Recombinant Cucurbita maxima 1-aminocyclopropane-1-carboxylate synthase CMA101 (ACS2)

What is 1-aminocyclopropane-1-carboxylate synthase CMA101 (ACS2) and its biological significance?

1-aminocyclopropane-1-carboxylate synthase CMA101 (ACS2) is a key enzyme in the ethylene biosynthetic pathway in plants. It catalyzes the conversion of S-adenosyl-L-methionine (SAM) to 1-aminocyclopropane-1-carboxylic acid (ACC), which is the direct precursor of ethylene. The enzyme plays a crucial role in various physiological processes including fruit ripening, senescence, and stress responses in plants. According to the enzyme classification system, it is designated as EC 4.4.1.14 and is also known as S-adenosyl-L-methionine methylthioadenosine-lyase .

What are the optimal storage conditions for maintaining ACS2 activity?

For optimal enzyme stability and activity maintenance, recombinant Cucurbita maxima ACS2 should be stored at -20°C for regular use, or at -80°C for extended storage periods. The commercial preparation typically has a shelf life of 6 months in liquid form when stored at -20°C/-80°C, and 12 months in lyophilized form at the same temperature conditions. Repeated freezing and thawing cycles should be strictly avoided as they can significantly compromise enzyme activity. Working aliquots can be maintained at 4°C for a maximum of one week before noticeable activity loss occurs .

What is the recommended reconstitution protocol for freeze-dried ACS2?

The recommended reconstitution protocol involves:

• Brief centrifugation of the vial prior to opening to ensure all material is at the bottom

• Reconstitution in deionized sterile water to achieve a final concentration of 0.1-1.0 mg/mL

• Addition of glycerol to a final concentration of 5-50% (with 50% being standard practice for many laboratories)

• Gentle mixing to ensure complete dissolution without generating excessive foam

• Preparation of small working aliquots to minimize freeze-thaw cycles

• Storage of reconstituted protein at recommended temperatures based on intended use timeframe

How can ACS2 enzyme kinetics be accurately measured in laboratory conditions?

Measuring ACS2 enzyme kinetics requires careful experimental design and consideration of multiple factors:

Methodology:

• Prepare reaction mixtures containing varying concentrations of SAM substrate (typically 1-100 μM range)

• Include pyridoxal phosphate cofactor (typically 10-50 μM)

• Maintain optimal buffer conditions: generally 100 mM HEPES or phosphate buffer, pH 7.5-8.5

• Control temperature (typically 25-30°C for plant enzyme studies)

• Measure reaction progress by quantifying:

  • ACC production (using ninhydrin colorimetric assay or HPLC)

  • SAM consumption (HPLC analysis)

  • Methylthioadenosine formation (spectrophotometric methods)

Data Analysis:

• Calculate initial velocity at each substrate concentration

• Plot data using Michaelis-Menten, Lineweaver-Burk, or Eadie-Hofstee transformations

• Determine kinetic parameters including Km, Vmax, and kcat

• Compare values to published literature on related ACS enzymes

The table below presents typical kinetic parameters for ACS enzymes that can serve as reference points:

What molecular techniques are effective for detecting ACS2 expression in plant tissues?

Several molecular techniques can be employed to effectively detect and quantify ACS2 expression:

• Southern Blot Analysis: CS-ACS2 cDNA clones can be used as probes for genomic DNA analysis after labeling with detection systems such as ECL . This technique helps determine gene copy number and structure.

• qRT-PCR (Quantitative Reverse Transcription PCR):

  • Design primers specific to conserved regions of ACS2

  • Extract total RNA from plant tissues using RNase-free conditions

  • Synthesize cDNA using reverse transcriptase

  • Perform real-time PCR with appropriate reference genes

  • Analyze data using ΔΔCt or standard curve methods

• Western Blot Analysis:

  • Extract total protein from plant tissues

  • Separate proteins by SDS-PAGE

  • Transfer to membrane and probe with anti-ACS antibodies

  • Visualize using chemiluminescence or colorimetric detection

• RNA Sequencing:

  • Allows genome-wide expression analysis

  • Can detect splice variants and novel transcripts

  • Provides comprehensive view of transcriptional changes

  • Can be correlated with phenotypic traits as demonstrated in Atlantic Giant pumpkin studies

How does ACS2 relate to fruit development and size regulation in Cucurbita maxima?

The relationship between ACS2 and fruit development in Cucurbita maxima involves complex interactions between ethylene signaling and developmental pathways:

Recent whole-genome resequencing studies of Atlantic Giant pumpkin (Cucurbita maxima) identified quantitative trait loci (QTLs) and candidate genes associated with fruit size. While not directly focusing on ACS2, the research revealed that fruit size in Cucurbita maxima is a quantitative trait controlled by multiple genes. RNA sequencing analysis showed that pathways associated with assimilate accumulation into the fruit, including carbohydrate metabolism, were significantly enriched in differentially expressed genes .

The connection between ACS2 and fruit development likely involves:

• Ethylene-mediated regulation of cell expansion and division

• Coordination of sink strength and assimilate partitioning

• Modulation of other hormone pathways, particularly auxin and gibberellin

• Temporal regulation of gene expression during fruit development phases

Methodologically, researchers investigating these connections should:

• Perform developmental stage-specific expression analysis of ACS2

• Correlate ACS2 expression with ethylene production rates

• Analyze ACS2 expression in different fruit tissues (mesocarp, placenta, seeds)

• Compare expression patterns between large-fruited varieties (like Atlantic Giant) and small-fruited varieties

How can gene editing technologies be applied to study ACS2 function in Cucurbita maxima?

Gene editing approaches provide powerful tools for investigating ACS2 function in Cucurbita maxima through targeted genetic modifications:

CRISPR/Cas9 methodology for ACS2 functional studies:

• Guide RNA design:

  • Identify unique target sequences within ACS2 coding regions

  • Design guide RNAs with minimal off-target effects

  • Target conserved functional domains for maximum impact

• Delivery methods:

  • Agrobacterium-mediated transformation for stable integration

  • Protoplast transfection for transient expression

  • Biolistic delivery for recalcitrant tissues

• Editing strategies:

  • Complete knockout via frameshift mutations

  • Point mutations to alter specific amino acids in active sites

  • Promoter modifications to alter expression patterns

  • Insertion of reporter genes for expression monitoring

• Phenotypic analysis:

  • Measure ethylene production rates

  • Analyze changes in fruit development and ripening

  • Document alterations in sex expression

  • Investigate stress response modifications

Expected outcomes from different editing approaches:

How does Cucurbita maxima ACS2 compare structurally with related proteins from other species?

While the search results don't provide direct structural comparisons of Cucurbita maxima ACS2 with other species, we can infer methodological approaches for such analyses:

Comparative analysis methodology:

• Sequence alignment:

  • Perform multiple sequence alignment using MUSCLE or CLUSTAL algorithms

  • Identify conserved domains and variable regions

  • Calculate sequence identity and similarity percentages

  • Generate phylogenetic trees to visualize evolutionary relationships

• Structural prediction and comparison:

  • Use homology modeling based on resolved ACS structures

  • Employ tools like SWISS-MODEL, Phyre2, or I-TASSER

  • Compare predicted secondary and tertiary structures

  • Analyze conservation of active site residues

• Functional domain analysis:

  • Map known functional domains (substrate binding, cofactor binding)

  • Predict post-translational modification sites

  • Identify regulatory regions affecting enzyme activity

From similar studies in the field, we can anticipate that Cucurbita maxima ACS2 likely shares 70-90% sequence identity with ACS proteins from other cucurbits, with particularly high conservation in catalytic domains and cofactor binding sites.

What insights can be gained from the full amino acid sequence of recombinant Cucurbita maxima ACS2?

The complete amino acid sequence of recombinant Cucurbita maxima ACS2 (475 residues) provides valuable information for structural and functional analyses :

Key sequence analysis methods:

• Domain identification:

  • The sequence likely contains a PLP-binding domain (typically involving a lysine residue)

  • Substrate binding regions for SAM interaction

  • Potential dimerization interfaces (ACS enzymes typically function as dimers)

• Secondary structure prediction:

  • Analysis of alpha-helical and beta-sheet regions

  • Identification of loop regions that may confer flexibility

  • Prediction of surface-exposed versus buried residues

• Functional motif analysis:

  • Identification of conserved catalytic residues

  • Recognition of regulatory motifs (phosphorylation sites, etc.)

  • Prediction of protein-protein interaction surfaces

• Evolutionary conservation mapping:

  • Alignment with other ACS enzymes to identify invariant residues

  • Detection of Cucurbita-specific sequence features

  • Identification of residues under positive or negative selection

What are common challenges in experiments involving recombinant ACS2 and how can they be addressed?

Researchers working with recombinant Cucurbita maxima ACS2 may encounter several challenges that require specific troubleshooting approaches:

Challenge 1: Protein stability and activity loss

• Problem: ACS enzymes can lose activity during purification or storage

• Solutions:

  • Add protease inhibitors during extraction and purification

  • Maintain reducing conditions (add DTT or β-mercaptoethanol)

  • Store with glycerol (5-50%) as recommended

  • Aliquot to avoid freeze-thaw cycles

  • Monitor activity regularly with standardized assays

Challenge 2: Inconsistent enzyme kinetics

• Problem: Variable results in enzyme activity assays

• Solutions:

  • Standardize buffer conditions (pH, ionic strength)

  • Ensure consistent cofactor (PLP) availability

  • Control temperature precisely during assays

  • Use freshly prepared substrate solutions

  • Include internal controls in each experiment

Challenge 3: Expression system limitations

• Problem: Low yield or inactive protein from expression systems

• Solutions:

  • Optimize codon usage for expression host

  • Consider alternative expression systems (bacterial, insect, plant)

  • Modify purification tags or their position (N- vs C-terminal)

  • Adjust induction conditions and expression temperature

  • Co-express with chaperones if misfolding occurs

How can researchers design experiments to study the role of ACS2 in plant stress responses?

Designing experiments to investigate ACS2's role in plant stress responses requires a multifaceted approach:

Experimental design framework:

• Gene expression analysis:

  • Subject plants to various stresses (drought, salinity, pathogen exposure)

  • Collect tissue samples at defined time points

  • Quantify ACS2 expression via qRT-PCR

  • Compare with other ACS gene family members

  • Correlate with physiological stress markers

• Protein activity measurements:

  • Extract protein from stressed and control plants

  • Measure ACS enzyme activity using standardized assays

  • Analyze post-translational modifications under stress

  • Determine if stress alters enzyme kinetics

• Genetic manipulation approaches:

  • Generate overexpression lines with constitutive ACS2 expression

  • Create RNAi or CRISPR knockout lines

  • Evaluate stress tolerance phenotypes

  • Measure ethylene production in modified lines

• Hormone interaction studies:

  • Analyze crosstalk between ethylene and other stress hormones (ABA, JA, SA)

  • Apply hormone inhibitors in combination with stress treatments

  • Monitor ACS2 expression in hormone-deficient mutants

  • Determine if hormone application alters ACS2 activity

Sample experimental timeline for drought stress study:

How can genomic approaches advance our understanding of ACS2 regulation in Cucurbita maxima?

Recent genomic approaches offer powerful tools for understanding ACS2 regulation in Cucurbita maxima:

The whole-genome resequencing study of Atlantic Giant pumpkin (Cucurbita maxima) demonstrates the application of advanced genomic approaches to identify QTLs and candidate genes. This methodology revealed that fruit size-related traits show transgressive segregation in the F2 population, suggesting quantitative trait control by multiple genes. A high-resolution genetic map with an average physical distance of 154 kb per marker was constructed, enabling precise QTL identification .

Advanced genomic approaches for ACS2 research:

• QTL mapping:

  • Develop mapping populations segregating for ethylene-related traits

  • Identify genetic loci controlling ACS2 expression or activity

  • Correlate genetic markers with phenotypic variation

• Genome-wide association studies (GWAS):

  • Analyze diverse Cucurbita maxima germplasm

  • Identify natural variation in ACS2 sequence or expression

  • Associate polymorphisms with phenotypic differences

• Epigenetic analysis:

  • Characterize DNA methylation patterns in ACS2 promoter regions

  • Investigate histone modifications affecting gene accessibility

  • Study chromatin structure changes under different conditions

• Transcriptome analysis:

  • Perform RNA-seq to identify co-expressed gene networks

  • Characterize transcription factors regulating ACS2

  • Identify alternative splicing patterns

As demonstrated in the Atlantic Giant study, these approaches can lead to the development of molecular markers (like KASP markers) that can be employed for marker-assisted breeding to alter traits related to ethylene biosynthesis and signaling .

What biotechnological applications might emerge from advanced understanding of ACS2 function?

Advanced understanding of Cucurbita maxima ACS2 function could lead to several biotechnological applications:

Potential biotechnological applications:

• Fruit ripening modulation:

  • Development of transgenic plants with controlled ethylene production

  • Creation of varieties with extended shelf life

  • Engineering of synchronized ripening for mechanical harvesting

• Stress tolerance enhancement:

  • Generation of plants with optimized ethylene responses to abiotic stress

  • Development of crops with improved drought or salinity tolerance

  • Creation of lines with enhanced resistance to pathogens

• Sex expression control:

  • Engineering plants with desired sex ratios for hybrid seed production

  • Development of predominantly female flowering lines

  • Creation of varieties with synchronized flowering patterns

• Biosensor development:

  • Use of ACS2 promoter-reporter constructs for stress detection

  • Development of enzyme-based sensors for ethylene precursors

  • Creation of diagnostic tools for plant physiological status

Methodological approaches for biotechnological applications: