Recombinant Oryza sativa subsp. japonica Cytokinin dehydrogenase 5 (CKX5), partial

What is Cytokinin dehydrogenase 5 (CKX5) and what is its primary function in rice development?

Cytokinin dehydrogenase 5 (CKX5), also known as cytokinin oxidase 5 or OsCKX5, is one of 11 identified cytokinin oxidase/dehydrogenase enzymes in rice (Oryza sativa) . This enzyme (EC 1.5.99.12) catalyzes the irreversible degradation of cytokinins through oxidative cleavage, playing a critical role in maintaining cytokinin homeostasis within the plant . By regulating cytokinin levels, CKX5 contributes to various developmental processes including leaf and root growth, inflorescence architecture, fertilization, and grain weight .

What are the key specifications of commercially available recombinant OsCKX5?

Recombinant Oryza sativa subsp. japonica Cytokinin dehydrogenase 5 (CKX5) is typically produced as a partial protein with high purity (>90%) in expression systems including E. coli, yeast, baculovirus, or mammalian cells . The commercial product is generally supplied in liquid form containing glycerol to maintain stability. Storage recommendations include keeping the protein at -20°C for long-term storage, with working aliquots at 4°C for up to one week to prevent degradation through repeated freeze-thaw cycles .

How is CKX5 genetically characterized in the rice genome?

CKX5 is encoded by the CKX5 gene located on chromosome 1 of the rice genome. The gene has multiple identifiers including:

• Gene name: CKX5

• Gene synonyms: LOC_Os01g56810, P0413G02.1, P0490D09.33

• Other associated names: Os01g0775400, OsCKX5

This genetic information is crucial for designing primers, expression constructs, and gene-editing strategies in research applications.

How can researchers analyze the expression patterns of OsCKX5 in different rice tissues?

Comprehensive analysis of OsCKX5 expression can be conducted using the following methodological approaches:

• RNA sequencing (RNA-seq) to quantitatively assess transcript abundance across different tissues and developmental stages

• β-glucuronidase (GUS) staining using promoter-reporter constructs to visualize spatial expression patterns at the tissue level

• Quantitative RT-PCR (qRT-PCR) for targeted validation of expression in specific tissues

• In situ hybridization to detect mRNA localization at the cellular level

Research has revealed that each OsCKX gene, including OsCKX5, exhibits a unique tissue-specific expression pattern, which contributes to their specialized functions in different plant organs and developmental stages .

What methodologies are most effective for generating and analyzing ckx5 mutants in rice?

CRISPR/Cas9 technology has been successfully employed to generate ckx5 mutants for functional studies . Based on research approaches described in the literature, the following methodology is recommended:

• Guide RNA design: Target conserved regions encoding catalytically important domains of CKX5

• Transformation protocol: Use established rice transformation methods, typically Agrobacterium-mediated

• Mutation screening: Employ a combination of PCR, restriction enzyme digestion, and sequencing

• Validation of mutations: Confirm mutations at both DNA and transcript levels

• Phenotypic analysis: Assess changes in plant architecture, cytokinin content, and developmental parameters

• Higher-order mutant generation: Cross with other ckx mutants to study functional redundancy

This systematic approach allows researchers to effectively study CKX5 function while accounting for potential functional overlap with other family members .

How do CKX5 and other CKX family members exhibit functional overlap and subfunctionalization?

Research demonstrates that the 11 CKX family members in rice display both functional overlap and subfunctionalization . Single ckx mutants, including ckx5, often show subtle phenotypes compared to higher-order mutants due to functional redundancy. Specifically:

• Single ckx5 mutants exhibit altered responses to TOR kinase inhibition, suggesting a specific function in TOR-dependent pathways

• While ckx5 single mutants show some resistance to the TOR inhibitor AZD8055, the confidence intervals overlap with wild-type in some studies, indicating partial redundancy with other CKX enzymes

• The ckx1/3/5 triple mutant displays distinct phenotypes not observed in single mutants, confirming partial functional overlap

These observations highlight the importance of studying both individual CKX genes and their combined functions to fully understand cytokinin regulation in rice.

How does CKX5 function interact with the TOR kinase pathway in regulating shoot development?

CKX5 appears to play a role in the TOR kinase signaling pathway that regulates shoot development in rice:

• CKX5 has a specific effect on the TOR-dependent transcriptome, as observed in ckx5 single mutants

• Treatment with the CKX inhibitor INCYDE (75 nM) makes wild-type seedlings resistant to even 0.5 μM of the TOR inhibitor AZD8055, indicating that CKX activity mediates TOR's control of shoot growth

• While ckx5 mutants show some resistance to TOR inhibition, this resistance is limited to specific concentration ranges (between 0.5-1 μM AZD8055), suggesting a concentration-dependent interaction

This interaction between CKX5 and TOR kinase represents an important regulatory mechanism connecting nutrient sensing (via TOR) with hormonal control of shoot development (via cytokinin degradation).

What are the recommended experimental conditions for analyzing CKX5 activity in vitro?

When working with recombinant CKX5 protein for enzymatic assays, the following experimental conditions are recommended:

When analyzing CKX5 activity in plant tissues, researchers should consider using:

• Low concentrations of cytokinin (100 nM tZ and iP) to detect subtle differences in CKX sensing

• ARR5 expression as a sensitive readout of cytokinin response

• Pre-incubation with TOR inhibitors like AZD8055 to study pathway interactions

How can recombinant CKX5 be utilized to study cross-talk between cytokinin and other hormone pathways?

Recombinant CKX5 provides a valuable tool for investigating hormone cross-talk in rice:

• Transcriptome analysis: Compare gene expression profiles in wild-type and ckx mutants to identify genes at the intersection of cytokinin and other hormone pathways. Studies have shown that ckx mutants affect expression of genes involved in both auxin and cytokinin pathways

• Pharmacological approach: Use recombinant CKX5 in combination with hormone inhibitors/analogs to:

  • Study the effect of CKX5-mediated cytokinin degradation on auxin transport and signaling

  • Investigate interactions with the TOR pathway using inhibitors like AZD8055

  • Examine potential crosstalk with stress hormones like ABA

• Protein-protein interaction studies: Use recombinant CKX5 to identify potential interacting partners from other hormone pathways through techniques like:

  • Co-immunoprecipitation followed by mass spectrometry

  • Yeast two-hybrid screening

  • Bimolecular fluorescence complementation

These approaches can reveal how CKX5 functions within the broader hormonal regulatory network in rice.

What role might CKX5 play in rice-pathogen interactions and stress responses?

While specific research on CKX5's role in rice-pathogen interactions is limited in the provided literature, several lines of evidence suggest potential involvement:

• Cytokinins are known regulators of plant immunity and stress responses

• A genome-wide association study investigating the reciprocal adaptation of rice and Xanthomonas oryzae pv. oryzae (Xoo) identified 318 rice quantitative resistance genes, which may include genes involved in hormone metabolism

• The complex genetic interaction system between rice and pathogens likely involves hormone signaling pathways including cytokinin regulation

Researchers investigating CKX5's role in stress responses should:

• Examine CKX5 expression patterns under various biotic and abiotic stress conditions

• Generate and analyze ckx5 mutants for altered disease resistance phenotypes

• Test whether recombinant CKX5 activity is affected by stress-related factors

How do mutations in the CKX5 gene impact rice yield components and agronomic traits?

Research on CKX family mutants provides insights into how CKX5 might affect rice yield:

Understanding the specific contribution of CKX5 to these traits requires comprehensive phenotypic analysis of single and higher-order mutants under various growing conditions.

How might targeted modification of CKX5 be utilized for rice improvement?

Based on current understanding of CKX5 function, several strategies could be employed for rice improvement:

• Tissue-specific modulation: Using tissue-specific promoters to alter CKX5 expression only in specific organs (panicles, roots) to fine-tune cytokinin levels where beneficial

• Precision genome editing: Creating specific allelic variants of CKX5 that maintain homeostatic function while optimizing agronomic traits

• Exploiting TOR-CKX5 interactions: Developing breeding strategies that optimize the balance between TOR signaling and cytokinin degradation to enhance plant architecture and yield potential

• Combined approaches: Stacking optimized CKX5 variants with modifications in other cytokinin-related genes for synergistic effects on plant development and stress tolerance

These approaches require detailed knowledge of CKX5's specific contribution to rice development and careful consideration of potential pleiotropic effects.

What experimental approaches could reveal the structural basis of CKX5 substrate specificity?

Understanding the structural basis of CKX5 substrate specificity would advance both basic research and applied efforts. Recommended approaches include:

• Protein structure determination:

  • X-ray crystallography of recombinant CKX5 alone and in complex with substrates/inhibitors

  • Cryo-electron microscopy for larger complexes

  • Homology modeling based on known structures of related CKX enzymes

• Structure-function analysis:

  • Site-directed mutagenesis of predicted substrate-binding residues

  • Enzyme kinetics with various cytokinin substrates to determine specificity constants

  • Thermal shift assays to assess protein stability upon substrate binding

• Computational approaches:

  • Molecular docking simulations with different cytokinin substrates

  • Molecular dynamics to understand protein flexibility and substrate interactions

  • Quantum mechanical calculations of the reaction mechanism

This structural information would provide insights into the molecular basis of CKX5 function and guide efforts to develop specific inhibitors or engineered variants.