Ca 2+ ug g -1 DW Ca 2+ ug g -1 DW Ca 2+ ug g -1 DW Supplemental Data. Fu et al. Plant Cell. (213). 1.115/tpc.113.113886 A 5 4 3 * Col- cau1 B 4 3 2 Col- cau1 ** * * ** C 2 1 25 2 15 1 5 Shoots Roots * * Col- cau1 RRS-7 Lov-1 D 1 21d 28d 38d 5d CAU1 Actin2 CAS Actin2 Supplemental Figure 1. CAU1 affects calcium accumulation. (A) Calcium accumulation in shoots and roots of 28 days old plants grown in hydroponics supplemented with.5 mm Ca 2+. n=3. (B) Calcium accumulation in shoots of 21, 28, 38 and 5 days old plants grown in soil. n=3. (C) Calcium accumulation in shoots of 28 days old Col-, cau1, and late-flowering ecotypes RRS-7 and Lov-1 grown in hydroponics supplemented with.5 mm Ca 2+. n=3. (D) CAU1 and CAS expressions in Col-, RRS-7 and Lov-1. *P <.5, ** P <.1 in t tests. 1
Leaf number Supplemental Data. Fu et al. Plant Cell. (213). 1.115/tpc.113.113886 A B C D E F 4 3 ** 2 1 Col- cau1 G H Supplemental Figure 2. cau1 mutant shows pleiotropic phenotypes. (A-C) Partial chlorosis in cau1 leaves diminished gradually with development, as indicated by seedlings at 14 days (A), 21 days (B) and 32 days (C) of age. (D-F) Late flowering observed in cau1. (D) 42 days old wild-type, (E) 42 days old cau1 mutant, (F) Rosette leaf numbers of wildtype Col- and cau1 mutant at bolting. Values are mean ± SD, n = 25. (G) Chlorosis in young cauline leaves and altered shoot morphology in cau1. (H) A close-up photo of one lateral shoot from (G) shows chlorosis in young cauline leaves. **P <.1. Bars =.5 cm in (A-C); or 1cm in (D, E, G, H). 2
Supplemental Data. Fu et al. Plant Cell. (213). 1.115/tpc.113.113886 A Z value (SD from average) 4 3 2 1-1 -2-3 -4 Al Ca Fe Cu Zn Ba Pb B Col- Col- cau1 CAU1 Actin2 35S/CAU1-cau1 CAU1 Actin2 C D E Col- cau1 35S/CAU1-cau1 F 35S/EYFP: CAU1-cau1 Supplemental Figure 3. Complementation assay of the cau1 mutant. (A) Comparative profiling of ion accumulation in wild-type (black open circles, n>3) and the cau1 mutant harboring construct 35S/CAU1-pBI121 (35S/CAU1-cau1, gray lines, n>5). Blue and red lines outline ion-profiles of wild type control and the transgenic 35S/CAU1-cau1 plant respectively. (B) RT-PCR analysis of CAU1 expression in wild-type Col-, cau1 or 35S/CAU1-cau1. (C-F) Visual phenotypes for Col- (C), cau1 (D), 35S/CAU1-cau1 (E) and cau1 harboring 35S/EYFP:CAU1- pmon53 (35S/EYFP:CAU1-cau1, F). Bar = 1cm in (C-F). 3
Water loss rate (%) CAS relative expression Survival rate (%) Stomatal aperture Supplemental Data. Fu et al. Plant Cell. (213). 1.115/tpc.113.113886 A B C Supplemental Figure 4. Stomatal closure, drought tolerance and CAS expression in 35S/CAU1-cau1. D F 1 8 6 4 2 7 6 5 4 3 2 1 E a b b Col- cau1 35S/CAU1-cau1 Col- cau1 35S/CAU1-cau1 3 6 12 18 24 3(min).7.6.5.4.3.2.1 G 5 4 3 2 1 a a b Col- cau1 35S/CAU1-cau1 Col- cau1 35S/CAU1-cau1.5mM 1mM (A-C) Drought tolerance in WT, cau1 and 35S/CAU1-cau1 plants. Watering of 1-day-old WT (A), cau1 (B) and 35S/CAU1-cau1 (C) plants were withheld for 2 weeks, and then rewatered and allowed recovery for 1 week. (D) Survival rates of plants in (A-C). Values are mean ± SE from three different experiments. n=5. (E) Stomatal aperture (width/length) were determined using 22 days old plant rosette leaves. Values are mean ± SD (n 12). (F) Walter loss rate was determined using detached leaves from WT, cau1 and 35S/CAU1-cau1 plants. Values are mean ± SE from three replicated experiments and each contains six detached leaves. n=6. (G) CAS expression in response to Ca 2+ treatments. 3-week-old plants grown in hydroponics were treated with,.5, 1 mm Ca 2+ for 4 days. Values are mean ± SD and normalized to Actin2, n=3. ANOVA analyses were performed to determine statistical difference at P <.5. Bar=2 cm in (A-C). 4
Indo-1 signal intensity Stomatal aperture Stomatal aperture Supplemental Data. Fu et al. Plant Cell. (213). 1.115/tpc.113.113886 A C.6.5.4.3.2 -Ca +Ca Col- cau1 35S/CAU1-cau1 Col- cau1 35S/CAU1-cau1 Rel. Indo-1 intensity B D.7.6.5.4.3 4 3 2 1 Col- cau1 35S/CAU1-cau1.1 1 5 (um) (µm) a b b Col- cau1 35S/CAU1-cau1 Supplemental Figure 5. CAU1 complements stomatal closure and [Ca 2+ ] o signaling phenotype in the cau1 mutant. (A, B) [Ca 2+ ] o -induced stomatal closure. The stomatal aperture assay was performed as described in Methods, n > 3. (C) Quantification of the relative Indo-1 fluorescence intensity indicating [Ca 2+ ] cyt in wild type, cau1 and 35S/CAU1-cau1 plants using Indo-1 staining and UV confocal assay. The first row showed fluorescence images, and the second row showed the merged fluorescence and bright-field images. [Ca 2+ ] cyt was calibrated and pseudo-color-coded according to the color scale. (D) Quantification of fluorescence from assays as represented in (C), n=4 cells. Lowercase letters above each bar indicate whether averages were statistically different at P <.5 by t tests. Bars=5 µm in (C). 5
Supplemental Data. Fu et al. Plant Cell. (213). 1.115/tpc.113.113886 Col- cau1 Col- cau1 Col- cau1 Col- cau1 Actin2 PHOT1 PLC3 PIS1 CAS CAX1 PLC4 PIS2 PIP2A CAX3 PLC5 5PTase1 PIP3 CBL1 PLC6 5PTase2 PLC1 PIP5K1 PLC7 5PTase13 PLC2 PIP5K2 PLC8 CVP2 PLC9 FRA3 Supplemental Figure 6. Analysis of alternative splicing of Ca 2+ homeostasis or signaling pathway genes. RT-PCR amplification of genes involved in the IP3 pathway or Ca 2+ homeostasis. Plants were grown in soil to 28 days of age. 6
% Germination Stomatal aperture (%) Stomatal aperture (%) A B C 12 11 1 9 8 7 6 12 11 1 9 8 7 6 1 Col- cas-1 cau1 cau1 cas-1 1 1 1 ABA (nm) Col- cas-1 cau1 cau1 cas-1 1 1 1 ABA (nm) Col- cau1 cas-1 cau1 cas-1 Supplemental Data. Fu et al. Plant Cell. (213). 1.115/tpc.113.113886 Supplemental Figure 7. cau1 is hypersensitive to ABA. (A, B) Stomatal assays were performed as described in Methods with minor modification: intact epidermes were placed in a low external Ca 2+ solution buffered to.2 µm free Ca 2+ (A), or a high external Ca 2+ solution buffered to.2 mm free Ca 2+ (B) and incubated under light for 2 h. Subsequently, the epidermal peels were transferred to the same buffer supplemented with, 1, 1 or 1 nm ABA respectively, and incubated under light conditions for 2 h. The stomatal apertures under ABA exposure were normalized to those under control condition (without ABA). n=3. (C) Seed germination analysis. 5 seeds of Col-, cau1, cas-1 and cau1 cas-1 respectively were plated onto ¼ PNS minimal medium plates supplemented with ABA at indicated concentrations. Germination rates were scored 7 d later. Data are mean ± SD. 8 6 4 2.1 1 5 1 2 ABA (µm) 7
Supplemental Data. Fu et al. Plant Cell. (213). 1.115/tpc.113.113886 Supplemental Table1. Transcriptomic analysis of Ca 2+ homeostasis genes, Ca 2+ signaling pathway genes, and genes known to be regulated by CAU1. AGI Index number Alias Exp. 1 (Fold change) Exp. 2 (Fold change) At5g114 FLC 2.83 2.6 At5g236 CAS 4.92 2.15 At2g3817 CAX1 2. 1.39 At3g1332 CAX2 1.15-1.15 At3g5186 CAX3 2.14 1.17 At5g149 CAX4 1. -1.3 At1g5573 CAX5 1.7-1.3 At1g5572 CAX6 1.7-1.2 At1g896 CAX11 1.87 1.46 At1g563 IP3 1.23 1.1 At4g181 IP5PII -1.8-1.2 At3g5342 PIP2A -1.52-1.25 At2g3717 PIP2B -1.41 1.48 At4g351 PIP3 1.23 1.6 At5g737 IPK2A -1.23 1.2 At5g6176 ATIPK2Β -1.15-1.15 At1g547 CVP2 1.23 1.39 At4g3869 PLC1-1.23-1.28 At3g851 PLC2 1. -1.37 At5g5867 PLC3 1.7-1.4 At5g5868 PLC4 5.28 1.5 AT5G5869 PLC5 1.87-2.17 At1g645 PLC6 1.36 1.7 At4g3869 PLC7-1.23-1.28 At3g4729 PLC8 1.41-1.43 At3g4722 PLC9-1.28 1.41 At4g175 PLC1 1.32 1.12 At4g3492 PLC11 1.74-1.15 At4g3493 PLC12 1.15-2.33 At4g3853 PLC13 1.23-2.4 At4g3869 PLC14-1.23-1.28 At3g5594 PLC15 1.5 1.29 At2g2687 PLC17 2.14 1. At1g1889 CDPK1 1.23 1.2 At1g3567 CDPK2-1.6-1.3 At4g2365 CDPK6-1.41-1.6 At5g2358 CDPK9 1.9-1.52 8
Supplemental Data. Fu et al. Plant Cell. (213). 1.115/tpc.113.113886 At5g1945 CDPK19-1.15-1.25 At2g2698 CIPK3 1.15 1.26 At1g114 CIPK9-1.41-1.9 At2g259 CIPK16-1.8-1.79 At1g4826 CIPK17 1.7-1.33 At1g2923 CIPK18-1.8-2.22 At1g327 CIPK23 1.7-1.6 At4g17615 CBL1-1.8-1.3 At5g5599 CBL2 1.52 1.53 At4g2657 CBL3 1.26 1.18 At5g2427 CBL4 1.34 1.13 At4g1635 CBL6 6.96-1.37 At4g2656 CBL7 1.7-1.45 At1g6448 CBL8 4.59 3.66 At5g471 CBL9 1.7-1.25 At4g33 CBL1 1.57-1.69 At5g5313 CNGC1 1. 1.17 At2g4643 ATCNGC3 1.32-1.22 At5g5425 ATCNGC4 1.23 2.16 At5g5794 ATCNGC5-1.2-1.15 At2g2398 ATCNGC6 1.1 1.27 At1g1599 ATCNGC7 2.46 2.8 At1g1978 ATCNGC8 1. 1.14 At4g356 ATCNGC9 1.15-1.6 At1g134 ATCNGC1-1.8-2.38 At2g4644 ATCNGC11 1.32-1.92 At4g11 ATCNGC13-1.15-1.19 At2g2461 ATCNGC14-2. -1.54 At2g2826 ATCNGC15 1.87 2.12 At3g481 ATCNGC16 1.62-1.4 At4g336 ATCNGC17 1.23-1.32 At3g1769 ATCNGC19-1.15 1.42 At1g2777 ACA1 1.52-1.6 At3g5733 ACA2 1.41-1.33 At2g4156 ACA4-1.8-1.11 At3g2118 ACA9 1.15 1.12 At5g5711 ACA8-1.8 1.34 At4g299 ACA1 1.7-1.2 At1g781 ECA1 1. -1.3 At3g285 SKOR 1.52-1.82 At5g3778 CAM1 1. -1.37 At2g4111 CAM2 1.74 1.74 At3g568 CAM3 1. 1.18 9
Supplemental Data. Fu et al. Plant Cell. (213). 1.115/tpc.113.113886 At1g6641 CAM4-1.23-1.2 At2g273 CAM5 1.41 1.38 At5g21274 CAM6.77.85 At3g4381 CAM7 1.52.93 At4g1464 CAM8 1.23 1.8 At3g5192 CAM9-1.23-1.52 At3g155 NAC3-2.46-7. At3g553 CRK 1.41 1.76 At2g4114 CRK1 1. -1.8 At4g2313 CRK5 1.23-1.56 At4g2314 CRK6 1.23 1.1 At4g2318 CRK1-1.32-1.67 At4g2319 CRK11-1.41 1.13 At2g2665 AKT1-1.75-2. At1g412 MRP5 1. 1.15 At3g411 GLR1-1.41 1.48 At4g356 TPC1 1.7-1.2 At4g3592 MCA1 1.41 1.27 At5g375 GORK 1.7 1.33 At3g4578 PHOT1 1.32 1.7 At5g5814 PHOT2 1.15 1.2 At1g924 NAS3 3.25 7.26 At2g432 ZIP7 7.46 2.13 At3g1625 NDF4 3.3 4.76 At1g232 FRO3-2. -3.39 At1g53 ZIP5-3.3-2.64 Note: Leaves of 4-week-old plants grown in soil (Exp 1) or hydroponics (Exp 2) were used to extract RNA and subject to hybridization to Arabidopsis whole genome ATH1 arrays. Data were extracted and normalized according to manufacturer s standard protocol. Fold change represents data from cau1 against data from Col-. - represents signal decrease in cau1 leaves. 1
Supplemental Data. Fu et al. Plant Cell. (213). 1.115/tpc.113.113886 Supplemental Table 2. List of primer sequences. Primer names Forward primer Reverse primer CAU1-1 ATGCCGCTCGGAGAGAGA CTAAAGGCCAACCCAGTA CAU1-2 TGTTTCCTTCTTCTTCCTCG TGTCTTACCAGTCCCTCTGA CAU1-3 GGATCCATGCCGCTCGGAGAGAGA GAGCTCCTAAAGGCCAACCCAGT CAU1-4 CTCGAGATGCCGCTCGGAGAGAGA GAATTCCTAAAGGCCAACCCAGT CAU1-5 ATGCCGCTCGGAGAGAGA AGTCCCTCTGAGGTATCAT Actin2 CCCTGTTCTTCTTACCGAG CCACATCTGCTGGAATG CAS-RT ATGGCTATGGCGGAAATG GCAAAGAAGGTCAAGCGTT CAS-QP ATGCTGCAAAGACAGTAACA GCAAAGAAGGTCAAGCGTT Actin2-QP AGGTATCGCTGACCGTATGAG CATCTGCTGGAATGTGCTGA CAS-salk TGTGCTACATCTGTTACTGT TATGGCTATGGCGGAAATGGCA LBa1 N/A ATTTTGCCGATTTCGGAAC PLC1-QP GATGAAAGAATCATTCAAAGTGTG TTGCCAAACCTCGCGGGTCA PLC2-QP ATGTCGAAGCAAACGTACAAAGTG ATGATGCACCTTATGGAGAGCA PLC3-QP ATGTCGGAGAGTTTCAAAGTGTG TCATGATGCACCTGGCCAGACA PLC4-QP ATGTTCTTGGTCCATCAAAACATG TTTTGATGGACCTGATCGGCGA PLC5-QP ATGAAGAGAGATATGGGGAGTTAC TCTTGATGCACCTTACTATCAA PLC6-QP ATGGATGGTTTGAAGATTTC TCTTGATGCACGTGAGGGGTA PLC7-QP ATGTCGAAGCAAACATACAAAGTC TCTTGATGCACCTCAAGAGAAGA PLC8-QP ATGTTAGTTACGAGGCGATGGG TCATGATGAACCTGGTCACCGA PLC9-QP ATGGTGAATTTAAGAAAGAAGTTTG ATGACGAACCTGGTCCTGAT FRA3-QP ATGGAAGATCGTCAAAACGA AGTCCATAACTGTCTCTCAGT PIP5K1-QP ATGAGTGATTCAGAAGAAGA TCCACTTGGCCATGAGAAT PIP5K2-QP ATGATGCGTGAACCGCTTGT TCCATTCTCCCAGATTTGAA 5PTase1-QP ATGGCGGAAGTACGATCACG AGCACAGATTCTGATTGCTT 5PTase2-QP ATGAAAACAAGACGTGGGAA CTTGAAACCCGATAATGTA 11
Supplemental Data. Fu et al. Plant Cell. (213). 1.115/tpc.113.113886 5PTase13-QP ATGGATTCGCTAATTATCGA AGAATCTCCCAGTCTGCGT CVP2-QP ATGAGAGAAG AGAAATCCAA TCTTGGAATCCGAGTACATA PIS1-QP ATGGCTAAAAAGGAGAGACC AATGAGAGGAAGACCAAACT PIS2-QP ATGGCTAAACAGAGACCGGC AAGACAGGCTGTGCTGACT PHOT1-QP ATGGAACCAACAGAAAAACC TGGAGATTGTCTCCGGTGAT CAX1-QP ATGGCGGGAATCGTGACAGA TCCGTTTTTCAAGTATCCCA CAX3-QP ATGGGAAGTATCGTGGAGCC CCGGTTGCTGCATATTTTAA Intron flanking primers Primer names Forward primer Reverse primer CAS ATGGCTATGGCGGAAATGGCA TCAGTCGGAGCTAGGAAGGAA PIP2A ATGGCAAAGGATGTGGAAGC GACGTTGGCAGCACTTC PIP3 ATGTCGAAAGAAGTGAGCGA ATTGGTTGCGTTGCTTC PLC1 GATGAAAGAATCATTCAAAGTGTG CTAACGAGGCTCCAAGACAAAC PLC2 ATGTCGAAGCAAACGTACAAAGTG TCACACAAACTCCACCTTCACG PLC3 ATGTCGGAGAGTTTCAAAGTGTG TCAACGAAACGTATAAGGAGGATC PLC4 ATGTTCTTGGTCCATCAAAACATG TCAGACAAACTCGAAGCGCATAAG PLC5 ATGAAGAGAGATATGGGGAGTTAC TTAAAGAAAGTGAAACCGCATGAG PLC6 ATGGATGGTTTGAAGATTTC TCATGGAAATTTCCGAGCTT PLC7 ATGTCGAAGCAAACATACAAAGTC TCACACAAACTCCAACCGCAC PLC8 ATGTTAGTTACGAGGCGATGGG CTAAGACCACTTAAAACGTGTGAG PLC9 ATGGTGAATTTAAGAAAGAAGTTTG CTAAGACCACTTAAAACGTGTGAG PHOT1 ATGGAACCAACAGAAAAACC TCAAAAAACATTTGTTTGCA CAX1 ATGGCGGGAATCGTGACAGA TTAACCCGTTTTAACTTTAT CAX3 ATGGGAAGTATCGTGGAGCC TTAACGTGAGAAAACTTCTC FRA3 ATGGAAGATCGTCAAAACGA CTATGGACTGTGCAAGTTCT CBL1 ATGACAACTGGCCGACCAAA TCAGTCTTCAACCTCAGTGT PIP5K1 ATGAGTGATTCAGAAGAAGA TTAGCCCTCTTCAATGAAGA PIP5K2 ATGATGCGTGAACCGCTTGT TTAGCCGTCTTCGATGAAGAT 12
Supplemental Data. Fu et al. Plant Cell. (213). 1.115/tpc.113.113886 PIS1 ATGGCTAAAAAGGAGAGACC TCAAGGCTTCTGCTGCTTCT PIS2 ATGGCTAAACAGAGACCGGC TCAAGGCTTCTTATGCTGTT 5PTase1 ATGGCGGAAGTACGATCACG TTAGGCGTCAAGGCCTTGAA 5PTase2 ATGAAAACAAGACGTGGGAA TCAAAAAGAAGGTTCAGGAT 5PTase13 ATGGATTCGCTAATTATCGA TCACCGGCTTTTACCTCGTC CVP2 ATGAGAGAAG AGAAATCCAA CTAGAAGAAGCTGAGCTC ChIP quantitative PCR primers Primer names Forward primer Reverse primer CAS-1 TGTTTGTTTGTTATTGTTTTGGGTA ATAAAAAAAAAAAGTTAAGACAAAA CAS-2 TGATCTGTTTTTTAACAATGGT GGCTCAACAAACAAAAACAA CAS-3 ACTGAATCCAACTTGTACTT GAACTCATAACAACATAGCC CAS-4 CTGATATGAAATTTGCTGTG TAGTAATGCAAAAGCAAAGG CAS-5 TTTGCTTTTGCATTACTATG TGAATATCACCACTTGGATC CAS-6 TAATAACCTTCTAGAGAGCC CTTATAACAAAAGTGTCTTT CAS-7 TCATTGGTCTTCCTGTGTCC TGAAGTTTTGCTCCAAGGAA CAS-8 TTCCTTGGAGCAAAACTTCA CATATTTTAACAGCCCCTCT CAS-9 TTCTCTACAATGTTCTTATA AACAAGCAAGCTCACCATTT CAS-1 TAGCATAGTTTAAATGGTGA ATTAACTTTCCCATGATTGA CAS-11 TTTTACTAGTAGACGGTTGA ATTGGACTGATGTTGGTTTA CAS-12 TTAAGATACTTGGTTTTGTA GAAGCGGTTCAACCTCCGCT CAS-13 TTTGATTACCAATCGAACTG TACTACAGAATAAGAGCAAC CAS-14 CACCTCTGATATTACTGCTA AAGATGGATTCTTATTGGTC CAS-15 TCAAATCCCCTTCCTTCAAT CAGAGAAAGAGACAAAGAAT 13
Supplemental Data. Fu et al. Plant Cell. (213). 1.115/tpc.113.113886 CAS-16 TCGCATTGATTTGTAAAAAA TGTCTCAGACTCAGTGTCTT CAS-17 AAATCGTCTCCTCTCTCACT GAACTTGGTTGATTGTTTTC CAS-18 AAGAAACTGGTTCTAGTGTA GCAAGCTTCATAGCTTCTTC CAS-19 ACAGTAACAGATGTAGCACA GAAACGCAGCACCAGCAGCA TUB8 ATAACCGTTTCAAATTCTCTCTCTC TCCGATCTGGTTTCCGCATTGGCCA 14