FolEau (Functional ecoGenomics of water transport in the leaf under fluctuating water regime)
Leader : Jean Stéphane VENISSE
We want to contribute to solving the problems posed by climate change to forest crop production. It should be noted that ligno-cellulosic production is the main economic income from forestry operations. This is also the case for poplar, which is our model tree and for which the selection of registered varieties is based on criteria that do not currently include water saving in the tree. In addition, early selection, whose main objective is to identify the most promising genotypes at a juvenile stage, requires accurate information on key gene families. Our work on the study of the genes involved in water movements in the leaf helps to meet this demand.
Some basics to understand
In the ecosystem, photosynthesis, which consumes CO2 and water, produces the sugars essential to all life on Earth. In this system, the tree constitutes a remarkable hydraulic interface between soil and atmosphere: it uses the energy released by the passage between water-liquid and water-steam (perspiration) to maintain the water flow. For example, a 15-year-old poplar tree needs to evaporate 250 litres of water per day in summer for its growth. Maximizing water flows, which would be a natural adaptive trait, would require a compromise of physiological responses between at least three key parameters: stomatal sensitivity, xylem vulnerability to cavitation and the ratio of root-leaf exchange surfaces. In the short term, the level of regulation would be perspiration, while in the longer term, ontogenic, the key parameter would be the leaf area.
At the population level, trees, non-domesticated species, have a high adaptive and evolutionary potential due to their high genetic diversity, the size of each population and the high gene flows between populations. On the one hand, the phenotypic plasticity of the traits (i.e. adaptation to heterogeneous environmental conditions) allows trees to respond quickly to fluctuating conditions. On the other hand, it is combined with genetic adaptation (i.e. genetic changes in a population in response to selection) which is slower and requires several generations.
In the whole tree, it seems that the organs furthest from the soil's water source are the first to be sacrificed in the event of drought. The water stress we apply aims to remain within a reversible response range, for example by approaching the point of leaf abscission without reaching it in order to preserve function and address regulatory mechanisms. Among the resistances that oppose the transport of water, we can distinguish six interfaces when the water rises in the tree: the passage from soil to root (symplastic, transmembrane and apoplastic); from root to stem (apoplastic); from stem to petiole (apoplastic); from petiole to leaf vein (apoplastic); from vein to leaf mesophyll (symplastic and transmembrane) and from mesophyll to atmosphere (symplastic and apoplastic). Overall, in conditions of moderate drought, resistance to water flow is low in the stem and is distributed in similar proportions between roots and leaves and influences general metabolism, photosynthesis and transpiration.
At the cellular level, the molecular basis for drought tolerance is based on a large number of candidate genes. Among the classes of proteins involved in drought response, aquaporins (AQPs) are excellent candidates for the regulation of water homeostasis. Indeed, water can pass from one cell to another bidirectionally, particularly via membrane AQPs. The existence of metabolically regulated AQPs is clearly formulated in foliar stoma response models. In poplar, we have observed modulations of expression of plasmalemic AQP genes (PIP). Proven functional involvement, for example in banana trees, with a plasma aquaporin gene (PIP1;2) built under the control of the dehydrin promoter (DHN-1) conferring a higher recovery capacity to a water deficiency. In Populus trichocarpa, tonoplastic AQPs (TIPs) would contribute to water exchange between xylem vessels and cells of the perivascular sheath (Bundle Sheath Extension, BSE), allowing foliar hydraulic conductance to be recovered after water stress. In poplar, we have shown opposite AQP expression patterns in drought conditions.
How to address the problem
The central hypothesis we formulate is that some genotypes would carry a combination of genes or allelic variants for water transport that are more conducive to an appropriate response, but this advantage would only occur under certain given environmental constraints. To this end, we are seeking to understand the molecular mechanisms of water transport in the leaf in order to identify their genetic signatures (gene families, regulatory profiles). These signatures would be conducive to the identification of individuals in a population that embraces the ideotypes sought. The aquaporin family (AQP) is the initial foundation of our research. In this perspective, it can be expected that differences in ecophysiological and molecular responses will appear depending on the ease with which individuals can use the available water. For example, the plasticity of the recycling component of PIP AQPs would be the way for plants to respond to rapid variations in water availability; knowing that plasticity leads to acclimatization, itself leading to adaptation. To move forward, three research actions are in place.
1- Aquaporin regulation networks
A complex subject that involves several biological models. We seek to describe the gene regulatory networks implemented in relation to the aquaporin family by mobilizing three experimental systems: the first is based on samples of dissected ribs of 6 black poplar genotypes subjected to controlled and reversible drought and on a kinetic study on the same genotypes during the installation of the same drought. We have complete transcriptomic data currently being excavated (A Gousset-Dupont, B Fumanal, M Garavillon-Tournayre, M Vandame & C Savel). The second is based on the study of the Trichoderma-Fusarium interaction, where what interests us is that Trichoderma diverts water resources from its prey to its benefit, in a mycoparasitic interaction, by deregulating aquaporins (JS Venisse, C Savel, G Pétel & P Rockel-Drevet), which also leads us to look more closely at the endophyte microbiota in the overall response of the black poplar to drought (B Fumanal). The third is based on work carried out on ear blight by Fusarium in wheat (collaboration L Bonhomme & F Rocher). This time a plant-pathogen interaction showed that the main component of the response was the reallocation of wheat resources (sugar and water) to the pathogen. Is this a reprogramming of water use similar to the one we are studying between Trichoderma and Fusarium?
2- Functional diversity of aquaporin genes in poplar
When observed in more than 1% of individuals in a population, the most interesting mutations are SNVs (single nucleotide variation, also called SNPs). Most are neutral, i.e. without any detectable phenotypic effect. For others, association studies aim to identify correlations between SNV and phenotype, but evidence of causality remains a challenge given the biological complexity of individuals. However, genetic variants impact the sequence and alter the phenotype through changes that affect: the level of gene expression (promoters & UTR), alternative splicing (intron-exon junctions), non-coding RNAs (miRNA & lnc-RNA) and the 3D shape or binding properties of a given protein (3D atomic structure). In this context, we plan to describe the natural variability of sequence composition, targeting functional impact SNVs, in poplar aquaporins (B. Fumanal, P. Rockel-Drevet & P Label). It turns out that the natural variability of AQPs is high in plants in general and more particularly in non-domesticated species such as forest trees. For example, Populus trichocarpa 54 genes and Brassica napus 121 genes, compared to Homo sapiens 13 genes or Escherichia coli 2 genes. The diversity of AQPs also addresses the relative specificity of the transported molecule, which may be small solutes other than the water molecule. It also focuses on pore selectivity mechanisms where redundancy is evident, particularly in plant AQPs (collaboration D Auguin, JS Venisse & R Mom).
3- The structure-function relationship in aquaporins
We hypothesize that, under sequence differences, different physical properties of pore selectivity would rest when no particularity is detected in the structure of gene promoters. These differential selectivity properties are suspected from primary sequence differences in the units characterizing the atomic structure of the pore. We propose to explore two aspects of the structure-function relationship in aquaporins. On the one hand, by studying structural differences on six AQPs (four plants, and two positive human and bacterial controls) by modelling atomic structures of the six pores inserted in their respective protein context and by evaluating the pore selectivity mechanism of tree AQPs from the perspective of the role of membrane potential (P Label, R Mom, & D Auguin collaboration). This membrane potential is particularly dependent on the ion concentration on either side of the plasmalemma. On the other hand, by deepening the role of the ionic environment. Indeed, water stress is related to ion concentrations in cell solutes. AQPs are sensitive to high ionic concentrations that disturb both the electrostatic membrane balance and the hydrophobic forces that guarantee the globular structure. Molecular dynamics studies report a link between the electrostatic environment and the functioning of AQPs. The side chains of amino acids involved in the selective pore would act as voltage-dependent selectors and switch between two structural states: high or low, the second corresponding to a closed AQP pore. A research line will target the study of the structural consequences of the phosphorylation state of PIP2 isoforms where the first cytosolic loop (B-loop) and the C-terminal end of the protein carry several phosphorylation sites whose state is impacted by a water deficiency (R Mom).