Understanding the Biostimulant Action of Vegetal-Derived Protein Hydrolysates by High-Throughput Plant Phenotyping and Metabolomics: A Case Study on Tomato

Abstract

Designing and developing new biostimulants is a crucial process which requires an accurate testing of the product effects on the morpho-physiological traits of plants and a deep understanding of the mechanism of action of selected products. Product screening approaches using omics technologies have been found to be more efficient and cost effective in finding new biostimulant substances. A screening protocol based on the use of high-throughput phenotyping platform for screening new vegetal-derived protein hydrolysates (PHs) for biostimulant activity followed by a metabolomic analysis to elucidate the mechanism of the most active PHs has been applied on tomato crop. Eight PHs (A–G, I) derived from enzymatic hydrolysis of seed proteins of Leguminosae and Brassicaceae species were foliarly sprayed twice during the trial. A non-ionic surfactant Triton X-100 at 0.1% was also added to the solutions before spraying. A control treatment foliarly sprayed with distilled water containing 0.1% Triton X-100 was also included. Untreated and PH-treated tomato plants were monitored regularly using high-throughput non-invasive imaging technologies. The phenotyping approach we used is based on automated integrative analysis of photosynthetic performance, growth analysis, and color index analysis. The digital biomass of the plants sprayed with PH was generally increased. In particular, the relative growth rate and the growth performance were significantly improved by PHs A and I, respectively, compared to the untreated control plants. Kinetic chlorophyll fluorescence imaging did not allow to differentiate the photosynthetic performance of treated and untreated plants. Finally, MS-based untargeted metabolomics analysis was performed in order to characterize the functional mechanisms of selected PHs. The treatment modulated the multi-layer regulation process that involved the ethylene precursor and polyamines and affected the ROS-mediated signaling pathways. Although further investigation is needed to strengthen our findings, metabolomic data suggest that treated plants experienced a metabolic reprogramming following the application of the tested biostimulants. Nonetheless, our experimental data highlight the potential for combined use of high-throughput phenotyping and metabolomics to facilitate the screening of new substances with biostimulant properties and to provide a morpho-physiological and metabolomic gateway to the mechanisms underlying PHs action on plants.

Yield and Nutritional Quality of Vesuvian Piennolo Tomato PDO as Affected by Farming System and Biostimulant Application

“…The legume-derived PH biostimulant obtained through enzymatic hydrolysis contains 75% of free amino acids and peptides, 22% of carbohydrates and 3% of mineral nutrients. The detailed aminogram of the product along with the phenolics, flavonoids and elemental composition were reported by Rouphael et al [31] and Paul et al [25]. The PE biostimulant obtained by fermentation of tropical plants contains 54% of free amino acids and peptide, 17% carbohydrate, 23% mineral nutrients, 6% vitamins and 0.22% phytohormones as reported in detail by Rouphael et al [33] and Caruso et al [32].…”

“…Tropical plant extract (PE) and especially legume-derived protein hydrolysates (PHs) obtained from vegetal origin proteins have been drawing interest in world agricultural areas, compared to animal-derived ones, due to both their higher agronomic value [16] and no use constraints in organic farming. Moreover, PE or PH application to leaves and/or roots reportedly elicit physiological processes, thus resulting in enhancement of growth [17,18], production and quality [18,19], tolerance to abiotic stressors, such as drought, soil and water salinity, extreme temperature, nutrient deficiency, soil acidity and alkalinity [11,[20][21][22][23][24][25]. Notably, PE or PHs also encourage plant activity of key enzymes involved either in N or C metabolism [12,24,26,27].…”

Changes in Biochemistry and Yield in Response to Biostimulants Applied in Bean (Phaseolus vulgaris L.)

“…Paul et al [118] found that tomato plants treated with protein hydrolysates were characterized by a higher content of, e.g., low-molecular-weight phenolic compounds, phytohormones (polyamines), hydroxy-carotenoids, poly-hydroxy fatty acids, and membrane lipids (glycoand phospholipids). They suggest that the metabolic changes caused by treating plants with protein hydrolysates can be correlated with a relatively small number of processes that converge toward the ROS-related (reactive oxygen species-related) plant signaling network.…”

“…They suggest that the metabolic changes caused by treating plants with protein hydrolysates can be correlated with a relatively small number of processes that converge toward the ROS-related (reactive oxygen species-related) plant signaling network. Increasing the content of secondary metabolites, such as phenols and carotenoids, which play a key role in protecting plants against oxidative stress [62,102,119], suggests fine-tuning of ROS signaling in plants after the application of protein hydrolysates [118]. In addition, the use of animal protein hydrolysates had a positive effect on the content of protein, phenols, and flavonoids in bananas [120], and vegetal protein hydrolysates stimulated an increase in the content of phenolic compounds and anthocyanins in grape [55].…”

Biostimulants Application in Horticultural Crops under Abiotic Stress Conditions

“…However, it is important to underline that the biostimulant activity must not depend on the product's nutrients or natural plant hormones content. The mechanisms activated by biostimulants are often difficult to identify and are still under investigation [58]. High-throughput phenotyping and omic technologies seem to be useful approaches to understand biostimulants activity and hypothesize a mode of action [59][60][61].…”