CHARACTERIZATION OF PROTEOMIC CHANGES IN CROPS DURING METABOLIC ADAPTATION TO DIFFERENT NITROGEN INPUTS

Nitrogen availability is one of the major factors that influence plant growth, morphology, and metabolism and, hence, crop productivity. In agricultural soils, nitrogen is present in different forms, both inorganic (nitrate and ammonium) and organic (amino acids, short peptides and urea), with variable and heterogeneous distribution. Nowadays, improving knowledge about the nitrogen nutrition in plants is crucial to address the urgent need for a more sustainable agricultural production. In the last years, the “-omics” approaches provided a holistic perspective of the molecular mechanisms underlying plant metabolic adaptations to different nitrogen inputs. Among these, proteomics was largely and successfully applied to analyze various aspects, including the role of post-translational modifications and enzyme isoforms. The aim of this PhD project was to obtain new insights about the biochemical events during sensing and adaptation to different availabilities of nitrogen forms in crops, through an approach based on the integration of physiological, metabolic and proteomic evaluations. The first research activity consisted in an extensive literature revision about plant nitrogen nutrition and plant proteomics, which led to the publication of a review article. This activity highlighted that the majority of the information derives from studies conducted in Arabidopsis and in model crops, such as maize, rice and tomato. Nevertheless, analogies and peculiarities remain to be verified in other crop species. For instance, nitrogen nutrition has been only slightly investigated in perennial plants, and proteomics has been rarely applied in this context. At the same time, although in herbaceous species great progress has been made in understanding plant metabolic responses to inorganic nitrogen forms, several aspects await a clear elucidation. A representative case consists in the fact that a clear overview of the role of specific subcellular compartments during nitrate and/or ammonium nutrition is still fragmentary. Moreover, the literature revision pointed out an increased interest in understanding the relevance of organic nitrogen forms as nutrients influencing plant growth and development. Although it is recognised that plants are able to take up organic forms of nitrogen, such as amino acids, their actual contribution to plant nitrogen nutrition is currently unknown. Starting from these considerations, during the PhD three studies have been designed and conducted, leading to the publication of an article and the preparation of two manuscripts. The first study was devoted to the analysis of nitrogen metabolism in a perennial woody species. In particular, it was aimed at investigating the biochemical and proteomic responses to nitrate in a grapevine rootstock genotype. Indeed, even though grapevine has been adopted as a model perennial species, little is known about the biochemical roles played by roots in nitrogen acquisition. Moreover, this topic has never been addressed through a proteomic approach. The aim of the second study was to deepen the knowledge about the responses to nitrate, ammonium or their co-provision in maize seedlings, adopting a novel proteomic approach. Specifically, key proteins involved in these biochemical mechanisms are localized in different cellular compartments and their functionality is deeply affected by the kind of inorganic source. Hence, a subcellular proteomic approach was applied to obtain new insights about the roles of distinct organelles in the ability of roots to manage different inorganic nitrogen availabilities. Finally, the third study aimed to investigate whether maize plants are able of using and metabolizing amino acids as nitrogen source, when they were externally supplied as a mixture that mimics soil conditions. Considering the complexity of the amino acid metabolism in plants, the application of a proteomic approach was chosen as a useful holistic strategy to obtain a comprehensive overview and to gain new information. In particular, the physiological, biochemical and proteomic changes in roots and leaves were compared to those associated with nitrate availability as a reference inorganic nitrogen source. Overall, these studies provide novel information about how plants perceive and adapt to different nitrogen availabilities. According with the literature, nitrate influenced several aspects acting as either a nutrient, an osmolyte and a signal molecule. In addition, it has been possible to highlight that, in comparison to herbaceous species, specific responses to nitrate occurred in grapevine. Moreover, the subcellular proteomic investigation allowed to appreciate how nitrate and ammonium have different, and sometimes contrasting, effects on the cell organelles functionalities, especially with regard to mitochondria and vacuoles. Finally, new hints about the metabolic pathways involved in amino acid-based nutrition were provided. In particular, the provision of amino acids to the maize plants impacted on the carbon and energy metabolism in roots and influenced the translocation of amino acids to shoot. In conclusion, these studies confirm that the different nitrogen sources have distinct and significant effects on plant growth and physiology, and also put in evidence some interesting peculiarities related to plant species and developmental stages. Moreover, they underline the key role of roots in response to nitrogen forms, providing new evidence that amino acid metabolism represents a key point in the carbon/nitrogen balance in plants.