PYR/PYL/RCAR family members are major in-vivo ABI1 protein phosphatase 2C-interacting proteins in Arabidopsis
Version of Record online: 9 NOV 2009
© 2009 The Authors. Journal compilation © 2009 Blackwell Publishing Ltd
The Plant Journal
Volume 61, Issue 2, pages 290–299, January 2010
How to Cite
Nishimura, N., Sarkeshik, A., Nito, K., Park, S.-Y., Wang, A., Carvalho, P. C., Lee, S., Caddell, D. F., Cutler, S. R., Chory, J., Yates, J. R. and Schroeder, J. I. (2010), PYR/PYL/RCAR family members are major in-vivo ABI1 protein phosphatase 2C-interacting proteins in Arabidopsis. The Plant Journal, 61: 290–299. doi: 10.1111/j.1365-313X.2009.04054.x
- Issue online: 8 JAN 2010
- Version of Record online: 9 NOV 2009
- Received 3 August 2009; revised 2 October 2009; accepted 8 October 2009; published online 9 November 2009.
Figure S1. Identification of ABI1-interacting proteins in Arabidopsis. YFP–ABI1 and associated proteins purified from stable transgenic Arabidopsis plant lines expressing YFP–ABI1 or control YFP in the abi1-3 T-DNA mutant background treated without ABA (a) and with exogenous ABA (b). Eluates from YFP control and YFP–ABI1 purifications were analysed and visualized by silver staining. Proteins were subsequently identified by LC-MS/MS. Stars indicate predicted YFP and YFP–ABI1 bands. Arrow heads: Two of the major protein bands associated with YFP–ABI1 show molecular weights similar to SnRK2s and Bet VI protein family members.
Figure S2. ABA-induced interaction between ABI1 and PYR1 in Arabidopsis. Total protein extracts (Input) from YFP–ABI1 and YFP plants that were grown on MS plates for 3 weeks. Plants were incubated for 2 h in water (−ABA samples). After pre-incubation for 2 h in water, 3-week-old plants were treated with 100 μm ABA for 48 h (+ABA samples). After co-immunoprecipitation using anti-GFP beads, input and immunoprecipitated samples were detected with anti-GFP and anti-PYR1 antibodies. The PYR1, ABI1–YFP, YFP and ACTIN bands are indicated by triangles. Non-specific bands are indicated by closed stars.
Figure S3. Tandem mass spectrum of peptides from proteins ABIP8 and ABIP11 listed in Tables S3 and S4 that showed one unique sequence to the corresponding proteins. The protein identifications were further supported by identified peptides that, even though their sequence are non-unique to proteins ABIP8 (a) and ABIP11 (b), non-unique peptides were not found within that of any other identified ABI1-interacting proteins that passed the DTASelect filter; thus providing further support of their link to the ABIP8 and ABIP11 proteins. Such non-unique peptide identifications (see Table S1) were fully tryptic which provides additional confidence in the correctness of the sequence match (data not shown).
Figure S4. Transcription profiles of ABI1, PYR1/PYLs and SnRK2s in seedlings, guard cells and mesophyll cells. (a) 21-day-old seedlings, (b) 7-day-old seedlings grown on liquid medium treated with or without ABA (c) Guard cells and mesophyll cells isolated from plants treated with or without ABA. Data were obtained from (Leonhardt et al., 2004; Goda et al., 2008; Yang et al., 2008).
Figure S5. ABA-induced stomatal closure is impaired in pyr1pyl1pyl2pyl4 quadruple mutant plants. ABA-induced stomatal closure (20 or 50 μm [ABA]) in pyr1pyl1pyl2pyl4 quadruple mutant and wild type abaxial leaf epidermes treated with the indicated ABA concentrations for 1 h (n = 3 experiments, 30 stomata per experiment and condition; genotype and [ABA] blind experiments). Experiments in Figure S5 were performed in independent experiments from those shown in Figure 5a, confirming the strong ABA insensitivity.
Table S1. List of candidate ABI1-interacting proteins in YFP–ABI1 plants derived from LC-MS/MS data.
Table S2. List of candidate YFP-interacting proteins in control YFP plants derived from LC-MS/MS data.
Table S3. The number of unique and total peptides and spectrum count of candidate ABI1-interacting proteins co-purified with ABI1 from Arabidopsis plants in the absence of exogenous ABA.
Table S4. The number of unique and total peptides and spectrum count of candidate ABI1-interacting proteins co-purified with ABI1 from plants exposed to exogenous ABA.
Table S5. The unique sequence coverage of known ABA signalling proteins co-purified with ABI1 from Arabidopsis plants without addition of exogenous ABA.
Table S6. The unique sequence coverage of known ABA signalling proteins co-purified with ABI1 from Arabidopsis plants exposed to exogenous ABA.
Table S7. The unique sequence coverage of candidate ABI1-interacting proteins co-purified with ABI1 from Arabidopsis plants in the absence of exogenous ABA.
Table S8. The unique sequence coverage of candidate ABI1-interacting proteins co-purified with ABI1 from plants exposed to exogenous ABA.
Experimental procedures S1.
Purification of YFP–ABI1 interacting proteins by affinity column purification methods (Nishimura et al.).
Multidimensional protein identification technology (MudPIT).
Following digestion, proteins were pressure-loaded onto a fused silica capillary desalting column containing 3 cm of 5-μm strong cation exchange (SCX) followed by 3 cm of 5-μm C18 (reverse phase or RP material) pressure packed into a non-deactivated 250-μm inner diameter (i.d.) capillary. To complete sample assembly a 100-μm i.d. capillary consisting of a 10-μm laser pulled tip packed with 10 cm 3-μm Aqua C18 material (Phenomenex, Ventura, CA, USA) was attached to the filter union (desalting column–filter union–analytical column). The resulting split-columns were placed in-line with a ThermoFinnigan Surveyor MS Pump (Version 2.3; Palo Alto, CA, USA) and analysed using a customized four-step separation method (90, 120, 120 and 150 min respectively) (Diop et al., 2008).
Analysis of tandem mass spectra.
PYR1 expression and antiserum preparation.
Construction of the erecta+ pyr1/pyl1/pyl2/pyl4 quadruple mutant.
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|TPJ_4054_sm_628-09R1SchroederSMdataclean.doc||87K||Supporting info item|
|TPJ_4054_sm_Nishimura_et_al_Suppl_Figs_Revised.pdf||850K||Supporting info item|
|TPJ_4054_sm_Nishimura_et_al_TableS1.xls||16664K||Supporting info item|
|TPJ_4054_sm_Nishimura_et_al_TableS2.xls||8851K||Supporting info item|
|TPJ_4054_sm_SIFigurelegends-clean.doc||24K||Supporting info item|
|TPJ_4054_sm_TableS3-S8clean.doc||310K||Supporting info item|
Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.