Contributions of Iron to Programmed Cell Death in Plants

Iron is essential for all forms of life, but also potentially toxic. In cellular environments, FeII catalyses the Fenton reaction in presence of superoxide anion or hydrogen peroxide. As a result, highly reactive hydroxyl radicals arise that oxidise any biomolecules close by and trigger the development of programmed cell death (PCD). Phenolic secondary metabolites with oxygen and nitrogen functions, such as juglone, ()-catechin and 8-hydroxyquinoline, are competitive ligands of iron. These three secondary metabolites are variably phytotoxic and also induce PCD to a variable extent. By contrast, the plant hormone salicylic acid also is a phenolic ligand of iron. In living cells, ferric iron is available only if it is complexed by the Krebs cycle intermediate citric acid and nicotianamine. In assumptions that autoxidation of ferrous iron in the complex will induce PCD, we hypothesise that juglone will induce PCD most efficiently. Juglone is more pro-oxidative than ()-catechin. 8-Hydroxyquinoline is a strong competitive iron chelator. The three secondary metabolites have been characterised thoroughly in terms of their potential anti- and pro- oxidant activities. The aim of the proposed research is to explore if their mode of action, which is suggested by the chemical assays, also applies to the induction of PCD. Their activities will be compared to plant hormone with similar structure, salicylic acid, and to naturally occurring ligands of iron, citric acid and nicotianamine. Furthermore, several iron-protective chelators that are used in the medicinal treatments of iron-induced diseases will also be included for comparative reasons. The complex with ethylenediaminetertraacetic acid (EDTA) represents a control, where the complex is an efficient catalyst of the Fenton reaction. Protoplasts will be isolated from the leaves of field mustard (Brassica rapa), and cultured on Murashige-Skoog medium. The protoplast cultures will be treated with the mentioned metabolites, either in free from or in complex with iron. Various microscopic and biochemical tests will be performed to determine specific effects that are characteristic for PCD, amongst others, cell death by Evans blue and FDA-PI (fluorescein diacetate-propidium iodide), lipid peroxidation by malonyldialdehyde (MDA) concentrations, index of oxidative damage; ascorbate/dehydroascorbate ratio, changes in redox homoeostasis; and DNA damage (DAPI for nuclear DNA condensation, DNAse activity, DNA laddering for detection of double strand breaks).

Iron is essential for all forms of life, but also potentially toxic for the cell in excess concentration. In cellular environments, FeII catalyses the Fenton reaction in the presence of superoxide anion or hydrogen peroxide. As a result, highly reactive hydroxyl radicals arise that oxidise any biomolecules close by and trigger the development of programmed cell death (PCD). Phenolic secondary metabolites with oxygen and nitrogen functions, such as juglone, ()-catechin and 8-hydroxyquinoline, are competitive ligands of iron. These three secondary metabolites are variably phytotoxic and also induce cell death to a variable extent. The aim of the proposed research was to explore if their mode of action, which was suggested by the chemical assays, also applies to the induction of PCD. The effects of these allelochemical were compared to naturally occurring ligands of iron, and citric acid. The complex with ethylenediaminetetraacetic acid (EDTA) represents a control, where the complex is an efficient catalyst of the Fenton reaction. In assumptions that autoxidation of ferrous iron in the complex will induce PCD, we hypothesise previously that juglone will induce PCD most efficiently. Indeed, iron-juglone was found to be highly toxic as it inhibited plant growth very severely and induced sudden death of roots and plants started exhibiting wilting of shoots very soon after 10 M Fe-juglone supply. In the low concentration range 2-5 M, juglone was found to be better in Fe supply compared to 10:10 M Fe in complex with salicylic acid, 8-hydroxyquinoline and ()-catechin and it did not inhibit plant growth. These results suggest that juglone in the low concentration range (2-5 M) is better in the Fe-supply to plants but higher concentration is highly lethal for plant growth. The iron complexes with ()-catechin and 8-hydroxzyquinoline and salicylic acid (10:10 M) induces Fe-deficiency-like effect and plant leaves exhibited symptoms (interveinal chlorosis) close to Fe-deficient plant leaves. These plants also exhibited non-autolytic PCD similar to Fe-deprived plants as indicated by enhanced levels of reactive oxygen species, activation of DNase, alkaline protease and caspase-3-like activities in affected leaves of Brassica napus plants. Protoplasts isolated from young leaves of these plants exhibited nuclear condensation. These observations suggest that Fe in complex with salicylic acid, 8-hydroxyquinoline and ()-catechin exhibited almost similar behaviour in induction of PCD by inducing Fe-deficiency-like-effect in the Brassica napus plants. These observations were further substantiated by enhanced activity of root ferric-chelate reductase, decreased activity of catalase (a major heme-Fe enzyme representing the functional Fe status of plants) and decreased concentrations of chlorophyll and carotenoids in the leaves. Fe-citrate, Fe-EDTA and Fe-deferoxamine (10:10 M) were efficiently supplied iron to the plants and did not induce any toxic effects. In conclusion Fe in complex with phenolic aromatic ligands such salicylate, 8-hydroxyquinoline, and ()-catechin do not release Fe efficiently from their complexes in planta for their functional activity. Therefore, these plants exhibited Fe-deficiency-like effect and consequently induced PCD due to overproduction and accumulation of ROS in the cellular environment.