Despite the scientific evidence supporting their biostimulant activity, the molecular mechanism(s) underlying the activity of protein hydrolysates (PHs) and the specificity among different products are still poorly explored. This work tested five different protein hydrolysates, produced from different plant sources using the same enzymatic approach, for their ability to promote rooting in tomato cuttings following quick dipping. Provided that all the different PHs increased root length (45–93%) and some of them increased root number (37–56%), untargeted metabolomics followed by multivariate statistics and pathway analysis were used to unravel the molecular processes at the basis of the biostimulant activity. Distinct metabolomic signatures could be found in roots following the PHs treatments. In general, PHs shaped the phytohormone profile, modulating the complex interaction between cytokinins and auxins, an interplay playing a pivotal role in root development, and triggered a down accumulation of brassinosteroids. Concerning secondary metabolism, PHs induced the accumulation of aliphatic glucosinolates, alkaloids, and phenylpropanoids, potentially eliciting crop resilience to stress conditions. Here, we confirm that PHs may have a hormone-like activity, and that their application can modulate plant growth, likely interfering with signaling processes. Noteworthy, the heterogenicity of the botanical origin supported the distinctive and peculiar metabolomic responses we observed across the products tested. While supporting their biostimulant activity, these findings suggest that a generalized crop response to PHs cannot be defined and that specific effects are rather to be investigated.

Plant phenomics is becoming a common tool employed to characterize the mode of action of biostimulants. A combination of this technique with other omics such as metabolomics can offer a deeper understanding of a biostimulant effect in planta. However, the most challenging part then is the data analysis and the interpretation of the omics datasets. In this work, we present an example of how different tools, based on multivariate statistical analysis, can help to simplify the omics data and extract the relevant information. We demonstrate this by studying the effect of protein hydrolysate (PH)-based biostimulants derived from different natural sources in lettuce and tomato plants grown in controlled conditions and under salinity. The biostimulants induced different phenotypic and metabolomic responses in both crops. In general, they improved growth and photosynthesis performance under control and salt stress conditions, with better performance in lettuce. To identify the most significant traits for each treatment, a random forest classifier was used. Using this approach, we found out that, in lettuce, biomass-related parameters were the most relevant traits to evaluate the biostimulant mode of action, with a better response mainly connected to plant hormone regulation. However, in tomatoes, the relevant traits were related to chlorophyll fluorescence parameters in combination with certain antistress metabolites that benefit the electron transport chain, such as 4-hydroxycoumarin and vitamin K1 (phylloquinone). Altogether, we show that to go further in the understanding of the use of biostimulants as plant growth promotors and/or stress alleviators, it is highly beneficial to integrate more advanced statistical tools to deal with the huge datasets obtained from the -omics to extract the relevant information.

Sweet basil (Ocimum basilicum L.) is one of the most produced aromatic herbs in the world, exploiting hydroponic systems. It has been widely assessed that macronutrients, like nitrogen (N) and sulfur (S), can strongly affect the organoleptic qualities of agricultural products, thus influencing their nutraceutical value. In addition, plant-growth-promoting rhizobacteria (PGPR) have been shown to affect plant growth and quality. Azospirillum brasilense is a PGPR able to colonize the root system of different crops, promoting their growth and development and influencing the acquisition of mineral nutrients. On the bases of these observations, we aimed at investigating the impact of both mineral nutrients supply and rhizobacteria inoculation on the nutraceutical value on two different sweet basil varieties, i.e., Genovese and Red Rubin. To these objectives, basil plants have been grown in hydroponics, with nutrient solutions fortified for the concentration of either S or N, supplied as SO 4 2− or NO 3 − , respectively. In addition, plants were either non-inoculated or inoculated with A. brasilense. At harvest, basil plants were assessed for the yield and the nutraceutical properties of the edible parts. The cultivation of basil plants in the fortified nutrient solutions showed a general increasing trend in the accumulation of the fresh biomass, albeit the inoculation with A. brasilense did not further promote the growth. The metabolomic analyses disclosed a strong effect of treatments on the differential accumulation of metabolites in basil leaves, producing the modulation of more than 400 compounds belonging to the secondary metabolism, as phenylpropanoids, isoprenoids, alkaloids, several flavonoids, and terpenoids. The primary metabolism that resulted was also influenced by the treatments showing changes in the fatty acid, carbohydrates, and amino acids metabolism. The amino acid

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Protein hydrolysates (PHs) are employed in agriculture to increase the sustainability of farming systems, with positive results on crop productivity and response against environmental stressors. Nevertheless, the molecular mechanism(s) triggered by their specific activity is not clearly understood. In this work, five PHs obtained by enzymatic hydrolysis of different vegetal protein sources were tested for their root-promoting activity on tomato cuttings. All the treatments improved both root length and number when compared to negative controls. Distinctive metabolomic signatures were highlighted in response to treatments, indicating the triggering of different molecular processes in leaf tissues of tomato cuttings. PHs differentially modulated the biosynthesis of plant stress-protectants, such as alkaloids and phenylpropanoids. Moreover, metabolites involved in phytohormone biosynthesis were significantly impacted. In this context, a clear modulation of several compounds related to auxin homeostasis was observed. In addition, the differential modulation of SlIAA2 and SlIAA9 genes, which are involved in the IAA signalling pathway, might further suggest the auxin-like activity elicited by the PHs tested. Here we provide evidence that PHs can impact plant molecular level, positively affecting root development, most likely by affecting the signalling cascades activated in leaf tissues. The biostimulant activity was sustained by PH-specific response at the molecular level, likely ascribable to their heterogeneous botanical origins. In fact, our findings did not point out a clear universal response to PHs, and specific effects are to be investigated.