Recombinant Zinnia elegans Unknown protein 2

What are the major characterized peroxidase isoforms in Zinnia elegans and how are they purified?

The major basic peroxidase from Zinnia elegans (ZePrx) has been isolated from suspension cell cultures and characterized as having two primary isoforms with molecular weights of 34,700 (ZePrx34.70) and 33,440 (ZePrx33.44), as determined by matrix-assisted laser-desorption ionization time of flight analysis .

Purification methodology:
The purification process follows a multi-step chromatographic approach:

Starting from 18.5 L of spent medium, the partially glycosylated isoform (ZePrx33.44) can be obtained with a yield of 20% (5.2 mg), an Rz value of 3.03, and a specific activity of 11.1 μkat mg⁻¹ protein when assayed against 4-methoxy-α-naphthol. The fully glycosylated isoform, ZePrx34.70, can be obtained with a yield of 6% (2.5 mg), an Rz value of 3.00, and a specific activity of 6.6 μkat mg⁻¹ protein .

How can researchers histochemically characterize secondary cell wall formation in Zinnia elegans xylogenic cultures?

For proper identification of secondary cell wall (SCW) formation timing in Zinnia elegans tracheary element (TE) cultures, a double-staining procedure has been developed. This method employs a combination of calcofluor and auramine-O to simultaneously visualize cellulose and lignin, respectively .

The procedure allows researchers to effectively track the progression of cell differentiation:

• At 48 hours post-induction, TEs are typically not yet visible

• All cells at this stage, regardless of their developmental fate, show calcofluor staining of the primary cell wall

• Later time points reveal lignification patterns characteristic of differentiating TEs

This technique is particularly useful for determining optimal sampling times for studies focused on secondary wall deposition and autolysis during TE formation .

What genetic tools are available for expressing recombinant proteins from Zinnia elegans?

While the search results don't specifically address vector systems for Zinnia elegans, recombinant expression of plant proteins typically employs several expression systems:

• E. coli expression systems: Most commonly used for initial characterization due to high yield and simplicity, though proper folding and post-translational modifications may be limited for plant proteins .

• Yeast expression systems: Offer eukaryotic processing capabilities while maintaining relatively high yields and ease of culture.

• Baculovirus expression systems: Provide more complex post-translational modifications and are particularly useful for proteins requiring extensive glycosylation.

• Plant-based expression systems: Can be used for homologous expression of Zinnia proteins, though typically at lower yields than heterologous systems.

For Zinnia elegans proteins specifically, cDNA cloning and characterization approaches have been successfully employed, as demonstrated with the cloning of four full-length cDNAs coding for ZePrx peroxidases that differ only in their 5′-untranslated regions .

How do the catalytic properties of recombinant Zinnia elegans peroxidases compare to native isoforms in lignin polymerization studies?

ZePrx isoforms have demonstrated specialized catalytic capacity for lignin biosynthesis. Both ZePrx34.70 and ZePrx33.44 isoforms show distinctive substrate preferences, with sinapyl alcohol being the optimal substrate compared to other monolignols . This preference correlates with their biological function in lignification.

Kinetic analysis of these enzymes reveals:

• Both peroxidases are capable of oxidizing ascorbic acid, ferulic acid, and sinapyl alcohol in a reaction strictly dependent on H₂O₂

• Sinapyl alcohol demonstrates the highest catalytic efficiency among tested substrates

• Endwise polymerization of sinapyl alcohol by both ZePrxs yields highly polymerized lignins with polymerization degrees ≥87

These characteristics suggest that recombinant ZePrxs could maintain the catalytic properties necessary for lignin polymerization studies, though direct comparison data between recombinant and native forms is not provided in the search results.

What role do the N-terminal and C-terminal domains play in regulating the function of proteins involved in xylogenesis in Zinnia elegans?

In Arabidopsis, regulatory domains play critical roles in protein function during xylogenesis. For example, the PIRIN2 (PRN2) protein suppresses S-type lignin accumulation in a non-cell-autonomous manner in xylem elements . This type of regulation could involve terminal domains that mediate protein-protein interactions or subcellular localization.

Additionally, in pollen-specific proteins like AtRopGEF12, C-terminal domains have been shown to play inhibitory roles while N-terminal domains can have positive regulatory functions . If similar domain organization exists in Zinnia elegans proteins involved in xylogenesis, researchers might investigate:

• Whether C-terminal truncations alter protein function or localization

• If post-translational modifications (such as phosphorylation) of terminal domains regulate activity

• Whether terminal domains mediate interactions with other proteins in signaling cascades

How can high-throughput approaches be optimized for recombinant expression of Zinnia elegans proteins involved in programmed cell death during xylogenesis?

Based on the integration of information from several search results, optimization of high-throughput expression of Zinnia elegans proteins involved in programmed cell death (PCD) during xylogenesis could be approached as follows:

• Target selection:

  • Prioritize cysteine proteases, as they are implicated in PCD during tracheary element (TE) differentiation

  • Include expansins, which are differentially expressed during xylogenesis

  • Consider PIRIN-like proteins, which may regulate lignification processes

• Expression system optimization:

  • Develop Gateway-compatible entry clones similar to those used for C. elegans high-throughput pipelines

  • Create a stepwise automation process for protein expression screening

  • Implement ELISA-based solubility screening to rapidly identify promising candidates

• Solubility enhancement strategies:

  • Bioinformatic analysis indicates that protein hydrophobicity is a key determining factor for soluble expression

  • Consider expression of functional domains rather than full-length proteins for challenging targets

  • Test co-expression with interacting partners to improve folding and stability

• Functional validation methods:

  • Develop assays to measure caspase-like protease activity, which has been implicated in zinnia TE differentiation

  • Implement imaging-based screens to detect cellular structural changes associated with PCD

  • Establish in vitro systems to test enzyme activity on relevant substrates

What mechanisms regulate the differential expression of ZePrx isoforms in different tissues and developmental stages?

The ZePrx isoforms show tissue-specific expression patterns that suggest complex regulatory mechanisms. Western blot analyses using anti-ZePrx34.70 IgGs revealed that:

• ZePrx33.44 is expressed in tracheary elements, roots, and hypocotyls

• ZePrx34.70 is only expressed in roots and young hypocotyls

• Neither isoform is significantly expressed in leaves or cotyledons

The regulatory mechanisms likely involve:

• Transcriptional regulation:
  The four full-length cDNAs coding for ZePrxs differ only in their 5′-untranslated regions, suggesting differential transcriptional regulation or alternative promoter usage .

• Genomic organization:
  Phylogenetic analysis suggests that the four putative paralogous genes encoding ZePrxs could have arisen from duplication events. A neighbor-joining tree analysis shows that AJ880394 and AJ880395 cluster together, as do AJ880392 and AJ880393, indicating two separate gene duplication events from an ancestral gene .

• Intron-mediated regulation:
  All four genes consist of three exons and two introns (type 2 and type 3). The position where each exon is disrupted by the introns is conserved (-389 bp upstream of the end codon for intron 3 and -734 bp upstream of the end codon for intron 2). The base pair sequence of intron 2 is completely conserved in pairs of genes, further supporting the duplication hypothesis .

Advanced research could investigate how these regulatory elements respond to developmental signals and environmental cues to fine-tune peroxidase expression.

What approaches can be used to identify genes specifically involved in secondary wall deposition and autolysis during tracheary element formation in Zinnia elegans?

Researchers have successfully employed several complementary approaches to identify genes involved in secondary wall formation and autolysis during tracheary element (TE) formation in Zinnia elegans:

• Suppression subtractive hybridization (SSH):

  • This technique has been applied to zinnia TEs to identify hundreds of novel putative xylogenesis markers

  • SSH is particularly useful for capturing genes induced at specific time points, such as the onset of secondary wall formation and PCD

• Time-course sampling strategy:

  • To specifically isolate genes involved in secondary wall deposition and autolysis, cells should be harvested at three critical time points:
    a) Secondary wall-associated cellulose deposition
    b) Lignification
    c) Autolysis

• Macroarray analysis:

  • Comprehensive macroarray analysis of isolated genes helps determine their temporal expression during TE differentiation

  • This approach can classify genes based on their regulation by auxin and/or cytokinin, which are required for TE differentiation

• Multiplex in situ-reverse transcription-PCR (IS-RT-PCR):

  • This technique integrates gene expression data at the single cell level in xylem development both in vitro and in planta

  • It allows researchers to distinguish between genes expressed in differentiating TEs versus surrounding cells

How can researchers effectively purify and characterize recombinant peroxidases from Zinnia elegans for structural and functional studies?

Based on the methods used for native ZePrx, an effective protocol for purifying recombinant peroxidases would include:

• Expression system selection:

  • E. coli systems may be suitable for preliminary studies, but eukaryotic systems (yeast, insect, or plant) would be preferable for proper folding and post-translational modifications, particularly glycosylation which appears important for ZePrx function

• Purification strategy:

  • Implement a multi-step chromatographic approach similar to that used for native ZePrx:
    a) Initial capture by ammonium sulfate precipitation
    b) Hydrophobic interaction chromatography (Phenyl Sepharose)
    c) Size exclusion chromatography (Superdex 75)
    d) Cation exchange chromatography (SP Sepharose)
    e) Affinity chromatography (Concanavalin A for glycosylated forms)

• Characterization methods:

  • Spectroscopic analysis: Absorption spectra should show maxima at 403 nm (Soret band), 500 nm, and 640 nm, characteristic of high-spin ferric class III peroxidases

  • Glycosylation analysis: Differences between isoforms should be characterized using glycan-specific staining or mass spectrometry

  • N-terminal sequencing to confirm protein identity

  • Tryptic fragment fingerprinting by reverse-phase nano-liquid chromatography to compare recombinant and native forms

• Functional assays:

  • Enzyme kinetics with various substrates, particularly monolignols (p-coumaryl, coniferyl, and sinapyl alcohols)

  • Assessment of polymerization capacity using size exclusion chromatography to determine polymer length

  • Testing of oxidative activity using H₂O₂-dependent assays with various phenolic substrates

What experimental design would effectively elucidate the role of cysteine proteases and caspase-like enzymes in programmed cell death during Zinnia elegans xylogenesis?

An effective experimental design to investigate the role of cysteine proteases and caspase-like enzymes during Zinnia elegans xylogenesis would include:

• Inhibitor studies:

  • Apply the cysteine protease inhibitor E64 to xylogenic zinnia cultures to confirm involvement of cysteine proteases in TE formation

  • Test human caspase inhibitors, which have been shown to suppress TE formation, providing evidence that re-differentiation of cultured mesophyll zinnia cells is a PCD event involving caspase-like protease cascades

  • Use a panel of different protease inhibitors with varying specificities to identify which protease classes are most critical

• Gene expression analysis:

  • Perform time-course transcriptome analysis to identify upregulated protease genes during TE differentiation

  • Focus on cysteine proteases, which are highly redundant in function with at least three different isoforms identified in Zinnia elegans (including two previously isolated isoforms with 87% identity and a third isoform, CP7, exhibiting 50% identity with previously identified cysteine proteases)

  • Use qRT-PCR to validate expression patterns of identified proteases

• Protein localization and activity:

  • Develop fluorescent reporters for subcellular localization of protease activity during PCD

  • Employ high-resolution confocal microscopy to document cellular structural changes associated with re-differentiation

  • Use activity-based protein profiling to identify active proteases during different stages of TE formation

• Genetic manipulation:

  • Develop RNAi or CRISPR-based approaches to silence or knockout specific protease genes

  • Create overexpression lines for proteases of interest

  • Assess effects on TE formation, timing of PCD, and final xylem morphology

• Biochemical characterization:

  • Purify and characterize recombinant proteases

  • Identify natural substrates using proteomics approaches

  • Determine activation mechanisms and regulatory factors

This multi-faceted approach would provide comprehensive insights into the protease cascades governing PCD during xylogenesis in Zinnia elegans.

How do peroxidases from Zinnia elegans compare functionally to those from other plant species in lignin biosynthesis?

Zinnia elegans peroxidases (ZePrx) exhibit distinctive features that both align with and differ from peroxidases in other plant species:

• Structural comparison:

  • ZePrx proteins belong to class III peroxidases (Prxs), plant-specific heme oxidoreductases composed of approximately 300 amino acid residues

  • Like other plant peroxidases, ZePrx isoforms contain RING finger domains and BRCT repeat domains that are structurally conserved

  • ZePrx genes contain a gene structure typical of class 'a' peroxidase genes, with three exons and two introns (type 2 and 3), similar to peroxidase genes in Oryza and Arabidopsis

• Catalytic properties:

  • ZePrx shows high affinity for sinapyl alcohol, making it particularly effective in S-lignin formation

  • This contrasts with some other plant peroxidases that may preferentially oxidize coniferyl alcohol (G-lignin precursor)

  • The high polymerization degree (≥87) achieved by ZePrx with sinapyl alcohol indicates specialized functionality

• Tissue-specific expression:

  • ZePrx isoforms show distinct tissue-specific expression patterns, with ZePrx33.44 expressed in tracheary elements, roots, and hypocotyls, while ZePrx34.70 is only found in roots and young hypocotyls

  • This specialized expression differs from Arabidopsis PRN2, which is localized specifically in cells next to vessel elements and functions in non-cell-autonomous lignification of xylem vessels

• Evolutionary relationships:

  • While most plant species have large peroxidase multigene families (73 genes in Arabidopsis, 138 in rice, 93 in poplar) , ZePrx genes appear to have evolved through specific gene duplication events, creating pairs of closely related paralogs

  • This evolutionary pattern suggests specialized adaptation of ZePrx for specific functions in Zinnia elegans

How can knowledge from C. elegans recombinant protein expression strategies be applied to improve Zinnia elegans protein production?

Although C. elegans (a nematode) and Zinnia elegans (a plant) are taxonomically distant organisms, valuable lessons from C. elegans recombinant protein expression can be adapted for plant protein production:

• High-throughput pipeline optimization:

  • The robotic pipeline developed for C. elegans protein expression using Gateway cloning/expression technology could be adapted for plant proteins

  • The stepwise automation approach used for C. elegans could be particularly valuable for screening multiple Zinnia genes involved in xylogenesis

• Predicting expression success:

  • Bioinformatic analysis of C. elegans expression data indicates that protein hydrophobicity is a key determining factor for soluble expression

  • This insight could help prioritize Zinnia elegans target proteins or guide protein engineering efforts to improve solubility

• Expression screening methods:

  • The ELISA method developed for C. elegans protein detection, which identifies candidates with expression levels ≥2 μg/mL, could be adapted for Zinnia proteins

  • This high-throughput screening approach would enable rapid identification of successful expression constructs

• Scale-up strategies:

  • The pipeline used for C. elegans successfully transitioned from small-scale screening to 1L scale-up production for 590 proteins

  • Similar scale-up parameters could be established for Zinnia proteins that show promise in initial screens

• Host system selection:

  • While E. coli was effective for many C. elegans proteins, plant proteins often require eukaryotic systems for proper folding and post-translational modifications

  • The success rate in C. elegans (approximately 15% soluble proteins from tested ORFs) provides a benchmark for expected yields with Zinnia proteins

What insights from Arabidopsis PIRIN2 studies can guide research on lignification regulation in Zinnia elegans?

Research on Arabidopsis PIRIN2 (PRN2) offers several valuable insights that could guide investigation of lignification regulation in Zinnia elegans:

• Non-cell-autonomous regulation mechanisms:

  • PRN2 in Arabidopsis suppresses S-type lignin accumulation in a non-cell-autonomous manner

  • PRN2 promoter activity and PRN2:GFP fusion protein are localized specifically in cells adjacent to vessel elements

  • This suggests researchers should investigate whether Zinnia elegans also employs non-cell-autonomous mechanisms for regulating lignin composition in specific cell types

• Lignin composition analysis techniques:

  • Advanced analytical methods used in PRN2 studies could be applied to Zinnia:
    a) Pyrolysis-gas chromatography/mass spectrometry
    b) 2D-nuclear magnetic resonance
    c) Phenolic profiling
    d) Fourier transform infrared and Raman microspectroscopy for chemotyping individual xylem elements

• Transcriptional regulation of lignin biosynthesis:

  • PRN2 function appears to be mediated through regulation of lignin-biosynthetic gene expression

  • Similar transcriptional networks may exist in Zinnia elegans, suggesting genome-wide expression analysis during xylogenesis could identify key regulatory factors

• Cupin domain function:

  • PRN genes encode cupin domain-containing proteins that function as transcriptional co-regulators in humans

  • Identifying cupin domain-containing proteins in Zinnia elegans could reveal potential regulators of lignification

• G-type vs. S-type lignin regulation:

  • PRN2 specifically suppresses S-type lignin accumulation near xylem vessels to promote G-type enriched lignin composition in vessel elements

  • This lignin-type specificity could be investigated in Zinnia elegans, particularly given that ZePrx shows high affinity for sinapyl alcohol (S-lignin precursor)

What emerging technologies could advance our understanding of recombinant Zinnia elegans proteins in xylogenesis?

Several cutting-edge technologies could significantly advance research on recombinant Zinnia elegans proteins involved in xylogenesis:

• Single-cell transcriptomics and proteomics:

  • Application of single-cell RNA-seq to track gene expression changes during TE differentiation at unprecedented resolution

  • Integration with spatial transcriptomics to map expression patterns within developing vascular tissues

  • Single-cell proteomics to identify post-translational modifications specific to differentiating TEs

• CRISPR/Cas9 genome editing in Zinnia elegans:

  • Development of efficient transformation and regeneration protocols for Zinnia elegans

  • Application of CRISPR/Cas9 to create knockout lines for key xylogenesis-related genes

  • Prime editing or base editing approaches for precise modification of regulatory elements

• Cryo-electron microscopy for structural biology:

  • Structural determination of recombinant Zinnia proteins, particularly peroxidases, at near-atomic resolution

  • Analysis of protein complexes involved in lignin polymerization

  • Visualization of conformational changes during enzyme catalysis

• Advanced imaging of lignification:

  • Super-resolution microscopy combined with lignin-specific fluorescent probes

  • Label-free imaging techniques such as stimulated Raman scattering (SRS) microscopy for real-time visualization of lignification

  • Correlative light and electron microscopy to link molecular events with ultrastructural changes

• In vitro reconstitution systems:

  • Development of cell-free systems to reconstitute the lignification process

  • Biomimetic approaches to create artificial systems that recapitulate aspects of xylogenesis

  • Microfluidic devices to study cell-cell communication during vascular differentiation

How might synthetic biology approaches enable new applications of recombinant Zinnia elegans proteins?

Synthetic biology approaches offer exciting possibilities for harnessing recombinant Zinnia elegans proteins:

• Designer lignin biosynthesis:

  • Engineering peroxidases with altered substrate specificities to produce novel lignin polymers with desired properties

  • Creating synthetic regulatory circuits to control lignin composition and deposition

  • Developing orthogonal lignification systems that can be triggered by external stimuli

• Biomaterial production:

  • Using recombinant Zinnia peroxidases for in vitro synthesis of novel biomaterials with lignin-like properties

  • Engineering controlled polymerization systems for creating structured materials

  • Developing hybrid enzyme systems combining peroxidases with other biocatalysts for multi-component materials

• Plant metabolic engineering:

  • Transferring optimized lignification pathways from Zinnia to crop plants to improve biomass properties

  • Creating synthetic regulatory networks to control vascular development and wood formation

  • Engineering plants with altered lignin content for improved biofuel production

• Biosensor development:

  • Creating biosensors based on Zinnia peroxidases for detecting phenolic compounds or hydrogen peroxide

  • Developing cell-based sensors to monitor vascular differentiation processes

  • Engineering synthetic signaling pathways that respond to plant hormones involved in xylogenesis

• Programmed cell death applications:

  • Harnessing the PCD machinery from Zinnia xylogenesis for controlled elimination of specific cells

  • Engineering inducible PCD systems for biotechnology applications

  • Developing molecular tools based on cysteine proteases from Zinnia for targeted protein degradation