Characterization of Four Bifunctional Plant IAM/PAM-Amidohydrolases Capable of Contributing to Auxin Biosynthesis
Abstract
:1. Introduction
2. Results and Discussion
2.1. Identification of AMI1-like Proteins in Plant Genomes
2.2. Subcellular Localization of Selected Plant Amidases
2.3. Functional Analysis of the Selected Plant Amidases
Parameter | AtAMI1 | SbAMI1 | OsAMI1 | PtAMI1 | MtAMI1 |
---|---|---|---|---|---|
Amino acid identity to AtAMI1 [%] | 100 | 58 | 57 | 64 | 66 |
Intracellular localization | cytoplasm/nucleoplasm | cytoplasm/nucleoplasm | cytoplasm/nucleoplasm | cytoplasm/nucleoplasm | cytoplasm/nucleoplasm |
Specific activity (IAM) [pkat mg−1] | 3070 ± 520 | 2378 ± 324 | 375 ± 52 | 329 ± 22 | 37 ± 3 |
Temperature optimum [°C] | 37 | 45 | 27 | 37 | 35 |
pH optimum | 7.5 | 6 | 7.5 | 7.5 | 7.5 |
Calculated molecular weight [kDa] | 46 | 45 | 50 | 53 | 57 |
2.4. Occurrence and Auxin Activity of Phenyl-2-acetic Acid in Arabidopsis
Plant Age (Weeks) | Free PAA pmol (g FW)−1 | Free IAA pmol (g FW)−1 |
---|---|---|
Two weeks | 98 ± 6.4 | 35 ± 2.7 |
Six weeks | 23 ± 2 | 15 ± 1 |
3. Experimental Section
3.1. Plant Material and Plant Growth Conditions
3.2. RNA Isolation and RT-PCR
Primer Name | Sequence (5'–3') |
---|---|
OsAMI1-SacI-His2C-For | 5'-TATGAGCTCTATGGCGATGGCGGGTGGAG-3' |
OsAMI1-KpnI-His2C-Rev | 5'-TATGGTACCGTGATTGGAGGACCAAGTTTTAG-3' |
SbAMI1-SpeI-pUC-For | 5'-TATACTAGTATGGCGATGGGCGGCGATTAC-3' |
SbAMI1-SmaI-pUC-Rev | 5'-TATCCCGGGGAGAGAGGAGTCTGGTGAGC-3' |
MtAMI1-XhoI-His2B-For | 5'-TATCTCGAGATGGAAACAGCCTCAGACTATG-3' |
MtAMI1-XbaI-His2B-Rev | 5'-TATTCTAGATATTTTTCAATGTTATCATAAATACTC-3' |
PtAMI1-XhoI-His2B-For | 5'-TATCTCGAGATGGAACGAGACCCGGATTATG-3' |
PtAMI1-XbaI-His2B-Rev | 5'-TATTCTAGATATTTTTCAGTGATCTCAACCTG-3' |
MtAMI1-BglII-pUC-For | 5'-TATAGATCTATGGAAACAGCCTCAGACTATG-3' |
MtAMI1-SalI-pUC-Rev | 5'-TATGTCGACCTATTTTTCAATGTTATCATAAATACTC-3' |
3.3. Generation of Bacterial Expression Constructs
3.4. Preparation of GFP Amidase Fusion Constructs
3.5. Heterologous Expression of Recombinant Amidases
3.6. Transient Expression in Plants and Confocal Laser Scanning Microscopy
3.7. Assay for Amidase Activity
3.8. Root Growth Assay
3.9. Auxin Extraction and Purification
3.10. Quantification of Endogenous Phenyl-2-Acetic Acid and Indole-3-Acetic Acid from Arabidopsis thaliana
3.11. Gel Electrophoresis and Immunoblotting
3.12. Phylogenetic Analysis
3.13. Statistic Analysis
4. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Sánchez-Parra, B.; Frerigmann, H.; Alonso, M.-M.P.; Loba, V.C.; Jost, R.; Hentrich, M.; Pollmann, S. Characterization of Four Bifunctional Plant IAM/PAM-Amidohydrolases Capable of Contributing to Auxin Biosynthesis. Plants 2014, 3, 324-347. https://doi.org/10.3390/plants3030324
Sánchez-Parra B, Frerigmann H, Alonso M-MP, Loba VC, Jost R, Hentrich M, Pollmann S. Characterization of Four Bifunctional Plant IAM/PAM-Amidohydrolases Capable of Contributing to Auxin Biosynthesis. Plants. 2014; 3(3):324-347. https://doi.org/10.3390/plants3030324
Chicago/Turabian StyleSánchez-Parra, Beatriz, Henning Frerigmann, Marta-Marina Pérez Alonso, Víctor Carrasco Loba, Ricarda Jost, Mathias Hentrich, and Stephan Pollmann. 2014. "Characterization of Four Bifunctional Plant IAM/PAM-Amidohydrolases Capable of Contributing to Auxin Biosynthesis" Plants 3, no. 3: 324-347. https://doi.org/10.3390/plants3030324