Recombinant Arabidopsis thaliana CBS domain-containing protein CBSCBSPB3 (CBSCBSPB3)

What are CBS domain-containing proteins in Arabidopsis thaliana?

CBS domain-containing proteins (CDCPs) represent an evolutionarily conserved superfamily of proteins that contain varying numbers of cystathionine-β-synthase (CBS) domains. In Arabidopsis thaliana, CDCPs are organized into eight phylogenetic groups based on their domain architecture and sequence similarity. These proteins function as essential regulators in plant responses to various biotic and abiotic stressors, as well as in fundamental developmental processes. The CBS domain was originally discovered in archaebacteria and has since been identified across numerous species, demonstrating its evolutionary importance . The CBSX subfamily in Arabidopsis contains members localized to different cellular compartments, including CBSX1 and CBSX2 in chloroplasts and CBSX3 in mitochondria, where they regulate cellular redox status via thioredoxin activation .

How are CBS domain-containing proteins structured?

A typical CBS domain contains approximately 60 amino acid residues that fold into a structure consisting of two α-helices and three β-strands. These domains generally exist as tandem repeats in the polypeptide, particularly in pairs or quads, which form the functional regulatory unit. Beyond the CBS domain itself, CDCP family genes encode various additional functional domains such as CNNM (or DUF21), inosine-5′-monophosphate dehydrogenase (IMPDH), Phox and Bem1 (PB1), and voltage chloride channel (Voltage CLC) . This domain architecture diversity contributes to the functional versatility of CDCPs. The CBS domain mediates binding of adenosine-based molecules such as AMP, ATP, or S-adenosylmethionine (SAM), which can modulate protein activity through allosteric regulation mechanisms .

Where are CBS domain-containing proteins localized in plant cells?

CBS domain-containing proteins exhibit specific subcellular localizations that correlate with their biological functions. In Arabidopsis thaliana:

• CBSX1 and CBSX2 are localized to the chloroplast, where they activate thioredoxins in the ferredoxin-thioredoxin system (FTS)

• CBSX3 is found in mitochondria, where it regulates mitochondrial thioredoxin members in the NADPH-thioredoxin system (NTS)

This compartmentalization enables CBS domain-containing proteins to regulate specific redox processes in different organelles. The subcellular localization of CBSX1 has been confirmed through multiple experimental approaches, including GFP fusion protein analysis and database verification with resources such as The Arabidopsis Information Resource (TAIR), ChloroP prediction tools, the Plant Proteome Database, and Subcellular Localization of Proteins in Arabidopsis .

What is the evolutionary significance of CBS domain-containing proteins?

The evolutionary conservation of CBS domains across bacteria, plants, and animals underscores their fundamental importance in cellular function. Mutations in the CBS domains of human proteins have been linked to numerous hereditary diseases, including homocystinuria (cystathionine-β-synthase mutations), retinitis pigmentosa (inosine-5′-monophosphate dehydrogenase mutations), familial hypertrophic cardiomyopathy (AMP-activated protein kinase mutations), and myotonia congenital (chloride channel mutations) . In plants, the phylogenetic grouping of CDCP family genes varies among species, with eight groups identified in Arabidopsis thaliana and Triticum aestivum, nine in Oryza sativa and Glycine max, and fourteen major clades identified across eleven genomes from ten Oryza species . This diversification suggests evolutionary adaptation of CDCPs to fulfill species-specific regulatory needs in plant metabolism and stress responses.

How are recombinant CBS domain-containing proteins produced for laboratory studies?

The production of recombinant CBS domain-containing proteins involves several critical steps:

• Gene Cloning: The coding sequence of the target CBS domain protein is amplified from Arabidopsis thaliana cDNA using gene-specific primers designed based on genomic information from databases like TAIR.

• Expression Vector Construction: The amplified gene is cloned into an appropriate expression vector (e.g., pET series vectors for bacterial expression) containing the necessary regulatory elements and affinity tags (His-tag, GST-tag) for downstream purification.

• Heterologous Expression: The recombinant vector is transformed into a suitable expression host (typically E. coli BL21(DE3) for plant proteins). Expression conditions must be optimized for temperature (often 16-20°C for plant proteins), IPTG concentration, and duration to maximize soluble protein yield.

• Protein Purification: The expressed protein is purified using affinity chromatography (Ni-NTA for His-tagged proteins), followed by size exclusion chromatography to enhance purity and remove aggregates.

• Protein Validation: The purified protein is analyzed by SDS-PAGE, western blotting, and mass spectrometry to confirm identity and integrity before functional assays .

For CBS domain proteins specifically, researchers should consider including adenosine-based molecules (AMP, ATP) during purification to maintain structural stability, as these are natural ligands for CBS domains .

What techniques are used to study CBS domain-protein interactions?

Multiple complementary techniques can be employed to characterize the interactions of CBS domain-containing proteins:

• Yeast Two-Hybrid Screening: This approach can identify potential interacting partners from a library of proteins. For example, CBSX1 interactions with chloroplast redox regulators, including various thioredoxins (Trx f, Trx m, Trx x, and Trx y), were initially identified through yeast two-hybrid screens .

• In Vitro Pull-Down Assays: Using recombinant proteins, pull-down assays can confirm direct physical interactions between CBS domain proteins and their partners. This technique was used to validate the interaction between CBSX1 and multiple thioredoxins .

• Bimolecular Fluorescence Complementation (BiFC): This technique enables visualization of protein-protein interactions in living plant cells, providing evidence for interactions in a biologically relevant context .

• Surface Plasmon Resonance (SPR): SPR provides quantitative measurements of binding kinetics and affinity between CBS domain proteins and their interaction partners or ligands.

• Isothermal Titration Calorimetry (ITC): ITC can determine the thermodynamic parameters of CBS domain interactions with adenosine-based molecules and protein partners.

These methods should be used in combination to build a comprehensive understanding of CBS domain protein interaction networks.

How can researchers confirm the subcellular localization of CBS domain-containing proteins?

Confirmation of subcellular localization requires multiple complementary approaches:

It's important to use multiple methods, as some proteins exhibit complex accumulation patterns or may localize to multiple compartments under different conditions .

What experimental approaches are suitable for analyzing CBS domain-protein function?

Functional analysis of CBS domain-containing proteins can be approached through various experimental strategies:

• Loss-of-Function Mutations: CRISPR/Cas9-mediated gene editing can generate single, double, or triple mutants to assess the physiological importance of CBS domain proteins. This approach has been successful in creating cbf mutants in Arabidopsis to study their role in cold acclimation .

• Overexpression Studies: Transgenic plants overexpressing CBS domain proteins can reveal gain-of-function phenotypes and help establish the protein's role in specific pathways.

• Enzyme Activity Assays: For CBS domain proteins that regulate enzyme activity, in vitro assays can measure how these proteins affect the activity of target enzymes under different conditions. For example, CBSX1 has been shown to enhance thioredoxin enzymatic activity, particularly in the presence of AMP .

• Phenotypic Analysis: Comparing wild-type and mutant plants under various stress conditions (drought, salt, cold) can reveal the functional importance of CBS domain proteins in stress responses.

• Transcriptome Analysis: RNA-seq or microarray studies comparing gene expression profiles between wild-type and mutant plants can identify downstream genes regulated by CBS domain proteins.

• Metabolomic Analysis: Since CBS domain proteins often regulate metabolic enzymes, metabolite profiling can reveal altered metabolic pathways in mutant plants.

How do CBS domain-containing proteins regulate plant responses to environmental stressors?

CBS domain-containing proteins play crucial roles in plant stress responses through multiple mechanisms:

• Redox Regulation: CBS domain proteins like CBSX1 function as redox regulators by activating thioredoxins, which in turn modulate the activity of various enzymes involved in stress responses. This regulation helps maintain cellular redox homeostasis under stress conditions .

• H₂O₂ Level Modulation: CBSX1 directly regulates thioredoxins and thereby controls H₂O₂ levels, which act as signaling molecules in stress responses. By modulating H₂O₂ levels, CBS domain proteins can influence stress signaling cascades .

• Metabolic Adjustment: Through regulation of key metabolic enzymes like malate dehydrogenase in the Calvin cycle, CBS domain proteins help plants adjust their metabolism in response to environmental challenges .

• Stress-Responsive Gene Regulation: CBS domain proteins indirectly influence the expression of stress-responsive genes by modulating transcription factor activity through redox regulation.

• Cross-Talk with Hormone Signaling: CBS domain proteins interact with hormone signaling pathways involved in stress responses, creating integrated regulatory networks.

The importance of CBS domain proteins varies depending on the stress type and plant species. In Arabidopsis, CDCP family genes have been implicated in responses to cold, drought, salt stress, and pathogen infection .

What is the relationship between CBS domain-containing proteins and thioredoxin systems?

The relationship between CBS domain-containing proteins and thioredoxin systems reveals a sophisticated regulatory mechanism:

• Direct Physical Interaction: CBSX1 directly interacts with multiple chloroplast thioredoxins (Trx f, Trx m, Trx x, and Trx y) as demonstrated through yeast two-hybrid screens, in vitro pull-down assays, and bimolecular fluorescence complementation .

• Activation Mechanism: CBSX1 activates thioredoxins and further enhances their enzymatic activity in the presence of AMP, suggesting that CBSX1 functions as an adenosine-sensing regulator of thioredoxin activity .

• Organelle-Specific Regulation: CBSX1 and CBSX2 regulate chloroplast thioredoxins in the ferredoxin-thioredoxin system (FTS), while CBSX3 regulates mitochondrial thioredoxins in the NADPH-thioredoxin system (NTS). This organelle-specific action allows coordinated but distinct regulation of redox processes across cellular compartments .

• Downstream Enzyme Regulation: Through thioredoxin activation, CBS domain proteins indirectly regulate numerous enzymes involved in photosynthesis, carbon metabolism, and antioxidant defense. For example, CBSX1 affects Calvin cycle enzymes like malate dehydrogenase via regulation of thioredoxins .

• Redox Homeostasis: The interaction between CBS domain proteins and thioredoxins helps maintain redox homeostasis under both normal and stress conditions, making it a critical regulatory node in plant metabolism and stress responses .

This multi-level regulatory relationship enables fine-tuning of cellular redox status in response to metabolic needs and environmental challenges.

How do mutations in CBS domains affect plant development and stress tolerance?

Mutations in CBS domains can have profound effects on plant development and stress responses:

• Developmental Impacts:

  • Altered plant growth and morphology

  • Changes in flowering time and reproductive development

  • Modified cell wall composition and structure

  • Abnormal lignin deposition affecting tissue architecture

• Stress Tolerance Effects:

  • Reduced freezing tolerance due to impaired cold acclimation

  • Altered drought response capabilities

  • Compromised salt stress tolerance

  • Modified pathogen resistance

• Metabolic Consequences:

  • Disrupted redox balance affecting numerous metabolic pathways

  • Altered carbon allocation and energy metabolism

  • Changes in hormone biosynthesis and signaling

  • Modified secondary metabolite production

• Molecular Mechanisms:

  • Impaired binding of adenosine-based molecules (AMP, ATP, SAM)

  • Reduced ability to activate thioredoxins and other partner proteins

  • Disrupted protein-protein interactions affecting signal transduction

  • Altered subcellular localization affecting protein function

The severity and nature of these effects depend on the specific mutation, the affected CBS domain protein, and environmental conditions. For example, cbf triple mutants exhibit extreme sensitivity to freezing after cold acclimation and are defective in seedling development and salt stress tolerance, demonstrating the essential role of these proteins in multiple aspects of plant physiology .

What are the current challenges in CBS domain-protein research?

Several significant challenges exist in the field of CBS domain-protein research:

• Functional Redundancy: Many plant species contain multiple CBS domain-containing proteins with potentially overlapping functions, making it difficult to determine the specific role of individual proteins. This redundancy often necessitates the creation of higher-order mutants to observe clear phenotypes .

• Context-Dependent Activity: The activity of CBS domain proteins can vary depending on cellular conditions, making it challenging to establish consistent functional paradigms across different experimental setups.

• Complex Interaction Networks: CBS domain proteins participate in extensive protein-protein interaction networks that are difficult to fully characterize using current experimental approaches.

• Limited Structural Information: Despite their importance, detailed structural information on plant CBS domain proteins, particularly in complex with their interaction partners, remains limited.

• Integration of Multi-Omics Data: Effectively integrating transcriptomic, proteomic, metabolomic, and phenomic data to build comprehensive models of CBS domain protein function remains technically challenging.

• Translating Findings Across Species: While CBS domains are conserved, their specific functions may vary between species, making it difficult to translate findings from model organisms to crop plants.

• Regulatory Complexity: Understanding how environmental signals are integrated to modulate CBS domain protein activity requires sophisticated experimental designs that can capture dynamic regulatory processes.

Addressing these challenges will require innovative experimental approaches, advanced computational tools, and interdisciplinary collaboration.

How should researchers interpret contradictory findings in CBS domain-protein studies?

When faced with contradictory findings in CBS domain-protein research, researchers should follow these methodological approaches:

What bioinformatic tools are most effective for CBS domain-protein analysis?

Several bioinformatic tools are particularly valuable for analyzing CBS domain-containing proteins:

• Sequence Analysis Tools:

  • HMMER (using the PF00571 profile) for identifying CBS domains in protein sequences

  • MEME Suite for motif discovery and analysis within CBS domains

  • CD-Search, Pfam, and SMART for domain architecture verification

• Structural Prediction Tools:

  • AlphaFold2 for predicting protein structure

  • SWISS-MODEL for homology-based structural modeling

  • PyMOL or UCSF Chimera for visualizing and analyzing protein structures

• Localization Prediction:

  • ChloroP, TargetP, and WoLF PSORT for subcellular localization prediction

  • Signal peptide servers (SignalP) for identifying targeting sequences

• Phylogenetic Analysis Tools:

  • MEGA X for constructing phylogenetic trees of CBS domain proteins

  • IQ-TREE for maximum likelihood phylogenetic analysis

  • MrBayes for Bayesian inference of phylogeny

• Protein-Protein Interaction Prediction:

  • STRING database for predicting functional protein association networks

  • PSICQUIC for accessing multiple interaction databases

• Expression Analysis Tools:

  • BAR Expression Browser for analyzing expression patterns across tissues and conditions

  • eFP Browser for visualizing gene expression data

• Genome Browsers and Databases:

  • TAIR for Arabidopsis-specific information

  • Phytozome for comparative genomics across plant species

  • UniProt for curated protein information

These tools should be used in combination to develop a comprehensive understanding of CBS domain proteins at the sequence, structure, and functional levels.

How can researchers integrate multi-omics data in CBS domain-protein research?

Integrating multi-omics data for CBS domain-protein research requires a systematic approach:

• Data Collection Strategy:

  • Design experiments to collect matched samples for transcriptomics, proteomics, metabolomics, and phenomics

  • Include appropriate time points to capture dynamic responses

  • Use both wild-type and CBS domain protein mutants under multiple conditions

• Data Processing Pipelines:

  • Implement consistent quality control across all data types

  • Apply appropriate normalization methods for each data type

  • Use standardized identifiers to facilitate data integration

• Multi-layered Analysis Approaches:

  • Correlation network analysis to identify relationships between different omics layers

  • Pathway enrichment analysis across multiple data types

  • Machine learning methods to identify patterns across diverse datasets

• Integration Frameworks:

  • Use tools like mixOmics, DIABLO, or MOFA for multi-omics data integration

  • Apply systems biology modeling to integrate diverse data types

  • Develop custom computational pipelines tailored to CBS domain protein research questions

• Visualization Strategies:

  • Create multi-dimensional visualizations that represent relationships across data types

  • Develop interactive visualizations to explore complex integrated datasets

  • Use pathway visualization tools to map multi-omics data onto biological processes

• Validation of Integrated Findings:

  • Design targeted experiments to validate predictions from integrated data analysis

  • Use CRISPR/Cas9 to create specific mutations for hypothesis testing

  • Apply biochemical assays to confirm predicted molecular mechanisms

This integrated approach can reveal emergent properties of CBS domain protein function that would not be apparent from any single data type alone.

What statistical approaches are recommended for analyzing CBS domain-protein experimental data?

• Experimental Design Considerations:

  • Power analysis to determine appropriate sample sizes

  • Randomized complete block designs to control for environmental variation

  • Factorial designs to assess interaction effects between multiple factors

• Differential Expression Analysis:

  • Linear models (limma) for microarray data

  • DESeq2 or edgeR for RNA-seq data analysis

  • Appropriate multiple testing correction (e.g., Benjamini-Hochberg)

• Time Series Analysis:

  • STEM (Short Time-series Expression Miner) for clustering time series data

  • maSigPro for identifying significantly different temporal profiles

  • Functional data analysis for continuous time modeling

• Multivariate Statistical Methods:

  • Principal Component Analysis (PCA) for dimension reduction

  • Partial Least Squares Discriminant Analysis (PLS-DA) for separating experimental groups

  • PERMANOVA for testing multivariate responses to experimental factors

• Correlation Network Analysis:

  • Weighted Gene Co-expression Network Analysis (WGCNA) for identifying functional modules

  • Gaussian Graphical Models for inferring conditional independence relationships

  • Canonical Correlation Analysis for relating different data types

• Experimental Data Analysis:

  • Mixed-effects models for handling nested experimental designs

  • Non-parametric tests when data violate normality assumptions

  • Survival analysis for stress tolerance assays

• Reproducibility Considerations:

  • Cross-validation approaches for model validation

  • Bootstrapping for robust confidence interval estimation

  • Detailed reporting of all statistical methods and parameters

Proper statistical analysis enhances the rigor and reproducibility of CBS domain-protein research and facilitates comparison across different studies.

What are the promising areas for future research on CBS domain-containing proteins?

Several promising research directions could significantly advance our understanding of CBS domain-containing proteins:

• Structural Biology Approaches:

  • High-resolution structural studies of plant CBS domain proteins in complex with their interaction partners

  • Investigation of conformational changes upon ligand binding

  • Structure-guided protein engineering to modify CBS domain protein function

• Molecular Mechanism Exploration:

  • Detailed characterization of how adenosine-based molecule binding affects CBS domain protein activity

  • Investigation of post-translational modifications regulating CBS domain protein function

  • Exploration of potential RNA-binding capabilities of CBS domain proteins

• Synthetic Biology Applications:

  • Development of synthetic CBS domain-based sensors for monitoring cellular energy status

  • Engineering of CBS domain proteins with novel regulatory capabilities

  • Creation of synthetic regulatory circuits incorporating CBS domain proteins

• Environmental Adaptation Studies:

  • Comparative analysis of CBS domain proteins across plant species adapted to different environments

  • Investigation of CBS domain protein evolution in response to environmental pressures

  • Exploration of CBS domain protein roles in extremophile plants

• Crop Improvement Applications:

  • Targeted modification of CBS domain proteins to enhance stress tolerance in crops

  • Investigation of natural variation in CBS domain proteins across crop germplasm

  • Development of CBS domain-based molecular markers for breeding programs

These research directions could yield important insights into fundamental plant biology and potentially contribute to agricultural innovation.

How might CRISPR/Cas9 technology advance our understanding of CBS domain-protein function?

CRISPR/Cas9 technology offers powerful approaches to investigate CBS domain-protein function:

• Precise Gene Editing:

  • Generation of knockout mutants for individual or multiple CBS domain-containing genes

  • Creation of allelic series with specific mutations in CBS domains to assess structure-function relationships

  • Domain swapping between different CBS proteins to investigate domain-specific functions

• Multiplexed Mutagenesis:

  • Simultaneous targeting of multiple CBS domain genes to overcome functional redundancy

  • Creation of higher-order mutants by targeting genes in related pathways

  • Systematic mutagenesis of entire CBS domain-containing gene families

• Base Editing Applications:

  • Introduction of specific amino acid changes without double-strand breaks

  • Creation of synonymous mutations to study codon optimization effects

  • Modification of regulatory sequences affecting CBS domain protein expression

• Transcriptional Modulation:

  • CRISPRi for targeted repression of CBS domain gene expression

  • CRISPRa for activation of CBS domain genes

  • Temporal control of CBS domain gene expression using inducible CRISPR systems

• In Vivo Tracking:

  • CRISPR-based tagging of endogenous CBS domain proteins for live-cell imaging

  • Integration of reporter genes to monitor CBS domain protein expression

  • Visualization of protein-protein interactions involving CBS domain proteins

As demonstrated in studies of CBF genes, CRISPR/Cas9 can overcome limitations of traditional genetic approaches, enabling the creation of higher-order mutants that reveal essential functions not apparent in single mutants .

What interdisciplinary approaches could enhance CBS domain-protein research?

Interdisciplinary approaches can significantly advance CBS domain-protein research:

• Computational Biology and Artificial Intelligence:

  • Deep learning approaches for predicting CBS domain protein function

  • Molecular dynamics simulations to understand CBS domain conformational changes

  • Network analysis tools to map CBS domain protein regulatory networks

• Chemical Biology:

  • Development of small molecule modulators of CBS domain protein function

  • Chemical proteomics to identify novel CBS domain protein interactions

  • Metabolite profiling to understand the impact of CBS domain proteins on plant metabolism

• Advanced Imaging Technologies:

  • Super-resolution microscopy to visualize CBS domain protein localization and dynamics

  • FRET/FLIM analyses to study protein-protein interactions in living cells

  • Label-free imaging techniques to observe CBS domain protein activity in vivo

• Evolutionary Biology:

  • Comparative genomics to trace the evolution of CBS domain proteins across species

  • Ancestral sequence reconstruction to understand evolutionary innovations in CBS domains

  • Evolutionary rate analysis to identify selection pressures on CBS domain proteins

• Systems Biology:

  • Mathematical modeling of CBS domain protein regulatory networks

  • Flux balance analysis to understand metabolic impacts of CBS domain proteins

  • Agent-based modeling to simulate emergent properties of CBS domain protein function

• Plant-Microbe Interaction Studies:

  • Investigation of how CBS domain proteins influence plant-microbe symbioses

  • Examination of pathogen effectors targeting CBS domain proteins

  • Study of CBS domain protein roles in immune signaling

These interdisciplinary approaches can provide novel insights that would be difficult to achieve through traditional plant biology methods alone.

How might systems biology approaches contribute to CBS domain-protein research?

Systems biology offers powerful frameworks for understanding CBS domain proteins in the context of whole-plant function:

• Network Modeling:

  • Construction of gene regulatory networks centered on CBS domain proteins

  • Protein-protein interaction networks to map the CBS domain protein interactome

  • Metabolic network analysis to understand how CBS domain proteins influence plant metabolism

• Multi-scale Modeling:

  • Integration of molecular, cellular, and whole-plant level data

  • Spatiotemporal modeling of CBS domain protein activity

  • Linking molecular mechanisms to physiological outcomes

• Constraint-based Modeling:

  • Flux balance analysis to predict metabolic consequences of CBS domain protein perturbation

  • Kinetic modeling to understand dynamic responses

  • Genome-scale metabolic models incorporating CBS domain protein regulation

• Bayesian Network Approaches:

  • Causal inference to identify regulatory relationships

  • Dynamic Bayesian networks to model time-dependent processes

  • Integration of prior knowledge with experimental data

• Module-based Analysis:

  • Identification of functional modules in which CBS domain proteins participate

  • Cross-species module comparison to identify conserved functions

  • Module-based phenotype prediction

• Whole-Plant Physiological Integration:

  • Models linking CBS domain protein activity to whole-plant responses

  • Integration of environmental inputs with molecular responses

  • Prediction of plant performance under various environmental scenarios

Systems biology approaches can reveal emergent properties and provide testable hypotheses about CBS domain protein function that might be overlooked in reductionist approaches.