Supplemental Data. Osakabe et al. (2013). Plant Cell /tpc
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1 Supplemental Figure 1. Phylogenetic analysis of KUP in various species. The amino acid sequences of KUPs from green algae and land plants were identified with a BLAST search and aligned using ClustalW (Thompson et al. 1994). Phylogenetic analysis based on the neighbor-joining (NJ) method was performed using the MEGA5 package (Tamura et al. 2011). The confidence levels for the individual branches were determined by bootstrap analysis with 1000 replicates. AT, A. thaliana; POPTR, Populus trichocarpa; OS, Oryza sativa; Selmol, Selaginella moellendorffii; Pp, Physcomitrella patens; Cre, Chlamydomonas reinhardtii. 1
2 Supplemental Figure 2. The expression levels of the KUP/HAK/KT family genes, KUP2/SHY3,,, and an ABA-responsive potassium channel, GORK, in Arabidopsis shoots and roots during abiotic stress and ABA treatments. The value for KUP2/SHY3 at 0 h was set to 1.0. To normalize the expression levels, 18S rrna was amplified as an internal control. Error bars indicate SD (n = 3). 2
3 Supplemental Figure 3. The expression patterns of KUP/HAK/KT and Shaker family members under various stimuli in the public microarray data provided by Genevestigator ( 3
4 Supplemental Figure 4. The tissue-specific expression patterns of KUP2/SHY3,,, and GORK in the public microarray data provided by Genevestigator ( 4
5 Supplemental Figure 5. T-DNA insertion mutant plants of KUP and GORK genes. (A) T-DNA insertion mutant plants kup6-1 (SALK_086950), kup8-1 (SALK_001070), gork-2 (SALK_082258), and kup2-8 (SAIL_504_A07). (B) The expression levels of KUP2/SHY3,,, and GORK in the mutant plants as determined by RT-PCR. An asterisk indicates a non-specific band. (C) Ten-day-old kup6-1, kup8-1, kup2-8, gork-2, kup26, kup68, and kup6g plants grown on GM-agar plates. Bars = 0.5 cm (D) Leaf mesophyll cells of seven-day-old single mutant plants grown on GM-agar plates. Leaves were treated with chloral hydrate and then viewed with a microscope. Bar = 20 µm (E) IAA and ABA responses in the lateral root formation of kup6-1 and kup8-1 plants. Values are means and SD (n = 25). (F) Ten day-old kup6-1, kup8-1, kup2-8, gork-2, kup26, kup68, and kup6g plants grown on GM-agar plates. Bars = 0.5 cm 5
6 Supplemental Figure 6. The overexpressing transgenic Arabidopsis plants. (A) The expression levels of in 35S: transgenic plants (line; L1 L3). VC (vector control); control transgenic plants carrying the 35S vector. (B) Weights of 14-day-old 35S: plants grown on GM-agar plates. Values are means and SD (n = 20). 6
7 Supplemental Figure 7. The measurements of water-loss and drought tolerance of the KUP mutants. (A) Transpirational water loss of the single mutants of and, and the double mutant of KUP2/SHY3 and during water deficit stress. Water loss is expressed as a percentage of the initial fresh weight. Values are means and SD of five samples of three leaves each. (B) Dehydration tolerance test of the kup268 and kup68g mutants, grown in soil pots in which the seeds were sowed at the same time, exposed to water deficit stress by not watering them for two weeks. 7
8 Osakabe_Supplemental Fig. 8 bait prey SD-LTHA SD-LTH 30 mm 3-AT 10 mm 3-AT SD-LT SRK2E SRK2D SRK2I SRK2C SRK2F SRK2A SRK2B SRK2E SRK2D SRK2I SRK2C SRK2F SRK2A SRK2B Supplemental Figure 8. Yeast two-hybrid analysis of SnRK2s (pgbkt7) and C-terminal regions of and (pgadt7). 8
9 Supplemental Figure 9. ABA and NPA sensitivity in the LR formation of srk2 mutants and the in-gel kinase assay using wild-type plants with drought stress, ABA, and auxin treatments. (A) LR formation of aba2-2 and srk2 single- or multiple-mutants was tested in the presence of 30 µm ABA and 10 µm NPA. Values are means and SD (n = 12). (B) In-gel kinase assay of SRK2 using histone or recombinant GST-tagged -CT as substrates with proteins extracted from the wild-type 0, 30, and 60 min after treatment with drought stress and 50 µm ABA with or without pretreatment of 30 µm IAA. In the case of +IAA, the plants were treated with both ABA and IAA after the pretreatment. 9
10 Supplemental Figure 10. Model of osmotic regulation via KUPs and GORK under normal growth and water deficit stress conditions (left). Stomatal responses via and GORK (right). The KUP/HAK/KT family potassium transporter is involved in additional control systems in ABA-mediated stomatal closure. 10
11 Supplemental Table 1. Primer pairs used in RT-PCR, quantitative RT-PCR, and SCT and pro:gus construction. Gene Forward primer Reverse primer RT-PCR KUP2/SHY3 ggaaatgcatcaggtttggctgt tcagttccgcatctgcttctgc ccggtgattctgttgtggctaatgtg tgcttgacttccaactacagcagcg gcgacaccaaacacataagcaatgcatcag cccactagaaaccgttcctccggtttcaca GORK atcggagaattgaaaccga taatagaagatacaagcagcagtgt Actin ggaaaggatctgtacggtaac tgtgaacgattcctggac quantitative RT-PCR KUP2/SHY3 gggttttacgtctctgtaccaaa gattgagaaagtcccactgatga accatggaaatcgaatcagg cacctaagctttgatacgctaatg gaattatccatgtctaagcagcaa tgttccatagtgttgtagcgaga GORK ggacgcacaatcttgaacaat cccaatgtgaatcactatgtcag LBD18 cgtcgctcacatctttgct tgccaaatgggcttgtaagt LBD29 gctaggcttcaagatcccatc tgtgctgcttgttgctttaga 18SrRNA cctacggaaaccttgttacga cgcgagaagtccactaaacc -CT gaattcatggagctaacagaggcacg gtcgacatcataccgacttctaaagt pro:gus gcatctataattgactaactttcgatttaatgtg tctgatgatgactgaattcggcgtttt 11
12 References in the supplemental data Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S MEGA5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Mol Biol Evol. 28: Thompson JD, Higgins DG, Gibson TJ CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:
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