Co-Cultivation with Azolla Affects the Metabolome of Whole Rice Plant Beyond Canonical Inorganic Nitrogen Fertilization

Azolla spp. are floating ferns used for centuries as biofertilizers to enrich the soil with inorganic nitrogen and improve rice yields. However, the molecular interactions between Azolla and co-cultivated rice plants only recently started to be thoroughly investigated. In this study, we exploited an experiment in which rice plants were grown together with Azolla by maintaining a low and constant concentration of inorganic nitrogen. We employed a combination of non-targeted metabolomics, chemometrics, and molecular networking to dissect the impact of Azolla co-cultivation on the metabolome of rice roots- and leaves, as well as to annotate the metabolites released by Azolla into the growing medium. Our analyses showed that Azolla can synthesize and release a broad range of metabolites in the culture medium, mainly comprising small peptides (i.e., di- and tri-peptides) and flavonoids, that may have stimulated the rice plant growth. We also observed a systemic response in the upregulation of rice metabolites, first in the roots and then in the leaves. Metabolomics analysis indicated that during the first stages of co-cultivation, the impact of Azolla on rice mainly resulted in the accumulation of small peptides, lipids and carbohydrates in roots, as well as flavonoid glycosides and carbohydrates in leaves. Consistent with these results, transcriptomics analysis of rice roots indicated significant changes in the expressions of genes coding for small peptide and lipid transporters and genes involved in the pathways of amino acid salvage and biosynthesis. Overall, our study provides new insights into Azolla's beneficial and growth-promoting effects on rice. Understanding the molecular mechanisms by which Azolla functions as a biostimulant in rice co-culture will facilitate the development of more sustainable and environmentally friendly techniques to increase yields.