGTCTT-3'; Reverse, 5'- CAGCGATACCTGAGAACATAGTGG-3')


Supplemental data

Figure S1

Figure S1. Changes in cytosolic Ca2+ induced by cold in Arabidopsis etiolated seedlings. Aequorin luminescence is shown as the relative luminescence measured with the Auto Lumat LB9507 Luminometer. A, The changes in relative luminescence (RLU) indicate the changes in cytosolic Ca2+ concentration in response to cold in etiolated seedlings of the wild type. B, The mean changes in cytosolic Ca2+ of A. Values are means of three independent experiments (16 measurements each) with SDs.


Figure S2

Figure S2. PKS1 interacts with PIN1, CAM4, PHOT1 and PHOT2 by GST pull-down assays. A, PIN1 proteins labeled with biotinylated-Lys were pulled down by GST-PKS1 but not by GST. GST was used as a negative control. B, CAM4 proteins labeled with biotinylated-Lys were pulled down by GST-PKS1 but not by GST. C and D, phot1 and phot2 proteins were pulled down by GST-PKS1. GST was used as a negative control. (Left) The coomassie blue-stained gels. (Right) The chemiluminescent detection.


Figure S3

Figure S3. PKS1 interacts with CAM5 and CAM7. A, PKS1 interacted with CAM5 and CAM7 in the yeast two-hybrid system. Yeast strains containing pAS2-PKS1 as bait and pACT2-CAM5, pACT2-CAM6 and pACT2-CAM7 as prey, were grown for 48 h on synthetic defined (SD) medium that lacked Trp and Leu (up panel) and were assayed for LacZ expression by a filter-lift assay for β-galactosidase activity (β-gal, down panel). The empty vector pAS2 and pACT2 were used as negative controls. pAS2-OST1 and pACT2-H3 used as positive control. A blue color indicates interaction. B, PKS1 interacted with CAM5 and CAM7 in vivo as determined by bimolecular fluorescence complementation (BiFC). Up panel, bright-field images of the cells. down panel, fluorescence images under confocal microscopy.


Figure S4

Figure S4. Analysis of the expression of PKS1, PIN1 and CAM4 genes in the hypocotyl of Arabidopsis etiolated seedlings by RT-PCR. RT-PCR analysis of PKS1, PIN1 and CAM4 gene expression levels in wild type (WT), phot1, phot2, phot1phot2, PHOT1-OX, PHOT2-OX, phot1 PHOT2-OX and phot2 PHOT1-OX lines. Actin2 was used as an internal control and amplified for 22 cycles by PCR. PKS1, PIN1 and CAM4 were amplified for 28 cycles, 31 cycles and 31 cycles, respectively.

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