Plant-derived protein hydrolysates (PHs) are an important category of biostimulants able to increase plant growth and crop yield especially under environmental stress conditions. PHs can be applied as foliar spray or soil drench. Foliar spray is generally applied to achieve a relatively short-term response, whereas soil drench is used when a long-term effect is desired. The aim of the study was to elucidate the biostimulant action of PH application method (foliar spray or substrate drench) on morpho-physiological traits and metabolic profile of tomato grown under limited water availability. An untreated control was also included. A high-throughput image-based phenotyping (HTP) approach was used to non-destructively monitor the crop response under limited water availability (40% of container capacity) in a controlled environment. Moreover, metabolic profile of leaves was determined at the end of the trial. Dry biomass of shoots at the end of the trial was significantly correlated with number of green pixels (R² = 0.90) and projected shoot area, respectively. Both drench and foliar treatments had a positive impact on the digital biomass compared to control while the photosynthetic performance of the plants was slightly influenced by treatments. Overall drench application under limited water availability more positively influenced biomass accumulation and metabolic profile than foliar application. Significantly higher transpiration use efficiency was observed with PH-drench applications indicating better stomatal conductance. The mass-spectrometry based metabolomic analysis allowed the identification of distinct biochemical signatures in PH-treated plants. Metabolomic changes involved a wide and organized range of biochemical processes that included, among others, phytohormones (notably a decrease in cytokinins and an accumulation of salicylates) and lipids (including membrane lipids, sterols, and terpenes). From a general perspective, treated tomato plants exhibited an improved tolerance to reactive oxygen species (ROS)-mediated oxidative imbalance. Such capability to cope with oxidative stress might have resulted from a coordinated action of signaling compounds (salicylic acid and hydroxycinnamic amides), radical scavengers such as carotenoids and prenyl quinones, as well as a reduced biosynthesis of tetrapyrrole coproporphyrins.

Root exudation patterns are linked to, among other things, plant growth, plant-microbe interaction and the priming effect. In this work, two complementary metabolomic approaches (both liquid and gas chromatography coupled to mass spectrometry) were applied to investigate the modulation of root exudation imposed by two beneficial fungi (substrate treatment of Trichoderma atroviride AT10, substrate application of Rhizoglomus irregulare BEG72 and seed treatment with T. atroviride AT10) on wheat (Triticum aestivum L.). The inoculation with R. irregulare elicited significant increases (by 18%, 39% and 20%) in the shoot, root dry biomass and root-to-shoot ratio compared to untreated plants, whereas inoculation with T. atroviride, as a substrate drench or as a seed coating, exhibited intermediate values for these parameters. The metabolomic approach demonstrated a broad chemical diversity, with more than 2900 compounds annotated in the root exudates. Overall, the Orthogonal Projections to Latent Structures Discriminant Analysis (OPLS-DA) supervised modelling highlighted a distinctive modulation of the metabolic profile in the root exudates as a function of both fungal inoculation and means of application. Most of the differences could be ascribed to lipids (sterols and membrane lipids), phenolic compounds and terpenoids, siderophores and chelating acids, derivatives of amino acids and phytohormones, and as such, the interaction between the wheat roots and beneficial fungi resulted in a complex response in terms of root exudates, likely involving a cascade of processes. Nonetheless, the changes imposed by plant-microbe interactions can contribute to the support of the biostimulant effects of both T. atroviride and R. irregulare.

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.

Sweet basil (Ocimum basilicum L.) is an aromatic, tender leafy green cultivated for the fresh market and industrial processing. Like many other leafy vegetables, this species can be harvested more than once to increase productivity. Although the cut-and-come-again (CC) harvest strategy is widespread in basil, not much is known about the effect of the cut. In this work, we examined differences in biomass, physiology, nutrient use efficiency and antioxidant capacity of basil leaves from two successive harvests. Moreover, we also performed an untargeted metabolomics analysis to reveal variations in the leaf metabolite profile. The data indicated that the cut affected many of the parameters under investigation, including a modest reduction of yield (−20%), of nitrogen use efficiency (−21%) and of some quality attributes such as the antioxidant capacity (−16%) and the nitrate content in leaves (+48%). Moreover, leaves of successive cuts displayed a significant variation of the profile of bioactive compounds, characterized by an overall decrease of phenylpropanoids and an increase in terpenoids. Our data revealed the impact of CC harvesting strategy in basil, and that this strategy offers the possibility to yield leaves with different metabolomics profiles and quality attributes.