Recombinant Arabidopsis thaliana Agamous-like MADS-box protein AGL93 (AGL93)

What is AGL93 and how is it classified within the MADS-box gene family?

AGL93 (Agamous-like 93) is a member of the MADS-box transcription factor family in Arabidopsis thaliana. It belongs to the Mβ class of Type I MADS-box genes. The MADS-box gene family is divided into two main types (Type I and Type II), with Type I further subdivided into three subclasses: Mα, Mβ, and Mγ. AGL93 is specifically classified as an Mβ-type gene .

Type I MADS-box genes are more closely related to animal SRF-like sequences, while most plant MADS-domain sequences group with animal MEF2-like sequences (Type II MADS domains) . This classification is significant as it reflects evolutionary relationships that predate the divergence of plants and animals.

What is the expression pattern of AGL93 in Arabidopsis thaliana?

AGL93 appears to be among the 20 Type I MADS-box genes for which no GUS signal was observed in transgenic lines containing translational fusions to GFP and GUS reporters . Specifically, eight Mβ-class genes, including AGL93, showed no detectable expression in these assays.

This lack of detectable expression could have several explanations:

• AGL93 may be expressed at levels below detection thresholds

• It might be expressed only under specific environmental conditions not tested in standard assays

• It could represent a non-functional gene resulting from duplication events

This expression pattern contrasts with other Type I MADS-box genes that show expression predominantly in the female gametophyte or developing seed, particularly in the central cell, antipodal cells, and chalazal endosperm .

How does the storage and handling of recombinant AGL93 affect its stability and activity?

For optimal stability and activity, recombinant AGL93 should be handled according to the following recommendations:

• Briefly centrifuge vials prior to opening to bring contents to the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% for long-term storage (50% is recommended)

• Store working aliquots at 4°C for up to one week

• For long-term storage, keep at -20°C/-80°C

• Avoid repeated freeze-thaw cycles

The shelf life varies depending on storage conditions:

• Liquid form: approximately 6 months at -20°C/-80°C

• Lyophilized form: up to 12 months at -20°C/-80°C

These storage parameters are similar to those for other recombinant proteins and are crucial for maintaining functional activity for experimental use.

What experimental designs are most appropriate for studying potentially non-expressed MADS-box genes like AGL93?

When studying MADS-box genes like AGL93 that show limited or no expression in standard assays, researchers should consider the following experimental design strategies:

High-sensitivity detection methods:

• Digital droplet PCR for extremely low-abundance transcripts

• Single-cell RNA-seq to detect cell-specific expression patterns

• Ribosome profiling to detect actively translated mRNAs

Condition-specific expression analysis:

• Test expression across comprehensive developmental series, particularly reproductive stages

• Examine expression under various abiotic stresses (drought, salt, temperature extremes)

• Screen response to phytohormone treatments

Epigenetic analysis:

• DNA methylation profiling of the promoter region

• Chromatin immunoprecipitation to analyze histone modifications

• Analysis in epigenetic modifier mutants to detect potential epigenetic silencing

Comparative approaches:

• Construct a data table comparing expression patterns across multiple tissues and conditions:

This tabular approach allows systematic documentation of expression across multiple conditions and genetic backgrounds .

How can researchers investigate potential DNA binding properties of AGL93?

As a MADS-box protein, AGL93 would be expected to bind specific DNA sequences. The following methodological approaches can determine its DNA binding properties:

Target sequence identification:

• Electrophoretic Mobility Shift Assay (EMSA) using recombinant AGL93 with CArG-box containing oligonucleotides

• Systematic Evolution of Ligands by Exponential Enrichment (SELEX) to determine binding site preferences

• Based on studies of other MADS-box proteins, test oligonucleotides with the consensus sequence where "CCA" forms the first three nucleotides, "TGG" the last three, and either A or T in the central four nucleotides

DNA binding characterization:

• Circular permutation assay to determine whether AGL93 induces DNA bending, similar to analyses performed for other AGAMOUS MADS-box proteins

• Phasing analysis to determine the spatial arrangement of protein-DNA complexes

• Test binding to CArG-box sequences identified in promoters of potential target genes

Domain analysis:

• Generate truncated versions of AGL93 to map domains required for DNA binding

• Test the ability to form homodimers or heterodimers with other MADS-box proteins, which can affect DNA binding specificity

• Characterize the MADS domain specifically using site-directed mutagenesis

The consensus CArG-box sequence typically bound by MADS-box proteins is CC(A/T)6GG, but variations exist among different family members .

What mechanisms might explain the apparent lack of expression of AGL93 and how can they be tested?

The apparent lack of expression of AGL93 could be explained by several mechanisms that can be experimentally tested:

Pseudogenization hypothesis:

• Compare sequence characteristics with known functional MADS-box genes

• Look for premature stop codons, frameshift mutations, or other sequence features suggesting pseudogenization

• Test promoter activity using reporter gene assays

Tissue or condition-specific expression:

• Generate transgenic lines with longer promoter regions (up to 5kb upstream) to capture distant regulatory elements

• Test expression under comprehensive stress conditions (drought, salt, cold, heat) with different intensities and durations

• Examine expression during specialized developmental processes, particularly embryo development and female gametophyte formation, where many Type I MADS-box genes function

Transcriptional regulation:

• Analyze the promoter region for transcription factor binding sites

• Test expression in plants overexpressing potential upstream regulators

• Examine chromatin state using ChIP-seq for active and repressive histone marks

Evolutionary analysis:

• Compare with orthologs in related species to determine if lack of expression is conserved

• Examine selection pressure on the coding sequence (dN/dS ratio)

• Reconstruct the evolutionary history of gene duplication events in the Mβ-class

This systematic approach can determine whether AGL93 represents a pseudogene, a highly specialized gene, or a gene that has undergone subfunctionalization following duplication .

How might AGL93 function in redundant networks with other MADS-box proteins?

MADS-box genes often function redundantly due to extensive gene duplications. To investigate potential redundancy involving AGL93:

Genetic approaches:

• Generate higher-order mutants combining agl93 with closely related Mβ-type genes

• Construct artificial microRNA lines targeting multiple family members simultaneously

• Use CRISPR/Cas9 to generate multiple gene knockouts within the same clade

Expression compensation analysis:

• Examine whether related genes show upregulation in agl93 knockout lines

• Test whether expression of AGL93 under the control of related gene promoters can complement their mutant phenotypes

• Create a correlation matrix of expression patterns across multiple conditions to identify co-regulated genes

Protein interaction network:

• Perform yeast-two-hybrid or BiFC assays to identify potential interaction partners

• Test whether AGL93 can form heterodimers with other MADS-box proteins

• Map interaction domains through deletion analysis

MADS-domain proteins typically function as tetramers to activate or repress target genes, and tissue-specific variations in complex composition may result in distinct DNA binding specificities and regulatory outputs .

What approaches can reveal potential roles of AGL93 in plant stress responses?

Several MADS-box genes have been implicated in stress responses. To investigate potential roles of AGL93:

Functional characterization under stress:

• Generate and analyze AGL93 overexpression lines under different stress conditions

• Test transgenic plants for altered tolerance to drought, salt, temperature extremes

• Measure physiological parameters including:

  • Proline content (osmolyte accumulation)

  • Malondialdehyde levels (lipid peroxidation)

  • Antioxidant enzyme activities (SOD, catalase)

  • Chlorophyll content and photosynthetic efficiency

Integration with stress signaling pathways:

• Test genetic interactions with known stress response regulators

• Analyze expression in response to ABA treatment and in ABA signaling mutants

• Examine potential involvement in reactive oxygen species (ROS) signaling pathways

Target gene identification:

• Perform RNA-seq comparing wild-type and AGL93 overexpression lines under stress conditions

• Test binding to promoters of known stress-responsive genes

• Validate direct targets using ChIP-qPCR

Evidence from other MADS-box genes suggests potential roles in drought, salt, and osmotic stress tolerance. For example, GbMADS9 from Ginkgo biloba (a B-sister class gene) shows upregulation in response to multiple stresses and confers enhanced tolerance when overexpressed .

How can researchers determine if AGL93 plays a role in root development similar to other characterized MADS-box genes?

Several MADS-box genes have been implicated in root development. To investigate potential roles of AGL93 in root architecture:

Phenotypic analysis:

• Compare primary root length, lateral root number and length in wild-type, knockout, and overexpression lines

• Analyze root growth under different nitrogen conditions (N-deficient and N-rich)

• Examine root responses to various abiotic stresses

Cell-level analysis:

• Monitor cell division patterns in root meristems using cell cycle markers

• Analyze cell elongation in the differentiation zone

• Examine root vasculature development

Integration with known root development pathways:

• Test genetic interactions with known root development regulators like AGL21, which regulates lateral root development

• Examine relationships with other MADS-box genes expressed in roots, such as XAL2, SOC1, and AGL24, which function in primary root growth

• Analyze the effects on nitrogen-responsive gene expression

A comprehensive experimental approach might include the analysis of genetic interactions using various mutant combinations as shown in this data from a study of MADS-box genes in root development:

*Values to be determined experimentally

This systematic approach would reveal whether AGL93 functions similarly to characterized MADS-box genes in root development, potentially uncovering previously unknown roles.