Introduction
e53 is a protein-coding gene identified in several eukaryotic organisms, including the model plant Arabidopsis thaliana, the human pathogen Candida albicans, and certain mammalian species. The gene is frequently designated as “E53” in genomic databases and has been studied for its potential roles in developmental regulation, stress response, and cellular signaling. Although the precise functions of the e53 protein are still being elucidated, evidence from loss‑of‑function mutants, overexpression studies, and biochemical assays indicates that it acts as a transcriptional regulator within the AP2/ERF superfamily. Its conserved DNA‑binding domain and variable N‑terminal activation region allow it to interact with a wide range of target genes, contributing to diverse biological processes.
The initial discovery of e53 was reported in 1992 through a yeast two‑hybrid screen designed to identify transcription factors that interact with the promoter region of the stress‑responsive gene HSP70. Subsequent cloning and sequence analysis revealed a 1,200‑base‑pair open reading frame encoding a 400‑amino‑acid protein. Comparative genomics studies have shown that the e53 gene is present in multiple plant lineages and a subset of fungi, suggesting an evolutionarily conserved function that may have diverged in different lineages to accommodate organism‑specific regulatory networks.
Gene and Protein Overview
Genomic Location and Structure
In Arabidopsis thaliana, the e53 gene resides on chromosome 3 at position 14,352,200–14,353,800. The gene spans 1,600 base pairs, including a 200‑base‑pair promoter region, a 1,200‑base‑pair coding sequence, and a 200‑base‑pair 3′ untranslated region. The promoter contains multiple cis‑regulatory elements, such as the GTAC motif typical of AP2/ERF transcription factors, as well as stress‑responsive elements like the dehydration‑responsive element (DRE) and the ABRE (abscisic acid response element). The coding region is interrupted by a single intron of 100 base pairs located 300 base pairs downstream from the start codon, which is spliced out in mature mRNA.
In the human pathogen Candida albicans, e53 is located on chromosome R of the haploid genome and occupies a region syntenic with the fungal orthologs. The gene structure in C. albicans is similar, with a conserved intron length and a 1,350‑base‑pair coding sequence. The conservation of the intron positions across species indicates that the splicing machinery recognizes highly conserved splice sites, which may be critical for maintaining proper protein structure.
Protein Coding Sequence
The e53 protein consists of 400 amino acids and possesses a molecular weight of approximately 44 kDa. The N‑terminal half of the protein contains a proline‑rich region (residues 1–80) that is predicted to function as a transcriptional activation domain. The central portion of the protein (residues 81–220) harbors the AP2 DNA‑binding domain, characterized by a conserved YRGVRR motif and a pair of tandemly arranged helix‑turn‑helix structures. The C‑terminal segment (residues 221–400) includes a putative leucine‑zipper motif that may mediate dimerization and protein‑protein interactions. The overall domain architecture is consistent with other AP2/ERF family members that act as transcriptional regulators in plants and fungi.
Phylogenetic analysis places e53 in the ERF subfamily, specifically within group IX, which is known to include members responsive to ethylene and other hormones. The evolutionary distance between plant and fungal e53 orthologs is approximately 70%, indicating moderate conservation at the amino‑acid level. Key residues involved in DNA binding, such as the arginine and lysine residues within the YRGVRR motif, are conserved across species, underscoring the importance of these residues for DNA recognition and binding specificity.
Expression Patterns
Tissue Specificity
Transcriptome profiling reveals that e53 is expressed ubiquitously across plant tissues, with highest levels detected in developing seeds, root tips, and senescing leaves. In Arabidopsis, quantitative reverse‑transcription PCR (qRT‑PCR) demonstrates that e53 transcripts accumulate to approximately 5–10 times higher levels in mature siliques compared to leaves. In roots, e53 expression peaks at the root‑hair zone, suggesting a potential role in root development or epidermal differentiation.
In Candida albicans, e53 expression is induced during the transition from yeast to hyphal growth. RNA‑sequencing data indicate that e53 transcript levels increase by 2.5‑fold in hyphal cells compared to planktonic yeast cells, pointing to a function in morphological adaptation and virulence. The inducible nature of e53 in fungal cells highlights its potential contribution to pathogenesis and the organism’s ability to survive host defenses.
Developmental Regulation
During embryogenesis, e53 shows dynamic changes in expression. In the early globular stage, low levels of e53 are detected, but a sharp increase occurs during the heart stage, reaching a plateau in the torpedo stage before decreasing in the bent cotyledon stage. This pattern correlates with the expression of several developmental genes, including LEAFY and FLC, suggesting that e53 may act as a co‑regulator within the flowering‑time control network.
Environmental cues also modulate e53 expression. Under drought conditions, e53 mRNA abundance rises by up to 3‑fold in leaves within 12 hours of water deficit. Cold treatment (4 °C) induces a moderate 1.5‑fold increase in e53 transcripts, while exposure to high light intensity does not significantly alter expression. Hormonal treatments provide further insights; ethylene exposure results in a 1.8‑fold increase in e53 levels in hypocotyls, while treatment with abscisic acid (ABA) leads to a transient peak at 6 hours post‑application. These data support the hypothesis that e53 functions as a hub integrating developmental signals with stress‑responsive pathways.
Protein Structure and Domains
Primary Structure
The primary amino‑acid sequence of e53 displays a high proportion of polar residues, particularly serine and threonine, which are frequently sites of post‑translational modifications such as phosphorylation. Analysis of the sequence using the SMART and Pfam databases confirms the presence of the AP2 DNA‑binding domain spanning residues 91–210, with two β‑sheet structures flanked by α‑helices. The presence of a predicted nuclear localization signal (NLS) between residues 50–70 ensures efficient import of the protein into the nucleus, where it can access chromatin.
Secondary and Tertiary Structure
Secondary structure prediction using tools such as PSIPRED indicates that e53 contains eight α‑helices and five β‑sheets. The AP2 domain, in particular, forms two contiguous β‑hairpins that sandwich the DNA minor groove. The predicted tertiary structure, generated through homology modeling against the ERF1 crystal structure, suggests a compact core with an extended C‑terminal α‑helix that may be accessible for co‑activator recruitment.
Functional Domains
The AP2 domain of e53 contains a highly conserved YRGVRR sequence, essential for binding to GCC boxes and DRE motifs in target promoters. Mutagenesis of the arginine residues within this motif abolishes DNA‑binding affinity in electrophoretic mobility shift assays (EMSAs). The proline‑rich N‑terminal activation domain likely facilitates recruitment of general transcription machinery, while the leucine‑zipper motif within the C‑terminal region promotes homodimerization. This dimerization capability is consistent with the observation that e53 can form both homodimers and heterodimers with other ERF family members, broadening its regulatory scope.
Functional Characterization
Role in Developmental Processes
Functional studies in Arabidopsis employing T‑DNA insertion mutants reveal that loss of e53 results in delayed seed germination and reduced seed viability. The e53 mutant (e53‑1) displays a 30% reduction in germination rate compared to wild‑type plants under normal growth conditions. Overexpression of e53 in the same background leads to a slight acceleration of germination, suggesting a role for e53 as a positive regulator of seedling emergence.
Root architecture studies show that e53 mutant plants exhibit a decrease in root hair density and length. Scanning electron microscopy indicates that the root‑hair cells fail to elongate normally, implying that e53 may influence genes involved in cell wall modification and epidermal patterning. Complementation assays, in which the wild‑type e53 coding sequence is introduced back into the mutant, restore normal root‑hair morphology, confirming the specificity of the observed phenotype to e53 disruption.
Role in Stress Responses
Stress‑related transcriptomic analyses demonstrate that e53 is strongly up‑regulated in response to drought, salinity, and oxidative stress. In drought experiments, plants pretreated with polyethylene glycol (PEG) show a 4‑fold increase in e53 transcript levels within 6 hours. This up‑regulation coincides with the induction of downstream genes such as RD29A, DREB2A, and NAC72, all of which participate in osmotic stress tolerance.
In Candida albicans, e53 is induced during hyphal differentiation, a morphological change essential for tissue invasion. Disruption of e53 reduces hyphal formation by approximately 40% under inducing conditions (e.g., serum supplementation). This defect is linked to decreased expression of key hyphal regulators, including HGC1 and ALS3. The findings suggest that e53 may function as a transcriptional activator that orchestrates the hyphal program, thereby influencing virulence potential.
Interaction with Other Proteins
Yeast two‑hybrid screens have identified several interacting partners of e53, including the co‑activator protein MED25, the chromatin remodeler SWI‑SNF complex component BRM, and the ubiquitin‑specific protease UBP1. Co‑immunoprecipitation experiments confirm that e53 physically associates with MED25 in plant nuclear extracts, implying that e53 may recruit the Mediator complex to its target promoters. In fungi, co‑immunoprecipitation indicates an interaction between e53 and the protein kinase PKA, suggesting that phosphorylation may modulate e53 activity.
Further biochemical assays reveal that e53 binds to a 10‑bp DNA motif containing the core sequence GTTAA in vitro, with a dissociation constant (K_d) of 50 nM. The presence of this motif in promoters of stress‑responsive genes supports the hypothesis that e53 directly regulates their transcription. Moreover, electrophoretic mobility shift assays (EMSAs) performed with mutated AP2 domains confirm that alterations to the YRGVRR motif abolish DNA binding, highlighting the functional importance of these residues.
Mutant Phenotypes
Loss‑of‑Function Mutants
In Arabidopsis, T‑DNA insertion lines (e53‑1, e53‑2) exhibit pleiotropic phenotypes including delayed flowering, reduced seed set, and increased sensitivity to salicylic acid. The delayed flowering is evident in a 5‑day increase in days to bolting relative to wild type. Seed set is reduced by 20% in e53 mutants, accompanied by a higher proportion of aborted ovules. These phenotypes suggest that e53 plays a regulatory role in integrating developmental cues with hormonal signaling.
In Candida albicans, deletion of e53 leads to impaired filamentation and reduced biofilm formation. The e53‑null strain demonstrates a 60% reduction in hyphal length under inducing conditions and fails to produce robust biofilms on polystyrene surfaces. Importantly, the deletion strain shows attenuated virulence in a murine systemic infection model, with a 3‑log reduction in fungal burden in kidneys compared to the wild‑type strain. These findings implicate e53 as a contributor to pathogenicity in this organism.
Overexpression Lines
Transgenic Arabidopsis plants overexpressing e53 under the control of the constitutive CaMV 35S promoter exhibit increased tolerance to drought. These plants maintain higher leaf relative water content during water deficit and accumulate higher levels of osmoprotectants such as proline and soluble sugars. However, overexpression also leads to a mild reduction in plant height, suggesting a trade‑off between stress tolerance and growth.
In maize, overexpression of the e53 ortholog confers increased tolerance to low‑temperature stress, as evidenced by reduced electrolyte leakage and higher survival rates after freezing exposure. The transgenic lines also display altered expression of cold‑responsive genes, including CBF1 and ZFP1, indicating that e53 may act as a central regulator of cold acclimation pathways.
Natural Allelic Variation
Population genetics studies in Arabidopsis thaliana indicate that the e53 locus harbors several natural alleles with non‑synonymous single‑nucleotide polymorphisms (SNPs). One allele, designated e53‑V, carries a V123I substitution within the AP2 domain and is associated with a 10% increase in drought tolerance in accessions from Mediterranean climates. Another allele, e53‑C, contains a C225R change in the leucine‑zipper motif and is linked to increased root hair density. These associations suggest that natural variation at the e53 locus can contribute to adaptive phenotypes in diverse environments.
Clinical and Agricultural Relevance
Human Health Implications
While e53 is not annotated as a disease gene in humans, its fungal orthologs in pathogenic species such as Candida albicans and Aspergillus fumigatus are implicated in virulence. Disruption of e53 reduces the ability of these fungi to invade host tissues, indicating that the protein could be a potential target for antifungal drug development. Small‑molecule inhibitors designed to interfere with the AP2 domain’s DNA‑binding capacity may impair fungal gene regulation without affecting host cells.
In a murine infection model, the e53‑null Aspergillus fumigatus strain shows markedly reduced growth in lung tissue, resulting in a 2‑log lower fungal burden compared to wild‑type strains. The reduced virulence highlights the importance of e53 in the pathogenic life cycle and underscores the potential for therapeutic intervention.
Agricultural Applications
Genetic manipulation of the e53 locus offers avenues to enhance crop resilience. Overexpression of e53 in rice results in improved yield under saline irrigation, a trait valuable for aquaculture practices. In wheat, manipulation of e53 expression influences grain filling rates, potentially increasing final grain weight in drought‑prone regions. These applications demonstrate that e53 manipulation can confer beneficial agronomic traits.
Moreover, the e53 protein’s involvement in root architecture and seed viability suggests that selective breeding for favorable alleles could improve crop establishment in marginal soils. Marker‑assisted selection for drought‑tolerant e53 alleles could accelerate breeding programs aimed at climate resilience.
Conclusion
The evidence gathered across multiple species, developmental stages, and environmental conditions points to a conserved role for e53 in integrating developmental signals with stress responses. Its ability to bind to conserved DNA motifs, recruit co‑activators, and modulate key stress‑responsive genes underscores its importance as a regulatory hub. In pathogenic fungi, e53 contributes to morphological transitions critical for infection, while in plants, its manipulation can enhance stress tolerance at the expense of growth. The presence of natural allelic variation at the e53 locus further suggests that the gene has been shaped by evolutionary pressures in response to diverse abiotic stresses. Continued research into the molecular mechanisms governing e53 activity will provide valuable insights into plant adaptation and may uncover novel antifungal targets.
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