** * * * Col-0 cau1 CAU1. Actin2 CAS. Actin2. Supplemental Figure 1. CAU1 affects calcium accumulation.
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1 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) /tpc A * Col- cau1 B Col- cau1 ** * * ** C 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
2 Leaf number Supplemental Data. Fu et al. Plant Cell. (213) /tpc 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
3 Supplemental Data. Fu et al. Plant Cell. (213) /tpc A Z value (SD from average) 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
4 Water loss rate (%) CAS relative expression Survival rate (%) Stomatal aperture Supplemental Data. Fu et al. Plant Cell. (213) /tpc A B C Supplemental Figure 4. Stomatal closure, drought tolerance and CAS expression in 35S/CAU1-cau1. D F E a b b Col- cau1 35S/CAU1-cau1 Col- cau1 35S/CAU1-cau (min) G 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
5 Indo-1 signal intensity Stomatal aperture Stomatal aperture Supplemental Data. Fu et al. Plant Cell. (213) /tpc A C Ca +Ca Col- cau1 35S/CAU1-cau1 Col- cau1 35S/CAU1-cau1 Rel. Indo-1 intensity B D Col- cau1 35S/CAU1-cau (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
6 Supplemental Data. Fu et al. Plant Cell. (213) /tpc 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
7 % Germination Stomatal aperture (%) Stomatal aperture (%) A B C Col- cas-1 cau1 cau1 cas ABA (nm) Col- cas-1 cau1 cau1 cas ABA (nm) Col- cau1 cas-1 cau1 cas-1 Supplemental Data. Fu et al. Plant Cell. (213) /tpc 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 ABA (µm) 7
8 Supplemental Data. Fu et al. Plant Cell. (213) /tpc 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 At5g236 CAS At2g3817 CAX At3g1332 CAX At3g5186 CAX At5g149 CAX At1g5573 CAX At1g5572 CAX At1g896 CAX At1g563 IP At4g181 IP5PII At3g5342 PIP2A At2g3717 PIP2B At4g351 PIP At5g737 IPK2A At5g6176 ATIPK2Β At1g547 CVP At4g3869 PLC At3g851 PLC At5g5867 PLC At5g5868 PLC AT5G5869 PLC At1g645 PLC At4g3869 PLC At3g4729 PLC At3g4722 PLC At4g175 PLC At4g3492 PLC At4g3493 PLC At4g3853 PLC At4g3869 PLC At3g5594 PLC At2g2687 PLC At1g1889 CDPK At1g3567 CDPK At4g2365 CDPK At5g2358 CDPK
9 Supplemental Data. Fu et al. Plant Cell. (213) /tpc At5g1945 CDPK At2g2698 CIPK At1g114 CIPK At2g259 CIPK At1g4826 CIPK At1g2923 CIPK At1g327 CIPK At4g17615 CBL At5g5599 CBL At4g2657 CBL At5g2427 CBL At4g1635 CBL At4g2656 CBL At1g6448 CBL At5g471 CBL At4g33 CBL At5g5313 CNGC At2g4643 ATCNGC At5g5425 ATCNGC At5g5794 ATCNGC At2g2398 ATCNGC At1g1599 ATCNGC At1g1978 ATCNGC At4g356 ATCNGC At1g134 ATCNGC At2g4644 ATCNGC At4g11 ATCNGC At2g2461 ATCNGC At2g2826 ATCNGC At3g481 ATCNGC At4g336 ATCNGC At3g1769 ATCNGC At1g2777 ACA At3g5733 ACA At2g4156 ACA At3g2118 ACA At5g5711 ACA At4g299 ACA At1g781 ECA At3g285 SKOR At5g3778 CAM At2g4111 CAM At3g568 CAM
10 Supplemental Data. Fu et al. Plant Cell. (213) /tpc At1g6641 CAM At2g273 CAM At5g21274 CAM At3g4381 CAM At4g1464 CAM At3g5192 CAM At3g155 NAC At3g553 CRK At2g4114 CRK At4g2313 CRK At4g2314 CRK At4g2318 CRK At4g2319 CRK At2g2665 AKT At1g412 MRP At3g411 GLR At4g356 TPC At4g3592 MCA At5g375 GORK At3g4578 PHOT At5g5814 PHOT At1g924 NAS At2g432 ZIP At3g1625 NDF At1g232 FRO At1g53 ZIP 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
11 Supplemental Data. Fu et al. Plant Cell. (213) /tpc 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
12 Supplemental Data. Fu et al. Plant Cell. (213) /tpc PTase13-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
13 Supplemental Data. Fu et al. Plant Cell. (213) /tpc 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
14 Supplemental Data. Fu et al. Plant Cell. (213) /tpc CAS-16 TCGCATTGATTTGTAAAAAA TGTCTCAGACTCAGTGTCTT CAS-17 AAATCGTCTCCTCTCTCACT GAACTTGGTTGATTGTTTTC CAS-18 AAGAAACTGGTTCTAGTGTA GCAAGCTTCATAGCTTCTTC CAS-19 ACAGTAACAGATGTAGCACA GAAACGCAGCACCAGCAGCA TUB8 ATAACCGTTTCAAATTCTCTCTCTC TCCGATCTGGTTTCCGCATTGGCCA 14
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