Final Report Summary - ORG INT ISP (Organelle interactions in iron-sulphur protein biogenesis)
Project context and objectives
Iron-sulphur (Fe-S) proteins are crucial to life-sustaining processes such as respiration, photosynthesis and nitrogen fixation. These metalloproteins contain ancient but versatile co-factors, called Fe-S clusters, consisting of iron and acid-labile sulphide. Biogenesis of these simple inorganic cofactors is surprisingly complex and at least four distinct assembly pathways have been identified in the living world, namely the NIF (nitrogen fixation system), the SUF (sulphur mobilisation system), the ISC (iron-sulphur cluster assembly system) and the CIA (cytosolic iron-sulphur protein assembly) assembly pathways. Whereas the last decade was dedicated to the identification of individual components of the Fe-S protein assembly pathways, we do not know how these pathways interact in plants. In this research project, I have addressed the relationship between compartmentalised Fe-S cluster assembly machineries in plants. I have used reverse genetic approaches in the model plant Arabidopsis thaliana. The two main objectives that were proposed at the start of the project have been achieved.
Objective 1. To investigate the function of the mitochondrial ABC transporter AtATM3, in particular:
(i) its involvement in cytosolic Fe-S protein assembly, and
(ii) in chloroplast functions, and
(iii) to get clues about the substrate of AtATM3.
Objective 2. To assess whether impairment of one specific pathway affects the assembly of Fe-S proteins in other cell compartments of the plant cell (interaction map). To achieve this goal, biochemical assays have been used to monitor the assembly of Fe-S protein reporters in the mitochondria, the plastid or the cytosol, using selected mutants in each of the mitochondrial, the cytosolic and the plastid Fe-S protein assembly pathway.
Main results
Objective 1. To investigate the function of the ABC transporters of mitochondria (ATMs) in plants.
The model plant Arabidopsis thaliana has three ATM genes, namely ATM1, ATM2, and ATM3. Using a collection of insertional mutants, we showed that only ATM3 has an important function for plant growth. Additional atm3 alleles were identified among sirtinol-resistant lines (from Yunde Zhaos lab in San Diego, USA), correlating with decreased activities of aldehyde oxidases. These cytosolic enzymes convert sirtinol into an auxin analog, and depend on iron-sulphur (Fe-S) and molybdenum cofactor (Moco) as prosthetic groups. Arabidopsis atm3 mutants displayed defects in root growth, chlorophyll content, and seedling establishment. Analyses of selected metal enzymes showed that the activity of cytosolic aconitase (Fe-S) was strongly decreased across the range of atm3 alleles, whereas mitochondrial and plastid Fe-S enzymes were unaffected. Nitrate reductase activity (Moco, heme) was decreased by 50 % in the strong atm3 alleles, but catalase activity (heme) was similar to that of the wild type. These results indicate that AtATM3 is crucial for cytosolic Fe-S protein assembly, similar to its yeast and mammalian homologues, but not for mitochondrial or plastidic Fe-S proteins (Bernard et al., 2009).
Strikingly, in contrast to mutants in the yeast and mammalian orthologues, Arabidopsis atm3 mutants did not display a dramatic iron homeostasis defect and did not accumulate iron in mitochondria. Our data suggest that Arabidopsis ATM3 may transport (1) at least two distinct compounds or (2) a single compound required for both Fe-S and Moco assembly machineries in the cytosol, but not iron (Bernard et al., 2009).
Although a genetic screen was planned in the proposal (to find mutations that would suppress the chlorotic phenotype of atm3 mutants), we had to take the decision to give up this approach due to a lack of genetic information on the original mutant, and reports that such a genetic screen was already done by another research group.
However, with respect to the chloroplast function of ATM3, I was able to show that:
(i) weak atm3 mutants are also chlorotic (Bernard et al 2009);
(ii) that chlorosis is not a consequence of low activity of the Moco enzyme nitrate reductase. Spraying plants with reduced nitrogen salts did not rescue chlorosis in atm3 mutants, but it did rescue chlorotic Moco biosynthesis mutants, e.g. cnx5;
(iii) if ATM3 is present in the chloroplast envelope, it is below immunological detection levels, i.e. the majority (> 95 %) of ATM3 is associated with mitochondria. Therefore, ATM3 is likely to play an indirect role in chloroplast function.
Objective 2. To provide a map of interactions of compartmentalised Fe-S assembly pathways in plants.
Mitochondrial mutants
In an attempt to isolate T-DNA insertion lines affected for the mitochondrial Fe-S machinery, we screened specific lines obtained from stock centres by genotyping, and analysis of expression level of candidate genes. In particular, we isolated a complete knock-out of NFU4 (NiFu-like protein 4), with no apparent phenotype, suggesting a non-essential function or redundancy with NFU5. We also isolated hemizygote mutants of the essential gene NFS1 (nitrogen fixation gene 1), encoding a cysteine desulphurase, by crossing a homozygous mutant with decreased expression with a heterozygous knock-out line of the gene. In addition, we isolated a partial mutant of the scaffold ISU1 displaying a decrease in expression level.
Chloroplastic mutants
To assess whether or not assembly of cytosolic Fe-S proteins was also dependent on the plastid machinery, we analysed two different mutants known to be affected for the plastid machinery (CpNifS and nfu2). None of the three cytosolic Fe-S proteins studied here were affected in these mutants, suggesting that assembly of cytosolic Fe-S in plants does not rely on plastids.
Cytosolic mutants
To investigate the cytosolic pathway per se, and due to the essentiality of most of the genes involved in Fe-S protein assembly, we selected T-DNA insertion mutant lines with expected low expression level (T-DNA inserted in the promoter, 5' UTR (untranslated region) or 3 UTR). Candidate genes were identified according to their homology to previously characterised yeast assembly factors. Unfortunately, all T-DNA insertion mutants obtained from stock centres were either homozygous lethal or causing no disruption of the expression level. We thus decided to use down-regulated lines using RNA interference (constructed by Dr K. Bych). Surprisingly, down-regulation of AtNBP35 to around 20 % of wild-type level did not seem to affect the activity of cytosolic Fe-S proteins (aldehyde oxidase, xanthine dehydrogenase and cytosolic aconitase), indicating that NBP35 (cystolic Fe-S cluster assembly factor) might achieve a different function in plants, unless 20 % would be sufficient to fulfil its function (results to be published). This is a plausible hypothesis in the sense that AtNBP35 in the green lineage has previously been shown to work differently than its yeast homologous, i.e. without its CFD1 (cystolic Fe-S cluster assembly factor) partner (Bych et al. 2008).
During the course of this project, I have also shown that AtDRE2, one of the components of the CIA machinery, is an essential protein that binds an 2Fe-2S cluster and complements a yeast mutant (in collaboration with A. Pierik in Marburg).
Finally, we investigated a mutant in the Arabidopsis AE7 gene, isolated by Xiaofeng and co-workers in Shanghai, China, with respect to the activity of Fe-S enzymes. Whereas aldehyde oxidases and xanthine dehydrogenase were unaffected, the activity of mitochondrial and cytosolic aconitases was strongly decreased in this mutant. This observation reinforces the notion that some Fe-S assembly factors, in particular those acting downstream, may have a limited number of targets.
Potential impact
Iron-sulphur clusters are the most common use of iron in a plant, and iron-sulphur enzymes are key to photosynthesis and respiration. These two processes, located in the plastids and mitochondria, respectively, need to be coordinated to optimise plant growth, i.e. biomass. However, both photosynthesis and respiration compete for iron as a co-factor. Therefore, a thorough understanding of iron distribution, the use of iron in Fe-S proteins, and the interaction between mitochondria and chloroplasts can contribute to increase biomass, in a long term perspective. Investigating the iron content of crops is also important for iron nutrition, especially for vegetarians or people on a diet containing little red meat.
Iron-sulphur (Fe-S) proteins are crucial to life-sustaining processes such as respiration, photosynthesis and nitrogen fixation. These metalloproteins contain ancient but versatile co-factors, called Fe-S clusters, consisting of iron and acid-labile sulphide. Biogenesis of these simple inorganic cofactors is surprisingly complex and at least four distinct assembly pathways have been identified in the living world, namely the NIF (nitrogen fixation system), the SUF (sulphur mobilisation system), the ISC (iron-sulphur cluster assembly system) and the CIA (cytosolic iron-sulphur protein assembly) assembly pathways. Whereas the last decade was dedicated to the identification of individual components of the Fe-S protein assembly pathways, we do not know how these pathways interact in plants. In this research project, I have addressed the relationship between compartmentalised Fe-S cluster assembly machineries in plants. I have used reverse genetic approaches in the model plant Arabidopsis thaliana. The two main objectives that were proposed at the start of the project have been achieved.
Objective 1. To investigate the function of the mitochondrial ABC transporter AtATM3, in particular:
(i) its involvement in cytosolic Fe-S protein assembly, and
(ii) in chloroplast functions, and
(iii) to get clues about the substrate of AtATM3.
Objective 2. To assess whether impairment of one specific pathway affects the assembly of Fe-S proteins in other cell compartments of the plant cell (interaction map). To achieve this goal, biochemical assays have been used to monitor the assembly of Fe-S protein reporters in the mitochondria, the plastid or the cytosol, using selected mutants in each of the mitochondrial, the cytosolic and the plastid Fe-S protein assembly pathway.
Main results
Objective 1. To investigate the function of the ABC transporters of mitochondria (ATMs) in plants.
The model plant Arabidopsis thaliana has three ATM genes, namely ATM1, ATM2, and ATM3. Using a collection of insertional mutants, we showed that only ATM3 has an important function for plant growth. Additional atm3 alleles were identified among sirtinol-resistant lines (from Yunde Zhaos lab in San Diego, USA), correlating with decreased activities of aldehyde oxidases. These cytosolic enzymes convert sirtinol into an auxin analog, and depend on iron-sulphur (Fe-S) and molybdenum cofactor (Moco) as prosthetic groups. Arabidopsis atm3 mutants displayed defects in root growth, chlorophyll content, and seedling establishment. Analyses of selected metal enzymes showed that the activity of cytosolic aconitase (Fe-S) was strongly decreased across the range of atm3 alleles, whereas mitochondrial and plastid Fe-S enzymes were unaffected. Nitrate reductase activity (Moco, heme) was decreased by 50 % in the strong atm3 alleles, but catalase activity (heme) was similar to that of the wild type. These results indicate that AtATM3 is crucial for cytosolic Fe-S protein assembly, similar to its yeast and mammalian homologues, but not for mitochondrial or plastidic Fe-S proteins (Bernard et al., 2009).
Strikingly, in contrast to mutants in the yeast and mammalian orthologues, Arabidopsis atm3 mutants did not display a dramatic iron homeostasis defect and did not accumulate iron in mitochondria. Our data suggest that Arabidopsis ATM3 may transport (1) at least two distinct compounds or (2) a single compound required for both Fe-S and Moco assembly machineries in the cytosol, but not iron (Bernard et al., 2009).
Although a genetic screen was planned in the proposal (to find mutations that would suppress the chlorotic phenotype of atm3 mutants), we had to take the decision to give up this approach due to a lack of genetic information on the original mutant, and reports that such a genetic screen was already done by another research group.
However, with respect to the chloroplast function of ATM3, I was able to show that:
(i) weak atm3 mutants are also chlorotic (Bernard et al 2009);
(ii) that chlorosis is not a consequence of low activity of the Moco enzyme nitrate reductase. Spraying plants with reduced nitrogen salts did not rescue chlorosis in atm3 mutants, but it did rescue chlorotic Moco biosynthesis mutants, e.g. cnx5;
(iii) if ATM3 is present in the chloroplast envelope, it is below immunological detection levels, i.e. the majority (> 95 %) of ATM3 is associated with mitochondria. Therefore, ATM3 is likely to play an indirect role in chloroplast function.
Objective 2. To provide a map of interactions of compartmentalised Fe-S assembly pathways in plants.
Mitochondrial mutants
In an attempt to isolate T-DNA insertion lines affected for the mitochondrial Fe-S machinery, we screened specific lines obtained from stock centres by genotyping, and analysis of expression level of candidate genes. In particular, we isolated a complete knock-out of NFU4 (NiFu-like protein 4), with no apparent phenotype, suggesting a non-essential function or redundancy with NFU5. We also isolated hemizygote mutants of the essential gene NFS1 (nitrogen fixation gene 1), encoding a cysteine desulphurase, by crossing a homozygous mutant with decreased expression with a heterozygous knock-out line of the gene. In addition, we isolated a partial mutant of the scaffold ISU1 displaying a decrease in expression level.
Chloroplastic mutants
To assess whether or not assembly of cytosolic Fe-S proteins was also dependent on the plastid machinery, we analysed two different mutants known to be affected for the plastid machinery (CpNifS and nfu2). None of the three cytosolic Fe-S proteins studied here were affected in these mutants, suggesting that assembly of cytosolic Fe-S in plants does not rely on plastids.
Cytosolic mutants
To investigate the cytosolic pathway per se, and due to the essentiality of most of the genes involved in Fe-S protein assembly, we selected T-DNA insertion mutant lines with expected low expression level (T-DNA inserted in the promoter, 5' UTR (untranslated region) or 3 UTR). Candidate genes were identified according to their homology to previously characterised yeast assembly factors. Unfortunately, all T-DNA insertion mutants obtained from stock centres were either homozygous lethal or causing no disruption of the expression level. We thus decided to use down-regulated lines using RNA interference (constructed by Dr K. Bych). Surprisingly, down-regulation of AtNBP35 to around 20 % of wild-type level did not seem to affect the activity of cytosolic Fe-S proteins (aldehyde oxidase, xanthine dehydrogenase and cytosolic aconitase), indicating that NBP35 (cystolic Fe-S cluster assembly factor) might achieve a different function in plants, unless 20 % would be sufficient to fulfil its function (results to be published). This is a plausible hypothesis in the sense that AtNBP35 in the green lineage has previously been shown to work differently than its yeast homologous, i.e. without its CFD1 (cystolic Fe-S cluster assembly factor) partner (Bych et al. 2008).
During the course of this project, I have also shown that AtDRE2, one of the components of the CIA machinery, is an essential protein that binds an 2Fe-2S cluster and complements a yeast mutant (in collaboration with A. Pierik in Marburg).
Finally, we investigated a mutant in the Arabidopsis AE7 gene, isolated by Xiaofeng and co-workers in Shanghai, China, with respect to the activity of Fe-S enzymes. Whereas aldehyde oxidases and xanthine dehydrogenase were unaffected, the activity of mitochondrial and cytosolic aconitases was strongly decreased in this mutant. This observation reinforces the notion that some Fe-S assembly factors, in particular those acting downstream, may have a limited number of targets.
Potential impact
Iron-sulphur clusters are the most common use of iron in a plant, and iron-sulphur enzymes are key to photosynthesis and respiration. These two processes, located in the plastids and mitochondria, respectively, need to be coordinated to optimise plant growth, i.e. biomass. However, both photosynthesis and respiration compete for iron as a co-factor. Therefore, a thorough understanding of iron distribution, the use of iron in Fe-S proteins, and the interaction between mitochondria and chloroplasts can contribute to increase biomass, in a long term perspective. Investigating the iron content of crops is also important for iron nutrition, especially for vegetarians or people on a diet containing little red meat.