Biostimulant mediated modulations of physiological nutritional and yield traits in Oryza sativa L under nutrient deficiency A metabolomic perspective

Abstract

The utilization of chemical fertilizers presents substantial risks to the ecosystem andnovel biostimulant formulation serves as a multifaceted approach, for sustainableagricultural system. Characterizing new biostimulants, understanding howbiostimulants work, and developing efficient methods for abiotic stress tolerance isvery important. Two rice varieties (Hraswa and Manu ratna) were selected forevaluation based on their performance in nutrient-deficient conditions in response tothe application of different biostimulants. The experimental design was structuredacross four developmental stages—seedling, tillering, booting, and ripening—tocapture dynamic changes in physiology and metabolism. At each stage, parameterssuch as carbohydrate translocation, metabolite allocation, reactive oxygen species(ROS) generation, antioxidant enzyme activity, and chlorophyll a fluorescencebased physiological aspects were systematically assessed. The stage-wise evaluationprovided a comprehensive understanding of how biostimulant treatment modulatedplant responses under stress at different stages of its lifecycle. Among the fourstages, the booting phase emerged as particularly critical, showing pronouncedchanges in physiological performance and metabolic regulation. The booting stagemarks a crucial shift from vegetative to reproductive growth in cereals like rice. Thedevelopmental sequences that ultimately determines spikelet fertility and yieldpotential occurs at this period, when the panicle primordia rapidly differentiate andelongate within the flag leaf sheath. Numerous studies have emphasized thephysiological importance of this stage as plants are more vulnerable to abioticstressors such as temperature extremes, water shortages, and nutritional deficienciesduring this growth phase. Exposure to stress during booting can cause irreversibleyield loss by interfering with pollen viability, assimilate partitioning, and floralorgan development. Furthermore, the booting period marks the peak demand formineral elements, especially potassium and nitrogen, which are essential for thetransfer of carbohydrates and the synthesis of proteins. During this phase, lack ofnutrients results in sterility and inadequate grain filling. Booting stage analyses mayalso be used as a diagnostic window to evaluate source-sink dynamics, translocationpatterns, and nutrient absorption efficiency in both ideal and stressful conditions.Therefore, detailed analyses such as gas exchange measurements and ICP-MS-basednutrient profiling were carried out at this stage. Subsequently, yield traits and FTIRspectroscopy were employed to examine grain development and functional groupmodifications of leaf metabolites subjected to biostimulant application revealed thatamong the both varieties studied, the var. Manu ratna appeared more sensitive tonutrient deficiency, however biostimulant treatment induced more structural andbiochemical changes that improved its tolerance.Based on the observations, Manu ratna was selected for further in-depthmetabolomic investigations, since it displayed greater sensitivity to nutrientdeficiency but also exhibited marked improvement when treated with thebiostimulant. Advanced analytical approaches including GC-MS/MS, HR-LCMS,and amino acids profiling were employed to characterize metabolic shifts induced bybiostimulant application. These analyses revealed clear alterations in the abundanceof primary metabolites, secondary compounds, and amino acid pools, underscoringthe capacity of biostimulant treatment to reprogram central metabolic pathwaysunder nutrient deficient conditions. Metabolomic profiling of rice under control,stress, and biostimulant treatments provided crucial insights into the biochemicalreprogramming that accompanies stress adaptation and the ameliorative effects ofbiostimulants. Using complementary platforms (GC-MS/MS, HRLC-MS and aminoacids profiling), a wide range of metabolites—including amino acids, organic acids,fatty acids, sugars, and secondary metabolites—were identified, revealing distinctsignatures across treatments. The integration of multivariate analyses (PCA, PLS-DA, VIP scores, SAM) and pathway enrichment further highlighted treatment-specific metabolic regulation, underscoring the importance of biostimulants inrestoring metabolic balance under treatments. The findings underscore the potentialof tailored biostimulant formulations to optimize nutrient release, enhance plantresilience, and improve agricultural sustainability. This study establishes afoundational framework for the strategic design and application of biostimulantsacross diverse crops and agroecological conditions, supporting innovative andsustainable agricultural practices.