Seed Priming with Nanoencapsulated Gibberellic Acid Triggers Beneficial Morphophysiological and Biochemical Responses of Tomato Plants under Different Water Conditions

Abstract

Water deficit (WD) promotes great losses in agriculture, and the development of new sustainable technologies to mitigate the effects of this stress on plants is essential. This study aimed to evaluate the morphophysiological and biochemical alterations induced by the priming of tomato seeds with different formulations in plants under field capacity and WD conditions. In the first experiment, the treatments consisted of nanoparticles of alginate/chitosan and chitosan/tripolyphosphate containing gibberellic acid (GA₃) in different concentrations (0.5, 5, and 50 µg mL⁻¹ GA₃), in addition to control with deionized water. The alginate/chitosan (5 µg mL⁻¹ GA₃) provided the greatest gains in plant growth under field capacity. In addition, under WD this treatment reduced damage to photosystem II (−14%), stomatal conductance (−13%), and water loss (−38%) and increased the instantaneous carboxylation efficiency (+24%) and intrinsic water use efficiency (+12%). In the second experiment, the treatments were alginate/chitosan nanoparticles containing GA₃ (NPGA₃ 5 µg mL⁻¹), free GA₃ (GA₃ 5 µg mL⁻¹), nanoparticles without GA₃ (NP), deionized water (WATER), and non-primed seeds (CONT). Under WD, GA₃ and CONT maintained plant growth and lost water rapidly, reducing stomatal conductance (−87%) and net photosynthesis (−69%). In contrast, NPGA₃ decreased leaf area (−44%) and increased root-to-shoot ratio (+39%) when compared to GA₃, reducing water loss (−28%). Activation of protective mechanisms (e.g., superoxide dismutase and catalase activities) by WATER, NPGA₃, and NP treatments also resulted in lower susceptibility to WD compared to CONT and GA₃. The results highlight the positive effect of seed priming on plant response to WD, which was enhanced by the use of nanoencapsulated GA₃.

1. Introduction

Climate change and anthropogenic activities are the main factors that compromise adequate food production. Drought, extreme temperatures, salinity, and low fertility are the most influential abiotic stresses on agricultural crops [1]. According to Wing et al. [2], climate change could reduce global production by 11–25% by the end of this century.

Among the stress factors amplified by climate change, water deficit causes the greatest reductions in productivity, resulting in a major impact on food production when compared to other abiotic stresses [3]. Under the effect of water deficit, plants show reduced growth and photosynthetic activity, resulting in lower dry mass and, consequently, lower productivity [4]. In response to water deficit, plants show morphophysiological modifications that make them more tolerant to this stressful condition [5].

In this scenario, the exogenous application of phytohormones and modulators of their homeostasis can be used to minimize the effects of different abiotic stresses on plants [6]. For example, external application of gibberellic acid (GA₃) has demonstrated the ability to alleviate some negative impacts of environmental stresses, resulting in enhanced growth, improved photosynthetic efficiency, increased enzyme activity, altered gene expression, higher nutrient uptake, and greater yields in diverse crops facing challenging conditions. Additionally, GA₃ interacts with other plant growth regulators, influencing various metabolic processes in plants [7,8]. The protective effects of GA₃ can be triggered by different application methods, such as foliar spray [9,10], addition to the substrate [11], and seed priming [12].

Seed priming is a pre-sowing treatment with hydration followed by dehydration of seeds without allowing radicle protrusion. This treatment has been proven to be a promising technique for inducing rapid and uniform germination, high seedling vigor, and better plant establishment, crop yield, and tolerance to abiotic stresses. Several methods of seed priming have been successfully used in agriculture, such as priming with water (hydropriming), plant growth regulators, osmoprotectants, plant extracts, and inorganic salts [13]. Previous studies have demonstrated the great potential of seed priming with GA₃ in improving germination, early growth, and tolerance mechanisms of different plants under stresses, such as nickel toxicity [14], low temperature [15], salinity [16], and water deficit [17,18,19].

Despite the advantages of using phytohormones to minimize the effects of stress, the application of these molecules has limitations, such as low physicochemical stability and high susceptibility to degradation processes, such as photolysis and hydrolysis, modifying their structure and decreasing the biological activity [20]. In addition, the difficulty of defining the correct dosage according to the purpose of application is one of the main limitations in the use of these products in the management of agricultural crops.

Within this context, nanotechnology has been considered a potential solution to different problems faced in modern agriculture, leading to the development of new products and/or the improvement of conventional ones. Agri-nanotechnology is a strategy to optimize crop production under different climate conditions through the use of nanopesticides, nanofertilizers, nanoherbicides, and nanosensors for improving soil fertility, plant growth, protection against pests, abiotic stress tolerance, and post-harvest management [21,22].

In particular, polymeric nanoparticles for carrying bioactive molecules have been used to create controlled release systems for agrochemicals, aiming to protect these bioactive molecules against rapid degradation [21,22,23]. In addition, polymeric nanocarriers can promote greater solubility and efficiency of agrochemicals (with the consequent reduction in the application dose), as well as a decrease in toxicity in non-target organisms [23]. The use of these systems for plant growth regulators can make them viable as a management strategy to improve plant tolerance to different types of stress [5,6,7,8,9,10,11,12,13,14,15,16,17,18,19]. Furthermore, nanoparticles themselves have been tested as a seed-priming agent (nanopriming), taking advantage of the unique properties of nanomaterials to enhance the benefits of seed priming [24]. Their use allows greater penetration into the seed coat to release active ingredients, in addition to providing higher absorption of water and nutrients by the seed [25].

Among the possibilities, chitosan-based polymeric nanoparticles have been developed as carrier systems for the controlled release of different compounds, such as nitric oxide donors for protection against saline stress [26], photoinhibition [27], water deficit [28,29,30], and heavy metals [31], and nanopriming with copper ions, for growth promotion and protection against abiotic [32,33,34] and biotic stresses [35]. Even in its conventional form, applying chitosan has protected plants against the deleterious effects induced by water deficit [36].

In recent years, Pereira et al. [37] developed chitosan-based nanoformulations for encapsulating GA₃, aiming at enhancing its physical–chemical properties and biological efficacy. The alginate/chitosan and chitosan/tripolyphosphate nanoformulations exhibited distinct characteristics in terms of size, surface charge, and GA₃ release, leading to different biological activities in common bean plants after seed treatment. Another study by Pereira et al. [38] demonstrated that the priming of tomato seeds with these nanoformulations not only improved plant growth but also enhanced fruit production. However, the morphophysiological and biochemical responses of plants promoted by seed priming with these nanoparticles have not yet been tested against water deficit.

Thus, the objectives of the current work were divided into two experiments: (i) to identify, through the evaluation of morphophysiological parameters, the formulation and ideal dose of chitosan-based nanoparticles for priming tomato seeds, aiming at promoting plant growth and reducing the susceptibility to water deficit; (ii) based on the results of the first experiment, to compare, at the ideal dose, the best nanoencapsulated GA₃ treatment (nanopriming) to the treatments with water (hydropriming), GA₃ (priming with phytohormone), and nanoparticles without GA₃ (nanopriming), to determine the morphophysiological and biochemical alterations induced by the respective treatments on plants. The hypothesis tested was that the combination of chitosan nanoparticles and GA₃ would result in a synergistic effect, standing out among other types of priming, with more prominent effects on plant growth and tolerance to water deficit.

2. Materials and Methods

2.1. Preparation of Formulations

The alginate/chitosan (ALG/CS) nanoparticles were prepared according to the ionotropic pre-gelation method proposed by Sarmento et al. [39]. With the aid of a peristaltic pump, 3.75 mL of CaCl₂ (18 mM) was added to 59 mL of an alginate solution (0.063%, pH 4.9) for 60 min, keeping the solution under strong magnetic stirring. During this stage of the process, cross-linking of CaCl₂ on alginate occurs through ionic interactions, forming a structure called eggbox. Still under strong agitation, 12.5 mL of chitosan solution (0.07%, pH 4.6) prepared in aqueous acetic acid solution (0.5%) was added for a period of 90 min, forming a polyelectrolyte complex between the polymers. The ALG/CS nanoparticles had a hydrodynamic size of 313 ± 2.6 nm, PDI of 0.25 ± 0.0018, and zeta potential of −39 ± −3 mV. For the synthesis of ALG/CS nanoparticles containing GA₃, the plant hormone was added to the alginate solution until its complete dilution, in order to obtain a final concentration of GA₃ of 50 µg mL⁻¹.

Chitosan/tripolyphosphate (CS/TPP) nanoparticles were prepared by the gelation method proposed by Calvo et al. [40], with modifications. Initially, 10 mL of an aqueous solution of chitosan (0.2%, pH 4.5 with 0.6% acetic acid) was kept under strong agitation. Then, 6 mL of tripolyphosphate aqueous solution (0.1%, pH 4.5 at 4 °C) was added. The CS/TPP nanoparticles showed a hydrodynamic size of 118 ± 1, PDI of 0.26 ± 0.003, and zeta potential of +15 ± 1 mV. For the synthesis of CS/TPP nanoparticles containing GA₃, the plant hormone was added to the chitosan solution until its complete dilution, in order to obtain a final GA₃ concentration of 50 µg mL⁻¹.

To obtain the conventional (non-nano) GA₃ stock solution, the reagent was dissolved in water at a concentration of 50 µg mL⁻¹. Throughout the study, GA₃ obtained from Sigma Aldrich (St. Loius, MO, USA) was used.

2.2. Plant Material and Experimental Design

Commercial seeds of Solanum lycopersicum cv. cerasiforme (cherry tomato) from Isla Sementes Ltda (Porto Alegre, Rio Grande do Sul, Brazil) were used. In each experiment, 50 seeds per treatment were primed with 10 mL of the respective formulations, kept under constant agitation at 200 rpm in a TS-2000A horizontal circular shaker (Benchmark, Edison, NJ, USA). After 16 h of treatment, the seeds were dried at room temperature for 12 h. Then, the seeds were sown in Carolina Soil^® commercial substrate (sphagnum peat, expanded vermiculite, dolomitic limestone, agricultural gypsum, and NPK fertilizer). Thinning was performed one week after sowing, leaving only one plant per experimental unit. All experimental cultivation was carried out in a greenhouse without temperature and humidity control, belonging to the Department of Animal and Plant Biology of the State University of Londrina (UEL, Londrina, Paraná, Brazil).

Two distinct seed-priming experiments were carried out. The first aimed to select the best chitosan-based nanoparticle (CS/TPP or ALG/CS) and dose adjustment. In the second experiment, the optimal dose of the nanoencapsulated GA₃ treatment was used, comparing it with the treatments of GA₃ and nanoparticles without GA₃.

2.2.1. Experiment 1

Experiment 1 was performed at the end of the winter period of 2019, to test two formulations of chitosan-based nanoparticles (CS/TPP or ALG/CS) containing GA₃, previously diluted in deionized water to obtain three different concentrations of GA₃ (0.5, 5, and 50 µg mL⁻¹), as indicated in

Table 1. Treatments applied for seed priming in experiment 1, corresponding nanoparticle formulation, and GA₃ concentration in suspensions.

Treatments	Formulation	GA3 (µg mL−1)
WATER	Deionized water (control))	0.0
ALG/CS_GA3_50	Alginate/chitosan containing GA3	50.0
ALG/CS_GA3_5	Alginate/chitosan containing 10× diluted GA3	5.0
ALG/CS_GA3_0.5	Alginate/chitosan containing 100× diluted GA3	0.5
CS/TPP_GA3_50	Chitosan/tripoliphosphate containing GA3	50.0
CS/TPP_GA3_5	Chitosan/tripoliphosphate containing 10× diluted GA3	5.0
CS/TPP_GA3_0.5	Chitosan/tripoliphosphate containing 100× diluted GA3	0.5

. These concentrations were chosen based on a previous study of our group with tomato seeds [38].

Cultivation was carried out in polyethylene plastic bags (300 mL). The plants were irrigated frequently with water in order to keep the substrate at field capacity (63% gravimetric humidity). At 35 days after sowing (DAS), the physiological and morphological evaluations of half of the experimental units of each treatment (randomly selected) were carried out. The remaining experimental units were then subjected to water deficit by interrupting irrigation. Chlorophyll a fluorescence and gas exchange evaluations were performed again on the 10th day after the onset of water deficit. Water loss was monitored until the 22nd day after the beginning of the water deficit, when the morphological variables were measured. The average gravimetric humidity of the substrate at the end of the water deficit was 37%.

2.2.2. Experiment 2

Experiment 2 was carried out during the summer of 2022. Based on the results of experiment 1, ALG/CS nanoparticles at a dose of 5 µg of GA₃ mL⁻¹ were selected to compose experiment 2, which was made up of the treatments indicated in

Table 2. Treatments applied for seed priming for experiment 2 and concentration of GA₃ in the formulations.

Treatments	Seed Priming	GA3 (µg mL−1)
CONT	Seeds not subjected to priming (control)	0.0
WATER	Deionized water (control))	0.0
NPGA3	Alginate/chitosan nanoparticles containing GA3	5.0
GA3	Gibberellic acid (GA3)	5.0
NP	Alginate/chitosan nanoparticles	0.0

.

Cultivation was carried out in polyethylene plastic bags (1.2 L), following the conditions described above. After 30 days under frequent irrigation, half of the experimental units of each treatment (randomly selected) were used for morphophysiological and biochemical evaluations, to obtain field capacity condition data (63% gravimetric humidity of the substrate). Then, at 33 DAS, the remaining experimental units were subjected to water deficit, completely suspending irrigation. They were kept in this condition until the end of the experiment (40 DAS) to obtain the morphophysiological and biochemical data of the plants in water deficit. The average gravimetric humidity of the substrate at the end of the water deficit was 11%.

2.3. Physiological Analyses

The youngest fully expanded leaf was selected for the physiological analyses. Chlorophyll a fluorescence variables were determined using an OS1p portable fluorometer (Opti-Sciences, Hudson, NY, USA). Leaves were dark adapted for 15 min using FL-DC clips, and basal fluorescence (F₀) was measured using dim light modulated for 0.1 s (10% intensity). The leaves were then exposed to a light-saturating pulse (8250 µmol m⁻² s⁻¹) for 0.8 s to measure the maximum quantum yield of the photosystem II (F_v/F_m) [41]. The effective quantum yield of photosystem II (ΦPSII = $\mathsf{\Delta }\mathrm{F}$/F_m′) was measured at 10:30 a.m. on light-adapted leaves exposed to photosynthetically active radiation (PAR), quantified at the time of analyses. The basal fluorescence (F′) and the maximum fluorescence (Fm′) of the light-adapted leaves were determined before and after exposure to the light saturation pulse (8250 µmol m⁻² s⁻¹) for 0.8 s, respectively, and ΔF was calculated as the difference between Fm′ and F′.

The gas exchange variables were determined from 8:00 a.m. to 11:00 a.m. with the aid of a portable infrared gas analyzer model LI-6400 XT (LI-COR© Biosciences, Lincoln, NE, USA), connected to a 6 cm² chamber, adjusted for saturating photon flux density (1500 μmol m⁻² s⁻¹). The rate of net photosynthesis (A), the stomatal conductance (g_s), and the intercellular concentration of CO₂ (C_i) were determined. Subsequently, the intrinsic efficiency of water use (iWUE) was calculated by the ratio A/g_s and the efficiency of carboxylation (k) through the A/C_i ratio. In experiment 2, the leaf used in the gas exchange evaluations was detached from plants under stress to measure the water potential with the aid of a Scholander chamber, model SAPS II 3115 (Soilmoisture Equipment Corp, Goleta, CA, USA).

To estimate the total water loss by plants under water deficit (WL), the weight of the experimental units with plants and the experimental units without plants (with substrate only) was determined daily and Equation (1) was applied:

$$\mathrm{W}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{r} \mathrm{l}\mathrm{o}\mathrm{s}\mathrm{s} (\mathrm{g})=\left(\frac{\mathrm{W}\mathrm{i} \mathrm{p}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{t}-\mathrm{W}\mathrm{f} \mathrm{p}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{t}}{\mathrm{D}\mathrm{a}\mathrm{y}\mathrm{s} \mathrm{u}\mathrm{n}\mathrm{d}\mathrm{e}\mathrm{r} \mathrm{w}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{r} \mathrm{d}\mathrm{e}\mathrm{f}\mathrm{i}\mathrm{c}\mathrm{i}\mathrm{t}}\right)-\left(\frac{\mathrm{W}\mathrm{i} \mathrm{s}\mathrm{u}\mathrm{b}\mathrm{s}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e}-\mathrm{W}\mathrm{f} \mathrm{s}\mathrm{u}\mathrm{b}\mathrm{s}\mathrm{t}\mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e}}{\mathrm{D}\mathrm{a}\mathrm{y}\mathrm{s} \mathrm{u}\mathrm{n}\mathrm{d}\mathrm{e}\mathrm{r} \mathrm{w}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{r} \mathrm{d}\mathrm{e}\mathrm{f}\mathrm{i}\mathrm{c}\mathrm{i}\mathrm{t}}\right)$$

(1)

where Wi plant and Wf plant are the initial and final weights of the experimental units with plants, and Wi substrate and Wf substrate are the initial and final weights of the units without plants.

2.4. Morphological Analyses

The plants were removed from the plastic bags and individually placed over a fine mesh sieve, and the roots were washed under running water until all adhered substrate was removed. Shoot length (SL) and root length (RL) were determined using a ruler (cm). The leaf area (LA) of the plants was determined using a portable meter, model LI-3000C (LI-COR© Biosciences, Lincoln, NE, USA). To obtain the dry mass of the aerial part (SDW) and root (RDW), the materials were placed individually in paper bags and kept in an oven at 60 °C until constant mass. In experiment 2, the variables of shoot and root dry mass, field capacity (DW0), and water deficit condition (DW10) were used to calculate the relative growth rate (RGR) of the plants in the period from the end of field capacity (30 DAS) to the end and disassembly of the experiment (40 DAS). The RGR was calculated according to Equation (2).

$$\mathrm{R}\mathrm{G}\mathrm{R}=\frac{(\ln \mathrm{D}\mathrm{W}10-\ln \mathrm{D}\mathrm{W}0)}{\mathrm{N}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r} \mathrm{o}\mathrm{f} \mathrm{d}\mathrm{a}\mathrm{y}\mathrm{s}}$$

(2)

The RDW/SDW ratio was also calculated.

2.5. Biochemical Analyses

In experiment 2, leaf and root samples were collected from experimental units of each treatment (randomly selected), and immediately immersed in liquid nitrogen. The material was stored at −80 °C until use for biochemical evaluations.

The determination of H₂O₂ was performed as described by Alexieva et al. [42] and malondialdehyde (MDA) was determined using the thiobarbituric acid reactive substance (TBARS) assay, following the methodology described by Camejo et al. [43] and Heath and Packer [44], respectively. The lateral roots and leaves (0.1 g) were extracted with 1 mL of trichloroacetic acid (TCA, 0.2% in methanol) and centrifuged at 15,644× g for 5 min at 4 °C. H₂O₂ content was measured at 390 nm after the reaction with KI (1 M) in phosphate buffer (0.1 M, pH 7.5). Lipid peroxidation was estimated by the determination of MDA, which was measured by reading the absorbance at 535 and 600 nm of the supernatant after reaction with thiobarbituric acid in the presence of TCA at 60 °C. The proline content was determined after homogenization of the leaves and roots (0.1 g) in ethanol (70%). The diluted extract reacted with ninhydrin (1%) in acetic acid (60%) and ethanol (20%) in a water bath (95 °C for 20 min), and the absorbance was then read at 520 nm [30,31,32,33,34,35,36,37,38,39,41,42,43,44,45]. The measurements were performed on a SpectraMax Plus 384 microplate reader (Molecular Devices, Sunnyvale, CA, USA).

The antioxidant enzyme activities were determined using extracts obtained by homogenizing lateral roots and leaves (0.1 g) in 1 mL extraction buffer (1 mM EDTA, 0.1 M potassium phosphate buffer pH 7.5, 2% (w/v) polyvinylpolypyrrolidone), followed by centrifugation at 15,645× g (4 °C for 20 min). The ascorbate peroxidase activity (APX, EC 1.11.1.11) was determined according to Nakano and Asada [46] by monitoring the ascorbate consumption at 290 nm in the presence of H₂O_2. Enzyme activity was calculated based on the molar extinction coefficient (ε = 2.8 mM⁻¹ cm⁻¹). The catalase activity (CAT, EC 1.11.1.6) was determined according to Aebi [47] and Peixoto et al. [48] by following the decrease in the H₂O₂ absorbance at 240 nm. The enzyme activity was calculated based on the molar extinction coefficient (ε = 36 M⁻¹ cm⁻¹). The peroxidase activity (POD, EC 1.11.1.7) was determined, according to Peixoto et al. [48], by following the increase in the absorbance at 420 nm, resulting from the pyrogallol oxidation in the presence of H₂O₂ (ε = 2.47 mM⁻¹ cm⁻¹). The superoxide dismutase activity (SOD, EC 1.15.1.1) was determined according to Giannopolitis and Reis [49] by measuring the ability of the extract to inhibit the nitro blue tetrazolium chloride photoreduction. A unit of SOD was defined as the enzyme activity required to inhibit the nitro blue tetrazolium photoreduction by 50% (compared to the control). The measurements were performed on a Genesys 10S UV–Vis spectrophotometer (Thermo Scientific, Waltham, MA, USA).

2.6. Statistical Analysis

For both experiments, the experimental design was completely randomized. First, tests and graphical analyses of the residuals were performed to verify the normality, homogeneity of variance, and independence. For the first experiment, the averages of the replicates were subjected to a hierarchical heatmap cluster analysis. The resulting hierarchical grouping was submitted to tests to obtain the cophenetic correlation coefficient to validate the dissimilarity measure and chosen grouping method. In the second experiment, the results were submitted to analysis of variance (ANOVA) using the F test and, when significant, the means were compared using the Tukey test (p < 0.05). In the figures with different conditions, uppercase letters and lowercase letters indicate the differences between the means in the respective condition (water regime or plant organ), separately. In the water deficit condition, it was not possible to analyze g_s by parametric statistics. The variable was analyzed using the Kruskal–Wallis test, subsequently applying the Dunn test (p < 0.05) for multiple comparisons of mean ranks. In addition, the means of the replicates were subjected to a heat map hierarchical cluster analysis. The cluster analysis was carried out to verify which treatments were more similar to each other and to identify the subgroups of homogeneous regions concerning the morphophysiological and/or biochemical parameters of the plants. All analyses were performed using the R statistical program [50], using the packages easyanova, ExpDes.pt, stats, biotools, RColorBrewer, FactoMineR, and factoextra.

3. Results and Discussion

3.1. Experiment 1

According to Figure 1, the cluster analysis using the hierarchical method showed the groups formed by dissimilarity (cophenetic correlation coefficient of 0.82). Among the treatments tested, ALG/CS_GA₃_5 provided the greatest gains in plant growth, both in length (RL and SL) and in dry mass (RDW and SDW) in the condition without water restriction (Figure S1). During water deficit, ALG/CS_GA₃_5 promoted the best indicators of improved drought tolerance, ensuring damage reduction (evidenced by the decrease in F₀) and maintenance of the effective activity of photosystem II (higher ΦPSII), in addition to lower g_s (−13% compared to WATER) and higher k (+24%) and iWUE (+12%) (Figures S2 and S3). The treatment also provided the lowest water loss (−38%) from the plants compared to the control (Figure S4).

These data indicate that, in the ALG/CS_GA₃_5 treatment, the low stomatal opening did not negatively affect the photosynthetic activity of the plants, making them more efficient in the use of water and in the assimilation of CO₂. In addition, ALG/CS_GA₃_5 promoted the lowest F₀, combined with maintenance of ΦPSII compared to the other treatments. Under stress, low F₀ is indicative of greater tolerance, since the increase in F₀ can be caused by damage to the reaction center of photosystem II, decreasing the ability to transfer excitation energy from the antenna complex to the reaction center [51]. Corroborating this, the maintenance of effective photosystem II activity provided by ALG/CS_GA₃_5 is possibly a result of the activation of defense mechanisms in the plant during water deficit. Therefore, all physiological alterations contributed to reducing the total water loss from the plants, ensuring maintenance of the plant metabolism and, consequently, the integrity and development of plants under water deficit.

The present study is in agreement with data from Pereira et al. [38], showing that the concentration of the nanoformulation was a fundamental factor that directly influenced plant responses. Furthermore, the ALG/CS formulation was more efficient than the CS/TPP formulation, as also observed in Pereira et al. [37] where treatment with ALG/CS increased the number of lateral roots and the concentration of photosynthetic pigments in common bean plants. The results demonstrate that not only GA₃ encapsulation per se is responsible for the biological effect, as the composition of the nanocarrier (e.g., polymer chosen to encapsulate the plant hormone) affected the GA₃ bioactivity [37,38].

It is noteworthy that the effects induced by nanomaterials on plants are dependent on different factors, including the plant species, cultivation environment, time of exposure, and characteristics inherent to the nanomaterial (such as type, composition, size, shape, and charge) [52]. The nanoparticles tested in the present study have different compositions and colloidal characteristics, such as size and surface charge, factors that are decisive for the interaction of nanomaterials in plants, and consequently, their biological effects [53]. In particular, there is evidence that nanoparticles with a negative zeta potential (such as ALG/CS nanoparticles) are more mobile in plant tissues than those with a positive charge [54].

In short, with less damage to the photosynthetic apparatus, combined with efficiency in gas exchange and water use, the ALG/CS_GA₃_5 treatment provided an increase in plant growth, reflected in greater accumulation of dry mass and, consequently, the plant maintained good development even in conditions of water restriction. Therefore, the ALG_GA₃_5 treatment was selected to compose the subsequent experiment and compare the effects induced by nanoencapsulated GA₃ with conventional GA₃, in addition to comparisons with the effects of empty nanoparticles.

3.2. Experiment 2

According to Figure 2a, F₀ showed a significant difference between treatments only in field capacity, with WATER presenting a higher average than NP. For ΦPSII (Figure 2b), in field capacity, the WATER and NPGA₃ treatments had the lowest means, significantly differing from GA₃ and NP. In water deficit, the CONT and GA₃ treatments showed the highest values of ΦPSII, as well as of F_v/F_m (Figure S5). The net photosynthesis rate (A) was lower in WATER and NPGA₃ treatments at field capacity when compared to GA₃ and NP treatments (Figure 2c). However, in the water deficit condition, the CONT and GA₃ treatments showed a significant reduction in A (69%, on average) when compared to the other treatments. The g_s was lower in WATER in field capacity and, again, CONT and GA₃ showed a significant reduction in g_s (87%, on average) when compared to the other treatments in the water deficit condition (Figure 2d). Due to the very low g_s in CONT and GA₃ treatments, it was not possible obtain an accurate measurement of C_i, k, and iWUE.

The water potential did not differ significantly in the field capacity condition (Figure 3a). However, under water deficit, CONT and GA₃ showed the lowest values of leaf water potential (Figure 3a), in addition to being the treatments that resulted in greater water loss during the entire period of water deficit (Figure 3b) and presenting the most severe stress symptoms (Figure 3c).

Conducting experiment 2 in summer with high temperatures led to a rapid establishment of water deficit when compared with experiment 1, which was conducted in winter. In experiment 1, the variables F₀ and ΦPSII were efficient to identify the effects of water stress on plants. However, in experiment 2, the F₀ variable did not present a significant difference during water deficit. This may have been caused by the speed and intensity with which stress was established in experiment 2. In the case of ΦPSII, there was a reduction in the plants of the WATER, NPGA₃, and NP treatments under water deficit. Using chlorophyll fluorescence for phenotyping wild and cultivated tomato genotypes, Sperdouli et al. [55] observed a reduction in ΦPSII induced by severe water deficit. According to Urban et al. [56], while substantial decreases in F_v/F_m and ΦPSII values are indicators of photodamage, small reductions in these parameters can be interpreted in terms of photoprotection. In the present study, the minor decreases in F_v/F_m (−2%) and ΦPSII (−4%) under water deficit in WATER, NPGA₃, and NP treatments could be associated with the induction of protective mechanisms against stress [57].

In fact, evaluating all the physiological variables, the seed priming with water, NP, and NPGA₃ proved to be efficient in mitigating the effects of water deficit, while the CONT and GA₃ treatments showed much lower A, g_s, and leaf water potential under this condition, in addition to greater accumulated WL and more severe wilting symptoms. Stomata play an important role in responses to many biotic and abiotic stresses [58], with the change in g_s being a rapid response and the change in stomatal density a long-term response [59]. Although stomatal closure in response to stress contributes to a reduction in water loss and plant survival, it can also lead to a reduction in production and productivity in different crops. In a study with tomato plants, Nemeskéri et al. [60] observed that changes in g_s during the generative stages of development determined the fruit yield under both severe drought and well-watered conditions.

It is interesting to note that NPGA₃ had the lowest mean LA among the treatments (Figure 4), with reductions of 38, 44, and 48% compared to CONT, GA_3, and NP, respectively. Studies indicate that the regulation of gibberellin homeostasis and signaling (e.g., by seed priming with free GA₃) plays fundamental roles in many aspects of plant growth and development, including cell elongation, seed germination, leaf expansion, senescence, and head formation [12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61]. Thus, treatment with nanoencapsulated GA₃ might have altered the homeostasis and signaling of the hormone during tomato plant development. WATER also showed a reduction in leaf area compared to GA₃ and NP, which contributed to the decrease in water loss through transpiration and greater tolerance to drought.

In field capacity, there were no significant differences for RDW, but the NP treatment had the highest means of SDW and TDW, significantly differing from NPGA₃. On the other hand, the RDW/SDW ratio was higher in the NPGA₃ treatment compared to GA₃ and NP. In water deficit, the CONT and GA₃ treatments showed, on average, gains of 43% (RDW), 46% (SDW), and 45% (TDW). Similar to what was observed for the LA, the NPGA₃ treatment had the lowest mean SDW, reflecting the lowest shoot growth during the field capacity condition. The RDW/SDW ratio did not differ in treatments under water deficit (Figure 5). For NPGA₃, the lower shoot development did not negatively impact root development, since the RDW did not differ between treatments. In addition, the combined effect of decreasing shoot growth without negatively impacting root growth provided a greater ratio between the root system and plant area, making the plants more able to tolerate subsequent water stress. According to Kou et al. [62], increasing the root-part-area ratio under drought conditions is a strategy to avoid the effects of water shortage, allowing the allocation of photoassimilates to the root, promoting the efficient acquisition of water and nutrients.

The GA₃ and CONT treatments showed the highest RGR during the water deficit period, both for roots (99%) and shoots (67%), on average, when compared to the other treatments (Figure 6). In accordance with this, Ma et al. [63] demonstrated that the priming of Leymus chinensis seeds with GA₃ promoted plant growth when compared to the hydropriming treatment. However, under water deficit, this greater general development of the plants in the GA₃ and CONT treatments accelerated the depletion of water content available in the substrate. This condition may indicate lower efficiency in recognizing and activating water deficit mitigation mechanisms.

Thus, the GA₃ and CONT treatments maintained plant growth, causing them to lose water rapidly (−214% of water potential in the leaf, on average), causing reductions of 87% in stomatal conductance and 69% in net photosynthetic rate, on average, when compared to the other treatments. On the other hand, NPGA₃ induced a smaller leaf area (−44%) and, consequently, a higher root-to-shoot ratio (+39%) and less water loss (−28%) when compared to GA₃, which was the most susceptible treatment to water deficit. Despite the NPGA₃ treatment having a lower growth in field capacity, it was able to promote the maintenance of growth under the stressful condition of water restriction, having a higher RGR in relation to the CONT and NP treatments (Figure 6).

In the present study, physiological and water potential assessments were performed prior to the visual symptoms of wilting. However, CONT and GA₃ already showed a significant reduction in the water potential of the leaves, indicating that water stress was already triggering negative effects on the metabolism of those plants, which was later confirmed by the early wilting of the leaves in relation to the other treatments (Figure 3c). However, when nanoencapsulated with ALG/CS, GA₃ began to reduce the severity of stress, evidenced by the NPGA₃ treatment that presented the lowest WL, significantly differing from GA₃ and CONT.

It is important to highlight that seed priming with water also increased drought tolerance, mainly in relation to CONT. It is already known that seed priming not only promotes seed germination and improves plant growth and crop yield but can also increase tolerance against abiotic stresses [13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64]. Janmohammadi et al. [65] reported that hydropriming corn seeds significantly improved germination as well as seedling growth under water stress conditions. Cotton seeds with water priming obtained better parameters of germination, growth, and higher productivity in conditions of water scarcity [66]. In Cleistogenes songorica (a grass native to northern China), water-priming treatments alleviated the harmful effects of water stress by decreasing lipid peroxidation and ROS accumulation and increasing antioxidant enzyme activities [67].

More recently, nanoparticles have been tested as a seed-priming agent (nanopriming), taking advantage of the unique properties of nanoparticles (size, charge, and large surface area) that favor interaction with plant tissues and enhance the benefits of seed priming [24]. Moreover, they act as a shield to protect the active compounds against degradation and promote sustained release over time, factors that increase the biological activity of the active compounds compared to non-encapsulated systems [68]. Herein, we showed that NPGA₃ treatment stood out among other types of priming, leading to more prominent effects on plant growth and responses to water deficit, as discussed above. It is noteworthy that this nanoformulation is based on biodegradable and biocompatible polymers of natural origin and it elicited positive effects at low concentrations in seed treatment. Thus, this approach represents a viable, economically accessible, and sustainable management strategy for improving conventional techniques of seed priming [69].

In order to ratify these results, biochemical analyses of the plants were carried out under field capacity and at the end of the water deficit (Figure 7). In field capacity, there was no significant difference for oxidative stress markers (MDA and H₂O₂) in the root; however, in the evaluation of the leaves, CONT showed an increase of 80 and 120% in MDA and H₂O₂ levels, respectively, when compared to the other treatments. Under water deficit, NP showed the highest averages of MDA and H₂O₂ content in the roots, differing from NPGA₃ and CONT in MDA content and from CONT in H₂O₂ content. In leaves, GA₃ has the highest concentration of MDA, differing from WATER and NP. For proline, in field capacity, NP showed the highest values in the roots, differing from CONT and GA₃. In leaves, CONT had the highest average of proline content, differing from WATER, NPGA_3, and GA₃ treatments. Under water deficit, GA₃ and CONT showed the highest concentrations of proline in roots, with GA₃ having a content 150% higher than WATER, NPGA₃, and NP treatments. In leaves, the same pattern was observed, with CONT and GA₃ showing the highest values and GA₃ with an average increase of 70% compared to WATER, NPGA₃, and NP treatments (Figure 7).

In roots, the higher levels of MDA and H₂O₂ in the NP treatment may be associated with the eliciting effect of chitosan. Chitosan treatment is known to trigger defense responses against different stresses, inducing the synthesis of different intracellular messengers that act on stress signals, such as H₂O₂ and nitric oxide [70]. Corroborating these results, Gomes et al. [34] observed that seed priming with chitosan nanoparticles containing Cu²⁺ ions did not significantly increase H₂O₂ and MDA levels in corn seedlings (similar to what was observed here for NPGA₃), while treatment with nanoparticles without the active agent practically doubled the levels of both markers of oxidative stress.

In field capacity, the highest levels of MDA and H₂O₂ in the CONT leaves may be related to the susceptibility of the treatment to different types of stress to which the plants were submitted during the entire period of growth and development prior to the induction of water deficit. In turn, in the other treatments, seed priming contributed significantly to attenuating the effects induced by stress in the plants [25].

Proline can act as a metabolic signal to regulate metabolic pathways that affect growth and stress tolerance, acting as an osmoprotectant and antioxidant in many plant species [71]. Thus, it can be used as an indicator of the environmental stress imposed on many plant species, including tomato [72]. In the present study, the treatments that were more tolerant to water deficit (CONT, NPGA₃, and NP) tended to have higher concentrations of proline in roots under field capacity conditions, which may have contributed to protection from subsequent water stress. However, under water deficit, the proline content observed in the roots of the treatments most susceptible to deficit (GA₃ and CONT) became significantly higher, which suggests delayed induction of stress response. Similarly, in the leaves, the proline content was higher in the CONT and GA₃ treatments under water deficit, as well as MDA and H₂O₂ levels. In accordance with these results, Alian et al. [73] reported that the accumulation of proline in tomato plants under saline and water stress was related to symptom severity.

Based on data from biochemical stress markers (Figure 7), the analysis of antioxidant enzyme activity was concentrated on the roots and leaves of plants under water deficit (Figure 8). According to Figure 8, WATER and NPGA₃ showed the highest activities of root SOD, with WATER showing values, on average, 55% higher than CONT, GA₃, and NP treatments. For CAT, significant differences were observed between CONT, NPGA₃, and NP treatments. The NP treatment showed an activity 158% higher than what was seen in the CONT and NPGA₃ treatments. Thus, the increase in SOD or CAT activity in the WATER, NPGA₃, and NP treatments may be another factor that contributed to greater stress tolerance compared to the CONT and GA₃ treatments. For APX and POD activities, there were no significant differences between treatments. Performing evaluations of antioxidant enzyme activity in leaves under water deficit, it was possible to verify significant differences only in SOD activity, with CONT presenting an average reduction of 37% in relation to the other treatments (Figure 8).

Our results indicate that seed primings with water (WATER) and with ALG/CS nanoparticles (NP), as well as the synergism of the association between nanoparticles and gibberellic acid (NPGA₃), were efficient for activating the enzymatic antioxidant defense in the roots, contributing to mitigating the deleterious effects caused by water stress. Corroborating this, Gomes et al. [34] observed that priming corn seeds with chitosan-based nanoparticles was efficient to induce early enzymatic antioxidant activity in seeds, mitigating the effects of accelerated aging. In addition to seed priming, treatments with chitosan-based nanoparticles containing plant bioactive, either via substrate [30] or via a foliar route [33,34,35], efficiently contributed to increasing antioxidant enzymatic activity in plants and/or did not induce negative effects that would compromise this defense activation, conferring protection against abiotic and biotic stresses, respectively.

Furthermore, under water deficit, a high CAT activity and a trend towards an increase in APX were observed in the roots of the NP treatment plants, associated with higher levels of MDA and H₂O₂. Possibly, there is a level of oxidative stress generated concomitantly by nanochitosan and water restriction, which is controlled by the high activity of enzymes such as CAT and APX, preventing a harmful excess of ROS. H₂O₂ molecules, produced non-enzymatically or by SOD action, are removed by CAT and APX; however, the removal of low concentrations of H₂O₂ is mainly performed by APX, allowing the control of this ROS in the order of μm in different cell compartments [74].

Similar to what was seen in the roots of plants under water deficit, NP induced high CAT activity in the leaves of plants at field capacity. In addition, there is a tendency for proline levels to be higher both in the root and in the leaves, compared to the WATER, NPGA₃, and GA₃ treatments at field capacity. These results support the hypothesis that priming with nanochitosan has induced a certain level of oxidative stress in plants, even under field capacity conditions. Possibly, this initial stress activated antioxidant defense mechanisms in advance, giving the plant greater tolerance to subsequent water stress. Priming with water led to a reduction in POD activity and a downward trend in SOD and APX activities. The use of nanoparticles for priming seeds has been shown to be more efficient in increasing the level of antioxidant enzymes during the plant life cycle, resulting in a better response to stress [75].

The cluster analysis by the hierarchical method with the results of field capacity (Figure 9a) and water deficit (Figure 9b) showed the groups formed by dissimilarity in each water condition. Clusters were created using the Canberra distance as a measure of dissimilarity and the average linkage method as an agglomeration method. The resulting clusters showed a cophenetic correlation coefficient of 0.98 for both water conditions. In field capacity, the variables formed four distinct groups, with the highest values of g_s, LA, A, and ΦPSII in the NP and GA₃ treatments. CONT was positively associated with MDA, H₂O₂, and proline content in the leaves, while NP was associated with the highest values of TDW and root proline. NPGA₃ and WATER were positively associated with RDW/SDW and F₀, respectively. In water deficit, the variables formed two distinct groups: one formed by GA₃ and CONT (positive association with ΦPSII, proline in leaves and roots, TDW, WL, MDA in leaves, and RGR of both plant organs) and another by NP, NPGA₃, and WATER (positive association with g_s, A, and WP). The second group is subdivided with the positive correlation of WATER with SOD activity in the roots and of NP with CAT, H₂O₂, and MDA in the roots.

Thus, the analysis of Figure 9 enables us to summarize the results of the experiment. At field capacity, NP and GA₃ treatments led to greater photosynthetic activity and shoot growth. In addition, NP provided the activation of stress protection mechanisms (accumulation of proline in the root and greater CAT activity in the leaves), which may have contributed to the tolerance to the subsequent water deficit. Differently, the GA₃ treatment maintained growth during water stress, without activating defense mechanisms, resulting in greater susceptibility to water deficit. CONT showed the highest values of stress indicators in the leaves, which demonstrates the benefits of seed priming. Furthermore, priming with water had a positive relationship with TDW, RDW/SDW, and F₀. NPGA₃ altered the effects triggered by priming with water or nanochitosan in plants, with a reduction in shoot growth and a greater increase in RDW/SDW. These pre-stress changes contributed to greater tolerance to water deficit of plants treated with NPGA₃.

In the water deficit condition, GA₃ and CONT showed the highest values of variables related to plant growth, proline accumulation, leaf peroxidation, and water loss. Thus, the greater growth during water deficit may have resulted in a rapid loss of water by plants treated with GA₃ and CONT. In addition, the greater accumulation of proline both in the root and in the leaves is an indication that the activation of defense mechanisms may have occurred late, resulting in greater lipid peroxidation and early leaf wilting. NPGA₃ and WATER provided SOD activation in the roots, which, together with pre-stress morphological alterations, contributed to the maintenance of photosynthetic activity and leaf water potential of plants under water deficit. However, NPGA₃ stood out for maintaining growth at higher rates than WATER during the period of water restriction. This characteristic could be decisive for the plants to recover, under field conditions, from the limitations imposed by the water restriction and to re-establish full development more quickly, mitigating the losses in production caused by the period of water deficit. The early activation of antioxidant defense mechanisms provided by the NP treatment also contributed to the maintenance of photosynthetic activity and water potential in leaves of plants under water deficit. However, the NP treatment was related to high levels of H₂O₂ and MDA in the roots, which is indicative of high oxidative stress during water deficit, even with CAT induction.

4. Conclusions

Overall, this study demonstrates that seed priming using water or ALG/CS nanoparticles with GA₃ showed important beneficial effects on drought-stressed tomato plants, including SOD activation in the roots and maintenance of photosynthetic activity and leaf water potential. However, the treatment with nanoGA₃ stood out for maintaining higher growth rates during the water deficit condition. Priming with ALG/CS nanoparticles without the hormone also contributed to the maintenance of leaf physiological traits of plants under water deficit, but this treatment was less effective in protecting against oxidative stress.

The GA₃ nanocarrier system used in the present study is based on biopolymers of natural origin, biodegradable, and non-toxic, which showed here positive effects even at low concentrations in seed treatment. Thus, this approach represents a viable, economically accessible, and sustainable management strategy for improving conventional technique of seed priming, contributing to the reduction in production losses in the face of a climate change scenario. However, even though the morphophysiological and biochemical indicators show the efficiency of the treatments, further studies are needed on the impacts of treatments on fruit productivity and on the mechanisms involved in the nanoGA₃-induced responses.