Mémoire

Abstract

Cereals constitute a key foundation of the global food system, providing a considerable portion of the world’s calory intake. The production of qualitative grains relies on the proper coordination of the plant’s developmental cycle with their environment, among which nutrient availability plays a critical role. Plants evolved efficient uptake mechanisms to acquire nutrients from their substrate and, to limit variations in their internal content, they also developed complex mechanisms to maintain steady internal nutrient contents upon fluctuations in their supply, a process known as homeostasis. While knowledge of the homeostasis mechanisms of individual nutrients and their cross-interaction is accumulating in the model dicot species Arabidopsis, much less is known of these processes in Pooideae, a group of plants that include wheat, oats, and barley. In my thesis, I used Brachypodium distachyon (Brachypodium), a temperate grass closely related to wheat, barley and oats, as a model plant for investigating the interaction between nitrogen (N), a key macronutrient driving plant growth and productivity, and zinc (Zn), an essential micronutrient that plays structural and catalytic roles in numerous metalloproteins. Although N and Zn homeostasis have been studied independently in temperate grasses, the potential interactions between the homeostasis of these two nutrients remain poorly characterized. To this avail, we examined the morphological and molecular responses of Brachypodium to varying N and Zn concentrations using hyperspectral imaging, ionomic analysis (ICP-AES), and transcriptomic profiling (RT-qPCR). The experimental design used nine culture conditions that combined three nitrate concentrations (0.5, 2, and 8.5 mM) with three Zn concentrations (0, 2, and 20 µM). Plants were grown under a 16h photoperiod in hydroponic systems and harvested for molecular and ionomic profiling after 5 weeks. The results from these experiments showed: (i) At the phenotypic level, the biomass of root and shoot was modified both in response to changes in N availability and to Zn deficiency or excess. (ii) At the ionome level, Zn quantification in shoot and roots confirmed the effectiveness of the Zn deficiency and excess treatments. Certain ions displayed stronger responses to nitrogen variation (K, Mn), others responded primarily to Zn variation (Zn, Fe), while some exhibited more complex response patterns (Cu, Mg). In contrast, Ca and P did not show statistically significant responses. (iii) At the transcriptional level, NRTs, NiR, and Fd-GOGAT-encoding genes responded to the changes in N availability; however, our data also reveal that their expression was influenced by both Zn deficiency and excess. Similarly, ZIPs, bZIPs, NAS, and IRT1 showed the expected responsiveness to Zn concentrations, but also displayed differential expression across varying N levels, suggesting complex interaction patterns. (iv) Finally, we used ionomic profiling data, known media compositions, and hyperspectral images in order to build predictive models of the N and Zn status of the plants. Interestingly, we could obtain a robust clustering of plants according to their Zn content and according to the N culture supply. Collectively, these results demonstrate a complex interaction between N and Zn homeostasis, both at the phenotypic and at the molecular levels, opening up a perspective for future research projects. The second part of my thesis aimed at examining the impact of the circadian clock on N and Zn homeostasis mechanisms. As the circadian clock was shown to affect N homeostasis in rice and Zn homeostasis in Arabidopsis, we decided to generate transgenic lines in which the gene encoding for the key clock component CIRCADIAN-CLOCK ASSOCIATED1 (CCA1) is either knocked out or overexpressed. We used the modular cloning (MoClo) system to generate (i) two binary vectors containing distinct RNA guides targeting CCA1 exons by CRISPR-CAS9, and (ii) one binary vector for CCA1 overexpression. These constructs were inserted into Agrobacterium tumefasciens and used to transform Brachypodium calluses, which are currently being used to regenerate transformed plants. The resulting transgenic lines will be used in future projects to explore the circadian clock’s role in nutrient regulation.