31456690 OR Characterization of Protein Radicals in Arabidopsis Dotaz Zobrazit nápovědu
Oxidative modification of proteins in photosystem II (PSII) exposed to high light has been studied for a few decades, but the characterization of protein radicals formed by protein oxidation is largely unknown. Protein oxidation is induced by the direct reaction of proteins with reactive oxygen species known to form highly reactive protein radicals comprising carbon-centered (alkyl) and oxygen-centered (peroxyl and alkoxyl) radicals. In this study, protein radicals were monitored in Arabidopsis exposed to high light by immuno-spin trapping technique based on the detection of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) nitrone adducts using the anti-DMPO antibody. Protein radicals were imaged in Arabidopsis leaves and chloroplasts by confocal laser scanning microscopy using fluorescein conjugated with the anti-DMPO antibody. Characterization of protein radicals by standard blotting techniques using PSII protein specific antibodies shows that protein radicals are formed on D1, D2, CP43, CP47, and Lhcb3 proteins. Protein oxidation reflected by the appearance/disappearance of the protein bands reveals that formation of protein radicals was associated with protein fragmentation (cleavage of the D1 peptide bonds) and aggregation (cross-linking with another PSII subunits). Characterization of protein radical formation is important for better understating of the mechanism of oxidative modification of PSII proteins under high light.
- Publikační typ
- časopisecké články MeSH
Biomolecule (lipid and protein) oxidation products formed in plant cells exposed to photooxidative stress play a crucial role in the retrograde signaling and oxidative damage. The oxidation of biomolecules initiated by reactive oxygen species is associated with formation of organic (alkyl, peroxyl and alkoxyl) radicals. Currently, there is no selective and sensitive technique available for the detection of organic radicals in plant cells. Here, based on the analogy with animal cells, immuno-spin trapping using spin trap, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) was used to image organic radicals in Arabidopsis leaves exposed to high light. Using antibody raised against the DMPO nitrone adduct conjugated with the fluorescein isothiocyanate, organic radicals were imaged by confocal laser scanning microscopy. Organic radicals are formed predominantly in the chloroplasts located at the periphery of the cells and distributed uniformly throughout the grana stack. Characterization of protein radicals by standard immunological techniques using anti-DMPO antibody shows protein bands with apparent molecular weights of 32 and 34 kDa assigned to D1 and D2 proteins and two protein bands below the D1/D2 band with apparent molecular weights of 23 and 18 kDa and four protein bands above the D1/D2 band with apparent molecular weights of 41, 43, 55 and 68 kDa. In summary, imaging of organic radicals by immuno-spin trapping represents selective and sensitive technique for the detection of organic radicals that might help to clarify mechanistic aspects on the role of organic radicals in the retrograde signaling and oxidative damage in plant cell.
- MeSH
- cyklické N-oxidy chemie MeSH
- elektronová paramagnetická rezonance MeSH
- lipidy chemie izolace a purifikace MeSH
- oxidace-redukce MeSH
- oxidační stres účinky léků MeSH
- peroxid vodíku chemie MeSH
- peroxidy chemie MeSH
- proteiny chemie MeSH
- reaktivní formy kyslíku chemie MeSH
- spin trapping * MeSH
- spinové značení MeSH
- volné radikály chemie izolace a purifikace MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Background and Aims: The non-specific phospholipase C (NPC) is a new member of the plant phospholipase family that reacts to abiotic environmental stresses, such as phosphate deficiency, high salinity, heat and aluminium toxicity, and is involved in root development, silicon distribution and brassinolide signalling. Six NPC genes (NPC1-NPC6) are found in the Arabidopsis genome. The NPC2 isoform has not been experimentally characterized so far. Methods: The Arabidopsis NPC2 isoform was cloned and heterologously expressed in Escherichia coli. NPC2 enzyme activity was determined using fluorescent phosphatidylcholine as a substrate. Tissue expression and subcellular localization were analysed using GUS- and GFP-tagged NPC2. The expression patterns of NPC2 were analysed via quantitative real-time PCR. Independent homozygous transgenic plant lines overexpressing NPC2 under the control of a 35S promoter were generated, and reactive oxygen species were measured using a luminol-based assay. Key Results: The heterologously expressed protein possessed phospholipase C activity, being able to hydrolyse phosphatidylcholine to diacylglycerol. NPC2 tagged with GFP was predominantly localized to the Golgi apparatus in Arabidopsis roots. The level of NPC2 transcript is rapidly altered during plant immune responses and correlates with the activation of multiple layers of the plant defence system. Transcription of NPC2 decreased substantially after plant infiltration with Pseudomonas syringae, flagellin peptide flg22 and salicylic acid treatments and expression of the effector molecule AvrRpm1. The decrease in NPC2 transcript levels correlated with a decrease in NPC2 enzyme activity. NPC2-overexpressing mutants showed higher reactive oxygen species production triggered by flg22. Conclusions: This first experimental characterization of NPC2 provides new insights into the role of the non-specific phospholipase C protein family. The results suggest that NPC2 is involved in the response of Arabidopsis to P. syringae attack.
- MeSH
- Arabidopsis enzymologie imunologie mikrobiologie MeSH
- fosfatidylcholiny metabolismus MeSH
- fosfolipasy typu C fyziologie MeSH
- Golgiho aparát enzymologie MeSH
- imunita rostlin fyziologie MeSH
- klonování DNA MeSH
- konfokální mikroskopie MeSH
- kvantitativní polymerázová řetězová reakce MeSH
- nemoci rostlin imunologie mikrobiologie MeSH
- proteiny huseníčku fyziologie MeSH
- protoplasty enzymologie MeSH
- Pseudomonas syringae * MeSH
- reaktivní formy kyslíku MeSH
- regulace genové exprese u rostlin MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Dirigent (DIR) proteins were found to mediate regio- and stereoselectivity of bimolecular phenoxy radical coupling during lignan biosynthesis. Here we summarize the current knowledge of the importance of DIR proteins in lignan and lignin biosynthesis and highlight their possible importance in plant development. We focus on the still rather enigmatic Arabidopsis DIR gene family, discussing the few members with known functional importance. We comment on recent discoveries describing the detailed structure of two DIR proteins with implications in the mechanism of DIR-mediated catalysis. Further, we summarize the ample evidence for stress-induced dirigent gene expression, suggesting the role of DIRs in adaptive responses. In the second part of our work, we present a preliminary bioinformatics-based characterization of the AtDIR family. The phylogenetic analysis of AtDIRs complemented by comparison with DIR proteins of mostly known function from other species allowed us to suggest possible roles for several members of this family and identify interesting AtDIR targets for further study. Finally, based on the available metadata and our in silico analysis of AtDIR promoters, we hypothesize about the existence of specific transcriptional controls for individual AtDIR genes and implicate them in various stress responses, hormonal regulations, and developmental processes.
