The development of a sustainable agriculture and the use of plants for biomass energy need biochemical engineering to optimize plant metabolism.
This is currently impeded by our poor understanding of critical metabolic steps that control complex plant metabolic networks and by the lack of an integrative and dynamic view of plant metabolism.
It is a prerequisite to identify the metabolic bottlenecks that require improvement. Photosynthetic carbon assimilation is not a sufficient base from which to calculate growth, it is also necessary to consider other metabolic pathways including photorespiration, respiration, nitrogen assimilation, amino acid synthesis, NAD biosynthesis.
These processes interact within leaf cells, thus allowing the assimilation of both carbon and nitrogen but also leading to the liberation of both CO2 and NH3 that must be re-assimilated at a certain energetic cost or lost to the atmosphere.
A simplified scheme showing interactions between primary metabolic plant functions and highlighting C, N inputs and C, N outputs.
Terrestrial life on Earth relies on plant photosynthesis to produce oxygen and to assimilate CO2 into organic matter. The most abundant enzyme in plant leaves is the ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) that produces building blocks to make organic molecules within the plant, via its carboxylase activity. However, RuBisCO has also an oxygenase activity that produces 2-phosphoglycolate (2PG) and 3-phosphoglycerate (3PGA). The photorespiratory pathway (or C2-cycle) allows recycling of 2PG carbon molecules to produce 3PGA (that can be used for biosynthetic processes). This pathway has a cost since it consumes energy and reducing power and it liberates assimilated carbon and nitrogen as CO2 and ammonia. Therefore, the photorespiratory cycle has been described as “wasteful” to plant productivity, especially in C3-plants, and therefore it is believed to be a good target to manipulate with respect to improving plant yield. Photorespiration requires eight core photorespiratory enzymes distributed between chloroplasts, peroxisomes, mitochondria and the cytosol. The photorespiratory cycle interacts with several plant metabolic pathways and functions including photosynthesis, amino acid and N metabolism, respiration and C1 metabolism. Photorespiration is also linked to cell redox homeostasis due to large amounts of H2O2 produced in peroxisomes and NADH generated in mitochondria. There are also interactions with other C metabolism pathways since released CO2 can be re-assimilated, and the 3PGA generated at the end of the cycle can be used for biosynthetic processes. To co-ordinate these multiple interactions, plants must have evolved a complex network of regulatory processes. However, the regulation of the photorespiratory cycle alone is poorly understood while even less is known about how its interactions with other plant metabolic pathways and functions are coordinated.
A simplified scheme showing the compartmentalization of the photorespiratory cycle and interactions with other primary metabolic processes
Our major aims are to obtain a better understanding of the interactions between primary metabolic pathways, to highlight limiting steps, and new regulatory steps that allow plants to modify and adapt their metabolisms to fluctuating environmental conditions.
Such information will be useful to identify targets to improve plant performance (nutrient use efficiency/yield/biomass) and fitness and the data can be used to construct metabolic networks to predict plant metabolic responses to changing conditions.
Two principal research areas are developed :
- Signaling and regulation – to understand the role of regulatory proteins and metabolites on plant primary metabolism and growth and to discover new protein phosphorylation-associated regulations.
- Metabolic interactions and bypasses – to understand the interactions between photosynthesis, photorespiration, respiration, N-assimilation, amino acid biosynthesis and NADH synthesis.
To attain our goals we combine biochemical, physiological, and “omics” analyses (including phosphoproteomics, metabolomics), as well as cell and molecular biology. Purified recombinant proteins, selected mutant lines and stable isotope labeling are routinely used. Much of our work focuses on Arabidopsis thaliana but we hope to transfer interesting phenotypes to produce new crop prototypes.
Signaling and regulation
REGULATION OF PHOTORESPIRATION BY PROTEIN PHOSPHORYLATION
To date, little is known about how the photorespiratory cycle is regulated. Our previous work and information retrieved from public databases show that many photorespiratory enzymes can be phosphorylated. The reversible covalent addition of a phosphate group to a specific amino acid residue allows the modulation of protein function such as activity, sub-cellular localization, capacity to interact with other proteins, and stability. We aim to better understand the role of protein phosphorylation in the control of photorespiration and its impact on plant metabolism and growth. This work is financed by a 4-year ANR-funded project (Regul3P) in which complementary approaches including phosphoproteomics, recombinant proteins, biochemistry, targeted (LC-MS, HPLC) and non-targeted (GC- MS) metabolite analyses, gas exchange measurements and reverse genetics will be used.
The Regul3P project has the following major aims :
- To monitor the dynamics in photorespiratory enzyme phosphorylation state as a function of leaf photorespiratory activity.
- To understand the effect of phosphorylation on photorespiratory enzyme activity and kinetic properties.
- To evaluate the impact of photorespiratory enzyme phosphorylation/non-phosphorylation on plant physiology and metabolism.
- To identify protein kinases responsible for photorespiratory enzyme phosphorylation in peroxisomes.
Ongoing work and results
We are currently investigating the phosphorylation of several photorespiratory enzymes (GO, SHMT1 and hydroxypyruvate dehydrogenase (HPR)) as well as two enzymes that metabolize 3-PGA (3-phosphoglycerate dehydrogenase (PGDH) and phosphoglycerate mutase (PGAM)) and the nitrate-signaling protein NLP7 (in collaboration with the team of Ann Krapp at the IJPB INRA Versailles.
We are also currently finishing work on a project investigating the regulation of SnRK1 protein kinases by the two upstream SnAK kinases.
- Recently we showed that SnRK1, through inhibition of KRP6 biological function by phosphorylation, appears to play a cardinal function in the control of cell proliferation in Arabidopsis plants (Guérinier et al 2013 Plant J).
To investigate the regulation of photorespiratory (and respiratory) enzymes by protein phosphorylation, phosphoproteomics analyses of Arabidopsis leaves placed under light or dark conditions and at different CO2 and O2 concentrations to modulate photorespiratory activities have been carried out.
- This initially led to the identification of 2420 phosphopeptides (of 1552 proteins), while 264 phosphopeptides (203 proteins) showed a significant change in level between treatments with 22 phosphoproteins associated with “Metabolism”.
- A single respiratory enzyme phosphopeptide was detected in our analysis, while phosphopeptides were found for four different photorespiratory enzymes of which glycolate oxidase (GO) and serine hydroxymethyl transferase (SHMT1) exhibited photorespiratory-dependent changes in phosphorylation level.
Selected phosphoproteomics data concerning RuBisCO activase, protein translational machinery, chloroplast movement proteins and cellulose biosynthesis have been published recently (Boex-Fontevieille et al Plos One 2013 Plant Biol, Plant Mol Biol Rep, J Exp Bot 2014).
NAD(H) AS A SIGNAL
NAD has recently been shown to be involved in several signalling pathways associated with stress tolerance and/or defense responses. The mechanisms by which NAD influences plant gene regulation, metabolism and physiology are not clear and we aim to better understand the role of NAD as a signal.
NAD metabolism has a major role in many plant pathways and responses to the environment that involve different sub-cellular compartments
Ongoing work and results
We are using Arabidopsis plants with deregulated NAD biosynthesis and recycling genes to analyze the signalling role of NAD in the regulation of cell function.
Previously, Arabidopsis thaliana lines over-expressing E. coli NadC, encoding the NAD biosynthesis enzyme quinolinate phosphoribosyl transferase (QPT) were found to accumulate NAD when given quinolinate. These lines were used as an inducible system to determine the consequences of increased leaf NAD content on gene expression. This approach has already shown the importance of NAD in plant-pathogen signaling.
- Large-scale transcriptomic analyses indicated an NAD-promoted induction of pathogen-related genes such as the salicylic acid (SA)-responsive defense marker PR1. Comparison with transcriptomic databases showed that gene expression under high NAD content was similar to that obtained under biotic stress, eliciting conditions or SA treatment.
- Upon inoculation with the avirulent strain of Pseudomonas syringae pv. tomato Pst-AvrRpm1, the NadC lines showed enhanced resistance to bacterial infection and exhibited an ICS1-dependent build-up of both conjugated and free SA pools. We conclude that a higher NAD content is beneficial to plant immunity by stimulating SA-dependent signalling and pathogen resistance (Guérard et al 2011 Plant Physiol Biochem ; Pétriacq et al 2012 Plant J, Plant Signal Behav).
Metabolic interactions and bypasses
THE INTERACTION OF PHOTORESPIRATION WITH PLANT PRIMARY METABOLISM
Photorespiratory activity is modified 1) by altering the composition of air with respect to CO2 and/or O2 levels and 2) in plant lines with altered photorespiratory enzyme activities and an extensive picture of leaf metabolism/physiology is carried out to provide an integrated view of metabolic pathway interactions. Typical analyses include metabolite profiling by GC-MS, quantitative amino acid analyses by HPLC, flux analyses using 13C/15N/2H isotope-labeling coupled to NMR/IRMS/LC-MS analyses, measurement of energy metabolites (ATP/NAD/NADP etc), enzyme activities, gas-exchange and chlorophyll fluorescence. In the future we will study the effect of changing environmental cues (biotic and abiotic) on plant primary metabolism and metabolic flux in wild-type and selected mutant lines.
Ongoing work and results
We are currently studying GO and glutamate/glyoxylate aminotransgferase (GGT1) mutant lines to analyze how reduced photorespiratory fluxes impact primary metabolism and plant growth. In collaboration with the team of Mathilde Fagard at the IJPB, INRA Versailles we are interested also in the role of GO in plant pathogen defense.
The combined use of stable isotope labeling coupled to gas exchange measurements, IRMS and NMR analyses has led our team to publish several original results and conclusions concerning interactions between respiration, photorespiration and N-assimilation.
- In the light the TCA (or Krebs) ‘‘cycle’’ operates in both the reverse and forward directions to produce fumarate and 2-oxoglutarate (2OG), respectively (Tcherkez et al 2008 Plant Physiol).
- Day respiratory metabolism is enhanced under high photorespiratory (low CO2) conditions (Tcherkez et al 2010 Proc Natl Acad Sci USA).
- The remobilization of night-stored C-reserves plays a significant role in providing 2OG for Glu synthesis in illuminated rapeseed leaves (Gauthier et al 2010 New Phytol).
- Arabidopsis leaves with low 2OG production capacities exhibit a stimulated Lys metabolism bypass pathway to generate 2OG for Glu metabolism (Boex-Fontvieille et al 2013 New Phytol).
We have also studied the reaction mechanisms of two key enzymes involved in photorespiration (GO) and ammonium assimilation (glutamine synthetase ; GS) using recombinant proteins and stable isotopes. The analysis of the isotopic effects on their kinetic parameters has allowed a better understanding of the transition steps of their overall enzymatic reaction.
- For the GO of Arabidopsis and Maize, the measured isotope effects support a hydride transfer mechanism and indicate glycolate deprotonation to be only partially rate-limiting (Dellero et al 2015 J Biol Chem).
NAD AND PLANT METABOLISM
We are also interested in understanding the interaction between NAD and plant primary metabolisms. Little is known about NAD synthesis in plants, except the identification of at least two possible pathways. The manipulation of Arabidospsis thaliana NAD biosynthetic enzymes is being used to constitutively deregulate NAD production and to investigate changes in plant metabolism and growth.
Ongoing work and results
Present work aims to clarify (1) the importance of NAD synthesis and recycling pathways, and (2) the influence of NAD content on plant function and growth. Previously we have shown that NAD level impacts plant metabolism and growth by analyzing selected Arabidopsis mutants and carrying out metabolite profiling, enzyme activity analyses and measuring plant development. To date, it has been found that :
- NAD deregulation modifies the levels of several metabolites including aspartate-derived amino acids and NAD-derived nicotinic acid and NAD has a critical role in C/N interactions by affecting N assimilation under photorespiratory conditions.