Recombinant Arabidopsis thaliana 5'-adenylylsulfate reductase-like 7 (APRL7)

What is Arabidopsis thaliana APRL7 and what is its role in plant metabolism?

APRL7 (5'-adenylylsulfate reductase-like 7) is a protein expressed in Arabidopsis thaliana that belongs to the adenosine 5'-phosphosulfate reductase-like family. It's encoded by the gene At5g18120 (also known as MRG7.8) and is part of the plant's sulfur metabolism pathway. The protein shares structural similarities with adenosine 5'-phosphosulfate reductases but exhibits distinct functional characteristics. The full mature protein spans amino acid residues 24-289 of the native sequence .

While specific APRL7 functions aren't fully characterized in the provided search results, research approaches similar to those used for other Arabidopsis proteins (such as ARP7) suggest it may play roles in developmental processes. Targeted studies using knockout or knockdown approaches would help elucidate its specific metabolic roles, similar to approaches used for other Arabidopsis proteins .

How should recombinant APRL7 protein be stored and handled for optimal stability?

For optimal stability and activity, recombinant APRL7 protein should be handled according to these protocols:

When working with the lyophilized powder form, briefly centrifuge the vial before opening to bring contents to the bottom. Repeated freeze-thaw cycles significantly reduce protein stability and should be avoided .

What expression systems are optimal for producing recombinant APRL7?

The recombinant APRL7 protein is successfully expressed in E. coli expression systems when fused with an N-terminal His-tag . While the specific E. coli strain isn't mentioned in the search results, standard laboratory strains optimized for protein expression (such as BL21(DE3) or Rosetta) would likely be suitable.

For researchers planning to express APRL7, consider these methodological approaches:

• Clone the cDNA sequence encoding amino acids 24-289 into a prokaryotic expression vector

• Include an N-terminal His-tag for purification purposes

• Transform into an appropriate E. coli expression strain

• Optimize expression conditions (temperature, IPTG concentration, induction time)

• Purify using nickel affinity chromatography

• Verify purity by SDS-PAGE (>90% purity is achievable)

How can I design effective knockdown or knockout experiments for studying APRL7 function?

While the search results don't specifically address APRL7 knockdown experiments, effective approaches can be derived from methodologies used for other Arabidopsis proteins like ARP7. Based on these established protocols, consider:

RNA Interference (RNAi) Approach:

• Design a construct containing inverted repeats of APRL7 cDNA fragments (300-500bp) separated by an intron spacer

• Transform wild-type Arabidopsis plants using Agrobacterium-mediated transformation

• Select transformants using appropriate antibiotic resistance markers

• Verify knockdown efficiency by Western blot analysis using APRL7-specific antibodies

• Group resultant lines based on phenotypic severity (normal, moderate, strong) for comparative analysis

T-DNA Insertion Mutant Approach:

• Screen available T-DNA insertion collections for lines with insertions in the APRL7 gene

• Confirm homozygous/heterozygous status by PCR genotyping

• Analyze phenotypes and perform complementation tests with wild-type APRL7 to confirm specificity

What methods are most effective for purifying recombinant His-tagged APRL7?

For high-purity isolation of recombinant His-tagged APRL7 protein, implement this purification workflow:

• Cell Lysis Preparation:

  • Harvest E. coli cells expressing His-tagged APRL7 by centrifugation

  • Resuspend in lysis buffer containing appropriate protease inhibitors

  • Lyse cells via sonication or pressure-based disruption

• Affinity Chromatography:

  • Bind the His-tagged protein to Ni-NTA or similar affinity resin

  • Wash extensively to remove non-specifically bound proteins

  • Elute with an imidazole gradient or high imidazole concentration

• Quality Assessment:

  • Verify purity by SDS-PAGE (target: >90% purity)

  • Confirm identity by Western blot with anti-His antibodies or APRL7-specific antibodies

  • Quantify protein concentration using Bradford or BCA assay

• Buffer Exchange and Storage:

  • Exchange into Tris/PBS-based storage buffer with 6% trehalose (pH 8.0)

  • Aliquot to avoid repeated freeze-thaw cycles

  • Store at -20°C/-80°C for long-term stability

How can temporal gene expression profiling be used to understand APRL7 regulation during leaf development?

High-resolution temporal gene expression profiling provides valuable insights into gene regulation during plant development. To analyze APRL7 expression patterns throughout leaf development:

• Sample Collection Strategy:

  • Harvest specific leaf tissue (e.g., leaf 7) at multiple time points throughout development

  • Include both morning and afternoon sampling (e.g., 7h and 14h into light period) to capture diurnal variations

  • Extend sampling from early development through senescence (approximately 20-40 days after sowing)

• Expression Analysis Methods:

  • Extract RNA from collected samples with biological replicates (minimum 4 recommended)

  • Perform microarray or RNA-Seq analysis across the time series

  • Normalize data using appropriate statistical methods (e.g., MAANOVA package)

  • Generate mean expression values for each time point

• Data Interpretation:

  • Identify significant expression changes correlated with developmental stages

  • Analyze promoter motifs to identify potential regulatory elements

  • Compare expression patterns with physiological changes (e.g., hormone levels)

  • Cluster with co-expressed genes to identify functional modules

This approach would reveal whether APRL7 expression changes during leaf development and senescence, potentially identifying its role in developmental processes.

What approaches can be used to identify potential protein interaction partners of APRL7?

To identify protein interaction partners of APRL7, consider implementing these complementary approaches:

• Yeast Two-Hybrid Screening:

  • Clone APRL7 into a bait vector fused to a DNA-binding domain

  • Screen against an Arabidopsis cDNA library fused to an activation domain

  • Validate positive interactions through growth on selective media and reporter gene activation

  • Confirm interactions using directed Y2H with individually cloned candidates

• Co-Immunoprecipitation (Co-IP):

  • Express epitope-tagged APRL7 (His-tag can be utilized) in plant cells

  • Prepare lysates under non-denaturing conditions to preserve protein-protein interactions

  • Perform pull-down with anti-His antibodies or APRL7-specific antibodies

  • Identify co-precipitated proteins using mass spectrometry

• Bimolecular Fluorescence Complementation (BiFC):

  • Create fusion constructs of APRL7 and candidate interactors with split fluorescent protein fragments

  • Co-express in plant cells (protoplasts or via Agrobacterium-mediated transformation)

  • Visualize reconstituted fluorescence using confocal microscopy

  • Analyze subcellular localization of interaction complexes

These approaches would help establish the APRL7 interactome, providing insights into its functional roles in plant metabolism and development.

How can near-isogenic line (NIL) populations be used to study the genetic basis of APRL7 function in Arabidopsis?

Near-isogenic line (NIL) populations offer powerful tools for dissecting the genetic basis of quantitative traits in Arabidopsis. To apply NIL approaches to study APRL7 function:

• Development of NIL Population:

  • Identify accessions with natural variation in APRL7 sequence or expression

  • Introgress genomic regions containing APRL7 variants from donor accessions (e.g., Cape Verde Islands, Cvi) into a reference background (e.g., Landsberg erecta, Ler)

  • Confirm introgression lines through molecular marker analysis

  • Create multiple independent NILs with overlapping introgressions spanning the APRL7 region

• Phenotypic Analysis:

  • Evaluate NILs for phenotypes potentially related to APRL7 function

  • Measure traits with different heritability (e.g., developmental, physiological, metabolic)

  • Analyze multiple replicates (at least 6-8) to achieve adequate statistical power

  • Compare phenotypic differences between NILs and the reference background

• QTL Mapping:

  • Map quantitative trait loci associated with APRL7 function

  • Compare mapping resolution with recombinant inbred line (RIL) populations

  • Use NILs to detect smaller-effect QTLs that might be masked in RIL populations

This approach would help determine how natural variation in APRL7 contributes to phenotypic variation in Arabidopsis populations, providing insight into its evolutionary significance.

What are common challenges in expressing recombinant APRL7 and how can they be addressed?

When expressing recombinant APRL7 protein, researchers may encounter several challenges. Here are common issues and their solutions:

For recombinant APRL7 specifically, maintaining the proper storage conditions (Tris/PBS-based buffer with 6% trehalose at pH 8.0) and avoiding repeated freeze-thaw cycles are crucial for preserving activity .

How can I validate antibody specificity for APRL7 in Arabidopsis studies?

When developing or selecting antibodies for APRL7 studies, validation is essential to ensure specificity and reliability. Implement these validation steps:

• Western Blot Validation:

  • Compare protein detection in wild-type plants versus APRL7 knockdown/knockout lines

  • Include recombinant APRL7 protein as a positive control

  • Test cross-reactivity with other APRL family members

  • Verify single band detection at the expected molecular weight

• Immunoprecipitation Tests:

  • Perform IP followed by mass spectrometry to confirm target identity

  • Compare IP efficiency with pre-immune serum controls

  • Validate recovery of known APRL7 interaction partners

• Immunolocalization Controls:

  • Include peptide competition assays to confirm binding specificity

  • Compare localization patterns in wild-type versus knockdown tissues

  • Validate subcellular localization with cell fractionation studies

• Quantitative Applications:

  • Establish standard curves using purified recombinant APRL7

  • Verify linear detection range within physiologically relevant concentrations

  • Compare antibody performance across different tissue types

An approach similar to that described for ARP7 antibody validation, where specificity was confirmed using RNAi lines, would be appropriate for APRL7 antibodies .

What are the best methods for assessing APRL7 enzymatic activity?

While the search results don't provide specific enzymatic assays for APRL7, adenylylsulfate reductase-like proteins can be assessed using these methodological approaches:

• Spectrophotometric Assays:

  • Monitor consumption of NADPH at 340 nm

  • Measure formation of sulfite using standard coupling reactions

  • Determine kinetic parameters (Km, Vmax) under varying substrate concentrations

• Radiometric Assays:

  • Use 35S-labeled substrates to track sulfur transfer

  • Quantify conversion of 5'-adenylylsulfate (APS) to sulfite

  • Compare activity of wild-type versus mutant protein variants

• Coupled Enzyme Assays:

  • Link APRL7 activity to measurable output through secondary enzymes

  • Establish positive and negative controls to validate assay specificity

  • Optimize reaction conditions (pH, temperature, ion concentrations)

• Activity Comparison Table:

Verify assay results by comparing activity of properly stored protein versus samples subjected to multiple freeze-thaw cycles, which would be expected to show reduced activity .

How can high-resolution temporal transcriptomics be integrated with APRL7 functional studies?

Integrating high-resolution temporal transcriptomics with functional studies of APRL7 can provide comprehensive insights into its biological roles:

• Experimental Design Integration:

  • Collect tissue samples for both transcriptomic analysis and protein studies across the same developmental time points

  • Include multiple time points per day to capture diurnal regulation patterns

  • Extend sampling from early development through senescence (e.g., 22 time points over 39 days)

• Data Analysis Approach:

  • Compare APRL7 transcript levels with protein abundance to identify post-transcriptional regulation

  • Cluster co-expressed genes to identify potential functional associations

  • Correlate expression patterns with physiological changes (e.g., hormone levels, metabolite profiles)

  • Apply MAANOVA or similar statistical packages to normalize data and identify significant changes

• Functional Correlation:

  • Identify developmental stages with significant APRL7 expression changes

  • Target these specific stages for detailed protein function studies

  • Correlate expression with phenotypic changes in knockdown or overexpression lines

This integrated approach would reveal not only when and where APRL7 is expressed but also how its expression correlates with developmental processes and other gene networks.

What approaches can be used to study the role of APRL7 in plant stress responses?

To investigate APRL7's potential role in plant stress responses, consider these methodological approaches:

• Stress-Responsive Expression Analysis:

  • Expose plants to various stresses (drought, salt, cold, heat, pathogen, etc.)

  • Measure APRL7 expression changes at multiple time points using qRT-PCR

  • Compare with known stress-responsive marker genes

  • Analyze promoter regions for stress-responsive elements

• Functional Phenotyping of Genetic Materials:

  • Compare stress tolerance in APRL7 knockdown/overexpression lines versus wild type

  • Measure physiological parameters (ROS levels, membrane stability, photosynthetic efficiency)

  • Assess growth and survival rates under stress conditions

  • Document recovery responses after stress alleviation

• Metabolic Profiling:

  • Analyze changes in sulfur-containing metabolites in response to stress

  • Compare metabolite profiles between wild-type and APRL7-modified plants

  • Correlate metabolite changes with APRL7 expression levels

  • Measure flux through sulfur assimilation pathways using labeled precursors

• Hormone Interaction Analysis:

  • Investigate how plant hormones regulate APRL7 expression

  • Determine if APRL7 affects hormone levels during stress (particularly ABA, SA, and JA)

  • Examine phenotypes of APRL7-modified plants treated with exogenous hormones

  • Create double mutants with hormone-signaling pathway genes to study genetic interactions

This comprehensive approach would establish whether APRL7 plays significant roles in stress adaptation and the underlying mechanisms of its function.