Transgenerational responses to nitrogen deprivation in Arabidopsis thaliana

Living organisms are constantly exposed to environmental stresses, which may have negative impacts on their growth, development, and reproduction. Such conditions are often so crucial to determine the environmental and geographical distribution of species, and provide a selective evolutionary pressure on a given population. Different strategies can be adopted by living organisms to minimize stress influence. Because of their sessile nature, plants are expected to have evolved sophisticated mechanisms to cope with unavoidable adverse environmental conditions. As perturbations may occur repeatedly, it would be advantageous to plants to retain the \u201cmemory\u201d of past events and to use it to adapt to new environmental challenges (i.e. pathogens attack, drought stress). According to this, in Arabidopsis thaliana has been demonstrated that stress signals are usually transduced into effects on gene expression. Furthermore, it has been frequently reported that changes in gene expression induced by stresses in plants could be transmitted to the progenies stabilizing stress-dependent modifications; however, the explanation of this phenomenon remains still controversial. One of the major stress types to which plants are frequently exposed is nutritional stress. The most critical example of this kind of stress is the deprivation of nitrogen, the essential mineral element required in greatest amount for plant growth due to its role as a constituent of many primary metabolites, such amino acids, nucleic acids, pigments as well as secondary metabolites, such as amines, phytohormones, alkaloids. Symptoms of nitrogen deficiency are slow growth, chlorosis of leaves and their fall off, as well as woodiness of stems and accumulation of anthocyanin pigments. Nitrogen concentration can rapidly fluctuate in the soil because of leaching, volatilization of ammonia, soil denitrification and especially human activities. Thus, this leads plants to efficiently adapt by adjusting their acquisition mechanisms to ensure an adequate element supply, a proper plant development as well as an appropriate yield in crops. Moreover, it is known that nitrate, the main source of nitrogen for plants growing in aerobic soils, is a signal capable of eliciting the rapid expression of transporters and assimilatory enzymes as well as changes in root morphology. The aim of this PhD thesis was to evaluate the possible establishment of a \u201ctransgenerational stress memory\u201d, at physiological and transcriptomic level, using an experimental setup based on three successive generations of the model plant Arabidopsis thaliana (Col-0), subjected to nitrate deprivation. Using nitrate uptake measurements and genome-wide gene expression analyses (RNA-sequencing), it was observed an increase in the capability to take up the anion between the first and the second generation, involving a high-affinity and saturable transport system, paralleled by a considerable modulation of gene expression at the level of nitrate transporters (i.e. NRT2.4 and NRT2.5). In order to assess the occurrence of a \u201ctransgenerational memory\u201d, transcriptional analysis on the third generation showed an enduring down-regulation of genes involved in the response to light, like for example LHCB2 and LHCA3, which are members of photosystem light harvesting complexes. In the second part of the project, in order to reinforce the observed transcriptional patterns, we explored the possibility that the observed changes in the expression of nitrogen-related genetic pathways might be accompanied by epigenetic changes. Indeed, the epigenetic analysis, focused on the high-throughput detection of genome-wide DNA methylation, a potentially inheritable DNA modification, revealed changes, also termed differentially methylated regions (DMRs), between treated (N-deprived) and control plants which also involved genes pinpointed by the transcriptome analysis. However, the mechanism underlying the maintenance of expression changes in the progeny of stressed plants remains elusive and compels further investigation. The results of this study highlight the molecular changes associated with the plant response to an important nutritional stress in a transgenerational perspective. Thus, this exploratory work paves the way for new lines of research that may help breeding for high crop production by reinforcing the natural resilience and adaptive attitude of plants in the context of nitrogen requirement and consequently reducing the use of N-fertilizers, that represents an expensive and environmentally costly strategy to achieve desirable crop yields