Recombinant Arabidopsis thaliana Peptidyl-prolyl cis-trans isomerase PASTICCINO1 (PAS1)

What is PASTICCINO1 and what role does it play in Arabidopsis thaliana development?

PASTICCINO1 (PAS1) is a crucial regulatory protein in Arabidopsis thaliana that plays a significant role in controlling plant development, particularly in cell proliferation and elongation. PAS1 belongs to the immunophilin family of proteins, specifically resembling FK506-binding proteins (FKBPs). Mutations in the PAS1 gene result in developmental abnormalities including ectopic cell proliferation in cotyledons, extra cell layers in the hypocotyl, and abnormal apical meristem formation .

The developmental importance of PAS1 is evidenced by the pleiotropic phenotypes observed in pas1 mutants, which display disorganized rosettes with fused, vitreous leaves. These phenotypic characteristics indicate that PAS1 functions in controlling cell division and differentiation during plant development. Additionally, PAS1 appears to be involved in embryogenesis, further highlighting its critical role throughout plant development .

How is the PAS1 protein structurally organized?

The PAS1 protein contains several distinct functional domains that contribute to its activity and interactions:

• FKBP-like domain: Located at the N-terminus, this domain is characteristic of FK506-binding proteins and likely possesses peptidyl-prolyl cis-trans isomerase (rotamase) activity .

• Tetratricopeptide repeat (TPR) units: PAS1 contains three TPR domains in its C-terminal region. These domains typically mediate protein-protein interactions and are found in various proteins involved in forming multi-protein complexes .

• Nuclear localization signal: PAS1 contains a nuclear localization signal, suggesting it primarily functions within the nucleus .

The domain architecture of PAS1 resembles that of other high-molecular-weight FKBPs, particularly Arabidopsis ROF1 (FKBP62) and wheat wFKBP73, with which it shares significant sequence similarity (71% and 54% respectively). This structural organization is also similar to mammalian FKBP52 and FKBP51 proteins, suggesting evolutionary conservation of these proteins' functions .

What phenotypes characterize pas1 mutants?

PAS1 mutants (pasticcino1) display distinctive developmental abnormalities that provide insights into the protein's function:

The pas1-1 mutant exhibits severe developmental abnormalities in the hypocotyl with irregular cortex cell numbers and disorganized cell layers. The meristematic regions are particularly affected, with variable structures ranging from almost non-existent to extremely enlarged meristems that occupy the entire apical region .

Notably, pas1 mutants show an abnormal response to cytokinins. While cytokinin treatment (5 μM BA) severely inhibits growth in wild-type plants, pas1 mutants display hypertrophy of the apical region, indicating altered cytokinin sensitivity. This response appears to be cytokinin-specific, as treatment with other plant hormones (auxin, ethylene, gibberellic acid, abscisic acid, and brassinosteroids) does not induce similar effects .

What are optimal conditions for expressing and purifying recombinant PAS1 protein?

Recombinant expression of PAS1 requires careful optimization due to its complex domain structure. The full-length Arabidopsis thaliana PAS1 protein (1-635 amino acids) can be successfully expressed with an N-terminal His-tag for purification purposes . Based on available data and common practices for similar proteins, the following protocol is recommended:

Expression System Selection:

• E. coli BL21(DE3) is suitable for initial expression trials

• Baculovirus-insect cell systems may yield better results for functional studies requiring proper folding

• Yeast expression systems (P. pastoris) can be considered for large-scale production

Expression Conditions:

• For E. coli systems:

  • Induce at OD600 of 0.6-0.8

  • Use IPTG concentration of 0.1-0.5 mM

  • Grow at lower temperatures (16-20°C) post-induction to enhance solubility

  • Extend expression time to 16-20 hours at reduced temperatures

Purification Strategy:

• Cell lysis under native conditions using buffer containing:

  • 50 mM Tris-HCl (pH 8.0)

  • 300 mM NaCl

  • 10% glycerol

  • 1 mM DTT

  • Protease inhibitor cocktail

• Immobilized metal affinity chromatography (IMAC):

  • Use Ni-NTA resin for His-tagged protein

  • Include 20-40 mM imidazole in binding buffer to reduce non-specific binding

  • Elute with 250-300 mM imidazole gradient

• Secondary purification:

  • Size exclusion chromatography to separate aggregates and ensure homogeneity

  • Ion exchange chromatography as needed

• Buffer optimization:

  • Final storage buffer: 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 10% glycerol, 1 mM DTT

  • Aliquot and flash-freeze in liquid nitrogen for long-term storage at -80°C

When assessing protein quality, it's critical to verify both purity by SDS-PAGE and functionality through peptidyl-prolyl isomerase activity assays.

How can researchers effectively study PAS1 peptidyl-prolyl cis-trans isomerase activity?

As a member of the immunophilin family with FKBP domains, PAS1 is expected to possess peptidyl-prolyl cis-trans isomerase (PPIase) activity. Studying this enzymatic function requires specialized approaches:

Standard PPIase Activity Assay:

• Chymotrypsin-coupled assay:

  • Substrate: N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide

  • Monitor absorbance change at 390 nm (release of p-nitroaniline)

  • Measure reaction rates in the presence/absence of potential inhibitors (FK506, rapamycin)

• Protease-free NMR-based assays:

  • Use 13C-labeled peptide substrates containing proline

  • Monitor cis/trans isomerization by 2D NMR spectroscopy

  • Provides direct measurement without coupling to proteolytic reactions

Inhibition Studies:
Based on prior research with related proteins, researchers should test PAS1 sensitivity to immunosuppressant compounds:

• FK506 (tacrolimus)

• Rapamycin (sirolimus)

• Synthetic derivatives with modified functional groups

Preliminary work suggests FK506 affects Arabidopsis seedling development only at high concentrations without inducing pas1 mutant phenotypes. This may indicate either limited drug penetration or more complex interactions. Cell culture or protoplast systems may provide better experimental platforms for inhibitor studies than whole seedlings .

Substrate Specificity Analysis:

• Test various peptide substrates with different amino acids flanking the proline residue

• Compare kinetic parameters (kcat, KM) to identify optimal substrates

• Compare activity with other plant FKBPs (wheat wFKBP73, Arabidopsis ROF1) to identify unique characteristics

What approaches can be used to study PAS1 interaction with other proteins?

Understanding PAS1's interactions is crucial for elucidating its function in plant development. The presence of TPR domains strongly suggests involvement in protein-protein interactions. Several complementary techniques can be employed:

Yeast Two-Hybrid Screening:

• Use full-length PAS1 as bait against Arabidopsis cDNA libraries

• Alternatively, use individual domains (FKBP domain or TPR domains) to identify domain-specific interactions

• Validate positive interactions with targeted Y2H assays

Co-Immunoprecipitation:

• Generate antibodies against purified recombinant PAS1

• Alternative approach: express epitope-tagged PAS1 in Arabidopsis

• Perform pull-downs from plant tissues at different developmental stages

• Identify interacting partners by mass spectrometry

Bimolecular Fluorescence Complementation (BiFC):

• Fuse PAS1 to N-terminal fragment of fluorescent protein (e.g., YFP)

• Fuse candidate interactors to C-terminal fragment

• Co-express in Arabidopsis protoplasts or Nicotiana benthamiana leaves

• Visualize interactions through reconstituted fluorescence

Proximity-Dependent Biotin Identification (BioID):

• Generate fusion of PAS1 with biotin ligase (BirA*)

• Express in Arabidopsis

• Proximal proteins become biotinylated and can be purified with streptavidin

• Identify by mass spectrometry

Given PAS1's relationship to cytokinin signaling, particular attention should be paid to potential interactions with components of cytokinin signaling pathways, including receptors, phosphotransfer proteins, and response regulators.

How does PAS1 interact with the cytokinin signaling pathway?

PAS1's connection to cytokinin signaling represents one of the most intriguing aspects of its function. The evidence for this relationship comes from several observations:

• Expression regulation: PAS1 gene expression is upregulated in the presence of cytokinins (specifically benzyl adenine, BA), suggesting that cytokinins positively regulate PAS1 at the transcriptional level .

• Altered response in mutants: pas1 mutants exhibit hypersensitivity to cytokinins, displaying hypertrophy of the apical regions when treated with BA. This response is specific to cytokinins and not observed with other plant hormones .

• Differential sensitivity: In pas1-1 mutants, the cytokinin induction of PAS1 expression occurs at lower BA concentrations (0.1 μM) compared to wild-type plants, indicating altered sensitivity to the hormone .

To investigate the molecular mechanisms connecting PAS1 to cytokinin signaling, the following experimental approaches are recommended:

Cytokinin Response Analysis:

• Compare transcriptome profiles of wild-type and pas1 mutants with/without cytokinin treatment

• Analyze expression of known cytokinin response genes (type-A ARRs) in pas1 background

• Examine cytokinin-regulated phosphorylation cascades in pas1 mutants

Genetic Interaction Studies:

• Generate double mutants between pas1 and cytokinin signaling components:

  • Cytokinin receptors (AHK2, AHK3, AHK4)

  • Histidine phosphotransfer proteins (AHPs)

  • Response regulators (ARRs)

• Analyze phenotypes to determine epistatic relationships

Biochemical Interaction Analysis:

• Test direct interaction between PAS1 and cytokinin signaling components

• Investigate whether PAS1's peptidyl-prolyl isomerase activity affects folding or activity of cytokinin signaling proteins

What is the relationship between PAS1, PAS2, and PAS3 in regulating plant development?

The three PAS genes (PAS1, PAS2, and PAS3) appear to function in an interconnected regulatory network, as evidenced by their similar mutant phenotypes and molecular interactions:

Expression Relationships:

• PAS1 expression is altered in both pas2 and pas3 mutants:

  • In pas2-1 mutants, PAS1 mRNA is detectable without cytokinin treatment or at low concentrations (0.1 μM BA), but not at higher concentrations (5 μM BA)

  • In pas3-1 mutants, PAS1 mRNA is only detectable when plants are grown at low cytokinin concentrations (0.1 μM BA)

• These expression patterns suggest that both PAS2 and PAS3 genes are required for regulated expression of PAS1, particularly in response to cytokinins.

Experimental Approaches to Study PAS Gene Relationships:

• Transcriptional regulation analysis:

  • Characterize the promoter regions of all three PAS genes

  • Perform chromatin immunoprecipitation to identify transcription factors binding to PAS gene promoters

  • Create promoter-reporter constructs to monitor expression patterns

• Protein-protein interaction studies:

  • Test direct interactions between PAS1, PAS2, and PAS3 proteins

  • Identify shared interaction partners

• Genetic studies:

  • Analyze double and triple mutant combinations

  • Perform cross-complementation experiments (express each gene under control of the others' promoters)

How can researchers generate and characterize new pas1 alleles?

Creating new pas1 alleles is valuable for understanding structure-function relationships and domain-specific roles. Several approaches can be used:

CRISPR-Cas9 Gene Editing:

• Design guide RNAs targeting specific domains:

  • FKBP domain (N-terminal region)

  • Individual TPR repeats

  • Regions between domains

• Transformation protocol:

  • Agrobacterium-mediated transformation of Arabidopsis using floral dip method

  • Select transformants and screen for mutations using PCR-based methods

  • Confirm mutations by sequencing

• Phenotypic analysis:

  • Compare new alleles to existing pas1-1 (T-DNA insertion) and pas1-2 (point mutation) alleles

  • Evaluate domain-specific functions through targeted mutations

EMS Mutagenesis:

• Treat Arabidopsis seeds with ethyl methanesulfonate

• Screen M2 population for pas1-like phenotypes

• Sequence PAS1 gene in candidate mutants

• Perform complementation tests with known pas1 alleles

Complementation Analysis:
For functional validation, the wild-type PAS1 cDNA can be used to complement pas1 mutations. This approach has been successfully demonstrated with the pas1-2 mutant using the PAS1 cDNA under control of the cauliflower mosaic virus 35S promoter .

What methods are recommended for studying PAS1 subcellular localization?

Understanding PAS1's subcellular localization is crucial for elucidating its function. Sequence analysis suggests a nuclear localization, but comprehensive experimental verification is essential:

Fluorescent Protein Fusion Approaches:

• Generate C-terminal and N-terminal GFP/YFP fusions of PAS1

• Express under native PAS1 promoter to maintain physiological expression levels

• Transform into pas1 mutant background to ensure functionality

• Visualize in various tissues and developmental stages using confocal microscopy

Immunolocalization:

• Develop specific antibodies against PAS1 protein

• Perform immunohistochemistry on fixed Arabidopsis tissues

• Use co-staining with organelle markers to confirm localization

Biochemical Fractionation:

• Isolate subcellular fractions (nuclear, cytoplasmic, membrane, etc.)

• Detect PAS1 by Western blotting in each fraction

• Use appropriate controls for each fraction (histone H3 for nuclear, tubulin for cytoskeletal, etc.)

Dynamic Localization Studies:

• Investigate whether PAS1 localization changes:

  • During development

  • In response to cytokinin treatment

  • Under stress conditions

• Use inducible expression systems to track protein movement

What are the best approaches for analyzing PAS1 expression patterns?

Understanding where and when PAS1 is expressed provides crucial insights into its developmental roles:

Transcriptional Analysis:

• Quantitative RT-PCR:

  • Analyze PAS1 expression across tissues and developmental stages

  • Compare expression in response to various hormones and stresses

  • Include pas2 and pas3 mutants in analysis to understand regulatory relationships

• RNA in situ hybridization:

  • Develop specific probes for PAS1 mRNA

  • Perform on tissue sections to provide cellular resolution of expression patterns

Promoter-Reporter Constructs:

• Generate PAS1 promoter fusions to reporter genes:

  • GUS for histochemical staining

  • Luciferase for real-time monitoring

  • Fluorescent proteins for live imaging

• Analyze reporter expression:

  • During development

  • In response to cytokinins and other hormones

  • Under various environmental conditions

Expression Data Analysis:
Research has shown that PAS1 is expressed throughout the plant, including stems, leaves, flowers, siliques, and roots . In pas1-1 heterozygous plants, a PAS1-GUS translational fusion showed strong expression in apical and root meristematic regions, with no detectable expression in cotyledons, leaves, hypocotyls, or differentiated root tissues .

How can PAS1 research contribute to understanding plant hormone crosstalk?

PAS1's involvement in cytokinin responses and its potential connection to other hormonal pathways makes it a valuable model for studying hormone crosstalk in plants:

Hormonal Interaction Analysis:

• Cytokinin-auxin crosstalk:

  • PAS1 expression shows weak upregulation in response to auxin (picloram)

  • Analyze pas1 mutant responses to combined cytokinin and auxin treatments

  • Investigate expression of auxin-responsive genes in pas1 backgrounds

• Other hormonal interactions:

  • Although pas1 mutants don't show altered growth responses to other hormones (ethylene, gibberellic acid, abscisic acid, brassinosteroids) , molecular interactions may still exist

  • Examine expression of hormone-responsive genes in pas1 mutants

Signaling Component Analysis:

• Investigate potential role of PAS1 in modifying stability or activity of:

  • Cytokinin response regulators

  • Auxin response factors

  • Other hormone signaling components

• Test hypothesis that PAS1's peptidyl-prolyl isomerase activity might regulate conformational changes in hormone signaling proteins

Developmental Pathway Integration:

• Examine how PAS1 integrates hormone signals into developmental outputs:

  • Cell division control

  • Cell elongation

  • Meristem organization

What insights can PAS1 provide about immunophilin functions in plants?

PAS1 represents an important model for understanding the broader roles of immunophilins in plant biology:

Comparative Analysis with Other Plant Immunophilins:

• Compare with other Arabidopsis FKBPs:

  • ROF1 (FKBP62) shares 71% similarity with PAS1

  • Low-molecular-weight FKBPs (AtFKBP15-1, AtFKBP15-2)

• Cross-species comparison:

  • Wheat wFKBP73 shares 54% similarity with PAS1

  • Examine conservation of function across plant species

Evolution of Plant Immunophilins:

• Phylogenetic analysis of plant FKBPs to understand:

  • Evolutionary relationships

  • Domain acquisition/loss

  • Functional diversification

• Compare with mammalian homologs:

  • PAS1 shows 49-51% similarity to mammalian FKBP52 proteins

  • Analyze conservation of mechanism versus evolution of plant-specific functions

Novel Functions Beyond Immunosuppressant Binding:

• Investigate plant-specific roles of immunophilins:

  • Developmental regulation

  • Stress responses

  • Hormone signaling

• Compare immunophilin functions in organisms lacking adaptive immunity