Volume 1, Issue 2, Pages 238-248 (March 2008) Calcineurin-B-Like Protein CBL9 Interacts with Target Kinase CIPK3 in the Regulation of ABA Response in Seed Germination  Pandey Girdhar K. , Grant John J. , Cheong Yong Hwa , Kim Beom-Gi , Li Le Gong , Luan Sheng   Molecular Plant  Volume 1, Issue 2, Pages 238-248 (March 2008) DOI: 10.1093/mp/ssn003 Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 1 Physical Interaction of CBL9 and CIPK3. (A) CBL9–CIPK3 interaction in yeast two-hybrid system. Saccharomyces cerevisiae strain (PJ-69) containing CBL9–pGBT9.BS as bait was transformed with prey pGAD.GH (empty vector), CIPK3–pGAD.GH, CIPK3 kinase domain–pGAD.GH, CIPK13–pGAD.GH, respectively. Cells were grown in media containing SC–LT (top panel) or SC–LTH plus 1.5 mM 3-amino-1,2,4-triazole (bottom panel). (B) Interaction of CBL9 and CIPK3 assayed by immuno-precipitation. Total protein was extracted from transgenic plants containing CBL9–c-myc, CIPK3–HA, CIPK3-kinase domain–HA, or CIPK13–HA. Protein extracts were mixed for immuno-precipitation using either anti-HA or anti-c-myc antibodies (see Methods). The immuno-precipitated samples were subjected to Western blotting with either anti-c-myc (top panel) or anti-HA antibodies (bottom panel). Molecular Plant 2008 1, 238-248DOI: (10.1093/mp/ssn003) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 2 Isolation of cbl9cipk3 Double Mutant. (A) Diagrammatic representation of T-DNA insertion in cbl9 (Pandey et al., 2004) and cipk3 (Kim et al., 2003) mutants used for crossing to generate cbl9cipk3 double mutant. Several independently segregated double mutant lines were generated such as cbl9cipk3-1 cbl9cipk3-2 cbl9cipk3-3. (B) RT–PCR analysis of CBL9 and CIPK3 gene expression from cbl9 cipk3 and cbl9cipk3-1 double mutants and control (WsxCol-0) plants. RT–PCR was performed with CBL9- or CIPK3-specific primers. Expression of Actin2 was analyzed as a quantitative control. Molecular Plant 2008 1, 238-248DOI: (10.1093/mp/ssn003) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 3 Phenotypic Analysis of cbl9cipk3 Double Mutant. (A) Inhibition of germination and growth of young seedlings in control WsxCol-0, cbl9 cipk3 and cbl9cipk3-1 double mutant plants. Control seeds (WsxCol-0), cbl9 cipk3 and cbl9cipk3 double mutant seeds on MS agar or MS agar containing 0.7 μM ABA, 150 mM NaCl, or 375 mM mannitol were incubated at 4°C for 6 d before transfer to 23°C for germination. The photograph was taken on day 12 after transfer to 23°C. (B) Kinetics of seed germination on medium containing 0.7 μM ABA, 150 mM NaCl, or 350 mM mannitol for single mutant (cbl9 cipk3) and single wild-type background (Col-0 and Ws) and double mutant (cbl9cipk3-1) and Col-0×Ws background. (C) Germination score analysis of Col-0×Ws background; three different segregated lines of cbl9cipk3 mutant, i.e. cbl9cipk3-1 cbl9cipk3-2 and cbl9cipk3-3; two independent heterozygous lines of CBL9×CIPK3 (CBL9×CIPK3-1 and CBL9×CIPK3-2) after 3 d of growth at 23°C on medium containing 0.7 μM ABA, 150 mM NaCl, or 350 mM mannitol. Results in (B) and (C) are presented as average values and standard errors from three experiments (n = 100 for each condition). Molecular Plant 2008 1, 238-248DOI: (10.1093/mp/ssn003) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 4 Expression of Constitutively Active CIPK3 (T/D CIPK3) Rescued the ABA and Osmotic Stress Hypersensitivity of cbl9 Mutant. (A) RT–PCR analysis of CIPK3 levels in four homozygous cbl9::T/D CIPK3 transgenic lines (CK3-T/D2, CK3-T/D4, CK3-T/D5, CK3-T/D11) with CIPK3 gene-specific primers. ACTIN2 was used as loading control. (B) Seeds of cbl9 Col-0, and cbl9::T/D CIPK3 transgenic lines (CK3-T/D2, CK3-T/D4, CK3-T/D5, CK3-T/D11) were grown vertically on MS agar medium with or without 0.7 μM ABA, 150 mM NaCl, 375 mM mannitol. Photographs were taken after 12 d of growth at standard growth condition. (C) Germination scored after 3 d of growth on MS agar or MS agar containing 0.7 μM ABA, 150 mM NaCl, 375 mM mannitol. Three independent experiments were performed and values are means ± SD (n = 100 for each condition). Molecular Plant 2008 1, 238-248DOI: (10.1093/mp/ssn003) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 5 Analysis of Over-Expression of Wild-Type CIPK3 in the cbl9 Mutant Background. (A) Over-expression of CIPK3 was verified by RT–PCR (25 cycles). Four homozygous cbl9: CIPK3 (WT) were analyzed with empty vector-transformed plants as control. (B) Seeds of cbl9 empty vector control, and four homozygous CIPK3 (wild-type) over-expressing lines designated as CK3-WT-1, CK3-WT-2, CK3-WT-3, and CK3-WT-4, in the cbl9 mutant background were grown vertically on MS agar medium with or without 0.7 μM ABA, 150 mM NaCl, 375 mM mannitol and photographs were taken after 12 d of growth at standard growth condition. (C) Seed germination analysis after 3 d for four independent cbl9:CIPK3(WT) transgenic lines designated as CK3-WT-1, CK3-WT-2, CK3-WT-3, and CK3-WT-4 in the cbl9 mutant background, empty vector control and cbl9 mutant. Molecular Plant 2008 1, 238-248DOI: (10.1093/mp/ssn003) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 6 Over-Expression of Constitutively Active CIPK3 (T/D CIPK3) in Col-0. (A) RT–PCR (25 cycles) confirmation of four transgenic lines of CIPK3 (T/D CIPK3), CK3–T/D3, CK3–T/D7, CK3–T/D11, CK3–T/D13 in Col-0 in background. (B and C) Vertical growth analysis of Col-0 and four transgenic lines of CIPK3 (T/D CIPK3) CK3–T/D3, CK3–T/D7, CK3–T/D11, CK3–T/D13. Molecular Plant 2008 1, 238-248DOI: (10.1093/mp/ssn003) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 7 A Simplified Model for ABA Signaling in Seed Dormancy. Arrows depict ‘activation’ and intersecting lines indicate ‘inhibition’. See Discussion for detailed description. Molecular Plant 2008 1, 238-248DOI: (10.1093/mp/ssn003) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions