Ascorbate (ASC) fulfills many different roles in plants by acting as a versatile electron donor for a variety of enzymes, metals, radicals and other oxidized cellular components. Plants completely unable to synthesize ASC do not survive, while mutants defective in single steps of ASC biosynthesis contain residual levels of ASC and show pleiotropic effects. For instance, ASC-deficient mutants are delayed in flowering and precursors of ASC biosynthesis reverse the effect, indicating that ASC controls the timing of flower induction, though its mechanisms of action are unknown. Being an antioxidant, the role of ASC depends, besides its concentration, on its redox state and hence on the ASC/monodehydroascorbate (MDHA) ratio. The ASC/MDHA ratio results from the combined action of enzymatic and non-enzymatic reactions that use ASC as electron donor, and those that regenerate ASC from its oxidized forms (e.g. MDHA and dehydroascorbate reductases). Plant cells contain neutral compartments like the cytosol where both ASC-utilizing and ASC-regenerating enzymes are present, and acidic compartments like vacuole and apoplast where ASC and ASC-consuming reactions occur in the absence of ASC-regenerating systems. A large neglected family of plant membrane proteins related to cytochrome b561 (cyb561) and residing in different membranes like tonoplast, plasma membrane and possibly Golgi, have the features to connect cytoplasmic and extracytoplasmic ASC pools in plant cells. Our preliminar results show that knock out mutants of two members of the cyb561 family (the only ones studied so far) have symptoms that recall those of mutants of ASC metabolism, including the different timing of flower induction. A major goal of the TASCO project is to understand to which extent cyb561-related proteins affect ASC homeostasis, thereby explaining how flowering induction and other physiological process may be modulated by ASC-mediated trans-membrane electron transport in mesophyll cells. The TASCO project takes advantage of the electrophysiological approaches that we recently set up for the characterization of cyb561 proteins of the plasma membrane and tonoplast, which woud be impossible in other ways. We will measure Km for ASC and capability to reduce MDHA, or transition metals as alternative electron acceptors. The role of extracytoplasmic domains will also be analyzed. This thorough kinetic analysis will be interdisciplinary connected to the phenotypic analysis of mutants that is aimed in characterizing ASC homestasis in different conditions and subcellular compartments. Combination of kinetic analysis and chacterization of the ASC system in mutants with ASC-related phenotypes will help understanding the role of cyb561 proteins on ASC homeostasis and the pleiotropic effects of its perturbation. In particular, a compared transcriptomic analysis of delayed flowering mutants will set the basis for a more general understanding of the redox control on flower induction.
Trost, P. (In stampa/Attività in corso). The role of trans-membrane electron Trasport in ASCorbate hOmeostasis and pleiotropic effects of ascorbate in plants (TASCO).
The role of trans-membrane electron Trasport in ASCorbate hOmeostasis and pleiotropic effects of ascorbate in plants (TASCO)
paolo trost
In corso di stampa
Abstract
Ascorbate (ASC) fulfills many different roles in plants by acting as a versatile electron donor for a variety of enzymes, metals, radicals and other oxidized cellular components. Plants completely unable to synthesize ASC do not survive, while mutants defective in single steps of ASC biosynthesis contain residual levels of ASC and show pleiotropic effects. For instance, ASC-deficient mutants are delayed in flowering and precursors of ASC biosynthesis reverse the effect, indicating that ASC controls the timing of flower induction, though its mechanisms of action are unknown. Being an antioxidant, the role of ASC depends, besides its concentration, on its redox state and hence on the ASC/monodehydroascorbate (MDHA) ratio. The ASC/MDHA ratio results from the combined action of enzymatic and non-enzymatic reactions that use ASC as electron donor, and those that regenerate ASC from its oxidized forms (e.g. MDHA and dehydroascorbate reductases). Plant cells contain neutral compartments like the cytosol where both ASC-utilizing and ASC-regenerating enzymes are present, and acidic compartments like vacuole and apoplast where ASC and ASC-consuming reactions occur in the absence of ASC-regenerating systems. A large neglected family of plant membrane proteins related to cytochrome b561 (cyb561) and residing in different membranes like tonoplast, plasma membrane and possibly Golgi, have the features to connect cytoplasmic and extracytoplasmic ASC pools in plant cells. Our preliminar results show that knock out mutants of two members of the cyb561 family (the only ones studied so far) have symptoms that recall those of mutants of ASC metabolism, including the different timing of flower induction. A major goal of the TASCO project is to understand to which extent cyb561-related proteins affect ASC homeostasis, thereby explaining how flowering induction and other physiological process may be modulated by ASC-mediated trans-membrane electron transport in mesophyll cells. The TASCO project takes advantage of the electrophysiological approaches that we recently set up for the characterization of cyb561 proteins of the plasma membrane and tonoplast, which woud be impossible in other ways. We will measure Km for ASC and capability to reduce MDHA, or transition metals as alternative electron acceptors. The role of extracytoplasmic domains will also be analyzed. This thorough kinetic analysis will be interdisciplinary connected to the phenotypic analysis of mutants that is aimed in characterizing ASC homestasis in different conditions and subcellular compartments. Combination of kinetic analysis and chacterization of the ASC system in mutants with ASC-related phenotypes will help understanding the role of cyb561 proteins on ASC homeostasis and the pleiotropic effects of its perturbation. In particular, a compared transcriptomic analysis of delayed flowering mutants will set the basis for a more general understanding of the redox control on flower induction.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
