Understanding the biostimulant action of vegetal-derived protein hydrolysates by high-throughput plant phenotyping and metabolomics: A case study on tomato

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

Effects of vegetal- versus animal-derived protein hydrolysate on sweet basil morpho-physiological and metabolic traits

Despite scientific evidence supporting the biostimulant activity of protein hydrolysates (PHs) derived from vegetal or animal sources, the morpho-physiological and biochemical mechanisms underlying the biostimulant action of PHs from plant biomass or animal by-products are still poorly explored. Accordingly, we performed a greenhouse experiment for assessing the morphological, physiological and biochemical responses of sweet basil (Ocimum basilicum L.) to three nitrogen equivalent rates (0.05, 0.15, and 0.25 g N/kg) of an animal-derived protein hydrolysate (A-PH) and a vegetal-derived protein hydrolysate (V-PH). The V-PH and A-PH applications determined a quadratic-dose response regarding the number and area of leaves and the shoot fresh and dry weight, with the best results obtained using V-PH at the N equivalent rates of 0.05 and 0.15 g N/kg. Improvement of shoot fresh weight with V-PH foliar application at the rate of 0.15 g N/kg was associated with a higher leaf CO2 assimilation and water use efficiency, with a concomitant higher uptake and translocation of K, Mg, and S in leaf tissue. The excessive accumulation of Na, Cl, and some amino acids (e.g., proline) under A-PH applications above 0.05 g N/kg induced a rapid decrease in plant photosynthetic performance, growth, and biomass production. The plants treated with A-PH at a higher dosage appeared to activate an alternative pathway involving the synthesis of alanine and GABA for storing excess ammonia, buffering cytoplasmic acidosis, and counteracting the negative effects of Na and Cl at toxic levels. The above findings demonstrated the potential benefits of protein hydrolysate application in agriculture, especially of vegetal-derived PHs, and highlighted the need to understand dose-dependent effects in order to optimize crop response

Evaluation of a Legume-Derived Protein Hydrolysate to Mitigate Iron Deficiency in Plants.

Biostimulants play an important role in the development of management practices able to reach adequate productivity to meet the food demand of a growing world population, while following a sustainable agriculture model. This work aims to evaluate the effect of a protein hydrolysate derived from legume seeds by enzymatic hydrolysis on plant growth and also to verify its ability to mitigate Fe deficiency, a widespread problem significantly limiting plant growth and crop productivity. Experiments were performed with tomato (Solanum lycopersicum L.-cv. AKRAI F1) and cucumber (Cucumis sativus L.-cv. EKRON F1). The plants were grown hydroponically under adequate or limited Fe supply. Changes in shoot and root fresh weight, leaf relative chlorophyll content and the accumulation of macro- and microelements in shoots and roots were measured. Plant ability to cope with Fe deficiency was measured by evaluating the activity of root Fe3+-chelate reductase. Our results indicate that the foliar treatments with the protein hydrolysate did not significantly affect growth parameters when plants were grown in full nutrient solution. However, the biostimulant was able to improve the growth performance of Fe-deficient plants. Therefore, the protein hydrolysate can be a powerful tool to stimulate crop growth under Fe-deficient environments, leading to reduced fertilizer inputs with related environmental and economic benefits

Imidazolo [1,2-a] e 1,2,4-triazolo[4,3-a] chinossaline analoghe degli antifolici metotrexato e trimetrexato

Abbiamo progettato una nuova serie di chinossaline, nelle quali l’anello pirrolico è stato sostituito con un’anello imidazolico o con uno triazolico lasciando nelle posizioni 2, 5, 6, 7 e 8 dell’anello chinossalinico gli stessi sostituenti precedentemente esaminati. Di questi composti verranno descritti la sintesi e i risultati farmacologici relativi alla loro attività

Protein Hydrolysates from Animal or Vegetal Sources Affect Morpho-Physiological Traits, Ornamental Quality, Mineral Composition, and Shelf-Life of Chrysanthemum in a Distinctive Manner

Protein hydrolysates (PHs) are a prominent category of plant biostimulants, mainly constituted of amino acids, oligopeptides and polypeptides, obtained by partial hydrolysis of animal or plant protein sources. Despite scientific evidence supporting the biostimulant action of PHs on vegetables, the morphological, physiological, and shelf-life performances underlying the PH action on cut flowers are still poorly explored. Accordingly, the aim of this research is to assess the effects of three commercial biostimulants, one animal PH (PH A, Hicure®) and two plant PHs (PH V1, Trainer®and PH V2, Vegamin©), on two chrysanthemum (Chrysanthemum morifolium) cultivars (Pinacolada and Radost). In both cultivars, only the plant-derived PH (V1 and V2) treatments recorded significantly higher fresh plant biomass than the control (on average +18%, in both cultivars). The foliar application of the vegetal-derived PHs but not the animal one, particularly in Pinacolada, improved the status of plants, stimulating stem elongation and the apical flower diameter. In Pinacolada, applications with PH V1 resulted in a significant increase in nitrate and P concentration in leaves and Ca content in flowers compared with the control (+43%, +27%, and +28% for nitrate, P, and Ca, respectively). In Radost, PH A and PH V2 applications caused a significant reduction in nitrate concentration in both leaves and flowers compared with the control. One week after harvest, in both cultivars, PH A applications caused flower stems to wilt faster than the control. In contrast, plants treated with PH V1 revealed significantly slower flower stem senescence compared to the control. Flower wilting during vase life was correlated to a decrease in the K-to-Na ratio in flowers due to an inability to transport K to the flowers from the leaves rather than an increase in Na in the flowers themselves

Combining Molecular Weight Fractionation and Metabolomics to Elucidate the Bioactivity of Vegetal Protein Hydrolysates in Tomato Plants

The comprehension of the bioactive fractions involved in the biostimulant activity of plant derived protein hydrolysates (PH) is a complex task, but it can also lead to significant improvements in the production of more effective plant biostimulants. The aim of this work is to shed light onto the bioactivity of different PH dialysis fractions (PH1 < 0.5\u20131 kDa; PH2 > 0.5\u20131 kDa; PH3 < 8\u201310 kDa; PH4 > 8\u201310 kDa) of a commercial PH-based biostimulant through a combined in vivo bioassay and metabolomics approach. A first tomato rooting bioassay investigated the auxin-like activity of PH and its fractions, each of them at three nitrogen levels (3, 30, and 300 mg L 121 of N) in comparison with a negative control (water) and a positive control (indole-3-butyric acid, IBA). Thereafter, a second experiment was carried out where metabolomics was applied to elucidate the biochemical changes imposed by the PH and its best performing fraction (both at 300 mg L 121 of N) in comparison to water and IBA. Overall, both the PH and its fractions increased the root length of tomato cuttings, compared to negative control. Moreover, the highest root length was obtained in the treatment PH1 following foliar application. Metabolomics allowed highlighting a response to PH1 that involved changes at phytohormones and secondary metabolite level. Notably, such metabolic reprogramming supported the effect on rooting of tomato cuttings, being shared with the response induced by the positive control IBA. Taken together, the outcome of in vivo assays and metabolomics indicate an auxin-like activity of the selected PH1 fraction

Protein Hydrolysates from Animal or Vegetal Sources Affect Morpho-Physiological Traits, Ornamental Quality, Mineral Composition, and Shelf-Life of Chrysanthemum in a Distinctive Manner

Protein hydrolysates (PHs) are a prominent category of plant biostimulants, mainly constituted of amino acids, oligopeptides and polypeptides, obtained by partial hydrolysis of animal or plant protein sources. Despite scientific evidence supporting the biostimulant action of PHs on vegetables, the morphological, physiological, and shelf-life performances underlying the PH action on cut flowers are still poorly explored. Accordingly, the aim of this research is to assess the effects of three commercial biostimulants, one animal PH (PH A, Hicure®) and two plant PHs (PH V1, Trainer® and PH V2, Vegamin©), on two chrysanthemum (Chrysanthemum morifolium) cultivars (Pinacolada and Radost). In both cultivars, only the plant-derived PH (V1 and V2) treatments recorded significantly higher fresh plant biomass than the control (on average +18%, in both cultivars). The foliar application of the vegetal-derived PHs but not the animal one, particularly in Pinacolada, improved the status of plants, stimulating stem elongation and the apical flower diameter. In Pinacolada, applications with PH V1 resulted in a significant increase in nitrate and P concentration in leaves and Ca content in flowers compared with the control (+43%, +27%, and +28% for nitrate, P, and Ca, respectively). In Radost, PH A and PH V2 applications caused a significant reduction in nitrate concentration in both leaves and flowers compared with the control. One week after harvest, in both cultivars, PH A applications caused flower stems to wilt faster than the control. In contrast, plants treated with PH V1 revealed significantly slower flower stem senescence compared to the control. Flower wilting during vase life was correlated to a decrease in the K-to-Na ratio in flowers due to an inability to transport K to the flowers from the leaves rather than an increase in Na in the flowers themselves

A simple and accurate allometric model to predict single leaf area of twenty-one European apricot cultivars

Research in fruit tree physiology and breeding often requires accurate and non-destructive methods for estimating leaf area (LA). The development of unbiased allometric model from linear measurements [leaf length (L) and/or width (W)] to predict individual LA of apricot irrespective of cultivars is still lacking. The models were built using LA, L, and W data measured in 3,040 leaves collected on trees of nineteen apricot cultivars (calibration experiment). Model(s) were validated on 520 apricot leaves collected from the trees of two additional cultivars (validation experiment). LA prediction models based only on L measurements (L or L2) were not suitable for estimating LA of apricot. A significant improvement in LA prediction was observed when the model including W2 as an independent variable was adopted. However, the coefficients of one dimension LA model (W2) were affected by leaf shape (L:W ratio) and consequently were excluded. To develop an accurate LA model for apricot, independent of leaf shape groups, the product L×W was used as an independent variable. The linear model LA = 1.193 + 0.668 (L×W) exhibited the highest R2, the smallest mean square error (MSE) and predicted residual error sum of squares (PRESS). In the model validation, correlation coefficients showed that there was a highly reliable relationship between the predicted and the observed LA values, giving an underestimation of 2.9% in the prediction. The LA model using LW as independent variable can be successfully adopted in research on apricot, since it provides an accurate, simple and non-destructive estimation of LA across apricot cultivars without the use of any expensive device