In the context of climate change, it appears essential to unravel the mechanisms governing abiotic stress tolerance in higher plants, in order to build predictive models and use this knowledge to assist selection and design of stress tolerant crops. We have previously uncovered remarkable adaptations in seed mitochondria, which because of the ability of seeds to survive desiccation, display impressive tolerance to abiotic stress. In particular, seed mitochondria accumulate high levels of small heat shock proteins (sHSP) and late embryogenesis abundant proteins (LEA). The sHSP are the most widespread but less conserved HSP. They contribute to the molecular chaperone network that assists protein biogenesis and homeostasis under stress conditions (sHSPs are stress inducible). In eukaryotes, mitochondrial sHSP (M-sHSP) have only been identified in plants and insects. LEA proteins are highly hydrophilic proteins, generally intrinsically disordered, which accumulate in desiccation tolerant organisms, and whose functions still remain largely enigmatic. The MITOZEN project aims at deciphering the molecular function and physiological role of the mitochondrial sHSP and LEA proteins (M-sHSP and M-LEA) in the model plant Arabidopsis thaliana. The genome of Arabidopsis harbors 17 sHSP genes (including 3 M-sHSP) and more that 50 LEA genes, among which we have recently identified 5 M-LEA genes. The molecular functions of the M-sHSP and M-LEA will be explored using biochemical and biophysical approaches to study recombinant proteins produced in Escherichia coli. Their structural features and protective activities (oligomerisation, secondary structure, chaperone activities, membrane protection) will be examined in the context of temperature stress and dehydration using a large panel of techniques and in vitro assays. The goal is to determine the potential molecular functions of the different M-sHSP and M-LEA in the context of stress tolerance (desiccation in seeds, high temperature in seeds and plants). A reverse genetics approach will be developed in Arabidopsis to explore the role of M-M-sHSPs and M-LEAs in the physiology and development of plants. Single and multiple knock-out mutant lines will be constructed, as well as overexpressors using an inducible system. Their phenotypic characterization will focus on seed development and abiotic stress tolerance of plants, including mitochondrial function. The integration of data provided by these multidisciplinary approaches (bioinformatics, biochemistry and biophysics, genetics, physiology) will shed light on the function and importance of the different M-sHSP and M-LEA in the development and stress tolerance of plants. It will also increase knowledge about molecular chaperones and in particular with respect to their yet unexplored role in the context of dehydration, and will shed novel light on the function of LEA proteins.

Microalgae and plant leaves are promising sources of fatty acids and triacylglycerol (TAG) for alternative energies or for green chemistry. A major biological bottleneck is the inverse correlation between proliferation and oil accumulation, which compromises productivity. Therefore it is imperative to take an integrated approach and investigate potential signaling pathways that regulates the balance between proliferation and TAG accumulation. Increasing evidence from our work and others indicates that the TOR (Target Of Rapamycin) pathway is essential for regulating growth and TAG accumulation in response to nutrient availability in both plants and algae. Moreover, recent independent genetic screens from two partners of this project and other groups suggest that members of the small family (3 to 5 members) of DYRK (dual-specificity tyrosine-phosphorylation-regulated kinases) could be essential effectors of TOR-dependent regulation of proliferation and lipid accumulation in plant and algae. First, TOR was shown to control cell growth and proliferation in Arabidopsis by phosphorylating DYRK kinase YAK1 which is a growth repressor acting downstream of TOR. Second, two DYRKs, TAR1 and DYRKP were reported to regulate the accumulation of reserve compounds (starch and oil) in the green algae Chlamydomonas. Our central hypothesis is that interactions between DYRK and TOR coordinate lipid accumulation and cell growth in response to environmental cues (e.g., nutrient, light). This will be addressed in parallel in plant and algal models Arabidopsis and Chlamydomonas where large numbers of genetic and molecular tools and mutants are available. This project is organized in three work-packages (WPs) and built on key preliminary results. WP1 will use state of the art biochemistry methods to identify DYRKs that are phosphorylated in a TOR-dependent manner and are therefore acting downstream of TOR. WP2 is dedicated to the functional relationship between TOR and DYRK kinases. Mutants in DYRKs, TOR, and their doubles mutant will be generated and phenotypes in regards to lipid, protein and starch content relative to biomass, with a particular focus on lipids (Heliobiotec lipidomic platform). Recently developed genome editing methods using CAS9 variant will be used to generate some of these mutants, particularly Chlamydomonas mutants carrying new point mutations in the TOR gene that we have identified in Arabidopsis and modulate TOR activity. Finally, during WP3, we will develop genetic screens of suppressors of dyrk mutants, in order to identify effectors of DYRK functions related to proliferation and TAG accumulation. The TOR-DYRKcontrol project brings together three partners with complementary expertise in TOR signaling, biochemistry, lipid metabolism, genetics, genome editing and plant and algal biology. The use of both plant and algae should shed light on the evolutionary aspects of the TOR regulation, and bridging gaps on the lack of knowledge across different evolutionary lineages. This project will advance our knowledge in the understanding of synergy between TOR pathway, cell growth and carbon storage, and allow the further use of this knowledge to create algal/plant prototypes for improved lipid production. Therefore, this project addresses two urgent societal issues, energy shortage and global warming. It should contribute to the emergency of a greener economy, replacing fossil fuels by renewable sources for the production of lipids for food, transportation and chemical industry, while lowering impact of CO2 overproduction on global warming.

Maize farming requires high amounts of N fertilizer, with adverse environmental effects and insufficient agronomic sustainability. Certain maize genotypes can be colonized endophytically by atmospheric nitrogen (N2)-fixing bacteria, but the agronomic potential of endophytic N2-fixation is not fully exploited. Our hypothesis is that a scientific understanding of the mechanisms controlling these endophytic N2-fixing associations combined with an assessment of maize genetic diversity and specificity with regards to this interaction could be useful to optimize endophytic N2-fixation and exploit it in agriculture. The aim of the project is thus to better understand the interactions between bacterial endophytes fixing N2 and maize, in order to identify and select maize genotypes that will be able to use the fixed N more efficiently and thus will be less dependent on mineral N fertilization. This will be achieved by developing a multidisciplinary approach integrating molecular physiology, the assessment of whole-plant N responses to the endophytic interaction, molecular plant-microbe ecology and agronomy. We will characterize both at the physiological and molecular levels the atmospheric N2-fixing endophytic interaction using a large-scale integrated transcriptomic, proteomic and metabolomic approach implemented with two established Herbaspirillum and Azospirillum models of N2-fixing endophytic bacteria and 19 representatives of European and American maize genetic diversity. This will allow identifying the genetic and physiological determinants required for an efficient N2-fixing endophytic association. Such study, combined to a genome-scale metabolic modelling approach, will then help obtaining an integrated view on the plant’s response to the endophytic interaction and on its adaptation to temperate climatic conditions. A molecular screening will also be conducted to obtain effective endophytic N2-fixing bacteria for agronomic improvement of maize cultivation at lower N input under temperate pedoclimatic conditions. To this end, we will implement a novel molecular screening strategy, using not only microbial traits but also the plant biological markers of the ability of the plant to utilize the fixed N more efficiently. Production of innovative fertilizers based on inoculant technology will be then undertaken to assess under agronomic conditions, if the maize genotypes exhibiting the best endophytic N2 fixation also exhibit improved performance in terms of biomass and grain production. Such an agronomic evaluation will also be conducted with commercial hybrids known for their high performance under reduced N fertilization. The project focuses on maize, a crop of major economic importance both in France and worldwide. Maize is particularly relevant for this project for four reasons. First, it has a huge genetic diversity, allowing the improvement of both its agronomic and environmental performances in terms of N fertilizer usage. Second, maize is also a model crop particularly suited to perform integrated agronomic, physiological and molecular genetic studies during the whole plant developmental cycle. Third, many maize genotypes are colonized endophytically by N2-fixing bacterial endophytes. Thus, deciphering the relationships between maize physiological status and the provision of “free” N by the bacterial endophytes will deliver science to underpin and implement novel agricultural strategies aimed at reducing the use of N mineral fertilisers in maize farming, which will be facilitated by the industrial development in this project of fertilizer micro-granules serving as carriers for N-fixing endophytic inoculant.

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.

Circadian rhythms are a fundamental feature of living organisms and are maintained by a molecular oscillator called the circadian clock. In plants, this mechanism has been shown to control biotic and abiotic stress responses, flowering time, photosynthesis, growth or fitness, among many other traits of agricultural interest. The circadian clock in plants is only well characterized in Arabidopsis, but it is clear now that this mechanism as well as its inputs and outputs are not conserved in other plant species. For example, several of the core-clock proteins from Arabidopsis are missing in tomato, while some others are duplicated. This raises the question of whether the model of the circadian clock in Arabidopsis is sufficient to understand this important mechanism in plants, the primary source of food and oxygen for out planet. We have recently shown that tomato domestication was accompanied by a significant deceleration of circadian rhythms (Müller et al., 2016). We localized the two responsible mutations, found strong signals of selection for the underlying genes, and most importantly, showed that changes in circadian rhythms have an effect in plants grown under production environments. My ERC proposal in 2016 was dedicated to generate the tools needed to build the first model of the circadian clock in a crop, to compare it with what is known from Arabidopsis and to investigate its potential to enhance plant performance for agriculture. Tomato is one of the few diploid, fast growing, self-crossing crops and there is a vast amount of genetic, genomic and molecular resources for this species. Taking advantage of the variation already present for circadian rhythms in tomato and using the latest gene editing, genomics and molecular biology technologies available for this system will allow us to consolidate the field of circadian biology in crops. This year, my ERC grant has changed significantly in the light of our new results and it has into account the suggestions from the reviewers in 2016. In this Templin-ERC proposal I request funds for technical support to set up the basic genetic material and methods for my new ERC grant, and obtain the preliminary results that will ensure the feasibility of the project.