The FolEau team is interested in water movement within leaves. In all plants, water, a vital element, is drawn from the soil, transported to the leaves (photosynthesis), and then evaporated into the atmosphere. Our team is seeking to understand how water molecules are transported from the circulatory system (the veins) to ‘spongy’ cells on the surface, where the water evaporates. The transport pathways involved involve highly specialised proteins that allow water to pass through cell membranes. These are aquaporins. There are several hundred of them in trees, and variations in their protein sequence determine their performance as water transporters. We are currently working to discover the molecular determinants of this function and are trying to figure out how to exploit this variability to help select trees that are more tolerant to drought.
Our motivations
We want to contribute to solving the problems posed by climate change to forest crop production. It is important to note that lignocellulosic production is the main source of income for 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 take into account the tree's water economy. 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 genes involved in water movement in leaves helps to meet this demand.
A few basics to understand
In the ecosystem, photosynthesis, which consumes CO2 and water, produces the sugars that are essential for all life on Earth. In this system, trees act as a remarkable hydraulic interface between the soil and the atmosphere: they use the energy released by the transition from liquid water to water vapour (transpiration) to maintain the flow of water. For example, a 15-year-old poplar tree needs to evaporate 250 litres of water per day in summer in order to grow. Maximising water flow, 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 transpiration, while in the longer term, ontogenetic, the key parameter would be leaf area.
At the population level, trees, which are non-domesticated species, have a high adaptive and evolutionary potential due to their significant genetic diversity, the size of each population, and high gene flow between populations. On the one hand, phenotypic trait plasticity (i.e. adaptation to heterogeneous environmental conditions) allows trees to respond quickly to changing conditions. On the other hand, it combines with genetic adaptation (i.e. genetic changes in a population in response to selection), which is slower and requires several generations.
In the entire tree, it seems that the organs furthest from the soil 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 leaf abscission point without reaching it in order to preserve function and address regulatory mechanisms. Among the resistances that oppose water transport, six interfaces can be distinguished as water rises through the tree: the passage from the soil to the root (symplastic, transmembrane and apoplastic); from the root to the stem (apoplastic); from the stem to the petiole (apoplastic); from the petiole to the leaf vein (apoplastic); from the vein to the leaf mesophyll (symplastic and transmembrane); and from the mesophyll to the atmosphere (symplastic and apoplastic). Overall, under moderate drought conditions, water flow resistance is low in the stem and is distributed in roughly equal proportions between the roots and leaves, influencing overall metabolism, photosynthesis and transpiration.
At the cellular level, the molecular basis of drought tolerance relies on a large number of candidate genes. Among the classes of proteins involved in the drought response, aquaporins (AQPs) are excellent candidates for regulating water homeostasis. Indeed, water can pass from one cell to another in both directions, particularly via membrane AQPs. The existence of metabolically regulatable AQPs is clearly demonstrated in leaf stomatal response models. In poplar, we have observed modulations in the expression of plasmalemmal AQP (PIP) genes. Functional involvement has been proven, for example in banana trees, with a plasmalemmal aquaporin gene (PIP1;2) constructed under the control of a dehydrin promoter (DHN-1), conferring a greater capacity for recovery from water deficiency. In Populus trichocarpa, tonoplast AQPs (TIP) contribute to water exchange between xylem vessels and perivascular sheath cells (Bundle Sheath Extension, BSE), enabling recovery of leaf hydraulic conductance after water stress. In poplar, we have demonstrated opposite AQP expression patterns in drought conditions.
How to approach the problem ?
Our central hypothesis is that certain genotypes carry a combination of genes or allelic variants for water transport that are more conducive to an appropriate response, but that this advantage only manifests itself under certain environmental constraints. To this end, we are seeking to understand the molecular mechanisms of water transport in leaves in order to identify their genetic signatures (gene families, regulatory profiles). These signatures would be conducive to identifying individuals in a population that harbour the desired ideotypes. The aquaporin (AQP) family forms the initial basis of our research. From this perspective, we can expect to see differences in ecophysiological and molecular responses depending on the ability of individuals to exploit available water. For example, the plasticity of the recycling process that constitutes AQP PIPs would be the means by which plants respond to rapid variations in water availability, given that plasticity leads to acclimatisation, which in turn leads to adaptation. To move forward, three research actions are in place.
1- Aquaporin regulatory networks
This is a complex subject involving several biological models. We seek to describe the gene regulation networks involved in relation to the aquaporin family by using three experimental systems: The first is based on samples of dissected veins from six black poplar genotypes subjected to controlled and reversible drought, and on a kinetic study of the same genotypes during the onset of the same drought. We have complete transcriptomic data, which is currently being analysed (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 we are interested in the fact that Trichoderma diverts its prey's water resources to its own advantage, in a mycoparasitic interaction, by deregulating aquaporins (JS Venisse, C Savel, G Pétel & P Rockel-Drevet), which also leads us to take a closer look at the endophytic microbiota in the overall response of black poplar to drought (B Fumanal). The third is based on work carried out on Fusarium head blight in wheat (collaboration between L Bonhomme & F Rocher). This time, a plant-pathogen interaction showed that the main component of the response involved the reallocation of wheat resources (sugar and water) in favour of the pathogen. Is this a reprogramming of water use similar to that we are studying between Trichoderma and Fusarium?
2- The functional diversity of aquaporin genes in poplar
When observed in more than 1% of individuals in a population, particularly interesting mutations are SNVs (single nucleotide variations, also known as SNPs). Most are neutral, i.e. with no detectable phenotypic effect. For the others, association studies aim to identify correlations between SNVs and phenotype, but proving causality remains a challenge given the biological complexity of individuals. Nevertheless, genetic variants impact the sequence and alter the phenotype through changes that affect: gene expression levels (promoters & UTRs), alternative splicing (intron-exon junctions), non-coding RNAs (miRNAs & lncRNAs) 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 SNVs with functional impact, 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 particularly in non-domesticated species such as forest trees. For example, Populus trichocarpa has 54 genes and Brassica napus has 121 genes, compared to Homo sapiens with 13 genes and Escherichia coli with 2 genes. The diversity of AQPs also relates to the relative specificity of the transported molecule, which may be small solutes other than water molecules. It also relates to the selectivity mechanisms of the pore, where redundancy is evident, particularly in plant AQPs (collaboration between D Auguin, JS Venisse & R Mom).
3- The structure-function relationship in aquaporins
We hypothesise that, beneath sequence differences, there lie different physical properties of pore selectivity when no particularities are detected in the structure of gene promoters. These properties of differential selectivity are suspected based on differences in primary sequence in the motifs characterising the atomic structure of the pore. We propose to explore two facets of the structure-function relationship in aquaporins. First, by studying the structural differences in six AQPs (four plant AQPs and two positive controls, one human and one bacterial) by modelling the atomic structures of the six pores inserted into their respective protein contexts and evaluating the pore selectivity mechanism of tree AQPs from the perspective of the role of membrane potential (P Label, R Mom, & collaboration D Auguin). This membrane potential is particularly dependent on the concentration of ions on either side of the plasmalemma. On the other hand, by further investigating the role of the ionic environment. Indeed, water stress is linked in particular to ion concentrations in cellular solutes. AQPs are sensitive to high ion concentrations, which disrupt both the electrostatic balance of the membrane and the hydrophobic forces that maintain the globular structure. Molecular dynamics studies report a link between the electrostatic environment and AQP function. The side chains of the amino acids involved in the selective pore act as voltage-dependent selectors and switch between two structural states: high or low, the latter corresponding to a closed AQP pore. One line of research will focus on studying the structural consequences of the phosphorylation state of PIP2 isoforms, where the first cytosolic loop (loop B) and the C-terminal end of the protein carry several phosphorylation sites whose state is impacted by water deficiency (R Mom).
