Polyamines as priming agents and biostimulants against plant abiotic and biotic stress

By 2050, global food demand is projected to increase by approximately 100–110 %, posing an unprecedented challenge to agricultural systems and requiring a substantial enhancement of crop productivity. Concurrently, climate change projections predict a global temperature rise of about 2 °C, which is expected to exacerbate a wide range of abiotic and biotic stresses, including drought, soil salinization, and increased pressure from pests and pathogens. Traditionally, modern agriculture has relied heavily on genetic improvement, chemical fertilizers, and pesticides to sustain crop yields. However, the growing urgency to reconcile food security with environmental sustainability has accelerated the development and adoption of eco-friendly and sustainable agronomic strategies. In this context, biostimulants (BSs) have emerged as promising tools to enhance plant resilience and mitigate yield losses under adverse environmental conditions. BSs improve plant performance by promoting nutrient uptake, modulating stress-responsive pathways, and supporting growth and development. Among the most promising BSs, polyamines (PAs), including putrescine (Put), spermidine (Spd), and spermine (Spm), play multifaceted roles in plant stress tolerance. These compounds function as osmoprotectants, scavengers of reactive oxygen species (ROS), regulators of gene expression, and signalling molecules. Moreover, PAs serve as precursors of hydrogen peroxide (H₂O₂), a key signalling component in plant stress-response networks. Cucumber (Cucumis sativus L.), one of the oldest domesticated vegetable crops, was selected as the model species for this study due to its economic relevance and marked sensitivity to soil salinity. Salinity was chosen as the primary abiotic stress factor, as it represents a major and increasingly restrictive constraint for cucumber cultivation worldwide. In addition to abiotic stress, cucumber production is severely limited by biotic challenges. The whitefly Bemisia tabaci (Bemisia) is a major phloem-feeding pest that reduces photosynthate availability, compromises plant vigor, and promotes the development of sooty mold through honeydew deposition, ultimately impairing photosynthetic efficiency. Furthermore, powdery mildew, predominantly caused by obligate biotrophic fungi such as Golovinomyces cichoracearum, constitutes another critical threat, particularly in protected cultivation systems where environmental conditions favor rapid pathogen proliferation and disease spread. Despite extensive knowledge of PAs as multifunctional biomolecules, their relative effectiveness as priming agents under agriculturally relevant abiotic and biotic stress remains poorly understood. In this study, the molecular, cellular, and biochemical mechanisms underlying plant responses to salinity stress, insect herbivory, and fungal infection following exogenous PA application were investigated, using A. thaliana as a model species and C. sativus as a crop plant. 5 Experimental activities were conducted on seeds, seedlings, and adult plants across three research sites: Roma Tre University (Rome, Italy), C.R.O. Agrigeos S.r.L. (Acireale, Italy), and the School of Biosciences at Cardiff University (Cardiff, UK). This multi-site experimental framework enabled the integration of complementary expertise and infrastructures, thereby enhancing the robustness, reproducibility, and translational relevance of the results. The first objective of this study was to evaluate the contribution of PA seed priming to to stress tolerance during germination and early seedling development under salinity conditions. In Arabidopsis, seed priming with 1 mM Put consistently improved the germination rate (GR) and germination index (GI) under both optimal growth conditions and severe salinity stress (200 mM NaCl). Notably, the beneficial effects of 1 mM Put were maintained under saline conditions, resulting in enhanced germination kinetics during the early phases following seed imbibition. To further investigate the molecular mechanisms underlying the protective effect of 1 mM Put during germination and based on the optimal efficacy observed for the 1 mM Put treatment in the previous experiments, germination parameters in loss-of-function Atcuao mutant lines were analysed. These mutants carry loss-of-function mutations in genes encoding copper amine oxidases (CuAOs), key enzymes involved in PA oxidation, specifically in Put catabolism. This genetic impairment results in altered Put turnover, thereby allowing the assessment of how altered Put oxidation influences salt stress tolerance during early developmental stages. Specifically, loss-of-function mutants of AtCuAOβ (Atcuaoβ.2), AtCuAOγ1 (Atcuaoγ1.1), and AtCuAOγ2 (Atcuaoγ2.1) were examined. Data suggested that CuAO-dependent Put catabolism is required for the proper regulation of germination under both optimal and saline conditions. Under control conditions, Atcuaoγ1 and Atcuaoγ2 displayed altered germination dynamics and reduced germination efficiency compared to WT. Under salinity stress, these mutants exhibited increased sensitivity, characterized by delayed and incomplete germination. Notably, seed priming with 1 mM Put failed to restore germination performance in the Atcuao mutants and instead further exacerbated stress sensitivity. These findings indicate that unaltered Put oxidation is essential for the beneficial priming effect observed in WT plants, highlighting a critical role for CuAO-mediated Put catabolism in the regulation of germination under saline conditions. Experiments were then conducted on cucumber seeds, to evaluate the agricultural relevance of PA priming. Seed priming with 1 mM Put significantly mitigated the detrimental effects of severe salinity (200 mM NaCl), resulting in increased GR and final GI compared to unprimed stressed seeds. In contrast, priming with Spd and Spm, although less effective or even inhibitory of Put during germination, selectively promoted early root growth in Arabidopsis seedlings, more than Put priming. This observation highlights a developmental stage– and response-specific role of distinct PAs. 6 Collectively, these findings identified Put as the most effective priming agent during germination, thereby providing a strong rationale for its further evaluation in adult plants. The second objective of this study focused on the effects of Put priming on salinity tolerance in adult cucumber plants. Foliar application of 0.5 mM Put was selected based on preliminary dose response analysis and significantly mitigated salt-induced reductions in root growth, plant height, and total biomass, while also limiting salt-induced foliar damage. Physiological analysis revealed a recovery of photosynthetic efficiency, improved plant water status, maintenance of chlorophyll content, and enhanced stomatal regulation. Furthermore, biochemical and molecular analysis indicated reduced oxidative damage, increased antioxidant capacity, and modulation of stress responsive gene expression. Importantly, long-term agronomic assessments demonstrated significant improvements in fruit length and weight under salinity stress. The third objective of this study investigated PA-mediated resistance to Bemisia. Pre infestation treatments revealed a strong and persistent protective effect of foliar spray with 5 mM Put , which markedly reduced whitefly population density, delayed insect development, and increased mortality. further confirmed that Put retained protective activity even when applied after pest establishment. These effects were associated with an improved accessory photosynthetic pigment content in Put-treated plants and a marked alteration of their volatilome, as revealed by TD–GC×GC TOF–MS analysis. Indeed, Put priming induced distinct volatile organic compound (VOC) emission patterns, enhancing specific secondary metabolites involved in pest-defence signalling. Surprisingly, VOC-mediated effects were also observed in neighbouring Put-untreated plants, demonstrating that treatment of a single plant with Put is sufficient to confer indirect protection against pest infestation to adjacent, untreated plants. The fourth objective of this study investigated PA-mediated defense against powdery mildew pathogen G. cichoracearum. Among the tested PAs, 1 mM Spd emerged as the most effective priming agent, significantly reducing fungal incidence and colony expansion, achieving protection levels like the fungicide azoxystrobin. These findings confirm that PA-induced resistance is stress specific and that different PAs preferentially activate distinct defence pathways. Overall, this research systematically evaluated targeted PA–stress combinations to identify optimal priming strategies for major agricultural constraints, including soil salinity, insect herbivory, and fungal disease. The results establish Put, at optimized concentrations, as a robust and versatile BS capable of enhancing stress tolerance, physiological performance, and potentially crop yield. By integrating molecular, physiological, and agronomic analysis, this work provides a solid scientific foundation for the development of sustainable PA-based strategies aimed at improving crop resilience and supporting global food security.