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Signaling, regulation and metabolic interactions

Team MetaboActions / Michael Hodges

 

Improvement of crop potential requires the identification of specific parts of metabolism, which can be manipulated to achieve better outputs. When nutrient supply is such that existing yield potential is reached, the only way to increase production is to improve the efficiency by which nutrients are taken up and used. Metabolism may be manipulated either to achieve more carbon assimilation (per unit of nitrogen) at a lower energetic cost or to increase the biosynthesis of energy compounds (for example NAD) and/or the capacity of nutrient use. An understanding of the basic processes of metabolic pathways and how they relate to plant biomass and respond to environmental constraints is of importance to guide plant breeding and to drive genetic engineering.

 

The MetaboActions team has 3 current major research interests:

  • Phosphoregulation of primary metabolism proteins mainly centered on selected enzymes of the photorespiratory cycle.
  • NAD biosynthesis and recycling.
  • C3-plant acclimation to elevated CO2 .


An over-simplified scheme showing links between the Calvin cycle, glycolysis, the Krebs cycle, photorespiration, GS/GOGAT cycle and NAD. Note the involvement of 4 sub-cellular compartments. The 4 phosphorylated photorespiratory enzymes studied and the first enzyme of the NAD biosynthesis pathway are shown

 

Phosphoregulation of photorespiratory enzymes

Photorespiration begins when RuBisCO assimilates O2 instead of CO2 to produce “toxic” 2-phosphoglycolate (2PG) and “useful” 3-phosphoglycerate (3PGA). The photorespiratory cycle metabolizes 2PG to form 3PGA but in doing so it removes assimilated carbon and nitrogen to produce CO2 and ammonium while consuming energy. Deemed a wasteful process, photorespiration has been a target to improve photosynthesis and increase biomass. To date, the core photorespiratory genes and enzymes have been identified and characterized but little is known about how the photorespiratory cycle and its interactions with neighboring metabolic pathways are regulated.

The recent development of phosphoproteomics has led to a wealth of phosphopeptide data becoming available. So far, this has suggested that all but one of the 8 core photorespiratory enzymes can be phosphorylated. We are currently trying to decipher the function and role of phosphorylation sites associated with Arabidopsis thaliana phosphoglycolate phosphatase (PGLP1), glycolate oxidase (GOX1 & GOX2), serine hydroxymethyltransferase (SHMT1) and hydroxypyruvate reductase (HPR1).

To attain our goals we are following two strategies: The analysis of phosphorylation site mutated recombinant proteins and the functional complementation of mutant lines with synthetic genes to produce phosphorylation-mimic or non-phosphorylatable photorespiratory enzymes. This work is financed by the REGUL3P ANR project. .

We are also still involved in two minor phosphoregulation projects involving 3-PGA metabolism and nitrate-sensing by studying Ser82 phosphorylation of the glycolytic 3PGA mutase and Ser381 and Ser807 phosphorylations of the nitrate sensor NLP7, respectively.

 

NAD biosynthesis and recycling

We are also interested in understanding the interactions between NAD and plant primary metabolisms. Despite the important role of NAD in plant metabolism and stress signalling, little is known about NAD synthesis, except for the identification of at least two possible pathways. To test the importance of NAD in plant metabolism, we have identified and characterized several key Arabidopsis thaliana NAD biosynthesis/recycling genes and proteins including L-aspartate oxidase, quinolinate phosphoribosyltransferase and  nicotinate/ nicotinamide mononucleotide adenyl transferase (N(a)MNAT.

A better understanding of the biochemical properties of recombinant L-aspartate oxidase and N(a)MNAT has provided new knowledge of how NAD biosynthesis might be regulated by these key enzymes. In parallel, the importance of these genes on NAD homeostasis, pathogen defence, abiotic stress resistance, growth and development are investigated in T-DNA insertion mutants and overexpressing plants. In this way a correlation was observed between plant NAD content and aspartate oxidase expression and activity.


Arabidopsis L-apartate oxidase mutant and overexpressor (OE) rosettes compared to WT (Col 0)

The industrial value of this work has been protected (De Bont & Gakière, EU & US patent application) and an IDEX Prematuration grant from the University Paris-Saclay (EnergyCrop) has allowed us to begin testing the overexpression of Arabidopsis L-aspartate oxidase in tomato, colza            and rice.

 

C3 plant acclimation to climate change elevated CO2 levels

Crop yields must increase by over 70% in the next 30 years to sustain human requirements and this must be attained without any detrimental effects on nutritional quality. Such a challenge is even more ambitious when taking into account objectives to limit N-fertilizers in order to reduce costs and environmental damage.

A current major target to increase yield is to improve photosynthetic CO2 assimilation but such aims could be restricted by C3-plant acclimation to future eCO2 levels. For optimal plant growth and development, it is essential to obtain a simultaneous improvement of both C and N utilization efficiencies to maintain C/N balance. Acclimation to eCO2 leads to decreased stomatal conductance, a reduction in photosynthetic gene expression leading to less RuBisCO protein, and a lower N-content that affects seed quality.

It is now urgent to understand better how C3 plants adapt/acclimate to CO2 climate change levels in terms of their primary metabolism and to what extent epigenetic and transcriptional regulations play a role.

In the context of a new major research project we hope to turn our attention to the impact of predicted climate change induced elevated CO2 (eCO2) levels on photosynthesis and N-metabolism in the context of C3-plant acclimation that limits the beneficial effects of eCO2 on yield and N-content. To date, we have compared several Arabidopsis mutants affected in altered stomatal movement under eCO2. Preliminary data suggest that the observed better growth of several mutants under eCO2 compared to WT plants is not necessarily correlated to an improved photosynthetic activity.

MetaboActions is also involved in an INRA starter project called COSTOMAP to study the role of MAPK12 controlled stomatal movements in the context of eCO2. While, the metabolism-metabolome facility (PMM) is a partner of an EIG Concert Japan project (IRUEC) which aims to improve resource use efficiency in cereals, under climate change. In this project wheat and rice cultivars are challenged by high temperature and CO2 levels under different N fertilization conditions.

 

 Previous research highlights include:

  • Identification of C reallocation when the photorespiratory cycle is blocked leading to reduced photosynthetic activity and the biosynthesis of less RuBisCO protein to maintain C/N balance; Dellero et al (2015) Plant J & (2016) J Exp Bot. This involved the development of 13C tracing methods coupled to NMR analysis that is currently being used to unravel the biosynthetic pathway of a terpene for the perfume industry (SPS2020 Project PISTILL).
  • Evidence showing the crucial role of SnRK1 activation/phosphorylation by SnAK kinases in plant development; Guerinier et al (2013), Glab et al (2017) Plant J.
  • NAD energy metabolism acts as an integral regulator of multiple defense layers; Petriacq et al (2016) Plant Physiol. Increased NAD levels improve plant biomass and seed yield (De Bont & Gakière Patent).

 

Major approaches used:

  • Reverse genetics: Production and selection of genetically-modified plants (insertion mutants, artificial microRNA, overexpressors, complemented lines with mutated genes).
  • Recombinant proteins & enzymology: Production and purification of tagged-recombinant proteins (wild-type or modified by site-directed mutagenesis) and the analysis of their kinetic properties. 
  • Phenotyping & plant physiology: Analyses including metabolites (mainly primary metabolism & redox) by GC-MS, LC-MS/MS, HPLC, stable-isotope labeling (13C,15N) coupled to NMR, H+-NMR, gas exchange measurements (CO2 and H2O), enzymatic activities, and gene expression analyses (qPCR, RNAseq).
  • Proteomics and phosphoproteomics (in collaboration with M. Zivy (PAPPSO facility)).
  • Translational biology from plant models to crop plants.

 

The Metabolism-Metabolome facility

The PMM  is devoted to providing adapted analytical services. A major goal is to offer expertise to analyze plant metabolites by developing isotopic and metabolomics protocols to allow metabolic phenotyping of plant lines. In this way, PMM helps to characterize the consequences of abiotic/biotic stresses and specific mutations, and the determination of carbon, nitrogen and water use efficiencies using the adequate technologies. These include: Semi-targeted and targeted metabolite profiling by GC-MS and NMR; isotope analyses on total organic matter (EA-IRMS), gases (GC-IRMS) and extracts (LC-IRMS); analyses of cofactors (by LC-MS), amino acids (by HPLC) and untargeted metabolomics (combining LC-MS/MS and GC-MS). PMM has contributed to research topics including: water stress and C/N metabolisms in Medicago, cytochrome c oxidase Arabidopsis mutants, CO2 effects on C metabolism in wheat flag leaves, sugar accumulation in melon, natural 13C distribution in oil palm, metabolite profiling of maize inbred lines inoculated with N-fixing bacteria, MAP kinase defense responses and salicylic acid (SA) and C-reallocation in photorespiratoty mutants and enzyme reaction mechanism.

PMM is currently involved in several National and International projects. Ongoing projects include:

  • CYTOPHENO (ANR project, F Budard, IJPB INRA Versailles).
  • AMAIZING & Project Nitrogen Use Efficiency (“Investissement d'avenir”, B.Hirel, IJPB INRA Versailles).
  • PISTILL (SPS LABEX project, A. Boualem, IPS2).
  • Orchidomics (EU project, MA Sellosse, MNHN Paris).
  • Water stress tolerance (FSOV project, JC Deswartes, Arvalis).

In parallel, PMM is developing mycotoxin detection protocols within the frame of the FUSAKILL project (M. Dufresne, IPS2).

The recent acquisition of a LC-MS/MS will allow PMM to detect molecules that are difficult to measure by GC-MS and LC-MS. It will also open up the possibility to undertake fluxomics analyses after isotopic-labeling. Non-targeted metabolomics is being developed including secondary metabolites that interest the perfume industry (PISTILL & FRAGRANCE, A. Boualem, IPS2), and the rubber industry (CIRAD Montpellier). The platform is also committed to making all data accessible via a public repository (Metabolights) and it is developing new tools to enhance the presentation of results and analytic procedures.