Casein as protein and hydrolysate: Biostimulant or nitrogen source for Nicotiana tabacum plants grown in vitro?

In contrast to inorganic nitrogen (N) assimilation, the role of organic N forms, such as proteins and peptides, as sources of N and their impact on plant metabolism remains unclear. Simultaneously, organic biostimulants are used as priming agents to improve plant defense response. Here, we analysed the metabolic response of tobacco plants grown in vitro with casein hydrolysate or protein. As the sole source of N, casein hydrolysate enabled tobacco growth, while protein casein was used only to a limited extent. Free amino acids were detected in the roots of tobacco plants grown with protein casein but not in the plants grown with no source of N. Combining hydrolysate with inorganic N had beneficial effects on growth, root N uptake and protein content. The metabolism of casein-supplemented plants shifted to aromatic (Trp), branched-chain (Ile, Leu, Val) and basic (Arg, His, Lys) amino acids, suggesting their preferential uptake and/or alterations in their metabolic pathways. Complementarily, proteomic analysis of tobacco roots identified peptidase C1A and peptidase S10 families as potential key players in casein degradation and response to N starvation. Moreover, amidases were significantly upregulated, most likely for their role in ammonia release and impact on auxin synthesis. In phytohormonal analysis, both forms of casein influenced phenylacetic acid and cytokinin contents, suggesting a root system response to scarce N availability. In turn, metabolomics highlighted the stimulation of some plant defense mechanisms under such growth conditions, that is, the high concentrations of secondary metabolites (e.g., ferulic acid) and heat shock proteins.