bhaskaraet al. a t dna...jul 24, 2012 · aba (ng g fw-1)-0 2 4 6 8 0 h * * * * aba (ng g fw-1)...
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Bhaskara et al.
T‐DNA mutant Salk ID References
hai1‐1 Salk_142672 Zhang et al., Plant Journal (2012) 69: 667‐678Antoni et al., Plant physiology (2012) 158: 970–980
hai1‐2 Salk_108282 Guo et al., Mol Biol Rep (2010) 37:763–769
aip1‐1 Salk_090738 Lim et al., Plant Science (2012) 187: 83‐88
aip1‐2 CS381494 This study
hai3‐1 Salk_033011 Yoshida et al., Plant Physiology (2006) 140 :115–126,
hab1‐1 Salk_002104 Saez et al., Plant Journal (2004) 37:354‐369
hab2td Salk_000836 This study
ahg1‐3 Salk_095052 Nishimura et al., Plant Journal (2007) 50, 935–949
ahg3‐3 Salk_124564 This study
abi1‐3 (abitd) Salk_038866 This study
abi2‐2 (abi2td) Salk_015166 Yoshida et al., Plant Physiology (2006) 140 :115–126Rubio et al., Plant Physiology (2009) 150,1345‐1355
A
B
WT
hai1‐2
aip1‐1
hai3‐1
LP+RP
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WT hai1‐1 WT aip1‐2
C
Supplemental Figure 1: Clade A PP2C T‐DNA lines.A. Table listing the T‐DNA mutant lines used in this study. References for the first use of each T‐DNA line are
also shown. The ABI1 and ABI2mutants are referred to as abi1td and abi2td to clearly distinguish them from the dominant negative abi1‐1 and abi2‐1mutants.
B. Diagram of T‐DNA insertion sites and RT‐PCR analysis of HAI PP2C T‐DNA mutants. LB and RB:Left and right border of T‐DNA insertion: RP1 and RP2‐ Right genomic primers for 1st and 2nd alleles of the gene respectively: RT1F and RT1R‐ Forward and Reverse RT‐PCR primers used for 1st allele , RT2F and RT2R‐Forward and Reverse RT primers used for 2nd allele. Primer sequences are given in Supplemental Table 9. Numbers above the T‐DNA insertion indicates chromosome coordinates adjacent to the T‐DNA flanking sequence verified by sequencing the left T‐DNA border.
C. Example data of PCR genotyping of the hai1‐2aip1‐1hai3‐1 triple mutant (top) and hai1‐1 and aip1‐2 single mutants (bottom panels). LP+RP gene specific primers were used to amplify the gene fragment without insertion and LBb+RP primers were used to amplify the left T‐DNA border and flanking sequence.
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Supplemental Figure 2: Low water potential‐induced Pro accumulation HAI PP2C double and triple mutants.Single mutant data is from Figure 1 of the main text and is shown here for comparison. Experimental details and data presentation are as described for Figure 1 of the main text.
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Supplemental Figure 3: Salt stress‐induced Pro accumulation HAI PP2C double and triple mutants. Seven‐day‐old seedlings were transferred from control media to media containing the indicated concentrations of NaCl and Pro measured 96 h after transfer. Data are means ± S.E. (n= 9‐12) from three independent experiments. Asterisks indicate signficantdifference between mutant and Col wild type (p ≤ 0.05). Water potentials (w) of the different treatments are: 0 NaCl = ‐0.25 MPa; 50 mM NaCl = ‐0.5 MPa; 100 mM NaCl = ‐0.75 MPa; 150 mM NaCl = ‐1.0 MPa (Verslues and Sharma, 2010 Plant Cell & Env. 33: 1838‐1851) Note that salt stress elicits a lower level of Pro accumulation than an equivalent ѱw imposed by PEG or soil drying since there is less need for osmotic adjustment. This is because of ion uptake and thus the salt stress function of Pro accumulation is more related to ion toxicity than osmoregulation (Sharma and Verslues, 2010).
-1.5-1.2-0.9-0.6-0.3
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Supplemental Figure 4: Osmotic potential and seedling fresh weight of hai1, aip1 and hai3 double and triple mutants after low w treatment.A. Osmotic potential (sof single, double and triple mutants of the HAI PP2Cs.
Single mutant data is from figure 2 of the main text and is shown here for comparison to the double and triple mutant data. Data are means of measurements from three independent experiments and significant differences (p ≤ 0.05) are marked by *.
B. Fresh weight of double and triple mutants of the HAI PP2Cs. Data are means from three independent experiments. No significant differences were detected.
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hai1‐2 Col
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Supplemental Figure 5: Wild type (Col) and hai1‐2plants during soil drying.Representative plants from experiments to measure osmotic potential and relative water content (Figure 3 of main text) were photographed at the indicated times after the start of water with‐holding. Plants were 30 days old at the start of water with‐holding (Day 0).
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Supplemental Figure 6: Co‐expression clusters formed from genes down regulated in hai1‐2 relative to Col wild type.Dotted blue line indicates the coexpression cluster shown in Figure 4 of the main text.
Supplemental Figure 7: Quantitative PCR analysis of proline metabolism genes shows similar expression in wild type and hai1‐2.Expression of proline metabolism genes was measured at 0, 10 and 96 h after transfer of seedlings from control (‐0.25 MPa) to low w (‐1.2 MPa) using Taqman probe assays as previously described (Sharma and Verslues, 2010 Plant Cell & Env. 33: 1838‐1851). Data are means ± S.E. (n = 3‐4) and significant differences (p ≤ 0.05) are marked with an asterisk (*).
Abbreviations: P5CS1 = 1‐pyrroline‐5‐carboxylate synthetase 1 (At2g39800); P5CR = 1‐pyrroline‐5‐carboxylate reductase (At5g14800); PDH1 = proline dehydrogenase (At3g30775); P5CDH = 1‐pyrroline‐5‐caroxylate dehydrogenase (At5g62530)
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Supplemental Figure 8: Confirmation of ABA hypersensitive germination in mutants of the ABA‐signaling Clade A PP2Cs.Seed germination across a range of ABA concentrations for Col wild type and mutants of the ABA‐signaling Clade A PP2Cs. Germination was scored based on radicle emergence 4 days after the end of stratification. Data are means ± S.E. (n = 3‐4) from 3 independent experiments.
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Supplemental Figure 9: ABA induced Pro accumulation does not differ in HAI PP2C single mutants and is only slight increased in the triple mutant.Pro accumulation was measured 96 h after transfer of seven‐day‐old seedlings to plates containing the indicated ABA concentrations. Data are means ± S.E. (n = 4‐6) from 3 independent experiments.
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Supplemental Figure 10: Low water potential‐induced ABA accumulation of Clade A PP2C mutants.ABA contents were measured 0, 10 or 96 h after transfer of seedlings to ‐1.2 MPa. ABA quantification was performed by GC‐MS/MS (See Methods). Reduced ABA accumulation was observed in hab1‐1 and ahg3‐3, consistent with previous reports of reduced ABA accumulation in triple mutants of the ABA‐signaling PP2Cs. In contrast no reduction in ABA content was seen for the HAI PP2Cs and a significant increase was seen in the triple mutant at 96 h. Data are means ± S.E. (n = 5‐9) combined from 2 or 3 independent experiments. Significant differences (p ≤ 0.05) of mutant compared to wild type are marked (*).
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Supplemental Figure 11: Alignment of the C‐terminal regions containing the active site and PYL interaction sites of the HAI PP2Cs, ABI1, ABI2 and HAB1 shows sequence differences which may affect PYL interaction and substrate specificity of the HAI PP2Cs.See complete figure legend on next page.
Amino acids directly involved in PYL interaction of ABI1, ABI2 and HAB1.PYL‐interacting amino acids not conserved in HAI PP2Cs
Amino acids involved in H‐bonding between ABI1, ABI2 or HAB1 and PYLTrp which contacts ABA in the PYL binding pocketABA box involved in SnRK2 interaction PYL‐PP2C Van der Waals contact in ABI1 and ABI2
abi1‐1/abi2‐1 dominant negative mutation SnRK3 interaction domain
Continued from previous page.Supplemental Figure 11: Alignment of the C‐terminal regions containing the active site and PYL interaction sites of the HAI PP2Cs, ABI1, ABI2 and HAB1 shows sequence differences which may affect PYL interaction and substrate specificity of the HAI PP2Cs.
Alignment of C‐terminal regions of the HAI1 PP2Cs, HAB1, ABI1 and ABI2 containing the active site and PYL/SnRK2 interacting regions. Residues conserved among HAB1, ABI1 and ABI2 but different in the HAI PP2Cs are shaded in black. Residues conserved among the HAI PP2Cs but divergent in HAB1, ABI1 or ABI2 are shaded in red. The amino acid altered in dominant negative (abi1‐1 and abi2‐1) mutants is indicated by red triangle (Sheen 1998). Amino acids shown by structural studies to be involved in PYL binding are indicated by open red circles above the sequence and those directly involved in hydrogen bonding indicated by green dots below the sequence (Yin et al., 2009, Dupeuxet al., 2011). Green shading indicates the ABA box involved in tethering of SnRK2s to HAB1 (Soon et al., 2012).
The HAI1 PP2Cs differ from ABI1, ABI2 and HAB1 in several of the amino acids directly involved in PYL binding (these differences are indicated by filled red dots above the sequence). This includes the Phe306 (red star) which is involved in PYL‐PP2C van der Waals contact; and Thr239 and Asn301 of ABI1 necessary for intermolecular hydrogen bonding with PYL1 (Yin et al., 2009, Dupeux et al., 2011). Several of these changes surround the critical Trp300 of ABI1 (indicated by purple arrow) which contacts ABA in the PYL binding pocket. Also, several amino acids of the ABA box are not conserved in the HAI PP2Cs.
Together these differences likely explain the specific PYL interaction pattern of the HAI PP2Cs and the limited PYL interaction of HAI1. These differences also suggest different SnRK2 interaction as the PYLs and SnRK2s mimic each other in PP2C binding (Soon et al., 2012). In addition, the motif which determines SnRK3 interaction specificity of ABI1 and ABI2 (particularly Ala197 and Ala201; Ohta et al., 2003) is not conserved in the HAI PP2Cs.
References:Dupeux F, Antoni R, Betz K, Santiago J, Gonzalez‐Guzman M, Rodriguez L, Rubio S, Park SY, Cutler SR, Rodriguez PL, Marquez JA (2011) Modulation of Abscisic Acid Signaling in Vivo by an Engineered Receptor‐Insensitive Protein Phosphatase Type 2C Allele. Plant Physiol 156: 106‐116
Ohta M, Guo Y, Halfter U, Zhu JK (2003) A novel domain in the protein kinase SOS2 mediates interaction with the protein phosphatase 2C ABI2. Proc Natl Acad Sci U S A 100: 11771‐11776
Sheen J (1998) Mutational analysis of protein phosphatase 2C involved in abscisic acid signal transduction in higher plants. Proc Natl Acad Sci U S A 95: 975‐980
Soon F‐F, Ng L‐M, Zhou XE, West GM, Kovach A, Tan MHE, Suino‐Powell KM, He Y, Xu Y, Chalmers MJ, Brunzelle JS, Zhang H, Yang H, Jiang H, Li J, Yong E‐L, Cutler S, Zhu J‐K, Griffin PR, Melcher K, XuHE (2012) Molecular Mimicry Regulates ABA Signaling by SnRK2 Kinases and PP2C Phosphatases. Science 335: 85‐88
Yin P, Fan H, Hao Q, Yuan X, Wu D, Pang Y, Yan C, Li W, Wang J, Yan N (2009) Structural insights into the mechanism of abscisic acid signaling by PYL proteins. Nature Struct Molec Biol 16: 1230‐1236
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Supplemental Figure 12: Additional yeast two hybrid and western blot assays with HAB1 and HAI1.A. Quantitative yeast two hybrid assay comparing PYL5, PYL8, PYL9 and PYL10
interaction of HAI1 (full length) and HAB1 (N‐terminal deletion) over a range of ABA concentrations. Data are means ± S.D. from at least three independent yeast colonies.
B. Quantitative yeast two hybrid assay of HAI1 N‐terminal deletion interaction with PYL5, PYL8, PYL9 and PYL10.
C. Western blot using an antibody against the GAL4 DNA binding domain (GBD) to detect the PP2C fusion proteins in yeast. MaV203 is the untransformed yeast cells and “No PYL” indicates cells transformed with the GBD PP2C bait construct but not yet transformed with PYL activation domain prey construct. GBD/MaV203 cells transformed with PRY1 and PYL8 activation domain constructs are also shown. Ponceau red staining of the membrane is shown as a loading control. The HAI1 construct was consistently expressed at levels similar to the HAB1 construct indicating that the weaker interactions of HAI1 were not caused by a lower level of protein expression.
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