Recombinant Arabidopsis thaliana Dof zinc finger protein DOF4.7 (DOF4.7)

What is the basic structure of the DOF4.7 zinc finger domain?

DOF4.7 contains a conserved zinc finger (ZF) DNA-binding domain characteristic of the DOF family of plant-specific transcription factors. The DOF-ZF domain features four cysteine residues that coordinate a single zinc ion, as confirmed by atomic absorption spectroscopy showing a Zn:protein ratio of approximately 1:1 . Unlike other zinc finger domains in steroid hormone receptors and metazoan GATA ZFs, the DOF-ZF has a distinctively longer loop separating the zinc-coordinating cysteine pairs (the C-X2-C units) . This structural feature is critical for functionality, as shortening this loop by deleting seven or more residues or replacing it with that of zinc-binding unit from other proteins abolishes DNA binding activity .

How does DOF4.7 function as a transcription factor?

DOF4.7 functions as a nucleus-localized transcription factor that binds to specific DNA sequences called DOF cis-elements in the promoters of target genes . It exhibits both in vitro and in vivo binding activity to typical DOF cis-elements, specifically recognizing the sequence AAAG in the promoter regions . DOF4.7 can regulate gene expression by directly binding to these elements and recruiting other transcriptional machinery components. In the case of floral organ abscission, DOF4.7 acts as a transcriptional repressor of the abscission-related polygalacturonase gene PGAZAT, with overexpression of AtDOF4.7 resulting in down-regulation of this target gene .

What is the DNA binding specificity of DOF4.7?

DOF4.7, like other DOF family proteins, recognizes the core sequence AAAG in target gene promoters . Studies using microscale thermophoresis have demonstrated that DOF zinc finger domains interact approximately 100-fold more tightly with DNA probes containing two copies of the recognition sequence (double-motif probe) compared to those with only a single motif . This suggests potential cooperative binding or conformational changes that enhance affinity when multiple binding sites are present. The interaction with double-motif probes forms stable complexes with dissociation constants (Kd values) in the low micromolar range (approximately 2.3-2.5 μM for related DOF-ZF domains) .

What is the tissue-specific expression pattern of DOF4.7?

AtDOF4.7 exhibits a highly specific expression pattern primarily in the abscission zones of floral organs. RNA analysis and GUS reporter gene studies have shown that DOF4.7 transcripts are mainly detected in flowers and young siliques, with particularly strong expression at the base of all floral organs, especially in the abscission zones of petals, stamens, and sepals . Lower levels of expression are also observed in sepals, filaments, stigmatic papillae, tips of young elongating siliques, the base of pedicels, and the bases of leaf trichomes . This tissue-specific distribution pattern suggests a specialized role in the process of floral organ abscission.

How does DOF4.7 expression change during flower development?

The temporal expression pattern of AtDOF4.7 closely corresponds to the progression of floral organ abscission. GUS reporter analyses have revealed that DOF4.7 expression begins at the abscission zones of position 4 flowers (counting from the first flower with visible white petals) and continues until position 10, disappearing at position 11 where wild-type plants typically complete their flower abscission process . This temporal regulation correlates with the developmental stages when abscission zone cells undergo significant changes in preparation for organ shedding, suggesting DOF4.7's involvement in this programmed developmental process.

Where is DOF4.7 protein localized within cells?

AtDOF4.7 is predominantly localized in the nucleus, consistent with its function as a transcription factor . This nuclear localization is critical for its ability to directly regulate target gene expression by binding to promoter regions. Studies using fluorescence techniques such as BiFC (Bimolecular Fluorescence Complementation) have confirmed this nuclear localization, particularly when examining interactions with other proteins like MPK6 . The specific nuclear targeting is likely mediated by nuclear localization signals within the protein structure, allowing it to access DNA and participate in transcriptional regulation complexes.

What proteins interact with DOF4.7?

AtDOF4.7 engages in several important protein-protein interactions that influence its function in abscission regulation. Notably, it interacts with Arabidopsis ZINC FINGER PROTEIN2 (AtZFP2), another abscission-related transcription factor, suggesting they may work together in a transcriptional complex to regulate target genes . Additionally, DOF4.7 interacts with mitogen-activated protein kinase 6 (MPK6) as demonstrated by both yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays . This interaction occurs in the nucleus and is part of the signal transduction pathway regulating abscission. While interaction with MPK3 was detected in Y2H assays, it could not be confirmed by BiFC in Arabidopsis cells, suggesting potential differences in interaction affinity or specificity between these two MAPKs .

How is DOF4.7 regulated by phosphorylation?

DOF4.7 is regulated post-translationally by phosphorylation via the MAPK cascade, which is a key component of signal transduction in the abscission pathway. In vitro studies have demonstrated that DOF4.7 can be phosphorylated by components of the MAPK cascade, and this modification may modulate its activity or stability . The interaction between DOF4.7 and MPK6 observed in BiFC assays suggests that MPK6 is likely responsible for this phosphorylation in vivo . This post-translational regulation adds another layer of control over DOF4.7 function, potentially allowing for rapid responses to abscission-inducing signals without requiring changes in gene expression.

What is the relationship between DOF4.7 and the IDA signaling pathway?

AtDOF4.7 is integrated into the INFLORESCENCE DEFICIENT IN ABSCISSION (IDA)-mediated floral organ abscission pathway. Genetic evidence indicates that DOF4.7 and IDA operate in a common pathway regulating abscission . IDA, a peptide ligand, negatively regulates DOF4.7 at the transcriptional level, providing upstream control of DOF4.7 expression . The IDA signaling pathway activates a MAPK cascade involving MPK3 and MPK6, which can phosphorylate AtDOF4.7 in vitro . This phosphorylation may regulate DOF4.7 protein levels or activity in vivo. Collectively, these interactions position DOF4.7 as a downstream effector in the IDA-mediated signaling pathway that controls floral organ abscission.

How does DOF4.7 regulate floral organ abscission?

DOF4.7 plays a critical role in regulating floral organ abscission by controlling the expression of cell wall hydrolysis enzymes that are essential for organ separation. As a transcription factor, DOF4.7 directly binds to the promoter of the abscission-related polygalacturonase (PG) gene PGAZAT, which encodes an enzyme involved in breaking down cell wall components . Overexpression of AtDOF4.7 results in down-regulation of PGAZAT expression, suggesting that DOF4.7 functions as a transcriptional repressor in this context . By suppressing the expression of cell wall hydrolysis enzymes, DOF4.7 controls the timing and progression of the abscission process, particularly the dissolution of the middle lamella between adjacent cell walls in the abscission zone.

What is the phenotype of DOF4.7 overexpression plants?

Plants overexpressing AtDOF4.7 (35S::AtDOF4.7 lines) exhibit a distinctive ethylene-independent floral organ abscission deficiency phenotype . In these transgenic plants, floral organs such as petals, sepals, and stamens remain attached to the developing siliques much longer than in wild-type plants. Anatomical analyses of these overexpression lines revealed that while the formation of the abscission zone appears normal, the dissolution of the middle lamella between cell walls fails to occur properly, preventing the separation of abscission zone cells . This results in delayed or incomplete shedding of floral organs, demonstrating DOF4.7's important role as a negative regulator of abscission.

What are the optimal conditions for recombinant expression and purification of DOF4.7?

For recombinant expression of DOF4.7, particularly its DNA-binding domain, the protein can be expressed as a GST-fusion protein in E. coli BL21(DE3) cells . The fusion construct should include the DOF zinc finger domain with adequate flanking sequences to ensure proper folding. Expression is typically induced with IPTG at moderate temperatures (around 25°C) to enhance solubility. Purification is effectively achieved using glutathione affinity chromatography, where the GST-tagged protein binds to glutathione-agarose beads and can be eluted with reduced glutathione . For biochemical and structural studies requiring tag-free protein, the GST tag can be removed using PreScission protease. The purified DOF4.7 zinc finger domain should be stored in buffer containing reducing agents (DTT or β-mercaptoethanol) to maintain the integrity of the cysteine residues essential for zinc coordination.

What methods are used to study DOF4.7 DNA binding properties?

Several complementary methods can be employed to study DOF4.7's DNA binding properties:

• Gel Retardation Assays (EMSA): This technique detects the formation of protein-DNA complexes based on their reduced electrophoretic mobility compared to free DNA. For DOF4.7, oligonucleotide probes containing one or multiple AAAG core recognition sequences are incubated with purified protein before analysis by native polyacrylamide gel electrophoresis .

• Microscale Thermophoresis (MST): This approach measures changes in the movement of fluorescently-labeled DNA in microscopic temperature gradients upon protein binding. MST allows determination of binding affinities (Kd values) under equilibrium conditions. For DOF4.7, studies have shown approximately 100-fold stronger binding to double-motif probes compared to single-motif probes .

• Chromatin Immunoprecipitation (ChIP): To study in vivo DNA binding, ChIP assays can be performed using antibodies against DOF4.7 or epitope-tagged versions of the protein expressed in plants. This method identifies genomic regions bound by DOF4.7 under physiological conditions.

• Yeast One-Hybrid Assays: This technique can verify DOF4.7 binding to specific promoter sequences by detecting activation of reporter genes in yeast.

How can researchers effectively study DOF4.7 protein-protein interactions?

Researchers can employ several techniques to investigate DOF4.7 protein-protein interactions:

• Yeast Two-Hybrid (Y2H) Assays: This method has successfully demonstrated interactions between DOF4.7 and proteins like MPK3 and MPK6. The assay involves expressing DOF4.7 fused to a DNA-binding domain (BD) and potential interacting proteins fused to an activation domain (AD). Interaction is detected by activation of reporter genes in yeast cells .

• Bimolecular Fluorescence Complementation (BiFC): This in vivo technique directly visualizes protein interactions in plant cells. DOF4.7 and its potential interacting partner are fused to complementary fragments of a fluorescent protein (e.g., YFP). When the proteins interact, the fragments come together to reconstitute fluorescence, which can be observed using confocal microscopy. This method confirmed DOF4.7 interaction with MPK6 in the nucleus .

• Co-Immunoprecipitation (Co-IP): This biochemical approach can verify interactions in plant tissues using antibodies against DOF4.7 or epitope-tagged versions to pull down protein complexes.

• In Vitro Pull-Down Assays: Using purified recombinant proteins, direct physical interactions can be tested by immobilizing one partner (e.g., GST-DOF4.7) and detecting binding of the other protein.

• Protein Microarrays: For screening multiple potential interacting partners simultaneously, DOF4.7 can be tested against arrays of plant proteins to identify novel interactions.

How can DOF4.7 be utilized in crop improvement strategies?

DOF4.7's role in regulating floral organ abscission presents several potential applications for crop improvement:

• Fruit Retention Enhancement: Modulating DOF4.7 expression in fruit crops could reduce premature fruit drop by delaying or reducing abscission, potentially increasing yields. This would be especially valuable in crops where fruit or seed loss due to environmental stresses is a significant problem.

• Flower Longevity Extension: For ornamental crops and cut flowers, controlled overexpression of DOF4.7 could delay petal abscission, extending flower display time and post-harvest quality.

• Seed Shattering Resistance: In cereal crops where seed shattering (premature seed dispersal) reduces yield, manipulating DOF4.7 and related abscission pathway components could develop varieties with improved harvest characteristics.

• Cross-Species Applications: Comparative analysis of DOF4.7 orthologs across different plant species could reveal conserved and divergent aspects of abscission regulation, informing targeted genetic modifications in crops where abscission affects productivity.

Implementation would require fine-tuned genetic modifications, potentially using tissue-specific or inducible promoters to avoid unintended developmental effects, as DOF4.7 may have pleiotropic functions beyond abscission control.

What are the functional redundancies among DOF family proteins in abscission regulation?

The Arabidopsis genome contains approximately 36 DOF family transcription factors, raising important questions about potential functional redundancy in abscission regulation:

• Phylogenetic Analysis: While AtDOF4.7 has a specialized role in abscission, closely related DOF proteins may share partially overlapping functions. Comprehensive phylogenetic analysis coupled with expression profiling of all DOF family members in abscission zones would identify potential redundant factors.

• Multiple Mutant Analysis: Single mutants of DOF family members often show subtle phenotypes due to functional compensation by related proteins. Creating and analyzing higher-order mutants (double, triple, or quadruple) of DOF4.7 with its closest homologs would reveal the extent of redundancy.

• Domain Swapping Experiments: Chimeric proteins created by swapping domains between DOF4.7 and other DOF proteins could identify which structural features contribute to functional specificity versus redundancy in abscission regulation.

• Transcriptome Analysis: Comparing the target genes regulated by DOF4.7 with those regulated by other DOF family members expressed in the abscission zone would reveal overlapping and distinct regulatory networks.

Current evidence suggests that while DOF4.7 has a specific role in abscission, its function likely exists within a network of partially redundant transcription factors that collectively ensure robustness in abscission regulation.

How does post-translational modification affect DOF4.7 DNA binding specificity?

Post-translational modifications, particularly phosphorylation by the MAPK cascade, potentially regulate DOF4.7 function in several sophisticated ways:

• Phosphorylation-Dependent Affinity Changes: Phosphorylation may alter the binding affinity of DOF4.7 to its DNA recognition sequences. Comparing the DNA binding properties of phosphorylated versus non-phosphorylated DOF4.7 using techniques like microscale thermophoresis or EMSAs with phosphomimetic mutants (S/T to D/E substitutions) could reveal such changes.

• Binding Partner Selection: Phosphorylation may influence DOF4.7's interaction with other transcriptional regulators, potentially switching between different protein complexes. This could alter the spectrum of target genes regulated by DOF4.7 depending on its phosphorylation state.

• Protein Stability Regulation: MAPK-mediated phosphorylation may affect DOF4.7 protein stability and turnover rates, providing temporal control over its activity during the abscission process. In vivo data suggest that AtDOF4.7 protein levels may be regulated by phosphorylation .

• Nuclear-Cytoplasmic Shuttling: Phosphorylation could potentially regulate DOF4.7 subcellular localization, affecting its access to nuclear DNA targets.

Understanding these mechanisms requires integrated approaches combining in vitro biochemical studies with in vivo analyses in plants expressing phosphosite mutant versions of DOF4.7, coupled with phenotypic assessment of abscission timing and progression.