Recombinant Oryza sativa subsp. japonica Cyclin-dependent kinases regulatory subunit 1 (CKS1)

What is the basic structure and function of CKS1 in rice (Oryza sativa)?

CKS1 (Cyclin-dependent kinase regulatory subunit 1) in rice is a small protein (typically 79-150 residues) that serves as an essential regulatory subunit for cyclin-dependent protein kinases (CDKs). In eukaryotes, including rice, CKS proteins bind to CDKs to regulate cell cycle progression, particularly during the G1 and G2 stages of cell division . The CKS proteins exist as hexamers formed by three interlocked homodimers, creating a distinctive 12-stranded beta-barrel structure. This hexameric arrangement creates a central tunnel approximately 12Å in diameter, lined by six exposed helix pairs, potentially allowing for the binding of up to six kinase units. This structural arrangement suggests that CKS1 may function as a hub for CDK multimerization, facilitating the coordination of multiple CDK activities during cell cycle progression .

How does rice CKS1 differ from CKS proteins in other organisms?

While the fundamental function of CKS proteins is conserved across species, there are notable differences between rice CKS1 and its counterparts in other organisms. In yeasts such as Saccharomyces cerevisiae (gene CKS1) and Schizosaccharomyces pombe (gene suc1), only a single CKS isoform is known, whereas mammals possess two highly related isoforms, CKS1B and CKS2 . Rice (Oryza sativa) contains CKS proteins that maintain the core regulatory function but may have plant-specific interaction patterns with CDKs and cyclins that differ from their mammalian counterparts. These differences may reflect evolutionary adaptations to plant-specific cell cycle regulation mechanisms, particularly those related to developmental processes unique to plants.

What methodologies are commonly used to express recombinant rice CKS1?

For expressing recombinant Oryza sativa CKS1 protein, researchers typically employ the following methodological approach:

• Vector Selection: Bacterial expression vectors containing T7 or similar strong promoters are often selected for high-yield expression. For plant-specific post-translational modifications, plant-based expression systems may be preferred.

• Expression System: While E. coli BL21(DE3) remains the most common host for initial expression trials, insect cell systems (Sf9/Sf21) are utilized when proper folding is challenging. Plant-based systems such as Nicotiana benthamiana can provide more authentic modifications.

• Fusion Tags: Hexahistidine (His6) tags facilitate purification, while fusion partners like MBP (maltose-binding protein) or GST (glutathione S-transferase) can enhance solubility and provide additional purification options.

• Induction Parameters: Typical conditions include IPTG induction (0.1-1 mM) at reduced temperatures (16-25°C) to enhance protein solubility, with expression times ranging from 4-16 hours.

Given the small size of CKS proteins, they generally express well in prokaryotic systems, though experimental validation of functionality remains essential through binding assays with cognate rice CDKs.

How do researchers characterize CKS1 interactions with rice CDKs and cyclins?

Researchers employ multiple complementary approaches to characterize CKS1 interactions with rice CDKs and cyclins:

• Yeast Two-Hybrid Assays: This method has successfully demonstrated differential interactions between rice CDK inhibitors (ICK/KRPs) and various cyclins and CDKs. For example, OsiICK1 and OsiICK6 both interact with OsCYCD, but show differences in their interactions with CDKA .

• Co-Immunoprecipitation: This technique confirms in vivo interactions by pulling down protein complexes from plant cell extracts using antibodies against either CKS1 or its potential binding partners.

• Surface Plasmon Resonance: This provides quantitative binding kinetics data (Ka, Kd, KD) for protein interactions, allowing researchers to measure the strength of CKS1 binding to different CDKs and cyclins.

• Computational Analysis: Molecular modeling approaches can identify key interaction surfaces and predict the effects of mutations. For example, computational alanine scanning identifies critical residues at protein interfaces that contribute significantly to binding energy .

• FRET/BRET Assays: These techniques can determine protein-protein interactions in living cells by measuring energy transfer between fluorophores attached to potential interacting proteins.

Research has shown that rice CKS proteins, like their counterparts in other organisms, can interact with multiple cyclin-CDK complexes, suggesting a role in coordinating various aspects of the cell cycle.

What experimental approaches reveal the subcellular localization of rice CKS1?

To determine the subcellular localization of rice CKS1, researchers typically employ the following experimental approaches:

• Fluorescent Protein Fusions: Enhanced green fluorescent protein (EGFP) fusions with CKS1 allow direct visualization in living cells. For example, studies with rice ICK/KRP proteins have demonstrated that EGFP:OsiICK1 and EGFP:OsiICK6 are localized in the nucleus, with distinct subnuclear distribution patterns .

• Immunofluorescence: Using specific antibodies against CKS1, researchers can detect the endogenous protein's localization with high specificity, particularly useful when overexpression might alter normal localization patterns.

• Subcellular Fractionation: This biochemical approach separates cellular components (nucleus, cytoplasm, membrane fractions) followed by Western blot analysis to detect CKS1 in specific fractions.

• Co-localization Studies: Simultaneous detection of CKS1 with known subcellular markers or interaction partners (such as CDKs) provides context for understanding its functional localization during different cell cycle phases.

Based on studies of related proteins, rice CKS1 is expected to primarily localize to the nucleus, consistent with its role in regulating CDK activity during cell cycle progression. The specific subnuclear distribution pattern may provide insights into its functional associations with particular nuclear components.

How can researchers measure the effects of CKS1 on cell cycle progression in rice?

To assess the impact of CKS1 on cell cycle progression in rice, researchers can employ several methodological approaches:

• Flow Cytometry Analysis: This technique quantifies DNA content in cell populations, allowing determination of the proportion of cells in different cell cycle phases (G1, S, G2/M). Changes in these proportions upon CKS1 manipulation indicate specific effects on cell cycle progression.

• EdU/BrdU Incorporation: These thymidine analogs are incorporated into newly synthesized DNA, allowing visualization and quantification of S-phase cells, helping to determine whether CKS1 affects DNA replication rates.

• Transgenic Rice Lines: Generating rice plants with modified CKS1 expression (overexpression or knockdown) allows assessment of phenotypic effects. For instance, studies with ICK/KRP proteins have shown that their overexpression results in multiple phenotypic effects on plant growth, morphology, pollen viability, and seed setting .

• Kinase Activity Assays: In vitro CDK activity assays using histone H1 or other substrates can determine how CKS1 influences the catalytic activity of CDK-cyclin complexes isolated from rice cells.

• Transcriptome Analysis: RNA-seq of tissues with altered CKS1 expression can identify downstream genes affected by cell cycle perturbations, providing insights into the molecular consequences of CKS1 function.

These approaches collectively provide a comprehensive understanding of how CKS1 influences cell division and development in rice.

What computational approaches are used to identify potential binding interfaces of rice CKS1?

Advanced computational approaches for analyzing rice CKS1 binding interfaces include:

• Electrostatic Surface Potential Analysis: Tools like APBS (Adaptive Poisson-Boltzmann Solver) generate electrostatic surface potential maps that help identify complementary charged regions between CKS1 and its binding partners. Studies of similar protein interactions have shown that binding interfaces often display electrostatic complementarity, with negative regions on one protein corresponding to positive regions on its partner .

• Molecular Dynamics Simulations: These simulations provide insights into the dynamic behavior of protein interactions over time. Heat maps of interaction occupancy (percentage of occurrence during simulation) highlight key residues involved in stable binding. For similar interactions, residues like ARG 20, ARG 44, GLN 49, GLN 50, and SER 51 have shown high rates of interaction persistence .

• Computational Alanine Scanning: This approach systematically assesses the contribution of individual amino acids to binding energy by virtually mutating them to alanine. The resulting ΔΔG values indicate whether mutations lead to loss of binding (positive values) or gain of binding (negative values) .

• Hydrogen Bond Analysis: Computational tools can identify intermolecular hydrogen bonds at protein interfaces, assessing their strength based on bond length (typically around 3.5 Å for strong interactions) and categorizing them as backbone-backbone, backbone-side chain, or side chain-side chain interactions .

These computational approaches provide valuable insights for designing experiments to validate key interaction residues and for developing strategies to modulate CKS1 function.

What are the challenges in distinguishing functional effects of CKS1 from other cell cycle regulators?

Distinguishing the specific functional contributions of CKS1 from other cell cycle regulators presents several methodological challenges:

• Functional Redundancy: Plant genomes often contain multiple genes with overlapping functions. Creating combinatorial mutants or using inducible systems may be necessary to overcome compensation effects.

• Temporal Dynamics: Cell cycle regulation involves precisely timed interactions. Time-resolved experiments using techniques like FRET-FLIM (Fluorescence Lifetime Imaging Microscopy) or synchronization of plant cell cultures are needed to capture these dynamics.

• Context-Dependent Effects: CKS1 may function differently in various tissues or developmental stages. Tissue-specific or developmentally regulated gene manipulation using promoters like those used in studies of ICK/KRP proteins can help address this challenge .

• Separating Direct and Indirect Effects: Determining whether phenotypic changes result directly from CKS1 manipulation or from downstream effects requires careful experimental design, including rescue experiments and rapid induction systems.

• Distinguishing Scaffolding from Regulatory Functions: CKS proteins serve both as CDK binding partners and as adaptors for substrate recruitment. Mutations that specifically disrupt one function while preserving the other can help dissect these distinct roles.

Addressing these challenges requires complementary approaches, including genetics, biochemistry, and cell biology, to build a comprehensive understanding of CKS1's specific contributions to cell cycle regulation.

How can researchers effectively compare rice CKS1 function across different abiotic stress conditions?

To effectively compare rice CKS1 function across different abiotic stress conditions, researchers should implement the following methodological approaches:

• Controlled Stress Application: Establish standardized protocols for applying specific stresses (drought, salinity, temperature extremes, etc.) with precise control over intensity and duration to ensure reproducibility.

• Multi-level Analysis Framework:

• Inducible Expression Systems: Employ chemically inducible promoters (like those used for CRK10 expression that resulted in enhanced immunity ) to control CKS1 expression at specific times during stress application.

• Genetic Background Comparison: Evaluate CKS1 function in different rice varieties with varying stress tolerance, potentially revealing genotype-specific roles.

• Integration with Other Stress Response Pathways: Analyze potential crosstalk between CKS1-mediated cell cycle regulation and known stress response pathways, such as those mediated by NH1 in rice immune responses .

This comprehensive approach allows researchers to determine whether CKS1 functions as a general cell cycle regulator across stress conditions or has stress-specific roles.

What are common challenges in producing active recombinant rice CKS1 and how can they be addressed?

Researchers frequently encounter several challenges when producing active recombinant rice CKS1 protein:

• Protein Solubility Issues:

  • Challenge: Formation of inclusion bodies in bacterial expression systems

  • Solution: Optimize expression conditions by lowering induction temperature (16-18°C), reducing IPTG concentration (0.1-0.5 mM), or using solubility-enhancing fusion tags (MBP, SUMO, or Trx)

• Proper Folding:

  • Challenge: Misfolded protein lacking biological activity

  • Solution: Co-express with molecular chaperones (GroEL/GroES, DnaK/DnaJ/GrpE) or switch to eukaryotic expression systems for complex folding requirements

• Protein Stability:

  • Challenge: Rapid degradation during purification

  • Solution: Include protease inhibitors throughout purification, optimize buffer conditions (pH, salt concentration, additives like 10% glycerol), and conduct purification at 4°C

• Functional Validation:

  • Challenge: Confirming biological activity of purified protein

  • Solution: Design activity assays based on known CKS1 functions, such as in vitro binding assays with rice CDKs or functional complementation of yeast cks1 mutants

• Post-translational Modifications:

  • Challenge: Missing critical modifications in bacterial systems

  • Solution: Use plant-based expression systems (e.g., Nicotiana benthamiana transient expression) if specific modifications are required for activity

When designing expression constructs, researchers should consider including a cleavable tag and testing multiple expression conditions in parallel to identify optimal parameters for their specific research needs.

How should researchers design experiments to differentiate the roles of CKS1 in cell division versus stress response pathways?

Designing experiments to differentiate CKS1's roles in cell division versus stress response requires careful methodological considerations:

• Temporal Control Systems:

  • Utilize inducible expression systems (similar to those used for CRK10 ) to manipulate CKS1 levels at specific developmental stages or precise times after stress application

  • This approach minimizes confounding effects from constitutive manipulation

• Tissue-Specific Manipulation:

  • Target CKS1 expression/suppression to specific tissues using promoters with known expression patterns

  • Compare effects in actively dividing tissues (meristems) versus differentiated tissues with primarily stress response functions

• Mutation Design Strategy:

  • Create separation-of-function mutations based on:

    • Structural information about CKS1 binding interfaces

    • Computational alanine scanning to identify residues specifically involved in CDK binding versus other interactions

  • Test these mutations for differential rescue of phenotypes

• Comprehensive Phenotypic Analysis:

• Genetic Interaction Studies:

  • Create double mutants with known cell cycle regulators versus stress response factors

  • Epistasis analysis can reveal pathway-specific functions

This multifaceted approach can distinguish direct roles in cell cycle regulation from indirect effects or separate functions in stress response pathways.

What controls are essential when comparing wild-type and mutant versions of rice CKS1 in interaction studies?

When comparing wild-type and mutant versions of rice CKS1 in interaction studies, the following controls are essential to ensure reliable and interpretable results:

• Expression Level Verification:

  • Western blot analysis to confirm equivalent expression levels between wild-type and mutant CKS1

  • qRT-PCR to verify similar transcript levels in transgenic lines

  • This control prevents misattribution of phenotypes to protein function rather than expression differences

• Protein Integrity Controls:

  • Circular dichroism spectroscopy to verify proper folding of mutant proteins

  • Size-exclusion chromatography to confirm appropriate oligomeric state (hexameric for CKS proteins )

  • These analyses ensure that mutations disrupt specific interactions rather than general protein structure

• Negative Interaction Controls:

  • Include proteins known not to interact with CKS1 in binding assays

  • Use unrelated mutations that shouldn't affect the interaction being studied

  • These controls establish specificity of observed interaction changes

• Positive Interaction Controls:

  • Include well-characterized CKS1 interactors (like specific CDKs)

  • Compare results with known interacting domains from other organisms

  • These controls validate the experimental system's sensitivity

• In vivo Validation Strategy:

  • Corroborate binding assay results with in planta co-localization studies

  • Perform bimolecular fluorescence complementation to visualize interactions in plant cells

  • These approaches confirm that in vitro findings reflect cellular reality

By implementing these controls systematically, researchers can confidently attribute observed changes to specific amino acid substitutions rather than experimental artifacts, thereby establishing clear structure-function relationships for rice CKS1.

How might rice CKS1 function differ from model plant systems like Arabidopsis?

Rice CKS1 likely displays both conserved and species-specific functions compared to Arabidopsis, reflecting evolutionary adaptations to different plant life cycles and environmental challenges:

• Differences in Cell Cycle Regulation:

  • Rice, as a monocot, exhibits distinctive developmental patterns, particularly in root architecture and leaf venation, suggesting potentially unique roles for cell cycle regulators like CKS1

  • Evidence from studies of other cell cycle regulators, such as ICK/KRP proteins, demonstrates rice-specific phenotypic effects, including characteristic leaf rolling toward the abaxial side when overexpressed

• Stress Response Integration:

  • Rice, as a semi-aquatic crop often exposed to flooding, may have evolved specific mechanisms linking cell cycle regulation and hypoxia response

  • Similar to how rice cysteine-rich receptor-like kinases (CRK6 and CRK10) show specific roles in BTH-activated immune responses , CKS1 might have acquired rice-specific functions in biotic or abiotic stress responses

• Interaction Network Divergence:

  • The repertoire of CDKs and cyclins differs between rice and Arabidopsis, potentially creating species-specific interaction networks

  • Comparative protein-protein interaction studies would be necessary to map these differences comprehensively

• Regulatory Mechanisms:

  • Preliminary evidence from studies of rice ICK/KRP proteins suggests differences in subcellular localization patterns compared to Arabidopsis homologs, with rice OsiICK1 showing homogeneous nuclear distribution while OsiICK6 displays punctate subnuclear localization

Methodologically, these differences can be investigated through comparative genomics, cross-species complementation experiments, and side-by-side biochemical characterization of rice and Arabidopsis CKS proteins.

What approaches can reveal how CKS1 phosphorylation affects its function in rice cell cycle regulation?

Investigating the role of CKS1 phosphorylation in rice cell cycle regulation requires multiple complementary approaches:

• Phosphorylation Site Mapping:

  • Mass spectrometry analysis of purified native and recombinant CKS1 to identify phosphorylation sites

  • Comparison with phosphoproteomic databases to identify conserved or rice-specific phosphorylation patterns

  • Creation of a comprehensive phosphorylation site map with temporal dynamics during cell cycle progression

• Phosphomimetic and Phosphodeficient Variants:

  • Generate CKS1 mutants where potential phosphorylation sites are replaced with:

    • Glutamic acid (E) or aspartic acid (D) to mimic constitutive phosphorylation

    • Alanine (A) to prevent phosphorylation

  • Test these variants in binding assays with CDKs, cyclins, and other partners to determine effects on interaction strength

• Kinase Identification:

  • In vitro kinase assays with candidate cell cycle-related kinases

  • Pharmacological inhibition of specific kinase families in rice cell cultures

  • Immunoprecipitation coupled with kinase assays to identify endogenous CKS1-phosphorylating enzymes

• Functional Impact Assessment:

• Systems Analysis:

  • Integration of phosphorylation data with transcriptomics and interactomics to build comprehensive regulatory networks

  • Comparison with known phosphorylation-dependent regulatory mechanisms of other cell cycle proteins

These approaches collectively would provide mechanistic insights into how phosphorylation dynamically regulates CKS1 function throughout the rice cell cycle.

How can genome editing technologies be optimized to study CKS1 function in rice development?

Optimizing genome editing technologies for studying CKS1 function in rice development requires careful consideration of several methodological aspects:

• CRISPR/Cas9 Design Strategy:

  • Guide RNA Selection: Design multiple sgRNAs targeting conserved functional domains identified through computational analyses like those shown for CKS-related proteins

  • Off-target Minimization: Employ tools like Cas-OFFinder to predict and avoid potential off-target sites in the rice genome

  • Editing Efficiency Assessment: Use protoplast assays to pre-screen guide RNA efficiency before full plant transformation

• Advanced Editing Approaches:

  • Base Editing: For introducing specific point mutations without double-strand breaks, particularly valuable for creating phosphorylation site mutants

  • Prime Editing: For precise insertions, deletions, or substitutions to introduce epitope tags or reporter fusions at the endogenous locus

  • Multiplex Editing: For simultaneously targeting CKS1 along with interacting partners to study genetic interactions

• Tissue-Specific and Inducible Modifications:

  • Two-component Systems: Combine tissue-specific promoters with Cre-lox recombination for conditional knockout of CKS1

  • Heat-shock or Chemical Induction: Implement temporal control similar to systems used for CRK10 expression studies

  • Integration with CRISPR Interference: Deploy dCas9-based transcriptional repression under tissue-specific or inducible promoters

• Phenotypic Characterization Pipeline:

  • High-throughput Imaging: Automated phenotyping of growth parameters throughout development

  • Single-cell Transcriptomics: Analysis of cell-type specific responses to CKS1 modification

  • Live Cell Imaging: Implementation of fluorescent cell cycle markers to monitor division patterns in edited plants

• Homology-Directed Repair Enhancement:

  • Optimize Donor Template Design: Include at least 500-800bp homology arms for efficient integration

  • Cell Cycle Synchronization: Time transformation to S/G2 phases when HDR is most active

  • DNA Repair Modulation: Transiently suppress NHEJ pathway components to favor HDR

These optimized approaches would enable unprecedented precision in manipulating CKS1 function, allowing researchers to address fundamental questions about its role in rice development with minimal confounding effects.

What integrated approaches can best reveal the complete functional network of rice CKS1?

To comprehensively map the functional network of rice CKS1, researchers should implement an integrated multi-omics approach:

• Interactome Mapping:

  • Proximity-dependent biotin labeling (BioID or TurboID) to identify proteins in close proximity to CKS1 in living rice cells

  • Quantitative proteomics to identify interaction changes across cell cycle phases and stress conditions

  • Yeast two-hybrid screening against normalized rice cDNA libraries, following methods that successfully identified interactions between rice ICK/KRPs and cyclins

• Functional Genomics Integration:

  • CRISPR-based screens to identify genetic modifiers of CKS1 function

  • Synthetic genetic array analysis using rice mutant collections

  • Transcriptome profiling of CKS1 overexpression and knockout lines under various conditions

• Structural Biology Approaches:

  • Cryo-EM analysis of CKS1-containing complexes to understand the hexameric arrangement described for CKS proteins

  • Hydrogen-deuterium exchange mass spectrometry to map dynamic protein interactions

  • Computational modeling of interaction interfaces based on established methods for analyzing protein-protein interactions

• Systems Biology Integration:

• Comparative Analysis Framework:

  • Cross-species comparisons with Arabidopsis, maize, and other crop plants

  • Evolutionary analysis to identify conserved versus species-specific functions

  • Integration with existing plant cell cycle databases

This multi-layered approach would provide unprecedented insights into CKS1's role as a hub in rice cell cycle regulation and reveal novel connections to development, metabolism, and stress response pathways.

How might findings about rice CKS1 contribute to improving crop resilience and yield?

Research on rice CKS1 has significant potential to contribute to crop improvement strategies through several translational pathways:

• Enhanced Stress Tolerance Development:

  • If CKS1 functions similarly to the characterized CRK proteins in rice immune responses , precise modulation of its activity could enhance specific stress tolerance pathways without growth penalties

  • Understanding how CKS1 integrates cell cycle regulation with stress responses could enable engineering of crops with maintained cell division under suboptimal conditions

• Yield Component Enhancement:

  • Studies of rice ICK/KRP proteins have demonstrated that manipulation of cell cycle regulators affects key agricultural traits including seed setting and pollen viability

  • Strategic modification of CKS1 expression in specific tissues or developmental stages could potentially increase seed number, size, or filling rate

• Architecture Optimization:

  • Findings that overexpression of cell cycle inhibitors like OsiICK6 affects leaf morphology (causing rolling) suggest that CKS1 manipulation could be used to fine-tune plant architecture

  • Targeted expression could optimize canopy structure for light interception or planting density

• Translational Approaches:

• CRISPR-Based Approaches:

  • Development of allelic series targeting regulatory regions of CKS1 to create a range of expression levels

  • Precise modification of protein interaction surfaces to alter specific functions while maintaining others

Translating these research findings into agricultural applications will require careful optimization to ensure that beneficial effects on stress resistance or yield components are not offset by unintended consequences on other aspects of plant growth and development.

What key methodological innovations are needed to advance our understanding of CKS1 dynamics in living rice cells?

Advancing our understanding of CKS1 dynamics in living rice cells requires several methodological innovations:

• Live-Cell Imaging Technologies:

  • Development of minimally disruptive fluorescent tagging methods for endogenous CKS1

  • Implementation of super-resolution microscopy optimized for plant cell imaging

  • Adaptation of light-sheet microscopy for long-term imaging of rice tissues with subcellular resolution

• Biosensor Development:

  • Creation of FRET-based sensors to detect CKS1-CDK interactions in real-time

  • Development of activity-based probes to monitor CKS1 functional states

  • Design of optogenetic tools to modulate CKS1 interactions with temporal and spatial precision

• Single-Cell Technologies:

  • Optimization of protoplast isolation protocols preserving cell cycle state information

  • Adaptation of single-cell RNA-seq and proteomics for rice cells

  • Development of computational pipelines to integrate single-cell data with tissue-level phenotypes

• In Situ Structural Analysis:

  • Implementation of cryoEM tomography for visualizing CKS1-containing complexes in their native cellular environment

  • Development of proximity labeling methods with enhanced spatial resolution

  • Adaptation of in-cell NMR techniques for plant systems

• Multi-parameter Phenotyping Platforms:

• Temporal Control Systems:

  • Development of faster-responding inducible systems than those currently available

  • Creation of synthetic circuits for oscillating gene expression to probe cell cycle dynamics

  • Implementation of degradation-based approaches for rapid protein depletion