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 (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) show the features to potentially connect cytoplasmic and extracytoplasmic ASC pools in plant cells. These proteins reside in different membranes of plant cells, like the tonoplast, plasma membrane and possibly Golgi. 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 would be impossible to be done in other ways. These methods allow thorough kinetic analyses of cyb561 proteins in vivo and ex vivo. Combination of kinetic analyses and characterization of 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.
Results achieved
A major outcome of the project was the demonstration that plant vacuoles actively participate in redox processes. Using patch-clamp electrophysiology on isolated Arabidopsis mesophyll vacuoles, we identified a tonoplast electron transport system functioning as a reversible, Asc-dependent transmembrane monodehydroascorbate (MDHA) oxidoreductase. Electron transport across the tonoplast was shown to depend on the presence of Asc as electron donor and MDHA or ferricyanide as electron acceptors on opposite membrane sides. This activity was attributed to cytochrome b561 isoform A (CYB561A), a tonoplast-localized redox protein characterized by distinct Asc binding sites on the cytoplasmic and luminal sides. The affinities for Asc of these sites were consistent with the physiological Asc concentrations in the respective compartments. Functional analyses of CYB561A-deficient mutants revealed the complete loss of trans-tonoplast electron currents and a marked accumulation of anthocyanins under high light conditions, indicating a role of CYB561A in modulating redox-dependent stress responses. These results, which had already been partially acquired at the time of the submission of the present project, were finalized in the following months and are contained in Gradogna et al., 2023 (New Phytologist 238: 1957–1971) which was accepted during the interval between the project's submission and its acceptance, which lasted more than a year. Beyond this paper, the project contributed to the broader functional characterization of the cytochrome b561 protein family, highlighting their conserved role in mediating transmembrane electron transport and supporting key physiological processes such as flower induction and antioxidant response under stress. Different Arabidopsis knock out mutants of selected members of the cyb561 family were obtained and thoroughly characterized through electrophysiology and molecular phenotyping. These studies clearly showed that different cyb561 isoforms share a similar type of control, at the molecular level, on seemingly unrelated physiological processes. The transcriptomic analysis generated a short list of candidate genes involved in cyb561 intracellular signaling. These results are ready for submission. Further studies will hopefully be developed starting from the genes identified by the transcriptomic analysis. A further key achievement of the project was the identification and functional characterization of HYPERSENSITIVE TO LOW P1 (HYP1), a previously uncharacterized CYBDOM member of the cyb561 family which contains both DOMON and cytochrome b561 domains. HYP1 was shown to act as an ascorbate-dependent metalloreductase at the plasma membrane, capable of reducing ferric and cupric substrates both in heterologous systems and in planta. Importantly, HYP1 expression is induced under phosphorus (P) deficiency in the root apical meristem, where it plays a critical role in maintaining meristem integrity and cell elongation. Loss-of-function mutants exhibited increased iron accumulation and callose deposition in the root meristem, accompanied by extensive transcriptional reprogramming. The data demonstrate that HYP1 counteracts malate-induced iron over-accumulation in the root apoplast, thereby preventing iron-dependent inhibition of root growth under low P conditions. These results were published in Maniero et al. 2024 (Nature Communications 2024, 15(1):422). Overall, the project achieved its objectives by uncovering novel ascorbate-dependent electron transport mechanisms at both the tonoplast and plasma membrane levels, and by establishing their functional relevance in plant stress adaptation. These findings provide significant advances in our understanding of plant redox biology and identify key molecular components linking redox regulation to stress responses and plant development. Project related publications Tosato E, Di Franco E, Hassan SH, Gradogna A, Lagostena L, Picco C, Sparla F, Trost P, Carpaneto A. Electron currents mediated by tonoplast cytochromes b561. Eur Biophys J. 2026 Apr;55(2):275-282. Di Franco E, Milenkovic S, Lagostena L, Meucci M, Festa M, Gradogna A, Dietrich P, Filippini A, Ceccarelli M, Carpaneto A. Plant Vacuolar and Human Endolysosomal Two-Pore Channels: Similarities and Differences. Cells. 2026 Apr 11;15(8):675. Maniero RA, Picco C, Hartmann A, Engelberger F, Gradogna A, Scholz-Starke J, Melzer M, Künze G, Carpaneto A, von Wirén N, Giehl RFH. Ferric reduction by a CYBDOM protein counteracts increased iron availability in root meristems induced by phosphorus deficiency. Nat Commun. 2024 Jan 11;15(1):422. Gradogna A, Lagostena L, Beltrami S, Tosato E, Picco C, Scholz-Starke J, Sparla F, Trost P, Carpaneto A. Tonoplast cytochrome b561 is a transmembrane ascorbate-dependent monodehydroascorbate reductase: functional characterization of electron currents in plant vacuoles. New Phytol. 2023 Jun;238(5):1957-1971. Project related oral presentations at scientific conferences: UNA Europa ECR workshop (Edinburgh) – (7/6/2022) Invited talk: Cytochromes b561: transmembrane electron wires – Given by Paolo Trost XVI Italian Federation of Life Sciences (FISV) Congress – Portici (NA), Italy (14-16/09/2022) Short talk. “The likely role of Arabidopsis thaliana cytochromes b561 in intracellular ascorbate redox homeostasis and ROS-mediated signalling” – Given by E. Tosato. Workshop on Plant Biology SIBV 2023 – Bertinoro (FC), Italy (22-24/02/2023) Elevator pitch: “The molecular phenotyping of Arabidopsis thaliana mutants suggests a role for cytochromes b561 in the regulation of intracellular ascorbate redox homeostasis and ROS-mediated signalling”. Given by E. Tosato Julius-von-Sachs Summer Symposium, Julius von Sachs Institute of the University of Wuerzburg, Wuerzburg, Germany, 20 July 2023. Invited talk: "Electron currents in plant vacuoles: functional characterization of a cytochrome b561" given by A. Carpaneto European Biophysical Societies' Association (EBSA)-2023 Congress in Stockholm, Sweden, July 31-August 4th. Selected Speaker: "Functional characterisation of electron currents in plant vacuoles: direct recordings of cytochrome b561A activity." Given by A. Carpaneto. XVI International Conference on Reactive Oxygen and Nitrogen Species in Plants (POG 2024) – Antibes Juan-les-Pins, France (29-31/05/2024) Short talk: “CYB561-A and CYB561-B are likely involved in intracellular redox homeostasis and ROS-mediated signalling” Given by E. Tosato Workshop on Plant Biology SIBV 2024 – Bertinoro (FC), Italy (21-23/02/2024) Sketch talk: “CYB561-A and CYB561-B are likely involved in intracellular ascorbate redox homeostasis and ROS-mediated signalling”. Given by E. Tosato. XXVII Congresso della Società di Biofisica Pura ed Applicata, Genova, 16-20 Giugno 2024. Selected Speaker: "Direct recordings of electron currents in plant vacuoles mediated by cytochrome b561A activity." Given by A Carpaneto.Dettagli del progetto
Responsabile scientifico: Paolo Bernardo Trost
Strutture Unibo coinvolte:
Dipartimento di Farmacia e Biotecnologie
Coordinatore:
ALMA MATER STUDIORUM - Università di Bologna(Italy)
Contributo totale di progetto: Euro (EUR) 190.013,00
Contributo totale Unibo: Euro (EUR) 97.415,00
Durata del progetto in mesi: 24
Data di inizio
05/10/2023
Data di fine:
28/02/2026
