Recombinant Arabidopsis thaliana Zinc finger protein JAGGED (JAG)

What is the molecular structure of JAG protein and how is it classified?

JAG is a member of the zinc finger family of plant transcription factors in Arabidopsis thaliana. It has a structure similar to SUPERMAN, featuring a single C2H2-type zinc finger domain, a proline-rich motif, and a short leucine-rich repressor motif . The protein belongs to the C2H2 and C2HC zinc fingers superfamily . JAG contains an ethylene-responsive element binding factor-associated amphiphilic repression motif near its N-terminus, consistent with its predominantly repressive function in transcriptional regulation .

What phenotypes are associated with JAG loss-of-function mutations?

Loss-of-function mutations in the JAG locus result in Arabidopsis plants with characteristically abnormal lateral organs, including:

• Serrated leaves

• Narrow floral organs

• Petals containing fewer but more elongate cells

• Suppressed bract formation in leafy, apetala1, and apetala2 mutant backgrounds

These phenotypes indicate JAG's critical role in lateral organ development and patterning. Quantitative 3D imaging of floral organ primordia in jag mutants has revealed complex changes in cell behavior, including reduced proliferation, alterations in cell enlargement, changes in cell size homeostasis, and shifts in oriented anisotropic growth .

Where and when is JAG expressed in Arabidopsis?

JAG mRNA is localized to lateral organ primordia throughout the plant but is notably absent in the shoot apical meristem . JAG is activated in emerging organ primordia and in the distal region of immature organs . This expression pattern aligns with its function in promoting organ growth and patterning in developing lateral organs.

How can JAG protein be expressed and purified for in vitro studies?

Recombinant JAG protein can be produced using several expression systems:

• Expression Systems: JAG can be expressed in E. coli, yeast, baculovirus, or mammalian cell systems, with each offering different advantages for protein folding and post-translational modifications .

• Purification Method: Standard purification involves:

  • Expression with appropriate tags (His, GST, etc.)

  • Lysis under native or denaturing conditions

  • Affinity chromatography

  • Size exclusion chromatography

  • SDS-PAGE confirmation of purity (≥85% purity can be achieved)

• Quality Control: Purified JAG protein should be validated through:

  • Western blotting with JAG-specific antibodies

  • Mass spectrometry analysis

  • Functional DNA-binding assays

What approaches are effective for studying JAG binding sites in the genome?

ChIP-Seq (Chromatin Immunoprecipitation followed by high-throughput sequencing) has been effectively used to identify JAG binding sites throughout the Arabidopsis genome. The experimental approach includes:

• Plant Material Preparation: Using complemented jag-2 inflorescences with genomic JAG-GFP fusion (JAG:JAG-GFP) .

• ChIP Protocol:

  • Cross-linking plant tissue with formaldehyde

  • Isolating nuclei and sonication of chromatin

  • Immunoprecipitation using anti-GFP antibodies (for JAG-GFP fusions) or anti-HA antibodies (for HA-tagged JAG)

  • Reversal of cross-linking and DNA purification

• Data Analysis: JAG binding sites identified through ChIP-Seq are predominantly found within 1.5-kb regions upstream and downstream of coding sequences, confirming JAG's role as a transcription factor .

The table below summarizes ChIP-Seq findings for JAG binding sites:

A total of 1,634 genes contained binding peaks with a false discovery rate (FDR) of less than 1% in all three replicates and were selected as high-confidence JAG targets .

What genetic tools are available for studying JAG function in Arabidopsis?

Several genetic tools have been developed for JAG functional studies:

• Loss-of-Function Lines:

  • T-DNA insertion mutants (e.g., jag-2, jag-1)

  • CRISPR/Cas9-generated knockout lines

• Gain-of-Function Lines:

  • Constitutive overexpression lines (35S:JAG)

  • Inducible systems (e.g., JAG-GR, an estradiol-inducible system)

  • Tissue-specific expression using appropriate promoters

• Reporter Lines:

  • JAG:JAG-GFP fusion for protein localization studies

  • pJAG:GUS for promoter activity analysis

• Traffic Line (TL) System:

  • TLs containing seed-specific eGFP and DsRed markers linked to chromosomal regions of interest can be used to follow the inheritance of unmarked chromosomes containing JAG

  • These lines facilitate genetic analysis by providing a visual method for determining genotypes in segregating populations

What genes are directly regulated by JAG and how was this determined?

JAG directly regulates numerous genes involved in organ patterning and growth. These targets were identified through a combination of ChIP-Seq and transcriptome analysis:

• Methodology:

  • ChIP-Seq identified JAG binding sites throughout the genome

  • Transcriptome analysis (using Affymetrix ATH1 arrays) compared wild-type, jag mutants, and plants with inducible JAG activation

  • Overlapping these datasets identified functionally relevant direct JAG targets

• Key Direct Targets:

  • Meristem development genes: JAG directly represses CLAVATA1 and HANABA TARANU

  • Basal organ development genes: JAG represses BLADE ON PETIOLE 2 and the GROWTH REGULATORY FACTOR pathway

  • Cell cycle regulators: JAG directly represses KIP RELATED PROTEIN 4 (KRP4) and KRP2, which control the transition to S-phase

  • Cell wall modification genes: JAG regulates genes involved in tissue polarity and cell wall remodeling

• Transcriptional Role: JAG functions predominantly as a transcriptional repressor, with ChIP-Seq data showing significant enrichment for repressed genes (p = 3.90 × 10^-20) but not for activated genes (p = 1.37 × 10^-1) .

How does JAG regulate the cell cycle in developing organs?

JAG regulates the cell cycle primarily through direct control of specific cell cycle inhibitors:

• Direct Repression of KRP Genes:

  • JAG directly represses KRP4 and KRP2, which were the two genes with the highest ChIP-Seq scores among JAG targets

  • KRP2 and KRP4 encode cyclin-dependent kinase (CDK) inhibitors that control the transition to S-phase

• Functional Relevance:

  • The krp2 and krp4 mutations suppress jag defects in organ growth

  • These mutations also rescue abnormal petal epidermal cell morphology in jag mutants

  • This genetic interaction confirms that JAG's control of S-phase entry is a key mechanism by which it regulates organ growth

• Impact on Cell Behavior:

  • JAG promotes cell proliferation

  • It affects cell enlargement

  • It influences cell size homeostasis

  • It shifts growth toward oriented anisotropic growth

How does JAG interact with other developmental regulators in Arabidopsis?

JAG functions within a complex network of developmental regulators:

• Interactions with Organ Identity Genes:

  • JAG mediates between genes that control organ identity and the cellular activities required for organ growth

  • It forms a critical link between patterning genes and growth effectors

• Related Zinc Finger Proteins:

  • NUBBIN (NUB) is closely related to JAG and regulates stamen and carpel development together with JAG

  • JAGGED-LIKE (JGL) shares structural similarities with JAG

• Integration with Other Zinc Finger Proteins:

  • The C1-1i subclass of zinc finger proteins in Arabidopsis includes 33 members, many involved in plant development

  • Other related proteins like ZFP10, ZFP11, ZFP5, and ZFP3 regulate various aspects of plant growth and development

  • JAG is part of a larger network of zinc finger proteins that coordinate different aspects of development

How does JAG influence cellular anisotropic growth during organ development?

JAG's role in anisotropic growth involves complex regulation of cell wall properties and tissue polarity:

• Evidence from Imaging Studies:

  • Quantitative 3D imaging of floral organ primordia shows that JAG influences oriented anisotropic growth

  • Computer modeling of the changes in organ growth in response to JAG supports its role in polarized tissue growth

• Molecular Mechanisms:

  • JAG directly regulates genes involved in cell wall modification

  • It influences genes that control tissue polarity

  • Both processes are critical for directional cell expansion and anisotropic growth

• Experimental Approaches:

  • Characterization of cell geometry in JAG loss- and gain-of-function backgrounds

  • Analysis of cell wall composition and mechanical properties

  • Live imaging of growing organs combined with computational modeling of growth vectors

  • Examination of cytoskeletal organization using fluorescently labeled markers

What role does JAG play in tissue-specific recombination landscapes?

While JAG itself hasn't been directly implicated in controlling recombination, the study of recombination patterns in Arabidopsis provides context for understanding how JAG's effects might interact with genetic inheritance:

• Recombination in Arabidopsis:

  • F2 populations show distinct patterns of recombination with most plants carrying only one or two crossovers per chromosome pair

  • This results in inheritance of very large, non-recombined genomic fragments from each parent

  • Recombination frequencies vary between populations but consistently increase adjacent to centromeres

• Implications for JAG Research:

  • When studying JAG and its targets, researchers must consider how recombination patterns might affect genetic linkage

  • The use of Traffic Lines (TLs) with fluorescent seed markers can help track inheritance of JAG alleles and linked chromosomal regions

  • Understanding the recombination landscape is crucial for mapping JAG-related traits in segregating populations

• Segregation Distortion:

  • Over half of F2 populations show segregation distortion between parental alleles

  • This can affect the inheritance of JAG and its target loci

  • Potential causes include variation in seed dormancy and lethal epistatic interactions

What regulatory mechanisms control JAG expression itself?

The regulation of JAG expression involves multiple levels of control:

• Transcriptional Regulation:

  • JAG is activated in emerging organ primordia

  • Upstream transcription factors likely control its tissue-specific expression

  • Identifying these regulators requires techniques such as:

    • Promoter deletion analysis

    • Yeast one-hybrid screening

    • ChIP-Seq of candidate regulators

• Post-Transcriptional Control:

  • miRNA regulation might fine-tune JAG expression

  • RNA-binding proteins could affect JAG mRNA stability or translation

• Research Approaches:

  • Characterization of the JAG promoter and regulatory elements

  • Identification of transcription factors that bind the JAG promoter

  • Analysis of JAG expression in different genetic backgrounds and environmental conditions

  • Investigation of potential feedback loops in JAG regulation

How could JAG be utilized in synthetic biology applications for plant development?

JAG's capacity to regulate organ growth and patterning suggests potential applications in synthetic biology:

• Engineering Organ Growth:

  • Controlled expression of JAG could potentially modify leaf size and shape

  • Tissue-specific or inducible JAG expression systems might allow precise control of organ growth

• Genetic Toolkit Applications:

  • JAG-based synthetic transcription factors could be designed to control specific target genes

  • JAG DNA-binding domains could be fused to various effector domains for specific regulatory outputs

  • CRISPR-based approaches could be used to modulate endogenous JAG expression or target specificity

• Experimental Requirements:

  • Characterization of minimal functional domains of JAG

  • Development of orthogonal systems to avoid interference with endogenous JAG function

  • Precise control of expression levels and timing

  • Thorough phenotypic assessment of modified plants

• Regulatory Considerations:

  • Research involving recombinant JAG constructs in plants falls under institutional biosafety regulations

  • Creation of transgenic Arabidopsis plants with JAG modifications requires appropriate institutional biosafety committee (IBC) registration and approval

What regulatory considerations apply to research with recombinant JAG in Arabidopsis?

Research involving recombinant JAG constructs must comply with institutional and national guidelines:

• NIH Guidelines Compliance:

  • Creation of transgenic Arabidopsis with JAG constructs generally falls under Section III-D-4 of the NIH Guidelines

  • Requires IBC review and approval prior to initiation

  • The biosafety level (typically BL1 for Arabidopsis) must be determined and approved

• Documentation Requirements:

  • Protocol registration with institutional biosafety committee

  • Description of JAG constructs and expression systems

  • Risk assessment for modified plants

  • Containment procedures appropriate to the biosafety level

• Material Transfer Considerations:

  • Transfer of transgenic Arabidopsis lines containing JAG constructs between institutions may require material transfer agreements

  • Both sending and receiving institutions typically need appropriate approvals

What methodological controls should be included in JAG functional studies?

Robust controls are essential for reliable JAG research:

• Genetic Controls:

  • Multiple independent transgenic lines should be analyzed to rule out position effects

  • Complementation of jag mutants with wild-type JAG for rescue experiments

  • Empty vector controls for expression studies

  • Wild-type controls alongside mutants

• Expression Controls:

  • Verification of JAG expression levels by qRT-PCR

  • Protein expression confirmation by Western blot

  • Localization studies to confirm proper subcellular targeting

• Functional Validation:

  • ChIP-qPCR validation of selected target genes from ChIP-Seq data

  • Independent confirmation of gene expression changes using qRT-PCR

  • Genetic interaction tests through creation of double mutants

  • Phenotypic rescue experiments to demonstrate causality