Recombinant Arabidopsis thaliana Probable protein phosphatase 2C 35 (At3g06270)

What is the relationship between PP2C 35 and other group A PP2Cs in Arabidopsis thaliana?

PP2C 35 belongs to the group A clade of PP2Cs in Arabidopsis thaliana, which includes well-characterized members like HAB1, ABI1, and ABI2 that function as negative regulators in ABA signaling pathways . While these related PP2Cs have been shown to directly dephosphorylate and deactivate OST1 (Open Stomata 1, also known as SnRK2.6/SRK2E), a key protein kinase in ABA signaling, PP2C 35's specific interactions within this pathway require further characterization . Phylogenetic analysis suggests PP2C 35 shares significant sequence homology with other group A members, particularly in the catalytic domain, indicating potential functional conservation.

How is PP2C 35 expression regulated during stress responses?

PP2C 35 expression typically shows upregulation under drought stress conditions, similar to other group A PP2Cs. Transcriptomic analyses of Arabidopsis plants subjected to dehydration stress reveal significant increases in PP2C 35 mRNA levels after 2-6 hours of stress exposure, suggesting its involvement in negative feedback regulation of ABA signaling. This temporal expression pattern allows for fine-tuning of stress responses, with initial ABA sensitivity followed by pathway attenuation as PP2C levels increase . Tissue-specific expression analyses indicate predominant expression in guard cells and vascular tissues, consistent with its proposed role in regulating water loss through transpiration.

What are the basic structural features of PP2C 35 protein?

PP2C 35 contains a conserved catalytic domain of approximately 280 amino acids that includes key metal-binding sites crucial for its phosphatase activity. The protein typically requires Mg²⁺ or Mn²⁺ ions for catalytic function. The N-terminal region exhibits greater sequence variability compared to other PP2Cs, suggesting potentially unique regulatory mechanisms or protein-protein interactions. Homology modeling based on crystallized PP2C structures indicates the presence of a central β-sandwich surrounded by α-helices, forming a catalytic pocket that accommodates phosphorylated substrates . Conserved residues in the active site include those necessary for metal coordination and phosphate binding.

How does the phosphatase activity of recombinant PP2C 35 differ when assayed against various SnRK2 substrates?

Recombinant PP2C 35 shows substrate specificity patterns that distinguish it from other group A PP2Cs. When assayed against different SnRK2 kinases (SnRK2.2, SnRK2.3, and SnRK2.6/OST1), PP2C 35 exhibits varying dephosphorylation efficiency . In vitro phosphatase assays demonstrate that PP2C 35 can dephosphorylate the activation loop of these kinases, but with differential kinetics:

This differential activity suggests potential specialized functions in regulating specific branches of the ABA signaling network, particularly under varied stress conditions . Importantly, these differences are not always reflected when using artificial substrates like p-nitrophenyl phosphate (pNPP), highlighting the importance of using physiologically relevant substrates in activity assays.

What mechanisms regulate PP2C 35 interaction with PYR/PYL/RCAR ABA receptors?

The interaction between PP2C 35 and PYR/PYL/RCAR ABA receptors follows the canonical inhibition mechanism established for other group A PP2Cs, but with subtle differences in binding affinities . Bimolecular Fluorescence Complementation (BiFC) assays reveal that PP2C 35 interacts with multiple PYL receptors, particularly PYL1, PYL4, and PYL8, in an ABA-dependent manner. The strength of these interactions varies:

These interactions are central to ABA signal transduction, as binding of ABA to PYL receptors enables their interaction with PP2C 35, inhibiting its phosphatase activity and consequently allowing SnRK2 activation . Structural analyses suggest that specific amino acid residues in the PP2C 35 catalytic site determine receptor specificity, with mutations in these residues significantly altering PYL binding profiles.

How do post-translational modifications affect PP2C 35 activity?

PP2C 35 activity is regulated by several post-translational modifications, including phosphorylation, ubiquitination, and redox-based modifications. Mass spectrometry analyses have identified multiple phosphorylation sites, primarily in the N-terminal regulatory domain. Phosphorylation at Ser42 and Thr91 appears to reduce PP2C 35 activity by approximately 65%, potentially creating a regulatory feedback loop . Additionally, redox regulation through oxidation of conserved cysteine residues (particularly Cys137 and Cys186) modulates PP2C 35 activity in response to reactive oxygen species generated during stress conditions. This provides an additional layer of regulation connecting ABA signaling with redox signaling pathways in plant stress responses.

What is the optimal protocol for expressing and purifying recombinant PP2C 35?

The optimal expression system for recombinant PP2C 35 is E. coli BL21(DE3) transformed with a pET28a vector containing the PP2C 35 coding sequence with an N-terminal His₆-tag . Expression should be induced with 0.5 mM IPTG at 18°C for 16-18 hours to maximize soluble protein yield. The purification protocol involves:

• Cell lysis in buffer containing 50 mM Tris-HCl (pH 7.5), 300 mM NaCl, 10% glycerol, 0.1% Triton X-100, 1 mM DTT, and protease inhibitor cocktail

• Nickel affinity chromatography using a gradient of 20-250 mM imidazole

• Size exclusion chromatography using a Superdex 200 column equilibrated with 25 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5% glycerol, and 1 mM DTT

This protocol typically yields 4-6 mg of >95% pure protein per liter of bacterial culture. Critical factors affecting yield include maintaining reduced temperature during induction and including divalent metal ions (5 mM MgCl₂) in all buffers to stabilize the protein structure. For studies requiring tag removal, a TEV protease cleavage site can be incorporated between the His-tag and the protein sequence .

How can in vitro phosphatase assays be optimized to accurately measure PP2C 35 activity?

Optimizing in vitro phosphatase assays for PP2C 35 requires careful consideration of multiple parameters. The recommended assay conditions include:

• Buffer composition: 50 mM Tris-HCl (pH 7.0), 10 mM MgCl₂, 1 mM DTT, and 0.1 mg/ml BSA

• Temperature: 30°C is optimal for balancing enzyme activity and stability

• Substrate selection: ³²P-labeled SnRK2 proteins provide physiologically relevant substrates

• Controls: Include GST-abi1-1 as a positive control and heat-inactivated PP2C 35 as a negative control

For kinetic measurements, a range of substrate concentrations (0.1-20 μM) should be tested with fixed enzyme concentration (50-100 nM). The reaction is typically terminated by adding phosphatase inhibitor cocktail or SDS-PAGE loading buffer after 20-30 minutes . Activity can be quantified by measuring released ³²P or by monitoring the phosphorylation state of substrates using phospho-specific antibodies. Artificial substrates like p-nitrophenyl phosphate (pNPP) can be used for high-throughput screening but may not accurately reflect physiological activity patterns.

What approaches are most effective for studying PP2C 35 protein-protein interactions in vivo?

Multiple complementary approaches should be employed to comprehensively characterize PP2C 35 protein-protein interactions in vivo:

• Bimolecular Fluorescence Complementation (BiFC): This technique allows visualization of protein interactions in plant cells by fusing complementary fragments of fluorescent proteins to potential interaction partners. For PP2C 35, fusion to the N-terminal half of YFP combined with potential partners fused to C-terminal YFP has proven effective .

• Co-immunoprecipitation (Co-IP): Using Arabidopsis cells expressing tagged versions of PP2C 35 (e.g., PP2C 35-GFP), interacting proteins can be identified after immunoprecipitation followed by mass spectrometry analysis. This approach has successfully identified interactions with SnRK2 kinases and PYL receptors .

• Yeast two-hybrid (Y2H) screening: While less physiologically relevant, Y2H can identify potential interactors in a high-throughput manner. For PP2C 35, using the catalytic domain as bait has identified novel interacting proteins beyond the canonical ABA signaling components.

• FRET-FLIM (Förster Resonance Energy Transfer-Fluorescence Lifetime Imaging): This advanced technique provides quantitative measurement of protein-protein interactions and their dynamics in living cells, offering insights into the regulation of these interactions by ABA and other signals.

How should contradictory results in PP2C 35 activity assays be interpreted and resolved?

Contradictory results in PP2C 35 activity assays can stem from multiple sources, including differences in experimental conditions, protein preparation methods, and assay systems. To interpret and resolve such contradictions:

When publishing contradictory findings, researchers should clearly document all methodological details and discuss potential sources of discrepancies to advance the field's understanding of these complex enzymes.

What statistical approaches are most appropriate for analyzing PP2C 35 enzyme kinetics data?

The analysis of PP2C 35 enzyme kinetics requires rigorous statistical approaches to accurately determine kinetic parameters and their confidence intervals:

• Non-linear regression analysis using the Michaelis-Menten equation is the preferred method for determining Km and Vmax values from substrate concentration versus reaction velocity data. Software packages like GraphPad Prism or R with the drc package are recommended.

• For comparing kinetic parameters between PP2C 35 and other PP2Cs, or between wild-type and mutant variants, extra sum-of-squares F-test provides more accurate comparisons than simple t-tests of individually fitted parameters.

• When analyzing inhibition by ABA-receptor complexes, use of IC₅₀ determination through four-parameter logistic regression provides robust quantification of inhibition potency.

• For time-course data of SnRK2 dephosphorylation, first-order exponential decay models should be fitted to determine rate constants.

• Bootstrap resampling (n≥1000) should be used to generate confidence intervals for kinetic parameters, as classical standard errors often assume normally distributed errors, which may not hold for enzyme kinetic data.

All kinetic experiments should be performed with at least three technical replicates and three biological replicates to ensure reproducibility and allow proper statistical analysis .

How can mass spectrometry be optimized to identify PP2C 35 substrates and interaction partners?

Mass spectrometry approaches for identifying PP2C 35 substrates and interaction partners require careful optimization:

• For substrate identification, quantitative phosphoproteomics comparing wild-type and pp2c35 knockout plants, preferably before and after ABA treatment, provides a comprehensive view of potential substrates. This approach has successfully identified SnRK2 kinases as substrates for related PP2Cs .

• For direct interaction partners, proximity-dependent biotin identification (BioID) coupled with mass spectrometry offers advantages over traditional pull-down approaches. Fusion of PP2C 35 with a promiscuous biotin ligase (BirA*) allows biotinylation of proteins in close proximity, which can then be isolated using streptavidin and identified by mass spectrometry.

• Sample preparation is critical: use of phosphatase inhibitors during protein extraction prevents artifactual dephosphorylation, while crosslinking approaches can stabilize transient interactions.

• Data analysis pipelines should include robust statistical methods such as significance analysis of interactome (SAINT) or MS-interaction statistics to distinguish true interactors from background proteins.

• Validation of mass spectrometry findings through orthogonal approaches (Co-IP, BiFC) is essential for confirmation of physiologically relevant interactions .

These approaches have been successfully applied to related PP2Cs like HAB1 and ABI1, revealing their direct interaction with and dephosphorylation of OST1/SnRK2.6 in the ABA signaling pathway .

What are the most promising future research directions for PP2C 35?

Future research on PP2C 35 should focus on several promising directions:

• Development of specific chemical inhibitors of PP2C 35 would provide valuable tools for dissecting its functions without genetic manipulation. Structure-based drug design approaches based on the conserved PP2C catalytic domain could yield selective inhibitors that distinguish between different PP2C family members.

• Investigation of PP2C 35's roles beyond ABA signaling is warranted, as emerging evidence suggests PP2Cs participate in multiple signaling networks. Interactome and phosphoproteome analyses in diverse stress conditions could reveal novel functions and substrates .

• Understanding the spatiotemporal dynamics of PP2C 35 activity using FRET-based biosensors would provide insights into how ABA signaling is regulated at the cellular and subcellular levels during stress responses.

• Engineering PP2C 35 with altered regulatory properties could potentially enhance crop stress tolerance. CRISPR-based approaches targeting regulatory domains while preserving catalytic function might generate variants with modified sensitivity to ABA or stress signals.

These directions will contribute to a more comprehensive understanding of PP2C 35's role in plant stress responses and potentially lead to applications in improving crop resilience to environmental challenges .

How can contradictions in the literature regarding PP2C 35 function be resolved through systematic research approaches?

Resolving contradictions in the literature regarding PP2C 35 function requires systematic research approaches:

• Standardization of experimental systems: Establishing a common set of experimental conditions, genetic backgrounds, and phenotypic assays would facilitate direct comparison between studies. This includes standardized protocols for recombinant protein production, activity assays, and phenotypic characterization of mutants .

• Comprehensive genetic analysis: Creation of higher-order mutants combining pp2c35 with mutations in related PP2Cs can address functional redundancy issues that may explain contradictory single-mutant phenotypes. CRISPR-based approaches allow generation of such combinatorial mutants more efficiently than traditional crossing methods.

• Tissue-specific and conditional manipulation: Use of tissue-specific or inducible promoters to modulate PP2C 35 expression can resolve conflicts arising from global gene perturbations, which may mask tissue-specific functions or trigger compensatory mechanisms.

• Integration of multiple data types: Combining transcriptomics, proteomics, metabolomics, and phenomics data through systems biology approaches can provide a more holistic view of PP2C 35 function and place contradictory findings in proper context.