Fig. 1. Experimental plots placed on the benches in correspondence of the barcodes detected by the PlantEye F500 – Multispectral 3D laser scanners.

In this study, a greenhouse experiment was conducted to evaluate the morpho-physiological and metabolic responses of tomato plants treated with two PHs derived from Malvaceae (PH1) and Fabaceae (PH2) under repeated drought stress cycles. The goal was to investigate the mechanisms through which these biostimulants enhance drought tolerance in tomato plants. Fertigation was carried out once after transplanting and once at mid-crop cycle. Plants were grown using a drip irrigation system and subjected to four drought stress cycles, with irrigation stopped until visible wilting occurred. PH1 and PH2 were applied as weekly foliar sprays and compared to control plants sprayed with distilled water. A high-throughput phenotyping platform (PlantEye F500, Phenospex) was used to monitor digital biomass, 3D leaf area, and plant height, alongside spectral indices (NDVI, NPCI, PSRI, GLI, Hue°) to assess plant health. At the end of the trial, leaf samples were collected for metabolomics analysis to investigate biochemical changes linked to drought stress resilience induced by PH treatments.

Fig. 4. Slopes of the linear regression model (y = ax +b where a is the slope and b is the y-intercept) describing the relationship between Digital Biomass (y) and days (x) of the recovery period upon re-irrigation after each water stress event. PH1 = Malvaceae-derived protein hydrolysate; PH2 = Leguminosae-derived protein hydrolysate. Bars indicate standard errors of the means.

Phenotyping analysis revealed that PH1 improved plant recovery following drought stress, particularly in terms of digital biomass, 3D leaf area, and plant height. In addition to the positive correlation with plant height, the strong relationship between digital biomass and 3D leaf area indicated that leaf expansion was the main driver of biomass accumulation under stress conditions. While PH2 had a temporary positive effect during the first drought event, its performance weakened in following stress cycles, making it more like the control. Regression analysis confirmed that PH1-treated plants had the highest recovery rates, with slopes 48-75% higher than untreated plants across all stress events. This suggests that PH1 enhanced plant resilience and growth restoration capacity.
Spectral indices allowed for a better understanding of plant health throughout the crop cycle. NDVI values, associated with photosynthetic activity and plant health, were higher in PH1-treated plants, indicating improved stress tolerance. NPCI and PSRI, which are linked with chlorophyll content and leaf senescence, increased over time, indicating a natural chlorophyll accumulation that matched with NDVI results. GLI and Hue°, which were always in line with NDVI, strongly corresponded to reflectance in the green band (GLI) and green range (75:135, Hue°).

Fig. 6. Chemical enrichment analysis (ChemRICH) of statistically different annotated metabolites in Malvaceae-derived protein hydrolysate (PH1) treated leaves compared to control treatment at the end of the trial. Red dots = up-regulated metabolites; blue dots = down-regulated metabolites; purple dots = up-/down-regulated metabolites.

Metabolomics analysis identified several metabolites significantly influenced by PH1 treatment. Among these, dipeptides (e.g., Leu-Phe, PyroGlu-Val, Arg-Phe) showed the highest accumulation in PH1-treated plants. These compounds are associated with nitrogen reallocation, osmoprotection, and metabolic regulation under stress. Dipeptides containing glucogenic amino acids (e.g., Arg-Leu, Val-Pro) likely acted as carbon source, mitigating carbohydrate starvation caused by reduced photosynthesis during drought stress. PH1-treated plants also exhibited lower levels of oxidative stress markers, such as (R)-S-lactoylglutathione and scopolin, suggesting that dipeptides played a role in reducing ROS damage. The accumulation of phenolic compounds and fatty acids further indicated that PH1 promoted stress adaptation through antioxidant pathways.
This experiment demonstrates that PH1 is an effective biostimulant for enhancing drought stress recovery in tomato plants, primarily by promoting faster recovery, and modulating key metabolites involved in stress tolerance. The results suggest that dipeptides play a crucial role in mitigating water stress effects and maintaining metabolic balance. Future research should investigate whether PH1-derived peptides are directly absorbed by plants or promote endogenous peptide synthesis. The study additionally highlights biostimulants’ potential to improve crop resilience under stressful conditions and offers important insights for future biostimulant development and screening.