Biotechnological approaches for the synthesis of natural iron (Fe) chelators and hormone-like molecules for a sustainable correction of chlorosis in agricultural crops

Iron (Fe) has been recognized as one of the limiting factors in crop production due to its involvement in essential biochemical processes in plants. However, notwithstanding its abundance in soil, the bioavailability of Fe for plants is extremely low and can result in significant nutritional imbalances. This phenomenon is principally due to the insolubility of Fe in conditions of high pH and mineral composition, where it tends to precipitate in insoluble forms that are not available for plants. To overcome this limited bioavailability, the most frequent strategies adopted in agriculture involve the use of synthetic Fe chelates, such as Fe(III)-EDTA and Fe(III)-EDDHA, developed in the 1950s and still considered the most effective strategy to correct Fe deficiency in plants due to their capability to maintain Fe in a soluble form, even in the presence of high pH. Nonetheless, besides their cost for farmers, synthetic chelates are characterized by high mobility in soil solutions, low biodegradability and a strong affinity for heavy metals, thus representing an unsustainable tool for agriculture. In this Thesis, three novel approaches are proposed to provide innovative and sustainable strategies to address the challenges mentioned above, with a focus on the correction of Fe deficiency in plants. Thus, we present a bio-based methodology to develop low-impact plant-derived extracts enriched with phytosiderophores, natural Fe-chelating agents extruded by gramineous plants (Strategy II plants) when exposed to Fe deficiency. N. benthamiana plants (Strategy I) were transiently agroinfiltrated using a strain of A. tumefaciens (EHA105), transformed to express the maize ZmNAAT1 and ZmDMAS1 genes, involved in the biosynthesis of phytosiderophores and naturally absent in Strategy I plants. The content of these molecules in the leaves of agroinfiltrated plants was determined by using a new and improved UHPLC-QQQ-MS/MS analysis. Subsequently, the plant extract was administered at different concentrations to cucumber plants (C. sativus) grown under iron deficiency conditions in hydroponic cultivation. At the end of the treatments, different morpho-physiological parameters related to the plants' response mechanisms to Fe availability were measured, monitoring the regreening capacity of the extracts in comparison to synthetic Fe chelates. Data analysis revealed that ZmNAAT1 gene represents a potential candidate for the production of phytosiderophore, effective to overcome Fe deficiency and ensuring optimal development and regreening of the treated plants, comparable to that achieved with synthetic Fe-chelates. Secondly, an alternative approach involving the use of biostimulants has been proposed. These molecules, recently developed as low-impact tools for agriculture, have paved the way to new opportunities for recycling industrial by-products while improving nutrient use efficiency, enhancing tolerance to abiotic stress, and increasing the quality of crop production. Thus, to investigate an additional alternative and sustainable method for correcting Fe deficiency, the individual and simultaneous application of fulvic acid (FA) and collagen-derived protein hydrolysate (PH) was tested during plant recovery from Fe deficiency, evaluating their biostimulant effects on plant phenotype and physiology. The simultaneous treatment with the two products highlighted an increase in plant biomass and an enhancement of the root biochemical activities related to Fe acquisition, as confirmed by molecular analyses, thus demonstrating a enhanced effect between these two biostimulants of different nature. Finally, an evaluation of a new biostimulant derived from the leaf extract of M. oleifera has been proposed. Priming tests conducted on tomato seeds (S. lycopersicum) to verify and identify a potential biostimulant effect of the extract have provided a boost in root development and morphology. Then, the plant extracts tested on cucumber plants (C. sativus) grown under Fe deficiency conditions in hydroponic cultivation have showed the potential of M. oleifera as a low-impact tool to facilitate the recovery from iron deficiency.