Recombinant Medicago sativa Eukaryotic translation initiation factor 5A-1

What is the structural and functional conservation of eIF5A-1 in Medicago sativa compared to other plant species?

eIF5A is one of the most highly conserved proteins across eukaryotes, including plants. In Medicago sativa, eIF5A-1 shares significant sequence homology with other plant eIF5A proteins. The protein contains the characteristic lysine residue that undergoes hypusination, a unique post-translational modification essential for its function in translation elongation and termination. While initially classified as a translation initiation factor, research has demonstrated that eIF5A actually functions in translation elongation and termination, as well as mRNA turnover .

Comparative studies between Medicago sativa and related species like Medicago truncatula have shown conservation of key functional domains. The protein contains regions necessary for binding to ribosomes and mRNA. Unlike some organisms that have multiple functionally redundant eIF5A isotypes, the specific roles of Medicago eIF5A variants may differ in their expression patterns during development and stress responses, similar to findings in other systems where eIF5A isotypes show distinct functions .

What are the key post-translational modifications of recombinant Medicago sativa eIF5A-1 and how do they affect protein function?

The critical post-translational modification of eIF5A-1 in Medicago sativa, as in other eukaryotes, is hypusination. This unique modification occurs in a two-step process: first, deoxyhypusine synthase (DHS) transfers the 4-aminobutyl moiety from spermidine to a specific lysine residue in eIF5A to form deoxyhypusine; second, deoxyhypusine hydroxylase (DOHH) hydroxylates this intermediate to form hypusine .

The hypusinated form (eIF5A-Hyp) is essential for proper functioning in translation elongation and termination. Recent research suggests that the unhypusinated form (eIF5A-Lys) may have independent functions distinct from the hypusinated version, potentially including regulatory roles in cellular metabolism and stress responses . Studies in zebrafish and mouse models have demonstrated that accumulation of eIF5A-Lys (due to DHPS knockdown) has more significant impacts on gene expression than the complete loss of eIF5A, supporting the concept that eIF5A-Lys is not merely an inactive precursor but may have distinct biological roles .

How is eIF5A-1 gene expression regulated in Medicago sativa under different developmental and stress conditions?

In Medicago species, eIF5A-1 expression is differentially regulated across developmental stages and in response to environmental stressors. Research suggests that eIF5A-1 is particularly important during specific developmental transitions and stress responses . Comparative proteomic analysis of Medicago species under salt stress has identified differential expression patterns of various proteins, including translation factors .

Similar to findings in other organisms, eIF5A isotypes in Medicago may be differentially expressed during key developmental transitions. For instance, in Entamoeba species, eIF5A1 gene was upregulated during excystation while eIF5A2 was downregulated, suggesting isotype-specific roles during differentiation . In Medicago, expression analysis under various stress conditions (particularly salt stress) has revealed complex regulation patterns, with specific isotypes likely playing more prominent roles under different conditions .

Methodologically, researchers studying eIF5A-1 expression in Medicago should employ real-time quantitative PCR with isoform-specific primers, in combination with RNA-seq approaches for genome-wide expression analysis. Promoter analysis and chromatin immunoprecipitation (ChIP) can further elucidate the transcriptional regulation mechanisms controlling eIF5A-1 expression under different conditions.

What are the critical methodological considerations for producing functional recombinant Medicago sativa eIF5A-1 with proper post-translational modifications?

Producing recombinant Medicago sativa eIF5A-1 with appropriate post-translational modifications, particularly hypusination, presents significant technical challenges. The hypusination process requires the co-expression of functional deoxyhypusine synthase (DHS) and availability of the spermidine substrate.

For successful production of properly modified recombinant eIF5A-1, researchers should consider the following methodological approaches:

• Expression system selection: While E. coli is commonly used for recombinant protein production, it lacks the machinery for hypusination. Co-expression of Medicago sativa eIF5A-1 with its cognate DHS enzyme in E. coli can significantly enhance DHS activity, as demonstrated in studies with Entamoeba histolytica where DHS activity increased ~2000-fold when DHS1 was co-expressed with DHS2 . Alternatively, eukaryotic expression systems like yeast or insect cells may provide more native-like post-translational modifications.

• Hypusination optimization: Supplementation of expression media with spermidine can enhance hypusination. Verification of hypusination status through mass spectrometry is essential to confirm proper modification.

• Purification strategy: Multi-step chromatography approaches (affinity, ion-exchange, and size-exclusion) while maintaining conditions that preserve the hypusine modification are recommended. The hypusine modification can be sensitive to certain harsh purification conditions.

• Activity assessment: Functional assays to confirm activity of the recombinant protein, particularly its ability to participate in translation elongation, should be conducted using in vitro translation systems.

How do different isotypes of eIF5A in Medicago sativa contribute to distinct cellular functions and stress responses?

Medicago sativa, like other eukaryotes, likely possesses multiple eIF5A isotypes with potentially distinct functions. Research in other systems has demonstrated that different eIF5A isotypes can have specialized roles in cellular processes and stress responses. For example, in Entamoeba histolytica, two eIF5A isotypes were found to be functionally interchangeable for proliferation, but they showed differential expression during differentiation, with eIF5A1 upregulated during excystation while eIF5A2 was downregulated .

In Medicago species, differential expression of eIF5A isotypes may regulate specific mRNA subsets related to stress responses. Comparative proteomic analysis between Medicago sativa and Medicago truncatula under salt stress has identified numerous differentially expressed proteins, suggesting complex stress response mechanisms that may involve translational regulation by eIF5A variants .

To investigate isotype-specific functions, researchers should consider:

• Isotype-specific gene silencing or CRISPR-based knockout studies to assess the unique contributions of each variant

• Transcriptome and translatome analyses to identify mRNAs specifically regulated by each isotype

• Protein interaction studies to identify isotype-specific binding partners

• Complementation assays to determine the degree of functional redundancy between isotypes

What is the relationship between eIF5A-1 hypusination status and stress-responsive protein translation in Medicago sativa?

The hypusination status of eIF5A-1 potentially serves as a critical regulatory mechanism for stress-responsive protein synthesis in Medicago sativa. Recent research in other systems has demonstrated that the balance between hypusinated (eIF5A-Hyp) and unhypusinated (eIF5A-Lys) forms can significantly impact cellular responses to stress .

Studies using zebrafish and mouse models with induced knockdowns of deoxyhypusine synthase (DHPS) and eIF5A have provided evidence that accumulation of eIF5A-Lys (resulting from DHPS knockdown) has more profound effects on gene expression than the complete loss of eIF5A. RNA sequencing revealed that DHPS knockdown altered expression of 1158 genes, while eIF5A knockdown affected only 68 genes . Importantly, these changes were not in the same direction, suggesting that DHPS and eIF5A independently contribute to distinct cellular pathways.

For Medicago sativa under stress conditions, particularly salt stress, proteomic analysis has identified differential expression of various proteins involved in metabolism, energy production, and stress response . The table below shows examples of differentially expressed proteins in Medicago species under salt stress:

The translation of these stress-responsive proteins may be specifically regulated by the hypusination status of eIF5A-1, particularly those containing polyproline stretches that are known to require eIF5A-Hyp for efficient translation.

What are the most effective protocols for analyzing the interaction network of Medicago sativa eIF5A-1 in planta?

Investigating the interaction network of Medicago sativa eIF5A-1 requires comprehensive approaches to capture both stable and transient interactions. The following methodological protocols are recommended:

• Affinity Purification-Mass Spectrometry (AP-MS): Express tagged versions of eIF5A-1 (both hypusinated and unhypusinated forms) in Medicago cells or tissues, followed by affinity purification and mass spectrometry analysis. This approach can identify stable protein complexes associated with eIF5A-1. Differential analysis between hypusinated and unhypusinated forms can reveal modification-specific interactions.

• Proximity-dependent Biotin Identification (BioID): Fusion of eIF5A-1 with a biotin ligase allows for biotinylation of proteins in close proximity, enabling identification of transient or weak interactions that might be missed by traditional pull-down methods.

• Yeast Two-Hybrid (Y2H) Screening: While less physiological than in planta methods, Y2H can systematically identify direct binary interactions. Split-ubiquitin Y2H systems may be particularly useful for membrane-associated interactions.

• Co-immunoprecipitation with RNA Analysis (RIP-seq): Since eIF5A-1 functions in translation and mRNA metabolism, identifying associated mRNAs is crucial. RIP-seq can identify the mRNA targets that interact with eIF5A-1 complexes.

• Bimolecular Fluorescence Complementation (BiFC): For validating specific interactions in planta, BiFC allows visualization of protein interactions in their native cellular context.

For data analysis, researchers should employ network analysis tools to construct comprehensive interaction maps, with particular attention to interactions that differ between hypusinated and unhypusinated forms of the protein, or that are specific to stress conditions.

How can researchers effectively measure the hypusination status of eIF5A-1 in Medicago sativa tissues?

Accurately quantifying the hypusination status of eIF5A-1 in plant tissues is technically challenging but essential for understanding its functional state. The following analytical approaches are recommended:

• Mass Spectrometry-Based Quantification:

  • Targeted LC-MS/MS approaches with multiple reaction monitoring (MRM) can specifically quantify hypusinated peptides

  • Use of isotopically labeled peptide standards containing the hypusine modification enables precise quantification

  • Sample preparation should include optimized digestion protocols that preserve the hypusine modification

• Antibody-Based Detection:

  • Use of hypusination-specific antibodies for Western blotting

  • Isoelectric focusing followed by Western blotting can separate hypusinated and unhypusinated forms

  • Immunohistochemistry with anti-hypusine antibodies can reveal tissue-specific patterns of hypusination

• Functional Enzymatic Assays:

  • Measurement of DHS activity in plant extracts using radiolabeled spermidine

  • In vitro hypusination assays with recombinant DHS and plant-derived eIF5A-1

• 2D-PAGE Analysis:

  • Two-dimensional gel electrophoresis can separate eIF5A-1 variants based on charge differences resulting from hypusination

  • Subsequent mass spectrometry identification can confirm modification status

For accurate assessment, researchers should always include appropriate controls, such as samples treated with DHS inhibitors to generate unhypusinated controls, and consider the dynamic nature of this modification under different physiological conditions.

What gene editing strategies are most effective for studying eIF5A-1 function in Medicago sativa?

Given the essential nature of eIF5A-1 for cellular viability, sophisticated gene editing approaches are necessary to study its function without causing lethal phenotypes. The following strategies are recommended for Medicago sativa:

• Inducible CRISPR/Cas9 Systems:

  • Temporal control of gene disruption using chemically-inducible or tissue-specific promoters driving Cas9 expression

  • Multiple guide RNAs targeting different exons to enhance knockout efficiency

  • Selection of target sites that specifically disrupt the lysine residue required for hypusination

• Isotype-Specific Targeting:

  • Careful design of guide RNAs to target specific eIF5A isotypes while leaving others intact

  • This approach can reveal isotype-specific functions while maintaining viability through redundant isotypes, similar to the strategy used in studying Entamoeba histolytica eIF5A isotypes

• Base Editing Approaches:

  • Precision modification of specific codons (e.g., converting the hypusinated lysine to arginine) without introducing double-strand breaks

  • This can generate hypusination-deficient variants while maintaining protein expression

• RNAi and Antisense Approaches:

  • For species where CRISPR efficiency is limited, RNA interference targeting specific regions of eIF5A-1 mRNA can achieve partial knockdown

  • Small antisense RNA-mediated gene silencing has been successful in studying eIF5A in Entamoeba

• Hairy Root Transformation:

  • Agrobacterium rhizogenes-mediated transformation for rapid generation of transgenic roots to test constructs before whole-plant transformation

For monitoring editing efficiency, a combination of sequencing, protein expression analysis, and functional assays should be employed to confirm the expected modifications and their physiological consequences.

How does recombinant Medicago sativa eIF5A-1 influence the translation of specific stress-responsive mRNAs?

The selective influence of eIF5A-1 on the translation of specific mRNA subsets, particularly those involved in stress responses, is a critical area of investigation. Research has demonstrated that eIF5A-Hyp plays a specialized role in the translation of mRNAs containing polyproline motifs and certain secondary structures.

To investigate this function in Medicago sativa, researchers should employ:

• Ribosome Profiling (Ribo-seq): This technique provides genome-wide information on actively translating ribosomes, enabling identification of mRNAs whose translation is specifically affected by eIF5A-1 status. Comparing ribosome profiles between plants with normal and altered eIF5A-1 (either through genetic manipulation or treatment with DHS inhibitors) can reveal eIF5A-1-dependent translation events.

• Polysome Profiling: Analyzing the association of specific mRNAs with polysomes in the presence of native versus modified eIF5A-1 can identify transcripts whose translation efficiency is particularly dependent on eIF5A-1.

• In vitro Translation Systems: Reconstituted translation systems supplemented with purified recombinant eIF5A-1 (hypusinated or unhypusinated) can directly assess the role of eIF5A-1 in the translation of specific candidate mRNAs.

Research in other systems has identified specific pathways affected by eIF5A hypusination status. For example, studies in zebrafish showed that knockdown of DHPS affected genes related to mRNA translation, neurogenesis, and stress pathways, while eIF5A knockdown had minimal impact, suggesting that the unhypusinated form (eIF5A-Lys) has independent regulatory functions . In Medicago, specific stress-responsive proteins showing differential expression under salt stress (as shown in the proteomic data ) may be regulated at the translational level by eIF5A-1.

What is the role of Medicago sativa eIF5A-1 in the regulation of programmed cell death during stress responses?

Emerging evidence suggests that eIF5A plays important roles in regulating programmed cell death (PCD) pathways, particularly during stress responses. In Medicago sativa, eIF5A-1 may serve as a critical link between translation regulation and PCD activation during environmental stresses.

The potential mechanism involves:

• Selective Translation of PCD Regulators: eIF5A-1 likely regulates the translation of specific mRNAs encoding PCD pathway components. Research in other systems has shown that eIF5A influences the expression of genes involved in cellular apoptosis/proliferation, including casp8, tp53, and other critical regulators .

• Hypusination-Dependent Regulation: The hypusination status of eIF5A-1 may serve as a metabolic sensor that links polyamine metabolism to PCD pathways. Under stress conditions, alterations in polyamine levels can affect hypusination efficiency, potentially shifting the balance between cell survival and death programs.

• Stress Pathway Integration: eIF5A-1 likely integrates signals from multiple stress pathways, including those related to endoplasmic reticulum (ER) stress. Genes involved in protein folding and ER stress responses (atf4b, hsp90a) have been identified as differentially regulated by hypusination status in other systems .

In Medicago species, the differential expression of stress-related proteins under salt stress, including heat shock proteins (fold change of 72.44 ± 5.12) and chaperonins (fold change of 9.92 ± 0.56) , suggests that translation regulation by eIF5A-1 may be particularly important for stress adaptation and cell survival decisions.

To investigate this role, researchers should consider employing cell death assays, transcriptome analysis during stress exposure, and genetic manipulation of eIF5A-1 hypusination status, while monitoring markers of PCD activation.

How does the interplay between eIF5A-1 and deoxyhypusine synthase affect plant development and stress tolerance in Medicago sativa?

The functional relationship between eIF5A-1 and deoxyhypusine synthase (DHS) represents a critical regulatory node affecting plant development and stress responses in Medicago sativa. This interplay extends beyond the simple enzymatic modification of eIF5A-1 by DHS.

Key aspects of this relationship include:

• Complex Formation and Regulation: Similar to findings in Entamoeba histolytica where DHS activity increased ~2000-fold when DHS1 was co-expressed with DHS2 , Medicago DHS isoforms may form heteromeric complexes for optimal enzymatic activity. The composition and regulation of these complexes likely responds to developmental cues and stress signals.

• Differential Developmental Effects: Studies in model systems have demonstrated that disruption of DHS versus eIF5A can produce distinct phenotypes. In zebrafish, knockdown of DHPS resulted in significant developmental defects in pancreatic growth, while eIF5A knockdown had minimal impact . This suggests that the accumulation of unhypusinated eIF5A (eIF5A-Lys) due to DHS deficiency may have more significant developmental consequences than the complete loss of eIF5A.

• Metabolic Integration: The DHS-eIF5A-1 system serves as an integration point for polyamine metabolism and protein synthesis. In plants under stress, polyamine levels fluctuate significantly, potentially affecting DHS activity and subsequently eIF5A-1 hypusination status.

Research approaches to investigate this interplay should include:

• Co-expression analysis of DHS and eIF5A-1 genes across developmental stages and stress conditions

• Protein-protein interaction studies to identify regulatory partners affecting the DHS-eIF5A-1 relationship

• Metabolomic analysis to correlate polyamine levels with hypusination efficiency under stress

• Genetic manipulation of DHS versus eIF5A-1 to compare the distinct developmental and stress-responsive phenotypes

In Medicago species, the identification of differential protein expression patterns under salt stress provides potential downstream targets whose expression may be regulated through the DHS-eIF5A-1 axis, offering insight into how this system contributes to stress adaptation.