Cereals in general, and rice in particular, are the major source of nutrition for a growing world population. Many of these crops are grown at intensively utilized fields, in which soil nutrients need to be constantly resupplied by fertilization. Because of the high costs and energy demand, there is a need to reduce the use of fertilizers and adapt to a more sustainable form of agriculture. Crop plants that use nutrients more efficiently as the currently available lines, can help to reach these future goals. Potassium (K+) is the most important cationic nutrient and its transport has been studied intensively for the model plant Arabidopsis., but little is known about the transport proteins that channel K+ fluxes in the cereals. Our previous study (Nguyen et al., 2017, Plant Physiology) has revealed important differences in tissue localization and activation mechanisms of K+ efflux channels, between rice plants and Arabidopsis. In the proposed project we will focus on Shaker type K+ efflux channels and HAK/KUP K+ efflux transporters, which enable transport of K+ from the root to the shoot of rice plants and within the stomatal complexes in the leaves. We will pinpoint the cell types in which the selected K+ transport proteins are expressed and generate rice plants that lack functional versions of these proteins. These mutant lines will be compared with wild type rice plants, for their ability to grow, consume water and produce crop yield, at green house and field conditions. Moreover, we will use Arabidopsis guard cells and Xenopus oocytes to express the rice K+ efflux channels and transporters and characterize their biophysical properties, like ion selectivity and voltage-dependent activation. The specific roles of the selected K+ channels and transporters in xylem function and stomatal movements will be at the centre of our attention. We will introduce fluorescence-tagged versions of the K+ channels and transporters to study if the transport proteins show a polarized subcellular localization. The specific roles of these transporters will be further uncovered with single cell techniques, in which ion selective electrodes are applied. Our studies will provide insights into the functions of specific K+ efflux channels and transporters at the cellular level, as well as their importance for growth of rice plants at field conditions. It is likely that this knowledge will be valuable for breeding rice plants with a lower demand for K+ fertilizers, while maintaining a good nutritional quality of the grains. Such traits will be of prime importance for sustainable agriculture and future food security.

Every plant cell is surrounded by a wall, which is at the same time sufficiently strong to resist the turgor pressure and extensible to allow growth. Understanding how plants grow requires studying the architecture and the mechanical homeostasis of this polymer network. The objectives of HOMEOWALL are to combine cell biology, structural biology, soft matter physics and computational modeling to elucidate the nano- and mesoscale architecture of the plant cell wall, the phase transitions in wall polymers that underlie the growth process and the dual role of the recently discovered RALF/LRX/CrRLKL1 module in wall architecture and the control of the phase transitions in expanding cell walls. These data are expected to support a paradigm shift in the understanding of plant cell expansion and to provide new insights in the interactions between co-evolved polyelectrolytes, which are potentially of interest for the conception of new intelligent nanomaterials.

In eukaryotes, intracellular processes such as signaling rely on a network of ion flux between the different intracellular compartments. Although crucial, ion flux coordination between membranes in series is still poorly understood. Plants offer an ideal cellular/biophysical model, the guard cells, to investigate ion flux coordination between compartments in a highly relevant biological process, the control of stomata aperture. Guard cells regulate the aperture of the stomata pores and in this way regulate gas exchanges between the leaves and the atmosphere. Given their function at the interface with the atmosphere, stomata are essential elements for plant adaptation to the environment directly impacting plant biomass production and adaptation to stress conditions. To control the stomata pore aperture guard-cells are able to change their intracellular turgor. This changes of turgor induce changes of the guard cell shape modifying the stomata pore aperture. Massive and coordinated fluxes of ions across the two major membranes (i.e. plasma and vacuolar membranes) of guard cells delimiting two intracellular compartments (i.e. the cytosol and vacuole) modify intracellular turgor. Despite its importance for stomata responses to environmental stimuli, the coordination of ion fluxes between cellular membranes is still poorly understood. The Netflux proposal will explore the molecular/biophysical basis of ion flux coordination between cellular membranes and compartments using guard cells as model system. To achieve its objectives, the Netflux project involves three partners with complementary competences in order to develop an innovative and interdisciplinary approach. Combining genetics, cell imaging, electrophysiology and mathematical modelling we will tackle the coordination of ion fluxes during cellular responses. The project will exploit unique and original genetic resources from the partners to analyze, based on cutting edge experimental techniques and mathematical tools, the dynamics of ionic fluxes in compartmentalized living cells. Further, within the NetFlux we will set a unique and innovative forward genetic screen to uncover new important regulators of ion fluxes in guard cells. The strategy we propose will be a great opportunity to unravel new fundamental mechanisms operating in eukaryotic cells during cellular responses to the environment. In the context of guard cell physiology, the results we will obtain will open new perspectives in our understanding of the stomata functioning. This will be instrumental to combine plant biomass production with tolerance to stress conditions in the context of the future challenges that agriculture will have to face.

MYCOTRANS aims at producing basic knowledge on the functioning of symbiotic exchange between plant roots and fungal symbiont, a beneficial interaction crucial for plant nutrition. MYCOTRANS will focus on the symbiotic ectomycorrhizal (ECM) model association Pinus pinaster – Hebeloma cylindrosporum, because this fungal species is the only one easily transformable with Agrobacterium, enabling genetics studies to be performed. Several transcriptional studies have revealed mycorrhiza-induced fungal membrane transport systems involved in K, N and P nutrition, but surprisingly, a member of the CDF (Cation Diffusion Facilitator) family was identified as the most mycorrhiza-induced transporter. So far, we have studied transport of macronutrients as potassium and phosphate, but we hypothesize that this micronutrient transporter, as other significantly mycorrhiza-induced genes, plays an important role in the development, maintenance or functioning of the ECM association and might provide new keys for understanding the positive effects of mycorrhizal symbiosis on host plant nutrition. However, the molecular function, cellular and subcellular localization, regulation and physiological role in the mycorrhiza of this CDF transporter are unknown yet. Hence, MYCOTRANS objectives will be (i) to decipher physiological function, localization, and regulation of the highly mycorrhiza-induced fungal metal transporter by performing a molecular genetics and functional analysis, (ii) to analyze the role of mycorrhiza-induced genes for the fungal symbiosis by developing tools for genome editing (CRISPR/Cas) for the ECM fungus, and (iii) to discover new aspects of mycorrhizal regulation occurring specifically at the level of proteins by the analysis of the ECM proteome and phosphoproteome. To address these objectives, we will use key methodologies which are: (i) heterologous expression in yeast and Xenopus laevis oocytes of the cDNA encoding the metal (putatively Zn, Fe, Mn) transporter of the CDF family, to assess the properties of this transporter, such as its selectivity for several micronutrients; (ii) In situ hybridization and green fluorescent protein (GFP-) fused proteins for cellular and sub-cellular localization of the CDF transporter in yeast and ectomycorrhizae; (iii) production of new CRISPR/Cas vectors and KO fungal mutants to study the role of mycorrhiza-involved fungal genes, as this technique of genome editing will be much more efficient than the RNAi method previously used by Partner 1; (iv) use of an in vitro symbiosis-mimicking system, where the fungus is incubated in a liquid solution either alone or with host plant roots ensuring a cross-talk between both partners of the symbiosis but without the formation of ECM structures on the root; (v) extraction of fungal proteins and separation in three fractions: soluble, microsomal and plasma membrane proteins, to carry out proteome analysis in all protein fractions and phosphoproteome analysis of plasma membrane proteins, as a target of possible post-translational modifications exerted by the host-plant. Establishment of the CRISPR/Cas technique for ECM fungi will lift a technical barrier and provide the scientific community with these missing tools. In addition, the whole set of the expected results should give decisive insights into the actual physiological role of the mycorrhiza-induced genes coding for transport functions, especially those located in the Hartig net that will determine, in turn, the efficiency of the ectomycorrhizal symbiosis. Hence, MYCOTRANS should help us to find true symbiotic marker genes, making it possible to use mycorrhizal interactions for sound management of both croplands and forests taking care of ecosystem services rendered by mycorrhizal fungi.

Phosphate (Pi), a major cell component, is a strategically important plant resource due to the impending shortage of high-quality sources of this element. Diminishing the use of Pi in crop production requires a better understanding of the factors that stimulate Pi uptake. It has been widely observed that plants exposed to Pi and Zinc (Zn) deficiencies accumulate more Pi than when they are in more Zn-rich soils. Current data suggest that a specific signaling pathway is involved in the crosstalk between Pi and Zn homeostasis. Although some regulators have been identified recently, most of the early players involved in this pathway remain unknown. The PHLOWZ project aims to clarify the early events involved in this crosstalk, using live imaging of changes in Zn and Pi homeostasis and transcriptomics. PHLOWZ will combine these results with the power of GWAS studies and reverse genetics to identify new early players and regulatory nodes of the Zn/Pi pathway. The discovery of these regulators will have great significance for basic research and agronomical applications.