Maize (Zea mays L.) is the world’s third most significant cereal crop, after wheat and rice.  It can be grown in a wide range of soil types and climates. Maize is extremely susceptible to drought, especially in the early stages of development, even though it has a C4 photosynthetic pathway that allows for excellent water usage efficiency. The uptake of water and nutrients is significantly influenced by the morphological and biochemical properties of roots. However, in drought conditions, root growth is frequently severely hindered, which limits cell division, elongation and nutrient uptake, thereby decreasing overall plant vigor and resilience (Chassot and Richner, 2002).
       
Seed priming with phytohormones and polyamines has aroused as a promising and cost-effective approach for improving seedling establishment and enhancing root development under drought stress conditions. Various phytohormones, including melatonin, serotonin, salicylic acid and brassinolides, play important roles in stimulating cell expansion, promoting lateral root formation and increasing water absorption through mechanisms similar to auxin signaling. In addition, polyamines such as spermine and spermidine contribute to stress tolerance by maintaining cellular homeostasis, strengthening antioxidant defense systems and regulating osmotic balance (Sarropoulou et al., 2012). Several current studies have demonstrated the effectiveness of these compounds in improving drought tolerance in maize. For instance, melatonin-based seed priming has been shown to alleviate oxidative damage, increase the activity of antioxidant enzymes and improve seedling biomass under polyethylene glycol (PEG)-induced water stress conditions (Cao et al., 2021). Likewise, spermidine treatment has been reported to enhance root growth characteristics, including root length, surface area and overall root vigor, thereby improving maize seedling performance under both controlled and field drought environments (Dong et al., 2022).
       
Recent evidence provided by  Manavalagan et al., (2023) further supports the beneficial role of seed priming in drought stress management. The study showed that priming treatments alleviated the inhibitory effects of polyethylene glycol (PEG)-induced water deficit on maize germination and seedling vigor, emphasizing the value of priming techniques for promoting robust early growth and improving drought resilience in maize.
       
Despite these promising results, studies associated to compare the relative efficacy of phytohormones and polyamines in improving root morphology and root biochemical traits under PEG-induced drought stress remains limited. Addressing this knowledge gap, the present research was undertaken to assess and compare the impact of phytohormones (Melatonin, serotonin, salicylic acid, brassinolides) and polyamines (Spermidine, spermine) on root morphological and root biochemical responses of maize seedlings exposed to PEG-induced water deficit stress (Fig 1).

The current study used genetically pure COH(M) 8 hybrid maize seeds as its experimental material and was carried out at Tamil Nadu Agricultural University, Coimbatore during 2023-2024. In vitro testing was used to assess how seed priming affected the root morpho-architectural characteristics and root biochemical activities of COH(M)8 during drought stress under temperature 25±2°C with photoperiod 16 h light and 8 h darkness. The light intensity used was 1200 lux with RH OF 60-70 %. Drought stress was simulated in the laboratory using PEG 6000 (polyethylene glycol). To standardize the stress level, different osmotic potentials viz., -0.2 MPa, -0.4 MPa, -0.6 MPa, -0.8 MPa, -1.0 MPa were tested. The ideal osmotic potential for causing drought stress was determined to be -0.4 MPa based on 50% seedling mortality seen on seventh day (Manavalagan et al., 2023).
       
The optimum concentrations of 6 seed priming compounds were standardized by testing different levels of each compound melatonin (50 µM, 100 µM, 150 µM, 200 µM), serotonin (50 µM, 100 µM, 150 µM, 200 µM), spermine (50 µM, 100 µM, 150 µM, 200 µM), spermidine (50 µM, 100 µM, 150 µM, 200 µM), salicylic acid (50 ppm, 75 ppm, 100 ppm, 150 ppm) and brassinolides (0.1, 0.2, 0.5, 1.0 ppm). The optimized concentrations were melatonin (150 µM), serotonin (100 µM), spermine (50 µM), spermidine (50 µM), salicylic acid (100 ppm) and brassinolides (0.5 ppm), which is selected based on highest germination percentage and seedling vigour in comparison with hydropriming and non-primed control.  To evaluate the influence of seed priming on root morpho-architecture and root biochemical responses under drought stress, seeds primed with the optimized concentrations of phytohormones and polyamines were subjected to simulated drought conditions (-0.4 MPa PEG 6000). 
       
A factorial completely randomized design (FCRD) with four replications was used for the experiment. Melatonin (150 µM), serotonin (100 µM), spermine (50 µM), spermidine (50 µM), salicylic acid (100 ppm) and brassinolides (0.5 ppm) and distilled water (hydropriming) were used to prime 100 surface sterilized seeds for 12 hours using a 1:1 weight by volume seed to solution ratio. After priming, the seeds were cleaned with distilled water and allowed to dry in the shade at room temperature in order to restore their initial moisture content (Manavalagan et al., 2025).
       
Primed seeds with optimal concentrations employing phytohormones and polyamines were subjected to drought stress conditions in order to investigate the impact of seed priming on root morphological architecture and root biochemical activity under drought stress.
       
The primed and control (non-primed) seeds were placed on germination paper to allow for radicle emergence. Once radicle emergence occurred, uniformly germinated seedlings were carefully transferred on a sponge inside a 180 ml test tube, which contained the Hoagland nutrient solution with simulated drought stress by adding PEG 6000 of -0.4 MPa. The seedlings were allowed to grow for 15 days in the nutrient medium and each treatment was replicated for five times.
       
15 days after sowing, observations on root morphological architecture parameters viz., length, fresh and dry biomass, depth, width, maximum number of roots, root bushiness, root area, root volume was observed using GiA roots software (Fig 2), which is an acronym for general image analysis of roots, which was used to automate and simplify the thorough examination of root networks (Galkovskyi et al., 2012). Also the root biochemical assays included root activity (mg g^-1 h^-1 FW) (Comas et al., 2000), root carbohydrate content (g glucose equiv. g^-1 DW) (Nelson, 1944; Somogyi, 1926), root membrane leakage (%) (Huang et al., 2005), root invertase (nmol g^-1 FW), root sucrose synthase (µmol g^-1 FW) (Appeldoorn et al., 1997) and root ATPase activity (mg g^-1) (Forbush, 1983) were experimented.


 
Statistical analysis
 
The R-based program GRAPES (v1.0.0) was used to statistically analyze root morpho architectural and root biochemical characteristics. Analysis of variance (ANOVA) and Pearson’s correlation coefficients between the attributes were evaluated. ANOVA was led to assess treatment effects.  Mean comparisons under drought stress conditions were carried out using post-hoc tests-duncan’s multiple range test (DMRT) to find significant variances between treatments.

The outcomes of the present investigation revealed that, the root morphological architecture and root biochemical activity of maize hybrid COH(M) 8 under simulated drought stress conditions were considerably impacted by seed priming with various phytohormones and polyamines. Drought stress adversely affected root growth and metabolic activities of maize seedling. However, considerable variation was observed among the treatments in their ability to mitigate stress effects. For every root growth and root biochemical parameter, our analysis of variance and mean comparison data amply demonstrated the variations between the priming treatments (Fig 3).


       
Using specific phytohormones and polyamines to prime seeds, markedly alleviated osmolyte [PEG] simulated drought stress inhibition of maize root growth, with melatonin (150 µM) which preserved the root morphological and root biochemical attributes even when there is water deficit stress condition (Singh et al., 2020). Among the various treatments, melatonin primed seeds recorded the highest root length (28.9 cm), fresh root weight (0.845 g) and dry root weight (0.121 g), followed closely by serotonin (100 µM) (27.6 cm, 0.811 g and 0.093 g, respectively), whereas control recorded comparatively lower values of (18.5 cm, 0.324 g and 0.021 g) (Fig 4 and 5), indicating that melatonin enhanced both elongation and biomass accumulation; this likely reflects melatonin’s auxin-like activity that promotes cell elongation and cell wall loosening, thereby facilitating root extension and mass gain (Bleiss and Ehwald, 1993; Mukherjee et al., 2014).  Serotonin also played a crucial role in promoting root elongation and lateral branching (Pelagio-Flores et al., 2011). Correspondingly, melatonin primed seeds also developed greater root depth (3452 cm) and width (3.51 cm) followed by serotonin treated seeds (3316 cm, 3.23 cm), while the control exhibited the lowest values (2291 cm, 1.98 cm) (Fig 6 and 7), which is consistent with melatonin enhancing root penetration and lateral expansion through modulation of auxin transport and root meristem activity (Hernández-Ruiz and Arnao, 2008). Root branching traits were similarly enhanced with melatonin which is observed with maximum root number (753) and root bushiness index of (1.52), followed by serotonin (745, 1.48 respectively). In contrast, the control seedlings had the lowest root number (532) and bushiness (1.37) (Fig 6 and 7). This is also again, a pattern that supports the hypothesis that both melatonin and serotonin influence auxin-mediated lateral root initiation and subsequent branching patterns (Hernández-Ruiz and Arnao, 2008; Pelagio-Flores et al., 2011). 








       
In addition, enhanced overall root architecture in melatonin primed seedlings exhibited maximum root area (132.5 cm²) and root volume (1.35 cm³), followed by serotonin. These improvements suggest a robust root system with enhanced water and nutrient acquisition capacity (Manonmani et al., 2025) (Table 1) (Fig 7), which implies improved soil exploration capacity and potential water and nutrient uptake, mirroring stimulatory effects reported for melatonin in other species such as cucumber, cherry and sunflower (Mukherjee et al., 2014; Sarropoulou et al., 2012). Seed priming treatments showed significant influence on root biochemical parameters under drought stress (Fig 4-7).


       
Melatonin and serotonin priming maintained higher metabolic and stress buffering activity of roots under drought. Thus, melatonin (45.5 mg g^-1 h^-1 FW) and serotonin (43.2) recorded the highest root activity, compared to control (25.1) which indicated better metabolic activity and nutrient uptake efficiency and also indicating greater respiratory and nutrient-uptake capacity likely driven by preserved mitochondrial function and enhanced energy metabolism in primed roots (Altaf et al., 2022) (Fig 8). Melatonin primed roots accumulated more carbohydrate content (0.088 g glucose equiv. g^-1 DW), followed by serotonin (0.085), suggesting that priming improved osmotic adjustment and carbon reserve availability under water deficit, which supports continued growth and osmoprotection (Fig 9) (Zahedi et al., 2024).




       
Membrane integrity was improved evidence by lower electrolyte leakage in melatonin (1.15%) and serotonin (1.18%) compared to control (2.11%) which points to better preservation of cellular membranes, likely via enhanced antioxidant defenses and stabilization of membrane lipids, a mechanism frequently proposed for both melatonin and polyamines (Altaf et al., 2022) (Fig 9). Enzyme activities associated with carbohydrate metabolism and ion transport were also elevated in melatonin treatments (root invertase, root sucrose synthase and root ATPase activity of about 5.32 nmol g^-1 FW, 5.14 µmol g^-1 FW and 0.89 mg g^-1, respectively) with lower activities in control, indicating the priming sustained enzymatic systems that mobilize sugars and maintain ionic gradients required for growth under stress. Such upregulation in root enzymatic activity enhances sink strength of roots and supports osmotic and energetic demands during drought (Fig 10).   


 
Correlation analysis of root morpho architectural and root biochemical traits
 
Correlation analysis showed that root architectural and biochemical characteristics work together to control drought tolerance in maize (Fig 11). A broad root system enhances soil exploration, water extraction and nutrient acquisition during moisture shortage, as evidenced by the substantial positive connections found between root length and root biomass, depth, width, bushiness, root number, volume, ATPase activity and carbohydrate content. Plant water status in maintained and increased biomass accumulation is supported by this coordinated root development (Lynch, 2018; Uga et al., 2013).


       
By promoting ion transport, nutrient uptake and osmotic adjustment, ATPase activity positively linked with root growth and preserved cellular metabolism during drought. A higher carbohydrate content supported root growth by providing energy and osmoprotection (Ruan, 2014). On the other hand, elevated invertase activity, sucrose synthase activity and membrane leakage hindered root growth by compromising carbon use, membrane stability and metabolic efficiency. In general, after melatonin and serotonin seed priming, increased ATPase activity and carbohydrate buildup improved root functioning and drought resistance in maize.
       
Although PEG-induced osmotic stress is a reliable and reproducible approach for simulating drought under controlled conditions, it does not fully represent the complexity of natural field drought. Therefore, the beneficial effects of phytohormone and polyamine seed priming observed in this study should be validated under greenhouse and field conditions. Nevertheless, the improved root architecture, carbohydrate metabolism, membrane stability and antioxidant defense indicate the potential of these priming agents as cost-effective seed enhancement technologies and provide useful physiological traits for breeding drought-resilient maize cultivars.

The current study concludes that seed priming with phytohormone and polyamines particularly melatonin and serotonin effectively improve the root morphological architecture and root biochemical activities of maize seeds even under drought stress. Therefore, future research should concentrate on clarifying the molecular mechanisms such as the signaling pathways and gene regulatory networks involved in drought stress responses that underlie the advantageous effects of these priming agents. Validation of these findings under different soil types, climatic conditions and field environments will also be essential to assess their practical applicability in agricultural production systems. Furthermore, investigating the combined effects of different phytohormones and polyamines, together with the use of advanced root phenotyping and imaging technologies, may provide deeper insights into the mechanisms of drought adaptation and facilitate the development of maize cultivars with enhanced drought resilience.   

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.