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The Plants, Stress & Metals team

Metabolism and stress

Published on 10 January 2019
General frame

As we explore in greater depth the interaction of plants with their environment, metabolite analysis is more than ever required for gaining insight into still not well understood stress adaptation processes. With the advent of high-resolution techniques like MS, HPLC, and NMR that permit to simultaneously analyse a large number of metabolites, conventional approaches involving elaborate sample preparation to analyse predetermined metabolites are now less systematically used. In addition, in vivo NMR technique extensively used in our work during the last years, and for which we are leader, is unique for real time non-invasive global analyses. The examples summarized below correspond to researches in which our team was leader.

Main results

Drought stress in lichen
Adaptation to successive periods of desiccation and hydration is one of the lichen's requirements for survival in harsh environments. In the dehydrated state, respiration and photosynthesis of the foliaceous lichen X. elegans is below the threshold of detection by infrared gas analysis. Following hydration, respiration recovers within seconds and photosynthesis within minutes. In order to identify metabolic processes that may contribute to restart so quickly lichen physiological activity, we analysed the metabolite profile of lichen thalli step by step during hydration/dehydration cycles, using 31P- and 13C-NMR. It appeared that the recovery of respiration was anticipated during dehydration by the accumulation of important stores of gluconate 6-P (glcn-6-P) and by the preservation of nucleotide pool, whereas glycolysis and photosynthesis intermediates like glucose 6-P and ribulose 1,5-diphosphate disappeared. The important pools of polyols present in both X. elegans photo- and mycobiont contribute to protect cells constituents like nucleotides, proteins, and membrane lipids, and to preserve the intactness of intracellular structures during desiccation. We stated that glcn-6-P accumulated due to the activation of the oxidative pentose phosphate pathway, in response to cell need for reducing power (NADPH) during the dehydration-triggered down regulation of metabolism. Glcn-6-P was metabolised instantly after hydration, supplying respiration with substrates during the recovery of glycolysis and photosynthesis pools of intermediates. This helps lichens to profit of all hydration opportunities, thus favouring its growth in the highly contrasted mountain climate. (Aubert et al. 2007).

A new effect of Light stress in plant leaves
While analysing photoinhibition processes in alpine plant leaves (Streb et al. 2008) metabolic profiling experiments utilizing 31P-NMR led us to discover that the leaves of a number of herbs and trees accumulate in the light 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (MEcDP), an intermediate of the methylerythritolphosphate (MEP) pathway of isoprenoid synthesis, during hot days. Isoprenoids form one of the largest groups of natural products including essential metabolites such as sterols, carotenoids, prenyl chains of quinones and a variety of volatile organic compounds. All isoprenoids derive from precursors synthesized by two different pathways: the cytosolic mevalonate pathway in eukaryotic cells and the chloroplastic MEP pathway which was recently discovered in bacteria by M. Rohmer, plant plastids, and parasites like Plasmodium falciparum possessing residual chloroplast structures. Enzymes involved in the MEP pathway are hence potential targets for new antibacterial or antiparasitic drugs and high value compound producing plants
In spinach leaves MEcDP accumulation typically depends on irradiance and temperature. It was the only 31P-NMR-detected MEP pathway intermediate. Remaining in chloroplasts, MEcDP is a sink for phosphate in this organelle. The accumulation of MEcDP suggested that its conversion rate into 4-hydroxy-3-methylbut-2-enyl diphosphate (HMBPP), catalyzed by (E)-4-hydroxy-3-methylbut-2-enyl diphosphate synthase (GcpE, also called HDS), is limiting under oxidative stresses like those O2 and ROS produced by photosynthesis disassemble GcpE [4Fe-4S]-cluster. Nevertheless, as isoprenoid synthesis is not inhibited in this case, these damages are supposed to be continuously repaired. Cadmium blocked the MEP pathway and reinforces MEcDP accumulation. In vitro experiments performed in the lab of Michel Rohmer (Univ. Louis Pasteur, Strasbourg) show that Cd2+ does not react directly with fully assembled GcpE, but that it interferes with its reconstitution from recombinant GcpE apoprotein and prosthetic group. Our results suggest that MEcDP accumulation in leaves originates from both GcpE hypersensitivity to oxidative environment and limitation of its repair. We proposed a model wherein GcpE turnover represents a bottleneck of the MEP pathway in plant leaves exposed to high irradiance and hot temperature (Rivasseau et al. 2009).


Scheme showing how oxidative conditions reveal the effects of inhibitory factors on GcpE turnover.

A nutrient stress: the phosphate deficiency in plants
During the last year we discovered with the help of in vivo31P-NMR that the cytoplasmic Pi homeostasis of plant cells usually assumed to be in the millimolar range corresponds only to a transient homeostasis of Pi in organelles (ca 2 d), whereas the cytosolic Pi concentration remains low (60 µM) and can fluctuate rapidly according to Pi supply and cell metabolism. This important discovery should modify our view on the Pi deficiency signalling and on the Pi exchanges in plant cells. As a matter of fact, the drop of the tiny cytosolic-Pi pool in the case of deficient Pi supply may be considered as an early endogenous signal leading to the instant activation of cell Pi starvation rescue metabolism. It can also facilitate the efflux of phosphate from the vacuole, thus contributing to attenuate the potentially dangerous effects of a sudden deficiency in the plant Pi supply. Finally, the utilisation of the Pi analogue MeP as a tool to analyse the movements of Pi in plant cells, led us to conclude that the cytosolic Pi mediates Pi as well as MeP exchanges across the tonoplast and that these movements may happen via a Pi-channel according to the electrochemical gradient between cytosol and vacuole (Pratt et al. 2009).

Resistance to irradiation and metal accumulation of micro-algae growing in the storage pools of a nuclear reactor
We study micro-algae living in cooling pools of used nuclear elements. Few eukaryotic organisms are able to live in such environments where ionizing radiation and toxic compounds is usual! The most resistant of these micro-algae, which belongs to a new species, survives after irradiation at a dose of 20,000 Gy. Although this is a eukaryotic organism, its resistance to ionizing radiation (LD50 10 kGy) is comparable to the most radio-resistant bacteria known such as Deinococcus radiodurans. It also has the ability to accumulate metallic ions. Tested for the purification of nuclear waste, it decontaminated efficiently radionuclides such as 238U, 137Cs, 60Co and 110mAg.
This microalga is the ideal candidate for a biological process of cleaning up nuclear waste. As part of the Nuclear Toxicology program of the CEA, we study the origin of its remarkable properties and we are developing a decontamination process based on this alga, in collaboration with the Laue Langevin Institute.

Development of analytical methods
As a support to our own projects as well as to other teams' projects, we developed techniques of molecular and elemental analysis. Elemental analysis using inductively coupled plasma-mass spectrometry (ICP-MS) enabled the quantification of Cd, Ag, Co, Cu, Zn, As, Fe, Mn, Mg, etc. in simple or complex matrices such as plant extracts in order to determine enzyme composition, functionality of metal carriers, or impact of metals on plants and cells (Rivasseau et al. 2009). Concerning molecular analysis, we optimized and validated a capillary electrophoresis method to determine the main organic acids (citric, malic, succinic, oxalic, formic, fumaric, and acetic) contained in different plant tissues and cells, including sycamore, arabidopsis, buttercup, and pea. A rapid and efficient separation is obtained in 100 s with mean precision values of 0.2 % for migration times enabling accurate identification and 3.4 % for quantification. Cadmium effect on pea leaves metabolism was assessed (Rivasseau et al. 2006).


Key words

Heavy metal stress, heavy metal detoxification, cadmium, caesium, RMN, ICP-MS, proteomics, metabolomics, transcriptomics, vacuole, selenium binding protein, Arabidopsis, plant cells and algae cultures, uranium, phytoremediation, MeP pathway, light stress, phosphate deficiency