Recombinant Oryza sativa subsp. japonica Protein phosphatase 2C 35 (XB15), partial

What is the molecular function of XB15/OsPP2C35 in rice?

XB15, also known as OsPP2C35, functions as a protein phosphatase 2C that catalyzes the removal of phosphate groups from phosphorylated serine and threonine residues in target proteins. It belongs to the larger PP2C family in rice (Oryza sativa), which comprises 78 genes encoding 111 putative proteins . As a PP2C, XB15 likely acts as a negative modulator of protein kinase pathways involved in various cellular processes. The protein has been annotated with the EC number 3.1.3.16, confirming its catalytic function as a phosphatase . Within rice signaling networks, XB15's alternative name as "XA21-binding protein 15" suggests it interacts with XA21, which is likely involved in defense response pathways, similar to other characterized PP2Cs that modulate stress and developmental processes .

How is XB15/OsPP2C35 classified within the larger PP2C family?

XB15/OsPP2C35 belongs to the Mg²⁺- or Mn²⁺-dependent protein phosphatase (PPM) family, specifically within the PP2C group. The comprehensive genome-wide analysis of rice identified 78 PP2C genes (OsPP2Cs) that were classified into 11 subfamilies based on phylogenetic analysis . These classifications are supported by analyses of gene structures and protein motifs. While the specific subfamily of XB15/OsPP2C35 is not explicitly stated in the search results, its identification as a PP2C confirms its placement within this broader classification system. The PP2C family represents one of the largest phosphatase families identified in plants, highlighting its evolutionary and functional significance in plant biology .

What protein domains and structural features characterize XB15/OsPP2C35?

XB15/OsPP2C35 contains the characteristic PP2C catalytic domain, which is identifiable using tools such as SMART and Pfam . While the specific protein length of the recombinant XB15 product is described as "partial," suggesting it doesn't represent the full-length native protein, the complete protein would contain the PP2C catalytic core essential for its phosphatase activity . PP2C proteins typically require Mg²⁺ or Mn²⁺ ions for their catalytic activity, distinguishing them from other phosphatase families. Additionally, some PP2C proteins contain specific motifs outside the catalytic domain that contribute to their substrate specificity and regulatory functions . The complete analysis of protein motifs would require examination of the specific sequence and structural data for XB15/OsPP2C35.

What are the optimal storage and handling conditions for recombinant XB15/OsPP2C35?

The optimal handling of recombinant XB15/OsPP2C35 requires careful attention to storage temperature and buffer composition. For long-term storage, the recombinant protein should be maintained at -20°C/-80°C, with typical shelf life of 6 months for liquid formulations and 12 months for lyophilized preparations . To maximize stability, the recombinant protein should be reconstituted in deionized sterile water to a concentration of 0.1-1.0 mg/mL, with the addition of glycerol to a final concentration of 5-50% (typically 50%) before aliquoting for long-term storage . For working stocks, store aliquots at 4°C for no more than one week, and avoid repeated freeze-thaw cycles as they can dramatically reduce protein activity and stability . Prior to use, vials should be briefly centrifuged to bring contents to the bottom, particularly for lyophilized preparations. These storage parameters are critical for maintaining enzymatic activity and structural integrity of the recombinant XB15 protein.

What expression systems are most effective for producing active recombinant XB15/OsPP2C35?

Based on the commercial preparation information, yeast appears to be an effective expression system for producing recombinant XB15/OsPP2C35 with high purity (>85% as determined by SDS-PAGE) . Yeast expression systems offer several advantages for plant protein production, including proper protein folding, post-translational modifications, and relatively high yields. When establishing an expression system for XB15/OsPP2C35, researchers should consider that PP2C proteins require metal ions (Mg²⁺ or Mn²⁺) for their catalytic activity, so the expression and purification protocols should account for this requirement. Alternative expression systems such as E. coli or insect cells might also be considered depending on specific experimental requirements, though they would need optimization to ensure proper folding and activity of this plant phosphatase. The choice of expression tags and purification strategies should be evaluated based on their potential impact on the phosphatase activity and binding properties of XB15.

How can researchers verify the enzymatic activity of recombinant XB15/OsPP2C35?

To verify the enzymatic activity of recombinant XB15/OsPP2C35, researchers should employ phosphatase activity assays using suitable substrates. As a PP2C with EC number 3.1.3.16, XB15 catalyzes the dephosphorylation of phosphoserine/phosphothreonine residues . Standard assays include colorimetric methods using artificial substrates like p-nitrophenyl phosphate (pNPP) to measure phosphatase activity, where the release of p-nitrophenol can be monitored spectrophotometrically. For more physiologically relevant assessments, researchers can use phosphopeptides mimicking the natural substrates of XB15, particularly those derived from proteins in the XA21 signaling pathway. Activity assays should be conducted in buffer conditions containing Mg²⁺ or Mn²⁺ ions, which are essential for PP2C function. Additionally, comparative activity analysis with known PP2C inhibitors such as okadaic acid and calyculin A (which inhibit PP1 and PP2A but typically not PP2C) can help confirm that the observed phosphatase activity is specifically due to PP2C activity.

What signaling pathways involve XB15/OsPP2C35 in rice?

The designation of XB15 as an "XA21-binding protein 15" strongly suggests its involvement in XA21-mediated signaling pathways in rice . XA21 is a receptor-like kinase involved in disease resistance, and XB15 likely regulates this pathway through dephosphorylation. From the broader context of PP2C functions in plants, XB15/OsPP2C35 may participate in stress response signaling networks, potentially including abscisic acid (ABA) responses, similar to other characterized PP2Cs . Many plant PP2Cs act as negative regulators of protein kinase pathways activated by environmental stresses or developmental cues. While the specific subfamily of XB15 is not explicitly mentioned in the search results, certain PP2C subfamilies (particularly subfamily A) are known to play primary roles in stress tolerance, especially ABA responses, while subfamily D members may constitute positive regulators in ABA-mediated signaling . The precise pathways modulated by XB15 would require experimental verification through interaction studies, gene expression analyses, and phenotypic characterization of mutant lines.

How does XB15/OsPP2C35 expression vary across different rice tissues and stress conditions?

While the search results don't provide specific expression data for XB15/OsPP2C35, the comprehensive analysis of PP2C genes in rice indicates that most PP2C genes play functional roles in multiple tissues . The expression patterns of PP2C genes can provide insights into their functional roles. For subfamily A members, induced expression by diverse stimuli indicates their primary role in stress tolerance, particularly ABA response . Without specific data for XB15/OsPP2C35, researchers investigating its expression patterns should conduct qRT-PCR analyses across different tissues (roots, shoots, leaves, reproductive organs) and under various stress conditions (drought, salt, cold, heat, pathogen infection). RNA-seq data mining from existing rice transcriptome datasets would also provide valuable information about tissue-specific and stress-induced expression patterns. Additionally, promoter analysis using approaches mentioned in the search results could help identify putative upstream regulatory elements that control XB15 expression in response to specific stimuli .

How do evolutionary relationships among rice PP2Cs inform functional studies of XB15/OsPP2C35?

Evolutionary analyses of PP2C genes in rice and Arabidopsis have revealed both common and lineage-specific subfamilies, providing a framework for understanding the functional evolution of XB15/OsPP2C35 . The phylogenetic classification of PP2Cs into 11 subfamilies in rice (compared to 13 in Arabidopsis) suggests subfamily-specific functional specialization . By examining the closest homologs of XB15/OsPP2C35 within rice and in other plant species, researchers can infer potential functions based on evolutionary conservation. Gene duplication events, including whole genome and chromosomal segment duplications, have contributed significantly to the expansion of OsPP2Cs, with tandem or local duplication occurring less frequently in Arabidopsis than in rice . This evolutionary context suggests that XB15/OsPP2C35 may have arisen through duplication events and subsequently developed specialized functions. Comparative sequence analysis focusing on conserved motifs and divergent regions between XB15 and its closest homologs would highlight the structural features potentially responsible for its specific functions and interactions.

What methodologies are most effective for identifying XB15/OsPP2C35 substrates and interacting partners?

Identifying the substrates and interacting partners of XB15/OsPP2C35 requires a multi-faceted approach combining biochemical, proteomic, and genetic methods. As XB15 is named as an XA21-binding protein, XA21 is already a known interacting partner, though the functional significance of this interaction requires further characterization . To identify additional interacting proteins, researchers should employ co-immunoprecipitation (co-IP) coupled with mass spectrometry, using antibodies against XB15 or epitope-tagged versions of the protein expressed in rice cells. Yeast two-hybrid (Y2H) screening using XB15 as bait against rice cDNA libraries can identify direct protein interactions. For substrate identification, substrate-trapping mutants can be created by mutating the catalytic site of XB15 to generate a phosphatase that binds but doesn't dephosphorylate substrates. Phosphoproteomic approaches comparing phosphorylation profiles in wild-type and XB15-overexpressing or knockout plants would reveal potential substrates at a global level. Bimolecular fluorescence complementation (BiFC) and Förster resonance energy transfer (FRET) can validate and localize these interactions in planta. These complementary approaches would provide a comprehensive understanding of XB15's protein interaction network.

What are the structural mechanisms by which XB15/OsPP2C35 achieves substrate specificity?

The substrate specificity of XB15/OsPP2C35 is likely determined by specific structural features of its catalytic domain and potential regulatory domains. While the search results don't provide specific structural information for XB15, studies of PP2C proteins generally indicate that their catalytic domains contain a characteristic fold with metal-coordinating residues essential for phosphatase activity . The substrate specificity is often determined by regions outside the core catalytic site, including surface loops that interact with substrates. Some PP2C proteins contain additional protein motifs outside the catalytic domain that contribute to substrate recognition and binding . To fully understand the structural basis of XB15's substrate specificity, researchers should pursue X-ray crystallography or cryo-electron microscopy studies of XB15 alone and in complex with substrates or interacting partners. Molecular docking and molecular dynamics simulations can complement experimental approaches by predicting substrate binding modes and identifying key residues involved in substrate recognition. Mutational analyses targeting these predicted interaction sites would validate their functional significance. Understanding these structural mechanisms would potentially enable the design of specific inhibitors or activators of XB15 for agricultural applications.

How can researchers effectively use transgenic approaches to study XB15/OsPP2C35 function in planta?

Transgenic approaches provide powerful tools for studying XB15/OsPP2C35 function in rice plants. Researchers should consider multiple complementary strategies: overexpression using constitutive (e.g., CaMV 35S, Ubiquitin) or tissue-specific/inducible promoters; RNAi or CRISPR/Cas9 for gene silencing or knockout; and complementation of mutant lines with wild-type or mutated versions of XB15. When designing these constructs, epitope tags (e.g., HA, FLAG, GFP) should be incorporated to facilitate protein detection, localization, and purification. For promoter studies, the XB15 native promoter can be fused to reporter genes like GUS or luciferase to visualize expression patterns in different tissues and under various conditions. The choice of transformation method is critical, with Agrobacterium-mediated transformation being preferred for rice. Phenotypic analyses of transgenic plants should examine multiple independent transformation events and multiple generations to account for positional effects and ensure stable transgene expression. These plants should be evaluated under normal growth conditions and various stresses to uncover XB15's roles in development and stress responses. Molecular analyses including RT-qPCR, western blotting, and phosphoproteomic comparisons between wild-type and transgenic lines will provide mechanistic insights into XB15's function.

What are the critical quality control parameters for recombinant XB15/OsPP2C35 preparations?

Quality control for recombinant XB15/OsPP2C35 preparations should assess multiple parameters to ensure experimental reliability. Purity should be evaluated using SDS-PAGE, with commercial preparations typically achieving >85% purity . Protein identity confirmation using mass spectrometry and/or western blotting with specific antibodies is essential. Enzymatic activity assays using appropriate substrates should verify that the recombinant protein is functional. Stability testing under various storage conditions and time periods helps establish optimal handling protocols. Endotoxin testing is critical if the protein will be used in cellular assays, as contaminating endotoxins can trigger cellular responses unrelated to the protein's activity. Batch-to-batch consistency should be monitored through standardized assays comparing activity and physical parameters across production runs. While commercial preparations like the one described in search result undergo manufacturer quality control, researchers producing their own recombinant XB15 should implement these quality control measures to ensure reliable and reproducible experimental results.

How should researchers design experiments to study XB15/OsPP2C35 interactions with potential substrates?

Designing experiments to study XB15/OsPP2C35 interactions with potential substrates requires careful consideration of multiple factors. In vitro phosphatase assays should use purified recombinant XB15 and phosphorylated substrates under conditions optimized for PP2C activity (including Mg²⁺ or Mn²⁺ ions). Kinetic parameters (Km, Vmax) should be determined for each potential substrate to assess relative affinities and catalytic efficiencies. Substrate specificity can be examined using peptide arrays containing various phosphorylated sequences to identify preferred recognition motifs. For in vivo validation, co-immunoprecipitation followed by western blotting can confirm physical interactions between XB15 and putative substrates in plant cells. Phosphorylation-specific antibodies or mass spectrometry can monitor changes in substrate phosphorylation status when XB15 activity is modulated. Bimolecular fluorescence complementation or FRET can visualize these interactions in living cells and determine their subcellular localization. Genetic approaches comparing phosphoproteomes between wild-type and XB15 mutant plants can identify physiologically relevant substrates. Control experiments should include catalytically inactive XB15 mutants and inhibitors of related phosphatases to ensure specificity. These complementary approaches provide robust evidence for genuine XB15-substrate relationships.

What computational tools and databases are most valuable for researching XB15/OsPP2C35?

Researchers studying XB15/OsPP2C35 should utilize a range of computational tools and databases to maximize their understanding of this protein. Sequence databases like UniProt (which contains XB15 under accession Q84T94) provide curated information about the protein's sequence, domains, and known functions. Structural databases such as PDB can identify homologous structures if XB15's structure hasn't been determined experimentally. For expression analysis, rice-specific gene expression databases including RiceXPro and Rice Expression Database compile transcriptomic data across tissues, developmental stages, and stress conditions. Phylogenetic analysis tools like MEGA, PhyML, or MrBayes can place XB15 in evolutionary context among other PP2Cs . Protein domain prediction tools including SMART and Pfam help identify functional domains beyond the PP2C catalytic domain . Promoter analysis tools like PlantCARE and PLACE can identify putative regulatory elements in the XB15 promoter region, providing insights into its transcriptional regulation . Protein-protein interaction databases such as STRING and BioGRID might contain predicted or experimentally verified interaction partners. Integrating data from these computational resources provides a comprehensive foundation for experimental studies of XB15 function and regulation.