Cochaperonin CPN20 negatively regulates abscisic acid signaling in Arabidopsis

Previous study showed that the magnesium-protoporphyrin IX chelatase H subunit (CHLH/ABAR) positively regulates abscisic acid (ABA) signaling. Here, we investigated the functions of a CHLH/ABAR interaction protein, the chloroplast co-chaperonin 20 (CPN20) in ABA signaling in Arabidopsis thaliana. We showed that down-expression of the CPN20 gene increases, but overexpression of the CPN20 gene reduces, ABA sensitivity in the major ABA responses including ABA-induced seed germination inhibition, postgermination growth arrest, promotion of stomatal closure and inhibition of stomatal opening. Genetic evidence supports that CPN20 functions downstream or at the same node of CHLH/ABAR, but upstream of the WRKY40 transcription factor. The other CPN20 interaction partners CPN10 and CPN60 are not involved in ABA signaling. Our findings show that CPN20 functions negatively in the ABAR-WRKY40 coupled ABA signaling independently of its co-chaperonin role, and provide a new insight into the role of co-chaperones in the regulation of plant responses to environmental cues. Electronic supplementary material The online version of this article (doi:10.1007/s11103-013-0082-8) contains supplementary material, which is available to authorized users.

Immunoprecipitation experiments were performed following the manufacturer's protocol. Cell lysates were pre-cleared with protein A/G Plus-agarose beads (Santa Cruz Biotechnology) and incubated with the anti-HA serum and the protein A/G Plus-agarose beads at 4℃ overnight in the extraction buffer. The beads were washed twice extensively with buffer A containing 50 mM Tris (pH 8.0), 150 mM NaCl, and 0.1% (V/V) Triton X-100 and buffer B containing 50 mM Tris (pH 8.0), and 0.1 % (V/V) Triton X-100 and then re-suspended in SDS-PAGE sample buffer. The immuno-precipitates were separated on a 12% SDS-PAGE and analyzed by immunoblotting with anti-MYC serum. For immunoprecipitation in Arabidopsis extracts, the total protein was re-suspended in the extraction buffer (3 mg/mL) containing 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1 % (V/V) Triton X-100, 10% (V/V) glycerol, 1 mM PMSF, and 1 μg/mL cocktail (Merck). The immunoprecipitation was done with the same procedures as described above except that the anti-ABAR or anti-CPN20 serum was used instead of the anti-Myc or anti-HA serum, and the beads were washed with the extraction buffer instead of the buffer A and buffer B.

Luciferase complementation imaging (LCI)
Luciferase complementation imaging (LCI) assay was used to detect protein-protein interaction in N. benthamiana leaves according to previously described procedures (Chen et al., 2008;Shang et al., 2010). The firefly Luc enzyme is divided into the N-terminal part (NLuc) and C-terminal part (CLuc). ABAR was fused with NLuc in pCAMBIA-NLuc vector, and CPN20, CPN20ΔNS or WRKY40 was fused with CLuc in pCAMBIA-CLuc vector respectively. Primers used for the vector construction were shown in Table S1. The constructs were mobilized into A. tumefaciens GV3101. Bacteria were suspended in infiltration buffer (0.2 mM acetosyringone, 10 mM MgCl 2 , and 10 mM MES) to identical concentrations (OD 600 = 0.6). Equal concentrations and volumes of bacteria were mixed and co-infiltrated into the 7-week-old N. benthamiana leaves using needleless syringes. After infiltration, plants were placed with 16 h-light/8 h-dark for 48 h at 24℃. The Luc activity was observed with a low-light cooled CCD imaging apparatus (Andor iXon). The mouse anti-full-length firefly Luc antibody (Santa Cruz Biotechnology) was used to immunodetect Luc fusion protein in transgenic tissues. All experiments were repeated at least five independent biological replicates.

Real-time PCR analysis
Total RNA was isolated from 10 day-old seedlings using a Total RNA Rapid Extraction Kit (BioTeke), treated with RNase-free DNase I (TAKARA) at 37℃ for 30 min to degrade genomic DNA and purified by using an RNA Purification Kit (BioTeke). A 2-μg aliquot of RNA was subjected to first-strand cDNA synthesis using M-MLV reverse transcriptase (Promega), and an oligo (dT21) primer. The primers of various ABA-responsive genes used for real-time PCR are listed in Table S1. Analysis was performed using the BioRad Real-Time System CFX96TM C1000 Thermal Cycler (Singapore). All experiments were repeated at least three times along with three independent repetitions of the biological experiments.

Phenotypic analysis
For the germination assay, about 100 seeds each from wild-type plants and mutants or transgenic lines were sterilized and planted in triplicate on MS medium. The medium contained 3% sucrose and 0.8% agar (pH5.9) and was supplemented with or without different concentrations of (±)ABA. The seeds were incubated at 4 for 3 days ℃ and placed at 21 under light conditions. Germination (emergence of radicals) was scored at the indicated times. ℃ For the seedling growth assay, the seeds were planted directly in ABA-containing MS medium or transferred 48 hours after stratification from the common MS medium to medium supplemented with different concentrations of ABA. Images were taken after 12 to14 days. For stomatal aperture assay, rosette leaves were used. To observed ABA-induced stomatal closure, leaves were immersed in solution containing 50 mM KCl and 10 mM MES-KOH (pH6.15) and exposed to a halogen cold light source for 3 hours. Subsequently, different concentrations of (±)ABA were added to the solution. Stomatal apertures were measured after 2.5 hours' ABA treatment. To study ABA-inhibited stomatal opening, leaves were immersed in the same solution in the dark for 3 hours before they were transferred to the cold light for 2.5 hours in the presence of ABA, and then apertures were recorded. For water loss assay, rosette leaves were detached and placed on filter paper. Water loss was evaluated by weighing excised leaves at the indicated times. For drought treatment, plants were grown on soil for about 2 weeks, and then drought was imposed by withdrawing irrigation until the lethal effect of dehydration was observed on the majority of the plants. Plants grown under a standard irrigation regime were used as a control.
We further tested whether the interaction partner of CPN20, CPN60 chaperonin, is involved in ABA signaling. Plant CPN60 includes two classes of subunits, CPN60α and CPN60β (Hill and Hemmingsen, 2001), in which Arabidopsis CPN60α is composed of two homologues, CPN60α1 and CPN60α2, and CPN60β of four homologues, CPN60β1, CPN60β2, CPN60β3 and CPN60β4 (Hill and Hemmingsen, 2001). These CPN60 subunits form different complex to function as molecular chaperons (Bonshtien et al., 2009). Previous reports showed that CPN60α1 is expressed at higher level than CPN60α2 (Weiss et al., 2009), and disruption mutation of CPN60α1 is lethal (Suzuki et al., 2009). We screened one T-DNA insertional knock-down mutant of CPN60α1 gene (SALK_082308), named cpn60α1, which shows developmental defects such as small size of seedlings and advanced flowering time (Fig. S11, A-C), but wild-type ABA responses (Fig. S11, D and E). We could not measure stomatal response to ABA in this mutant because of its too small size of mature leaves (Fig. S11C). In addition, we observed that CPN60α1 does not interact with ABAR (Fig. S11F). Given that the different α and β subunits of CPN60 form complexes to execute their functions in cells (Bonshtien et al., 2009), these data support the idea that CPN60 is not involved in ABA signaling. Arabidopsis total protein was immuno-blotted by anti-CPN20 serum and preimmune serum, showing that the anti-CPN20 serum recognizes specifically a 26-kD protein (CPN20). Mr, molecular mass markers; kD, kilodalton. The experiments were repeated three times with the same results. (B) Control images for GFP vector and mCherry vector: the green or red fluorescence are mainly distributed in cytosolic space, which is different from that of GFP-tagged CPN20 or mCherry-tagged chloroplast markers. The experiments were repeated three times with the same results. (C) Transient expression in Arabidopsis protoplasts shows that CPN20-mCherry and ABAR-GFP partly co-localize into the stroma and especially peripheral areas of the chloroplast (yellow areas). Bright, bright field; Merged, merged image of CPN20-mCherry and ABAR-GFP in bright field. The experiments were repeated three times with the same results.

Supplementary References
Supplementary Fig. 4. Identification of the cpn20 Mutants and CPN20-Overexpression Lines.
(A) T-DNA insertion sites in the three mutant alleles cpn20-1, cpn20-2 and cpn20-3 (with Col ecotype as background). Blue boxes and grey lines represent exons and introns, respectively. The T-DNA insertion sites are indicated by open arrowheads, and nt represents nucleotides. In the cpn20-1 mutant allele, the T-DNA was inserted into the promoter region at nt -393 to -376 upstream of the start codon ATG of CPN20 with an 18-bp fragment deleted. In the cpn20-2 mutant allele, the T-DNA was inserted into the promoter region at nt -301 to -265 upstream of the start codon ATG of CPN20 with a 37-bp fragment deleted. In the cpn20-3 mutant, the T-DNA was inserted into the fifth exon at nt 667 to 681 downstream of the start codon ATG of CPN20 with a 15-bp fragment deleted. (B) and (C) Real-time PCR and immunoblotting analysis of CPN20 expression in wild-type Col, homozygous mutants cpn20-1 and cpn20-2, and five CPN20-overexpression lines (OE2, OE3, OE4, OE5, and OE7). For the real-time PCR analysis, the value obtained from the 4-week old Col seedling after stratification was taken as 100%, and all the other values were normalized relative to this value. Each value for real-time PCR is the mean ± SE of three independent biological determinations. Immunoblotting was performed with anti-CPN20 serum in the total protein extracted from the leaves of the seedlings grown for 4 weeks after stratification. Actin was used as a loading control. The immunoblotting assays were repeated three times with the independent biological experiments, which gave the similar results. Supplementary Fig. 5. Quantification of Root Growth in Different Mutants and Transgenic Lines. Seedlings were transferred from the ABA-free medium to the medium supplemented with 0 or 5 μM (±)ABA, and the primary root length was measured 10 days after the transfer. (A) The statistics for the cpn20 mutants and CPN20-overexpression transgenic lines as described in Fig. 2C. (B) The statistics for the cpn20-1, cch single mutants and cpn20-1 cch (cpn20/cch) double mutant as described in Fig. 4C. (C) The statistics for the wrky40 mutant, the CPN20-overexpression (OE-CPN20) line in the Col background [OE-CPN20 (Col)], the OE-CPN20 lines in the wrky40 mutant background (OE-CPN20 (wrky40)) and OE-CPN20 wrky40 double mutant (OE-CPN20/wrky40) as described in Fig. 6B. (D) The statistics for the cpn20-1 mutant and ABAR-RNAi lines in the cpn20-1 mutant background [ABARi L1/L2 (cpn20-1)] as described in Fig. 4C. Each value in (A) to (D) is the mean ±SE of at least 30 seedlings. Supplementary Fig. 6. Status of the Detached Leaves of the Different Genotypes in a Water Loss Assay. The plants of wild-type Col, cpn20-1 mutant, two CPN20-overexpressing lines (OE2 and OE3) and abi2-1 mutant were subjected to a 6-hour period water loss assay as described in Fig. 2F. The experiment was repeated five times with similar results. Seeds were directly planted in the ABA-free or ABA-containing medium, and seedling growth was investigated 10 days after stratification. (F) Co-immunoprecipitation (Co-IP) assays in plants: ABAR was not co-immuno-precipitated with CPN60α. The anti-CPN60α1 serum was used in the Co-IP assay in the total protein from the wild-type Col plants. Immuno-precipitates (IP: anti-CPN60α1) was immuno-blotted with anti-ABAR serum (Blot: anti-ABAR). IP with preimmune serum was taken as a negative control.