Es wurden Second-Site-Mutanten identifiziert, die eine oder beide Stressreaktionen der Influenza-Mutante unterdrücken können. Die schützende Rolle von Tocopherol wurde in der Influenza-Mutante durch die Untersuchung des Zelltods in der Influenza/vte1-Doppelmutante und durch die Wirkung von exogenem Tocopherol in Influenza-Protoplasten nach einem Dunkel-/Hell-Zyklus bestätigt. Darüber hinaus wurde in dieser Arbeit der Zelltod der Influenza-Mutante mithilfe eines biochemischen Ansatzes charakterisiert.
Introduction
- Stress in plants
- The signalling of stress
- Biotic stress
- Abiotic stress
- ROS in plants
- Oxygen
- The ROS and their generation
- Singlet oxygen formation
- Avoiding production of ROS
- ROS scavenging mechanisms
- Photooxidation and singlet oxygen
- Modification of bio-molecules induced by singlet oxygen
- Signalling by singlet oxygen
- The flu system to study the singlet oxygen pathway
- The flu mutant
- Second-site mutagenesis of the flu mutant
Under high light, violaxanthin is converted to zeaxanthin and PsbS, a subunit of PSII, is protonated due to the low pH of the lumen (Li et al., 2000). We took advantage of the influenza mutant isolated in our group ( Meskauskiene et al., 2001 ), which is defective in the regulation of the chlorophyll biosynthetic pathway, to study singlet oxygen signaling. Constitutive acclimation to light stress induced by soldat10 suppresses singlet oxygen-mediated responses in seedlings of the double mutant soldat10/flu (Sánchez-Coll, 2006; Würsch, 2006).
Materials and Methods
Material
- Plant material
- Microorganisms
- Plasmid vectors
- Enzymes
- Oligonucleotides
- Genetic markers
- Chemicals
- Culture media
- Antibiotics
The cosmid encodes a neomycin phosphotransferase that confers kanamycin resistance to Agrobacterium tumefaciens and plants, a tetracycline resistance gene for bacterial selection, a Hind III restriction site to integrate the 9 to 25 kb fragment, a cos site to allow transfection (Meyer et al., 199). ). SSLP PCR markers were created by identifying small insertions/deletions that are polymorphic between Ler and Col (Jander et al., 2002), designing primers flanking each insertion/deletion, and testing for polymorphisms between Ler and Col.
Methods
- Culture methods
- Culture of Arabidopsis on MS plates
- Arabidopsis culture on soil
- DNA techniques
- Genomic DNA isolation
- PCR
- BAC complementation
- RNA techniques
- RNA extraction
- Analysis of RNA
- DNase treatment
- cDNA synthesis
- Transformation methods
- Preparation of VCS257 competent cells
- Transfection of E.coli VCS257 by the cosmid constructs
- Preparation of electro-competent Agrobacterium tumefaciens
- Electroporation
- Verification of the transformed Agrobacterium
- Transformation of Arabidopsis thaliana with Agrobacterium
- Plant growth
- Agrobacterium growth
- Transformation of Arabidopsis by the floral dip method
- Selection of positive transformants
- Pigment techniques
- Extraction of the pigments for carotenoid and chlorophyll
- Green-gel
- Isolation and solubilization of membrane sample
- Electrophoresis conditions
- Extractions of pigments from bands
- MS and NMR spectra
- Protoplast experiments
- Preparation of protoplasts
- Evans blue staining
The PCR mixture was prepared by adding to an Eppendorf tube 2 μl DNA solution, 2 μl BioTherm™ Mix Buffer, 1.5 units BioTherm™ DNA polymerase (Genecraft GmbH, Lüdinghausen, Germany), 25 pmol dNTP (Fermentas GmbH, St. Leon-Rot, Germany), 20 pmol of the specific forward and reverse primers and water to a final volume of 20 μl. The BAC DNA was isolated using the midiprep kit (Qiagen) as described in the manual, except that the water was preheated to 65°C before the elution of the adsorbed DNA from the column. The fragments larger than 9 kb were cut from the gel and extracted with the gel extraction kit (Macherey-Nagel).
During the elution step, the temperature of the elution buffer was increased to 65 °C, which allows for better elution efficiency. Twenty-five µl of three dilutions (1, 1/10 and 1/50) of the cosmid packaging reaction mixture in SM buffer and 25 µl of the corresponding bacterial cells were incubated for 30 minutes at room temperature. The cells were centrifuged again as above and the pellet resuspended in 500 ml of the same buffer.
Two ml of a 4 ml LB culture of the different colonies obtained after electroporation were spun off. A bacto-PCR with the corresponding primers used during the identification of the contig was used to confirm the presence of the insert. For the final subculture step, the 50 ml bacterial culture was poured into 1 l of the selective LB medium and grown to an OD600 of 2.
The Agrobacterium infiltration solution was supplemented with 500 μl of the detergent Silwet L-77, and the aerial parts of Arabidopsis were dipped for 2–3 seconds with gentle agitation.
Results
Genetic approach to study the effect of singlet oxygen
- Isolation of soldat/flu suppressor mutants
- Phenotypic and physiological analysis of soldat30/flu
- Phenotype of soldat30/flu at the rosette leaf stage
- Phenotype of soldat30/flu at the seedling stage
- Pchlide accumulation
- Map-based cloning
- Creating the mapping population
- Crude mapping
- Isolation of flu KO mutant
- Creating a second mapping population and fine mapping
- Complementation of the soldat30/flu phenotype
- Determination of the soldat30 gene
- In silico analysis of the soldat30 gene
- Gene and protein sequence soldat30
- The effect of the mutation on the maturation of 23S rRNA
- Analysis of tocopherols in soldat30/flu and their role in the singlet
- Measurement of the content of tocopherols in soldat30/flu
- The vte1 mutant
- The induction of cell death in the double mutant vte1/flu
- Tocopherols can attenuate the cell death in flu protoplast
- soldat30 has also an increased resistance to superoxide
- The effect of soldat30 in seedlings exposed to natural stress
- The effect of acclimatation on the tocopherol content of soldat30
A map-based cloning approach was used to identify the soldier30 gene responsible for the return of the influenza mutant. The first crude mapping indicated that the gene responsible for the soldier30 phenotype was located at the top of chromosome III (Figure 3.5), close to the location of the influenza gene. The predicted insertion of the T-DNA in the putative flu-KO mutant was detected using a PCR approach.
The insertion of the SOLDAT30 wild-type copy into the soldat30/flu mutant restored the original flu phenotype. To determine the effect of the soldier30 mutation on PNPase activity, the size of the 23S rRNA was determined. The synthesis of tocopherols starts from PDP and HGA. Tocopherol cyclase (TC) function is impaired in vte1.
The role of tocopherols in the flu mutant was investigated by comparing the phenotype of the vte1/flu double mutant with the flu single mutant under non-permissive conditions (Figure 3.19). The effect of the soldier30 mutation on seedlings was tested under high light and cold stress. The Fv/Fm values of the non-prestressed seedlings (np) were compared with those of prestressed (p) seedlings at the same time points (Figures 3.23 and 3.24).
The photosynthetic activity (Fv/Fm) was measured at and 9 days after the start of the stress treatment. The higher accumulation of tocopherols in soldat30 could be one of the reasons why the soldat30 mutation gave a higher resistance to light/cold stress. During the pretreatment (24 h, 120 μmol.m-2.s-1), the synthesis of tocopherol was upregulated more than 10-fold in both WT and in the soldier30 mutant.
The biochemical characterization of the effect of singlet oxygen
- Pigment accumulation in flu after a dark-light shift
- Spectrum of the unknown pigment in flu
- The localization of the pigment in different photosystem
- Accumulation of the Chl derivative under different stress conditions 80
- Accumulation of the Chl a derivative in soldat30/flu double mutant 82
- MS analysis
- NMR analysis of the Chl a derivative
The spectrum of the unknown pigment resembled that of chlorophyll a, so it was concluded that this unknown pigment must be a derivative of chlorophyll a. The content of the derivative of Chl a and b is reduced in all photosystem subcomplexes in the flu mutant compared to the wild type. Accordingly, WT seedlings were exposed to other oxidative stress conditions, and an HPLC analysis was used to determine the relative amounts of the Chl derivative.
Chlorophyll a was solubilized in acetone and irradiated with 100 μmol.m-2.s-1 in the presence of Rose Bengal (100 μM). This peak may correspond to the Chl a derivative formed in vivo in the influenza mutant after the dark/light switch. The effect of the soldier30 mutation on the accumulation of the Chl a derivative was investigated in the influenza background.
The mass-to-charge ratio of the main ionized product was 893, which corresponds to the molecular weight of chlorophyll a. The corresponding peak of the chlorophyll a derivative is 909.16 higher than the Chl a peak, which would correspond exactly to the MW of Chl a plus an additional oxygen atom. The arrows indicate the molecular weight of the chlorophyll molecule, which is 16 mass units higher for the chlorophyll derivative (MW of Chl a'=909.4) compared to Chl a (MW=893.6).
The analysis of several NMR spectra indicated that the additional oxygen is located at C132 of Chla, on the tetrapyrrole ring (Figure 3.37).
Discussion
- Oxidative stress and mode of action of ROS
- The flu system to study the singlet oxygen effects
- Comparison between soldat30, soldat8 and soldat10 mutants
- The mutation of the PNPase in soldat30 reverts the flu phenotype
- Scavenging effect of tocopherol in singlet oxygen-induced cell death 94
- The mutant flu/vte1 is more sensitive to singlet oxygen than flu
- The role of tocopherol in reversion of the flu phenotype
- Biochemical characterization of singlet oxygen-induced cell death
- The carotenoid content decreased after the release of singlet oxygen 97
- The chlorophyll a derivative accumulates in all photosystem
- The accumulation of the chlorophyll a derivative is induced by
- In vivo significance of the 13 2 -HO-Chl a
- Conclusion and future prospects
It is possible to manipulate the amount of singlet oxygen produced by varying the length of the dark period. 1O2-induced gene expression pattern almost to the level of the wild type (Lee et al., 2007). In rif10, the mutation led to an increased accumulation of key enzymes of the methylerythritol phosphate (MEP) pathway, which are involved in the synthesis of isoprenoids in chloroplasts.
Further analysis of ABA content after singlet oxygen release is needed to gain more insight into the role of ABA in influenza cell death. In the influenza system, a gradual accumulation of the Chl a derivative after a dark-light shift was observed in 5-day-old seedlings. It was previously shown that a T-DNA insertion resulting in the loss of function of this gene (rif) resulted in a significant increase in an enzyme involved in the MEP pathway.
The upregulation of the genes involved in the MEP pathway can induce an overaccumulation of an end product of this pathway, i.e. we cannot exclude that the mutation of PNPase in soldat30/flu acts at a different level, for example as a level more downstream of the signaling of the singlet oxygen. The analysis of the cell death after dark-light switching in the triple mutant will be compared with the cell death in the double mutants rif/.
The triple mutant could also be tested against the double mutants to further investigate the role of the PNPase in the acclimatory response.
Lutein is required for efficient quenching of chlorophyll triplets in the LHCII major antenna complex of higher plants and effective photoprotection in vivo under strong light. Crosstalk between abiotic and biotic stress responses: a current view from convergence points in stress signaling networks. Tocopherol is a singlet oxygen scavenger produced by the triplet states of chlorophyll in the PSII reaction center.
Enzymatic, but not nonenzymatic, 1O2-mediated peroxidation of polyunsaturated fatty acids forms part of the EXECUTER1-dependent stress response program in the influenza mutant of Arabidopsis thaliana. Identification and characterization of soldier8, a repressor of the cell death program activated by singlet oxygen in Arabidopsis seedlings. Plastid cues posttranscriptionally regulate the accumulation of key enzymes of the methylerythritol phosphate pathway in Arabidopsis.
Rate constants for the decays and reactions of the lowest electronically excited singlet state of molecular oxygen in solution. Identifizierung und Charakterisierung von soldat10, einem Unterdrücker des durch Singulett Sauerstoff induzierten Zelltodprogramms in Arabidopsis Keimlingen Diss., Eidgenössische Technische Hochschule ETH Zürich, Nr. Special thanks to: Chanhong for his great help regarding the daily technical problems and also for helpful discussions and sharing of ideas; Ais for our interesting evening discussions, for giving much appreciated insight into Asian cuisine and culture and for his friendship; Nuria for critical discussion and sharing ideas about our soldier mutants, but also for making life in the lab fun; Christophe for the time he spent with me discussing my project, how to increase the quality of the experiments and my presentations; Mena who showed me how to handle Arabidopsis plants and especially André who took care of the thousands of plants needed for this work; and finally all my colleagues and everyone else not mentioned by name.
I would especially like to thank Thomas Müller, who did the MS and NMR analysis of the chlorophyll a derivative, and Bilal and Florence who did the first spectrum analysis of the chlorophyll a derivative, but also for their valuable advice and encouragement during the bad. periods.
