Recombinant Oryza sativa subsp. japonica Calcineurin B-like protein 1 (CBL1)

What is Oryza sativa CBL1 and what are its basic characteristics?

Os-CBL1 is a calcium sensor protein belonging to the calcineurin B-like (CBL) family in rice (Oryza sativa). The protein plays a crucial role as a calcium signal relay in various physiological processes and stress responses. According to the available data, Os-CBL1 has the following characteristics:

• Molecular properties: The full-length mature protein consists of 213 amino acids

• Molecular weight: 24.485 kD

• Molecular formula: C1104H1706N280O333S8

• Theoretical isoelectric point (pI): 4.75, indicating it is an acidic protein

• Structural features: Contains EF-hand domains that bind calcium ions

• Sequence homology: Highly conserved among different plant species, with particularly high homology to CBL1 proteins in other gramineous crops like Sorghum bicolor, Zea mays, and Triticum aestivum

Os-CBL1 functions primarily as a calcium sensor that interacts with CBL-interacting protein kinases (CIPKs), particularly Os-CIPK23, to form signaling complexes that regulate various downstream targets, including ion channels and transporters .

How does CBL1 function in calcium signaling pathways in rice?

Os-CBL1 serves as a decoder of calcium signals in rice through the following mechanisms:

• Ca²⁺ binding: Os-CBL1 contains EF-hand motifs that undergo conformational changes upon binding calcium ions

• Formation of signaling complex: After Ca²⁺ binding, Os-CBL1 interacts with specific CIPKs, particularly Os-CIPK23

• Target regulation: The CBL1-CIPK complex modulates the activity of downstream targets

• Calcium dependency: The activation of Os-CBL1's target proteins is highly dependent on cytosolic Ca²⁺ concentration

Experimental evidence has shown that:

• An Os-CBL1 EF-hand mutation (E172Q in the fourth EF-hand) eliminated its ability to activate Os-AKT1 in HEK293 cells

• High cytosolic Ca²⁺ concentration enhances Os-AKT1 activity, and this enhancement is further increased in the presence of Os-CBL1-CIPK23 complex

• The regulatory mechanism is Ca²⁺-dependent, as demonstrated in heterologous expression systems

What are the optimal methods for expressing and purifying recombinant Os-CBL1?

Based on established protocols for Os-CBL1 and related proteins, the following methodologies are recommended:

Expression Systems:

• E. coli: The most common system used for Os-CBL1 expression

• Cell lines: HEK293 cells have been successfully used for functional studies of Os-CBL1

Expression and Purification Protocol:

• Vector selection: pUN1301 (modified pCAMBIA1301 with ubiquitin promoter) has been successful for expression

• Tag addition: Various tags can be used; tag type should be determined based on specific experimental needs

• Purification method: Affinity chromatography followed by size exclusion chromatography

• Protein storage: Store at -20°C/-80°C with 5-50% glycerol (50% is recommended as default)

Storage Conditions:

• Shelf life of liquid form: 6 months at -20°C/-80°C

• Shelf life of lyophilized form: 12 months at -20°C/-80°C

• For working solutions, store aliquots at 4°C for up to one week

• Avoid repeated freeze-thaw cycles

How can protein-protein interactions between Os-CBL1 and its targets be studied?

Several complementary approaches have been successfully used to study Os-CBL1 interactions:

Yeast Two-Hybrid Assays:

• Used to identify interactions between Os-CBL1 and various CIPKs

• Has confirmed interactions with Os-CIPK23, Os-CIPK3, and Os-CIPK19

• Procedure involves cloning the coding regions into appropriate vectors and testing for reporter gene activation

Bimolecular Fluorescence Complementation (BiFC):

• Methodology used in the search results:

  • Clone Os-CBL1 into vector pSPYCE (MR)

  • Clone Os-CIPK23 into vector pSPYNE (R) 173

  • Express construct pairs in Nicotiana benthamiana leaves

  • Observe YFP fluorescence after 5 days using confocal laser scanning microscopy

Heterologous Expression Systems:

• Coexpression in HEK293 cells to examine functional interactions

• Electrophysiological measurements to quantify the effect of Os-CBL1 on Os-AKT1 channel activity

In planta Validation:

• Transgenic complementation studies using Arabidopsis mutants (e.g., cbl1 cbl9)

• Analysis of phenotypic rescue and restoration of K+ content

What analytical methods can verify the interaction between Os-CBL1 and Os-CIPK23?

To definitively verify and characterize the interaction between Os-CBL1 and Os-CIPK23, researchers should employ multiple complementary techniques:

Electrophysiological Analysis:

• Patch-clamp techniques to measure Os-AKT1-mediated K+ currents in:

  • HEK293 cells expressing Os-AKT1 alone

  • HEK293 cells coexpressing Os-AKT1 with Os-CBL1-Os-CIPK23

• Results show that Os-CBL1-Os-CIPK23 complex enhances Os-AKT1-mediated inward K+ currents

Subcellular Localization Studies:

• Data from BiFC experiments indicate that Os-CBL1 is localized in plasma membrane and cytoplasm

• Co-localization of Os-CBL1, Os-CIPK23, and Os-AKT1 supports their functional interaction

Genetic Complementation:

• Os-CBL1 has been shown to rescue the low-K+-sensitive phenotype of Arabidopsis cbl1 cbl9 double mutants

• This cross-species functional complementation provides strong evidence for conserved CBL1 function

Functional Validation in transgenic lines:

• Os-CIPK23 RNAi lines exhibit similar K+-deficient symptoms as Os-akt1 mutant under low K+ conditions, confirming the functional relationship between these proteins

How does the Os-CBL1-CIPK23 complex specifically regulate Os-AKT1 activity in rice?

The regulatory mechanism of Os-AKT1 by the Os-CBL1-Os-CIPK23 complex involves several key steps and molecular interactions:

Activation Mechanism:

• Calcium binding to Os-CBL1 induces a conformational change

• Os-CBL1 recruits Os-CIPK23 and enhances its kinase activity

• Os-CIPK23 phosphorylates Os-AKT1, which increases channel activity

• Enhanced Os-AKT1 activity facilitates K+ uptake into rice root cells

Experimental Evidence:

• In HEK293 cells, Os-AKT1 alone can mediate inward K+ currents, but its activity is significantly enhanced when coexpressed with Os-CBL1-Os-CIPK23

• The magnitude of inward K+ currents in Os-akt1 mutant root cells is only 14% of that in wild-type cells at −180 mV

• Electrophysiological comparison of K+ currents in different genetic backgrounds:

Calcium Dependency:

• The activation of Os-AKT1 (with or without Os-CBL1-CIPK23) is dependent on cytosolic Ca2+ concentration

• A mutation in the fourth EF-hand of Os-CBL1 (E172Q) abolishes the ability of Os-CBL1-CIPK23 to activate Os-AKT1

What are the functional differences among CIPK proteins that interact with Os-CBL1?

Os-CBL1 interacts with multiple CIPK proteins, but these interactions display varying functional significance and activation potential:

Interaction Partners:

• Systematic screening of 25 rice CIPK genes identified three Os-CIPK proteins (Os-CIPK3, Os-CIPK19, and Os-CIPK23) that interact with Os-AKT1

Functional Comparison:

• Os-CIPK23 shows the strongest activation of Os-AKT1 in the presence of Os-CBL1

• Os-CIPK19 can also enhance Os-AKT1 inward K+ currents in the presence of Os-CBL1, but the activation is much weaker than that mediated by Os-CIPK23

• Os-CIPK3 interacts with Os-AKT1 but does not significantly enhance its activity

Transgenic Evidence in Other Plants:

• In Chinese cabbage (Brassica rapa ssp. pekinensis), three CIPK23 paralogs (BraCIPK23.1, BraCIPK23.2, and BraCIPK23.3) show functional differences in their ability to enhance potassium uptake

• When expressed in Arabidopsis, these paralogs confer different levels of resistance to potassium deficiency:

This functional diversity among CIPK proteins suggests evolutionary divergence and specialization in their regulatory roles.

How conserved is the CBL1-CIPK-AKT1 regulatory module across plant species?

The CBL1-CIPK-AKT1 regulatory module shows remarkable evolutionary conservation across diverse plant species, suggesting its fundamental importance in plant potassium nutrition:

Cross-Species Conservation:

• Rice Os-CBL1 and Os-CIPK23 can functionally complement Arabidopsis cbl1 cbl9 and cipk23 (lks1) mutants, respectively

• AKT1-like channels from different species (Arabidopsis, barley, grapevine) can be activated by CBL1-CIPK23 complexes

Functional Conservation Table:

Evolutionary Implications:

• The high degree of functional conservation suggests that this regulatory mechanism evolved early in plant evolution

• The ability of rice CBL1-CIPK23 to complement Arabidopsis mutants indicates that the core signaling mechanisms are preserved despite ~150 million years of evolutionary divergence

• The research indicates that "the function of CBL and CIPK proteins in K+ channel regulation may be conserved in different plant species"

How does Os-CBL1 contribute to abiotic stress responses in rice?

Os-CBL1 plays a critical role in multiple abiotic stress responses through regulation of ion homeostasis and other cellular processes:

Potassium Deficiency Response:

• Os-CBL1-Os-CIPK23 complex enhances Os-AKT1-mediated K+ uptake under low K+ conditions

• Os-akt1 mutant plants show decreased K+ uptake and display a low-K+-sensitive phenotype

• Disruption of Os-AKT1 significantly reduces K+ content, inhibiting plant growth and development

Salt Stress Response:

• Studies in sugarcane found that ScCBL1 (functionally similar to Os-CBL1) expression is up-regulated by NaCl treatment

• Expression of CBL1 in E. coli enhances tolerance to NaCl stress

• The regulatory mechanism appears similar to the SOS (Salt Overly Sensitive) pathway first described in Arabidopsis

Other Abiotic Stress Responses:

• In sugarcane, SsCBL1 gene expression changes under multiple stress conditions:

  • Low nitrogen, phosphorus, and potassium conditions

  • Drought stress

  • ABA treatment

Evolutionary Adaptation:

• The cell type-specific expression differences between indica and japonica rice varieties may reflect adaptations to different environmental conditions

• This adaptation might be particularly important for tropical japonica varieties, which have specific adaptations to their growing environments

What methodologies can be used to study Os-CBL1 expression patterns under stress conditions?

Multiple complementary approaches can be employed to comprehensively analyze Os-CBL1 expression patterns under various stress conditions:

Quantitative Real-Time PCR (qRT-PCR):

• Design primers specific to Os-CBL1 ORF

• Subject rice plants (e.g., variety ROC22) to various stress treatments:

  • Abiotic stresses: low N/P/K, drought, salt, ABA

  • Time points: typically 8h, 24h, 48h, and 96h

• Extract RNA from different tissues (roots, shoots, etc.)

• Perform qRT-PCR with appropriate reference genes

• Analyze relative expression changes

RNA-Seq Analysis:

• Perform transcriptome sequencing on multiple tissues

• Analyze differential expression of Os-CBL1 across different conditions

• Can reveal tissue-specific and stress-specific expression patterns

• Valuable for understanding co-expression networks with other signaling components

Single-Cell RNA Sequencing:

• More advanced technique to understand cell type-specific expression

• Can reveal expression differences between subspecies (e.g., indica vs. japonica)

• Protocol example from literature:

  • Isolate cells from root tips

  • Perform scRNA-seq

  • Filter cells (>1000 detected genes) and genes (detected in >10 cells)

  • Normalize and scale data

  • Perform clustering and identify cell types

  • Compare expression between varieties

Promoter-Reporter Assays:

• Clone the Os-CBL1 promoter region into a reporter vector (e.g., GUS or luciferase)

• Generate transgenic rice plants

• Subject plants to various stress conditions

• Visualize and quantify reporter activity

How can contradictory results in Os-CBL1 functional studies be reconciled?

When facing contradictory results in Os-CBL1 functional studies, researchers should consider several key factors that might explain the discrepancies:

Genetic Background Variations:

• Different rice varieties (indica vs. japonica) show significant cell type-specific gene expression differences

• The specific genetic background can influence the phenotypic outcomes of Os-CBL1 manipulation

Experimental System Differences:

• Heterologous expression systems (oocytes, HEK293 cells) may not fully recapitulate the native cellular environment

• In planta studies may show different results from in vitro or heterologous systems due to the presence of additional regulatory factors

Functional Redundancy:

• Multiple CBL proteins exist in rice, with potential functional overlap

• In the case of Os-AKT1 regulation, studies have shown that besides Os-CIPK23, Os-CIPK19 can also enhance Os-AKT1 activity, though to a lesser extent

• Consider the expression of multiple family members in the same tissues/conditions

Methodological Considerations:

• Protein expression levels in different experimental systems

• Different stress treatment protocols (duration, intensity)

• Sensitivity and specificity of detection methods

Reconciliation Strategies:

• Perform comprehensive genetic analysis using multiple alleles/mutants

• Use complementary approaches (in vitro, heterologous, and in planta)

• Consider tissue-specific, developmental stage-specific, and subcellular localization factors

• Validate key findings using multiple methodologies

What are the critical considerations for designing CRISPR/Cas9 experiments targeting Os-CBL1?

When designing CRISPR/Cas9 experiments to edit or modulate Os-CBL1 in rice, researchers should consider the following critical factors:

Target Site Selection:

• Focus on functionally critical regions such as:

  • EF-hand calcium-binding domains

  • CIPK interaction surfaces

  • N-myristoylation sites important for membrane localization

• Avoid regions with high sequence similarity to other CBL family members to minimize off-target effects

• Select target sites with low predicted off-target scores

Guide RNA Design:

• Design multiple gRNAs targeting different regions of Os-CBL1

• Ensure high on-target efficiency and minimal off-target potential

• Consider the GC content (40-60% ideal) and avoid polyT sequences

Functional Domain Considerations:

• The N-terminal region (residues 1-20) is important for targeting

• EF-hand domains (particularly the fourth EF-hand where E172Q mutation abolishes function) are critical for Ca²⁺ binding

• Consider making precise edits to conserved residues identified through alignment with other plant CBL1 proteins

Validation Strategy:

• Use T7 endonuclease or similar assays to verify editing efficiency

• Sequence multiple independent transgenic lines

• Confirm the absence of large deletions or rearrangements

• Verify the absence of editing at predicted off-target sites

• Assess protein expression levels by Western blotting

• Perform functional assays (K+ uptake, stress tolerance) to validate phenotypic effects

Controls and Complementation:

• Include appropriate controls (non-edited rice of same background)

• Prepare complementation constructs to rescue the phenotype and confirm specificity

• Consider creating tagged versions for protein localization studies

How can researchers effectively study the Os-CBL1-CIPK interaction specificity?

To effectively study the specificity of interactions between Os-CBL1 and various CIPKs, researchers should employ a multi-faceted approach:

Systematic Interaction Screening:

• Perform comprehensive yeast two-hybrid assays testing Os-CBL1 against all rice CIPKs

• Previous studies screened 25 rice CIPK genes and identified Os-CIPK3, Os-CIPK19, and Os-CIPK23 as interactors with Os-CBL1/Os-AKT1

• Compare interaction strengths using quantitative assays (e.g., β-galactosidase activity)

Structural Analysis:

• Perform homology modeling based on known CBL-CIPK structures

• Identify key interaction residues through sequence alignment and structural prediction

• Use site-directed mutagenesis to verify the importance of predicted interface residues

In Vitro Binding Assays:

• Express and purify recombinant Os-CBL1 and CIPK proteins

• Perform pull-down assays or surface plasmon resonance to quantify binding affinities

• Compare binding parameters (Kd, kon, koff) across different CIPK partners

Functional Validation:

• Test the ability of different Os-CBL1-CIPK pairs to activate Os-AKT1 in heterologous systems

• Quantify activation strength through electrophysiological measurements

• Previous work showed variation in activation potential:

  • Os-CIPK23 provides strong activation

  • Os-CIPK19 provides weak activation

  • Os-CIPK3 interacts but doesn't significantly activate

In Planta Confirmation:

• Generate transgenic plants expressing specific Os-CBL1-CIPK combinations

• Assess phenotypic outcomes under various stress conditions

• Compare with single gene manipulations to identify synergistic effects