Finessing, Extending and Developing an Overview of the Regulation of Ascorbate in plants (FEDORA)

Ascorbate is a key metabolite for all plants, from eukaryotic algae to angiosperms. It is the most abundant water-soluble antioxidant with major roles in photosynthesis, transmembrane electron transport, cell division, growth and tolerance of biotic and abiotic stress. It performs these diverse roles by serving as a major scavenger of active oxygen species generated as by-products of photosynthesis, in the dissipation of excess photonic energy through the water-water cycle and as a co-factor in the xanthophyll cycle. It serves as a cofactor for dioxygenases, which are active in the synthesis of the phytohormones, ethylene, abscisic acid and gibberellins and in the generation of hydroxyproline, important for decorating small signalling peptides like CLAVATA3 and arabinogalactan-proteins (AGPs), which are ubiquitous cell surface proteoglycans proposed to play essential roles in plant growth and development, including cell expansion, cell division, reproductive development and somatic embryogenesis. Consequently the regulation of ascorbate levels is key to a large number of physiological processes in the growth and development of all plants.

There is a vast body of literature describing the processes that influence ascorbate levels, but remarkably few papers describe the mechanisms of regulation, particularly at the molecular level. Needless to say, this lack of understanding of mechanism has limited our ability to engineer processes dependent on ascorbate through biotechnology or plant breeding for crop improvement. This limitation extends even to efforts to enhance ascorbate levels in fresh fruit and vegetables, despite the alarming reports of recent increases in the incidence of scurvy resulting from ascorbate/vitamin C deficiency. For example, in the UK between 2009 and 2014, hospital admissions related to scurvy went up by 27% due to malnutrition and obesity related to over-consumption of junk food diets.

All that was changed by the publication of two landmark papers, one by Laing et al., in The Plant Cell in 2015 demonstrating a unique negative feedback regulatory mechanism controlling the translation of GDP-L-galactose phosphorylase (GGP) an enzyme active in the Smirnoff-Wheeler pathway for ascorbate synthesis, and the other by Fenech et al., in Plant Physiology (2021) showing that GGP activity is the only significant controlling step determining ascorbate levels in plant tissues and shows almost linear control of flux through the Smirnoff-Wheeler pathway.

The aim of this application is to integrate our understanding of the regulation of ascorbate in plant cells. We aim to define the molecular mechanism for the negative feedback regulation of GGP translation and to understand how translational control of GGP is set within the boundaries defined by transcriptional control. We propose to understand how the control of GGP activity impacts flux to ascorbate. We propose to integrate our investigations of the mechanisms of regulation by studying primarily two plant species, Arabidopsis and tomato, to develop generic understanding of mechanisms for plants that could be broadly applicable. We propose to extend our understanding of the mechanism of negative feedback regulation of GGP to green algae through collaboration. To understand the physiological roles of ascorbate in different plant species we will investigate the phenotypic consequences of a series of GGP mutants with mis-regulated ascorbate levels, and determine whether similar or different phenotypic effects are observed in Arabidopsis.

FEDORA aims to integrate understanding of the regulation of ascorbate in plants.
1) We will define the molecular mechanism of negative feedback regulation of GGP translation using 5'uORF:luciferase reporters to identify the signal in plant cell and algal cultures.
2) We will use wheat germ in vitro translation to investigate the mechanisms of uORF translation and ribosome stalling that impair translation of GGP using Ribo-seq and modified ChIP to define the association between the uORF peptide and the stalled ribosome(s), leading to structural (low) resolution of the complex.
3) We will investigate how translational control of GGP is set within the boundaries defined by transcriptional control of GGP expression, by comparing proGGP:luciferase reporters with and without mutated uORFs in tomato and Arabidopsis.
4) We will use Arabidopsis lines expressing the proGGP muORF:luciferase reporters to establish a genetic screen for mutations affecting transcriptional regulators of GGP in Arabidopsis.
5) We will test whether transcriptional control by regulators already identified in tomato also operates in Arabidopsis, using T-DNA insertion mutants. We will use genome editing in tomato to develop required mutations in MoneyMaker genetic background.
6) We will investigate how the control of GGP activity impacts flux to ascorbate using targeted MRM analysis by LC-QQQ MS/MS.
7) We will investigate the physiological roles of ascorbate in different plant species by defining the phenotypic consequences of a series of GGP alleles with mis-regulated ascorbate levels, measuring ascorbate and glutathione, leaf number, leaf shape, flowering time, flower number, floral morphology/organ fusion, root biomass, fruit set, fruit weight, shelf life and resistance to necrotrophic pathogens,
8) We will determine whether similar or different phenotypes are observed in Arabidopsis by creating muORFAtGGP1/VTC2 and muORFAtGGP2/VTC5 double mutants by genome editing of Arabidopsis.