Supplementto“AtIQM1,a novel calmodulin-binding protein, is involved in stomatal movement in Arabidopsis”
Figure S1.Expression of a truncated IQM1 (amino acid 1–137) in E. coli.
The crude soluble proteins extracted from induced bacterial cells harboring an empty plasmid (i, negative control) ora plasmid containing a cDNA for the truncated IQM1 (ii), or His-tagged truncated IQM1 protein purified using metal affinity chromatography (iii) were separated on SDS-polyacrylamide gel and stained with Coomassie Brilliant Blue. M, protein molecular-weightstandard.
Figure S2. Subcellular localization of CaM5 proteinexpressed in onion epidermal cells.Images were taken 16 h after particle bombardment. Panels from top to bottom: GFP, GFP-CaM5 and GFP-CaM5 treated with 1Msucrosefor plasmolysis. Sampleswere stained without (i) and with(ii) 4',6-diamidino-2-phenylindole; arrows indicate cell nuclei; Bar = 50m.
Figure S3. Morphology of 4- (top panel) and 6-week-old (middle panel) plants of wild type and iqm1, and images of representative stomata in the leaves of 6-week-old plants grownin a light regime of 16-h lightand 8-h dark period(bottom panel).
The stomatal images were taken from the paradermal sections prepared from rosette leaves 3h after the onset of light period.
Figure S4. Drought tolerance assay of 4-week-old plants.
Plants were grown in soil in the same condition, then withheld from water for 24 d (top), and then rewateredfor one d (bottom).
FigureS5.Microarray analysis of iqm1-1 leaves.
(A) Functional categorization of genes up-regulated in iqm1-1.Two hundred and forty-three up-regulated genes found in iqm1-1 (Table S1) were classified according to their reported inducing stresses; 44% genes were responsive to either biotic and abiotic stressesor hormonal treatments (Table S3).Because one gene often responded to multiple stresses, it was counted repeatedly as a responding gene in multiple stresses. Total RNA prepared from rosette leaves of 6-week-old plants was subjected to microarray analysis.
(B) Semi-quantitative RT-PCR analyses of 5 genes chosen from genes up-regulated in iqm1-1with total RNA obtained as described above. WRKY: WRKY transcription factors; MAPKKK14: mitogen-activated protein kinase kinase kinase 14; RRTF1: redox-responsive transcription factor 1.-ATPase was used as a control.
FigureS6. Gene expression of marker genes in guard cell and mesophyll cell. Semi-quantitative RT-PCR analyses were performed with total RNA isolated from stomatal guard cells (GC) and mesophyll cells (MC) in the leaves of 6-week-old plants. MC marker: GLR2.7 (At2G29120); GC marker:GC1 (At1g22690). β-ATPasewas used as control.
FigureS7. Amino acid sequences of the RNA recognition motif of rice zinc finger CCCH domain-containing 16 (C3H16, residues 195 - 295) and N-terminal portion of trichosanthin (TS, residues 21 - 111) are aligned with that of IQM1. The IQ motif is shown in red and important residues in TS activity are shown in blue.
FigureS8. Effects of treatments with dehydration and ABA on IQM1gene expression.
(A) Semi-quantitative RT-PCR analyses.-ATPasewas used as a control.
(B) Quantitative RT-PCR analyses. Total RNA was extracted, treated with DNase, and purified with RNeasy columns(Qiagen, Valencia, CA, USA). 25 ng of total RNA was subjected to real time RT-PCRwith Onestep SYBR PrimeScript RT-PCR reactions (Takara), which was performed on an ABIPRISM 7000 sequence detection system (Applied Biosystems, Foster City, CA, USA). The cDNA fragmentsof IQM1 and actin 2 were amplifiedwith gene-specific primers (Table S5). The actin 2cDNA fragment was used as areference to normalizeamounts of the IQM1cDNA. Real time RT-PCR was performed in a 25-μl reaction mix. Thermal cycling conditionswere: 42°C for 5 min, 95°C for 10 s; 40 cycles of 95°C for 5 s, 60°C for31 s;then 95°C for 15 s, 60°C for 20 s and 95°C for 15 s forthe dissociation stage. The specificity of amplification was alsoconfirmed by melting curve analysis with one single peak at qRT-PCRreactions. Each sample was analyzed in triplicate andnormalized to endogenous control. The fold-changes of geneexpression between treated samples and control samples werecalculated using 2-Delta Delta Ct methodology (Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)). Method. Methods, 2001, 25: 402–408).
FigureS9. Powdery mildewinfectionassay.
Three-week-old Arabidopsis plants were inoculated with the powdery mildew pathogen, Erysiphe cichoracearum UCSC1. Fungal inoculum was prepared by touching infected Arabidopsis leaves. After all inoculations, plants were placed in a dew chamber in the dark at 100% relative humidity for 1 h and then were placed in a growth chamber under normal growthconditions. Photographs were taken 21 days after infection.
Table S2 Down-regulated genes in iqm1-1Gene id / Symbol / Oligo id / Ref seq_id / Fold change / Description
Sample / Mean
1 / 2
At5g26742 / EMB1138 / A005023_01 / NM_147920 / 0.56 / 0.44 / 0.5 / EMB1138 (EMBRYO DEFECTIVE 1138); ATP binding/ATP-dependent helicase
At1g69530 / EXP1 / At30006220 / NM_179538 / 0.52 / 0.48 / 0.5 / EXPA1 (ARABIDOPSIS THALIANA EXPANSIN A1)
At4g36360 / BGAL3 / At30021830 / NM_119799 / 0.33 / 0.66 / 0.49 / BGAL3 (beta-galactosidase 3); beta-galactosidase
At3g07460 / At30012855 / NM_202519 / 0.41 / 0.58 / 0.49 / Unknown protein
At5g13740 / ZIF1 / A018119_01 / NM_121377 / 0.63 / 0.35 / 0.49 / ZIF1 (ZINC INDUCED FACILITATOR 1); carbohydrate transporter/sugar porter
At2g16660 / A007073_01 / NM_127218 / 0.32 / 0.64 / 0.48 / nodulin family protein
At1g74670 / A002045_01 / NM_106125 / 0.51 / 0.44 / 0.47 / gibberellin-responsive protein, putative
At4g24780 / A021044_01 / NM_118611 / 0.4 / 0.54 / 0.47 / pectate lyase family protein
At5g56550 / OXS3 / A018348_01 / NM_125038 / 0.57 / 0.37 / 0.47 / OXS3(OXIDATIVE STRESS 3)，involved in tolerance to heavy metals and oxidative stress
At5g04160 / A023155_01 / NM_120498 / 0.45 / 0.48 / 0.47 / phosphate translocator-related
At4g34950 / A021114_01 / NM_119661 / 0.66 / 0.27 / 0.47 / nodulin family protein
At3g18290 / EMB2454 / A009777_01 / NM_112713 / 0.29 / 0.63 / 0.46 / EMB2454 (EMBRYO DEFECTIVE 2454), protein binding/zinc ion binding
At5g44020 / A018689_01 / NM_123769 / 0.5 / 0.42 / 0.46 / acid phosphatase class B family protein
At5g02260 / EXP9 / A024544_01 / NM_120304 / 0.53 / 0.39 / 0.46 / ATEXPA9 (ARABIDOPSIS THALIANA EXPANSIN A9)
At5g18020 / At30024093 / NM_121807 / 0.38 / 0.54 / 0.46 / auxin-responsive protein, putative
At3g45160 / A008951_01 / NM_114385 / 0.48 / 0.4 / 0.44 / unknown protein
At1g07050 / A001038_01 / NM_100579 / 0.42 / 0.44 / 0.43 / CONSTANS-like protein-related
At4g36570 / RL3 / A022632_01 / NM_119820 / 0.27 / 0.59 / 0.43 / RL3 (RAD-LIKE 3); transcription factor/DNA binding
At3g22231 / PCC1 / At30014557 / NM_113121 / 0.36 / 0.5 / 0.43 / PCC1 (PATHOGEN AND CIRCADIAN CONTROLLED 1)
At1g29440 / At30003093 / NM_102684 / 0.34 / 0.51 / 0.43 / auxin-responsive family protein
At2g30010 / A019839_01 / NM_128556 / 0.45 / 0.4 / 0.43 / unknown protein
Gene id / Symbol / Oligo id / Ref seq_id / Fold change / Description
Sample / Mean
1 / 2
At1g04040 / At30000354 / NM_100285 / 0.39 / 0.46 / 0.43 / acid phosphatase class B family protein
At4g16370 / OPT3 / A015260_01 / NM_117732 / 0.4 / 0.38 / 0.39 / OPT3 (OLIGOPEPTIDE TRANSPORTER); oligopeptide transporter
At5g13930 / CHS, / A017513_01 / NM_121396 / 0.3 / 0.48 / 0.39 / CHS (CHALCONE SYNTHASE); naringenin-chalcone synthase
At5g05250 / A018056_01 / NM_120607 / 0.35 / 0.36 / 0.36 / unknown protein
At1g29500 / A003399_01 / NM_102690 / 0.43 / 0.23 / 0.33 / auxin-responsive protein, putative
At4g36720 / HVA22K / A013634_01 / NM_119836 / 0.24 / 0.26 / 0.25 / HVA22K (HVA22-LIKE PROTEIN K)
At1g53480 / MRD1 / A002112_01 / NM_104226 / 0.14 / 0.15 / 0.14 / MRD1 (mto 1 responding down). Down-regulated in mto1-1 mutant that over-accumulates soluble methionine
At4g33050 / EDA39/ IQM1 / At30021448 / NM_119459 / 0.06 / 0.06 / 0.06 / EDA39 (embryo sac development arrest 39)/IQM1(IQ motif-containing protein 1); calmodulin binding
Table S4 Up-regulated chitin-responsive transcription factor and ubiquitin-ligase genes (Libault et al, 2007) in iqm1-1
Gene / Fold change / Function
Experiment / Mean
1 / 2
At5g47220 / 2.07 / 2.95 / 2.51 / AP2/ERE, ATERF2/ERF2 (ETHYLENE RESPONSE FACTOR 2)/transcription factor
At3g15210 / 4.75 / 3.62 / 4.19 / AP2/ERE, ATERF4/ATERF4/ERF4/RAP2.5 (ETHYLENE RESPONSE FACTOR 4)/transcription factor
At4g17490 / 4.96 / 1.89 / 3.43 / AP2/ERE, ATERF6 (ETHYLENE RESPONSE FACTOR 6)/transcription factor
At1g53170 / 3.06 / 2.59 / 2.82 / AP2/ERE, ATERF8/ERF8 (ETHYLENE RESPONSE FACTOR 8)/transcription factor
At1g28370 / 9.67 / 4.83 / 7.25 / AP2/ERE, ATERF11/ERF11 (ERF domain protein 11)/transcription factor
At2g44840 / 3.19 / 11.87 / 7.53 / AP2/ERE, ERF13/EREBP (ETHYLENE RESPONSE FACTOR 13)/transcription factor
At4g34410 / 34.77 / 25.64 / 30.2 / AP2/ ERF, RRTF1(REDOX RESPONSIVE TRANSCRIPTION FACTOR 1 /transcription factor
At1g64380 / 2.73 / 2.19 / 2.46 / AP2/ERF, transcription factor
At1g33760 / 4.9 / 6.46 / 5.68 / AP2/ERF, transcription factor
At5g51190 / 2.37 / 3.27 / 2.82 / AP2/ERF, transcription factor
At3g46080 / 3.37 / 4.61 / 3.99 / zinc finger (C2H2 type) family protein; nucleic acid binding/transcription factor/zinc ion binding
At3g19580 / 13.44 / 3.79 / 8.62 / zinc finger (C2H2 type) family protein; AZF2; transcriptional factor/zinc ion binding
At3g46090 / 3.49 / 4.06 / 3.78 / zinc finger (C2H2 type) family protein; ZAT7; nucleic acid binding/transcription factor/zinc ion binding
At5g04340 / 3.77 / 3.1 / 3.43 / zinc finger (C2H2 type) family protein; ZAT6; nucleic acid binding/transcription factor/zinc ion binding
At1g27730 / 3.51 / 3.48 / 3.5 / zinc finger (C2H2 type) family protein; ZAT10/STZ; transcription factor/zinc ion binding
At5g59820 / 2.26 / 3.29 / 3.29 / zinc finger (C2H2 type) family protein; ZAT12/RHL41; nucleic acid binding/transcription factor/zinc ion binding
At3g55980 / 8.65 / 3.15 / 5.9 / zinc finger (C3H-type) family protein; SZF1/transcription factor
At2g40140 / 3.09 / 4.02 / 3.56 / zinc finger (C3H-type) family protein; CZF1/ZFAR1/transcription factor
At3g52800 / 3.88 / 3.1 / 3.49 / zinc finger (AN1-like type) family protein; nucleic acid binding/zinc ion binding
At1g51700 / 2.16 / 2.4 / 2.28 / zinc finger(DOF-type) family protein; DOF1 (dof zinc finger protein 1); DNA binding/transcription factor
At4g31550 / 3.14 / 1.64 / 2.39 / WRKY11 (WRKY DNA-binding protein 11); transcription factor
At4g01250 / 5.8 / 5.1 / 5.45 / WRKY22 (WRKY DNA-binding protein 22); transcription factor
Gene / Fold change / Function
Experiment / Mean
1 / 2
At1g80840 / 8.3 / 8.38 / 8.34 / WRKY40 (WRKY DNA-binding protein 40); transcription factor
At4g23810 / 2.71 / 3.54 / 3.12 / WRKY53 (WRKY DNA-binding protein 53); transcription factor
At1g19180 / 2.82 / 1.91 / 2.37 / JAZ1(JASMONATE-ZIM-DOMAIN PROTEIN 1); response to jasmonic acid stimulus; transcription repressor
At1g17380 / 4.66 / 7.25 / 5.96 / JAZ5(JASMONATE-ZIM-DOMAIN PROTEIN 5); response to jasmonic acid stimulus; transcription repressor
At2g34600 / 3.3 / 5.69 / 4.5 / JAZ7(JASMONATE-ZIM-DOMAIN PROTEIN 7); response to jasmonic acid stimulus; transcription repressor
At1g32640 / 3.65 / 1.83 / 2.74 / MYC2/JAI1 (JASMONATE INSENSITIVE 1)/JIN1/ RD22BP1/ZBF1; DNA binding/transcription factor
*At1g20823 / 3.17 / 1.91 / 2.54 / zinc finger (C3HC4-type RING finger) family protein/protein binding/zinc ion binding/ubiquitin-ligase
*At3g46620 / 5.48 / 4.4 / 4.94 / zinc finger (C3HC4-type RING finger) family protein/protein binding/zinc ion binding/ubiquitin-ligase
*At5g59550 / 4.46 / 5.78 / 5.12 / zinc finger (C3HC4-type RING finger) family protein/protein binding/zinc ion binding/ubiquitin-ligase
*At5g66070 / 1.92 / 2.15 / 2.06 / zinc finger (C3HC4-type RING finger) family protein/protein binding/zinc ion binding/ubiquitin-ligase
*At2g35930 / 5.12 / 3.87 / 4.49 / U-box domain-containing protein
*At3g52450 / 4.2 / 4.17 / 4.19 / U-box domain-containing protein
*At1g66160 / 4.07 / 3.92 / 4 / U-box domain-containing protein
At3g49530 / 2.75 / 3.91 / 3.33 / ANAC062 (Arabidopsis NAC domain containing protein 62); transcription factor
At2g17040 / 3.03 / 1.67 / 2.35 / ANAC036 (Arabidopsis NAC domain containing protein 36); transcription factor
At5g24590 / 2.31 / 5.69 / 4 / ANAC091 (Arabidopsis NAC domain containing protein 91); transcription factor
At4g18880 / 2.56 / 2.6 / 2.58 / HSFA4A (Arabidopsis thaliana heat shock transcription factor A4A); DNA binding/transcription factor
At1g42990 / 3.13 / 2.72 / 2.92 / BZIP60 (BASIC REGION/LEUCINE ZIPPER MOTIF 60); DNA binding/bZIP transcription factor
At5g62470 / 2.67 / 3.12 / 2.9 / MYB96 (myb domain protein 96); DNA binding/transcription factor
*Ubiquitin-ligase genes respond to chitooctaose treatment
Table S5 Primers used in this studyExpriment / Gene/ID / Forward primer (Underlined isrestriction enzyme site) / Reverse primer (Underlined isrestriction enzyme site) / Restriction Enzyme Site
Yeast Two-Hybrid Assay / CaM5/AT2G27030 / CGCGAATTCATGGCAGATCAGCTCACCGATGATC / AGAGCGGCCGCTCACTTTGCCATCATAACTTTGAC / EcoRI; NotI
Mapping of IQ motif / IQM1/AT4G33050
Full-length cDNA(aa 1-488) / GTCAAGCTTATGGGTCTTGAAGTTGGGTCCTTATG / GAACTCGAGTTAACACTTTACAGCCCTAGGGCT / HindIII; XhoI
Full-length cDNA(aa 1-488) / GTCAAGCTTATGGGTCTTGAAGTTGGGTCCTTATG / GAACTCGAGTTAACACTTTACAGCCCTAGGGCT / HindIII; XhoI
Partial cDNA(aa 1-137) / GTCAAGCTTATGGGTCTTGAAGTTGGGTCCTTATG / ATACTCGAGCAGAGCTCCTCAACA ACGACCGCA / HindIII; XhoI
Partial cDNA(aa 100–488) / ATAAAGCTTCCTAGACCTGTCACCGAGCTTGAT / GAACTCGAGTTAACACTTTACAGCCCTAGGGCT / HindIII; XhoI
Partial cDNA(aa 138–488) / ATAAAGCTTGTGGAGGACTTTGGAAGGTGCAGC / GAACTCGAGTTAACACTTTACAGCCCTAGGGCT / HindIII; XhoI
Expression in E. coli / IQM1/AT4G33050
Partial cDNA(aa 1-137) / GTCAAGCTTATGGGTCTTGAAGTTGGGTCCTTATG / ATATGCGGCCGCCAGAGCTCCTCAACAACGACCGC / HindIII; NotI
CaM5/AT2G27030 / CGCGAATTCATGGCAGATCAGCTCACCGATGATC / AGAGCGGCCGCCTTTGCCATCATAACTTTGACAA / EcoRI; NotI
BiFC Assay / IQM1/AT4G33050 / GTCAAGCTTATGGGTCTTGAAGTTGGGTCCTTATG / ACGTTCTAGATTAACACTTTACAGCCCTAGGCGC / HindIII; XbaI
CaM5/AT2G27030 / CGCGAATTCATGGCAGATCAGCTCACCGATGATC / AGAGTCGACTCACTTTGCCATCATAACTTTGAC / EcoRI; SalI
Subcellular Localization / IQM1/AT4G33050 / AGCGCATGCCATGGGTCTTGAAGTTGGGTCCTTA / ATGTCGACAAACACTTTACAGCCCTAGGGCTTGG / SphI; SalI
CGCTCTAGAATGGGTCTTGAAGTTGGGTCCTT / ATACCCGGGTTAACACTTTACAGCCCTAGGGC / XbaI; SmaI
CaM5/ AT2G27030 / TTTCTAGAATGGCAGATCAGCTCACCGAT / ATCCCGGGTCACTTTGCCATCATAACTTT / XbaI; SmaI
Verification of theT-DNA Insertion Mutants / iqm1-1 / P1: ATACTCTGTCTCCTAGTT / P2: CCGTCTCCTATATCCAAC
iqm1-2 / P3: ATGGGTCTTGAAGTTGGG / P4: AAGTACCTCAAGGATCCC
the leftborder of T-DNA / LBb1:GCGTGGACCGCTTGCAACT
Complementation of iqm1 / IQM1/AT4G33050 / GCGCTCTAGAGAGAGGTCTTAAACCCTAAG / AGATCCCGGGGAGAAGCAAAGTCTTAGTAC / XbaI; SmaI
Histochemical GUS Assay / IQM1/AT4G33050 / GCGCTCTAGAGAGAGGTCTTAAACCCTAAG / ATTACCCGGGGACCACTAATATAACGAAAGG / XbaI; SmaI
Expriment / Gene/ID / Forward primer (Underlined isrestriction enzyme site) / Reverse primer (Underlined isrestriction enzyme site) / Restriction Enzyme Site
qRT-PCR / IQM1/AT4G33050 / ACCTACAGTTACTCCACCAGAGCCA / CTCAACAACGACCGCACAA
Actin2/AT3G18780 / TGACTACGAGCAGGAGATGGAA / CAAACGAGGGCTGGAACAA
RT-PCR in iqm1-1 / IQM1/AT4G33050 / RT-Fw: ATGGGTCTTGAAGTTGGGTCCTTATG / RT-Rev: TTAACACTTTACAGCCCTAGGCGC
WRKY22/AT4G01250 / GTAAGCTCATCAGCTACTACCAC / ACCGCTAGATGATCCTCAACAG
WRKY33/AT2G38470 / CTCCGACCACAACTACAACTAC / GGCTCTCTCACTGTCTTGCTTC
WRKY40/AT1G80840 / CCTCAGCTTACGGGAACTTCCACAC / ACTCCGCCAATAACCGCGCA
MAPKKK14/AT2G30040 / TCTCGCCACGTGTCTTCCTTCTCAA / CTCTCCCGAGTTCCGATAACCCCAC
PP2CA/AT3G11410 / CGACGTTACCGGGAAATCTA / TGGCAAGAACTCCAAGAACC
RRTF1/AT4G34410 / CGGCGCCGTGTCAGGGTTTT / ACTGGCGCTTGCACTTGCCT
rbohD/AT5G47910 / TAGTGTTGCTGGCGGCCGTG / GCCGGAGACGTTATTCCGGCG
rbohF/AT1G64060 / GTCGAGCCAGCAACCGCCAT / AAACTCTCGTCGTTGATTTGTGACCA
GPX3/AT2G43350 / CCTAGATCAAGCAGATGGGTCAAT / GTTGCAAGGAAATGCTAAGATCTC
AOX/AT3G22370 / AGCTATTGGGGTGTTGAACCTAAT / GTTACCTTTGTCAAGTTCCTTGAG
TAPX/AT1G77490 / GTACGTGACAATGTCTGTTTCTCT / TAGGATACTTGTCTTTGAGAGGCT
CSD2/AT2G28190 / TTAAAGCTCCGTCGAAAGCGTTGA / ATTGGCATTTATGTTTCCCAGGTC
APX1/AT1G07890 / ACATTGCTCTTAGGTTGTTGGACC / CTTTGTCAGAGACAAGCTGAAGAA
APX3/AT4G35000 / GTACTTGAAAGAGATCACTAAGGC / GTAGAAGACATCTCTGAGATGTTG
At3g14150 / CGAAGGTTTATTTTCAACCGAAGT / AGTTTCTCAGACCATATCTGTTGG
At4g18360 / GCTGGTCAAGTTGATCAATCACTT / AACCACTTAGTGCCATTGTAAGCT
CAT3/AT1G20620 / TGCTGATTTTCTCAGAGCCCCTGGT / CTCGGGGTAGTTGCCAGATGCAATG
HfsA4a/AT4G18880 / GTTTCATCGTTTGGAATCCGCCG / TCCGTCAACGGGTTTAACTGAGC
Zat7/AT3G46090 / CGGCTGCGGGGGAGATGAAC / AAGCCGTCACCGTCGTCGTC
Zat12/AT5G59820 / GTGGATGTCACGGCGGCGAA / GCGTGTAACCAACGCGCCAC
GC1/At1g22690 / TATGAAGAAGATGAATGTGGTGGC / TTATTGCCATGTGTACGGATACTG
GLR2.7/At2G29120 / ACCATAGTAGCAAACAGGTCATTG / TAATGTAGGCCACTTCGTCAAAAG
β-ATPase/AT5G08670 / CTGAATCAATCTCTTAAGCTG / GTGCAGAAAGTTCTACAGAACTAC