Enhancing cadmium and salinity stress tolerance in upland rice through priming: unraveling the impact of signaling molecules

ABSTRACT:

Priming associated with signaling molecules is an effective technique to promote uniform and rapid germination in the field, in addition to increasing stress tolerance. This study aimed to evaluate the effect of priming solutions on tolerance to cadmium and salinity stress in two upland rice cultivars, Douradão and Soberana. Five priming agents were tested: indole acetic acid (IAA), melatonin, sodium nitroprusside (SNP), hydrogen peroxide (H₂O₂) and chitosan. After treatment, the seeds were subjected to three germination conditions: control (no stress), saline stress and cadmium stress. Evaluations included seed moisture content, germination percentage, seedling growth and activity of antioxidant enzymes (superoxide dismutase - SOD, catalase - CAT and ascorbate peroxidase - APX). The results showed that the effects of priming varied according to the type of stress and the agent used. Under saline stress, H₂O₂ favored germination and root growth, while under cadmium stress, IAA, melatonin and H₂O₂ were more effective. Chitosan induced high SOD activity in the cadmium stress, and SNP stood out for salinity. H₂O₂ increased APX activity in the cultivar Soberana, while SNP was more effective for Douradão in both stress conditions. Catalase was activated by H₂O₂ and melatonin. The study concludes that SNP, H₂O₂ and IAA can improve stress tolerance during rice seed germination by activating antioxidant systems that favor growth under adverse conditions.

Index terms:
antioxidant system; germination; Oryza sativa L.; oxidative stress; seedling growth

RESUMO:

O priming associado a moléculas sinalizadoras é uma técnica eficaz para promover uma germinação uniforme e rápida no campo, além de aumentar a tolerância ao estresse. Este estudo visou avaliar o efeito de soluções de priming na tolerância ao estresse por cádmio e salinidade em duas cultivares de arroz de terras altas, Douradão e Soberana. Foram testados cinco agentes de priming: ácido indol acético (IAA), melatonina, nitroprussiato de sódio (SNP), peróxido de hidrogênio (H₂O₂) e quitosana. Após o tratamento, as sementes foram submetidas a três condições de germinação: controle (sem estresse), estresse salino e estresse por cádmio. Avaliações incluíram teor de água, porcentagem de germinação, crescimento de plântulas e atividade de enzimas antioxidantes (superóxido dismutase - SOD, catalase - CAT e ascorbato peroxidase - APX). Os resultados mostraram que os efeitos do priming variaram conforme o tipo de estresse e o agente utilizado. Sob estresse salino, o H₂O₂ favoreceu a germinação e o crescimento radicular, enquanto sob estresse por cádmio, o IAA, melatonina e H₂O₂ foram mais eficazes. Quitosana induziu alta atividade de SOD em cádmio, e SNP destacou-se para salinidade. H₂O₂ elevou a atividade de APX na cultivar Soberana, enquanto o SNP foi mais eficaz para Douradão em ambas as condições de estresse. A catalase foi ativada pelo H₂O₂ e melatonina. O estudo conclui que SNP, H₂O₂ e IAA podem melhorar a tolerância ao estresse durante a germinação de sementes de arroz, ativando sistemas antioxidantes que favorecem o crescimento sob condições adversas.

Termos para indexação:
sistema antioxidante; germinação; Oryza sativa L.; estresse oxidativo; crescimento de plântulas

INTRODUCTION

Rice (Oryza sativa L.) is cultivated globally, but faces adverse conditions, which can cause stress to plants. Climate change, intensified by human activity, increases the concentrations of salt or even heavy metals in the soil, such as cadmium (Cd), a highly toxic and mobile metal. The presence of heavy metals in the soil is a growing concern, and cadmium contamination puts plant development at risk, with enormous impact on humans (Wang et al., 2019). This metal, even in low concentrations, is highly toxic and easily absorbed by the roots, and can be transported to grains, depending on the genotype Cd contamination compromises rice development, causing morphophysiological and biochemical changes, such as chlorosis, growth inhibition, reduced biomass accumulation, and oxidative stress (Abedi, 2021).

Salinity is reported to drastically decrease rice production once grain development is compromised by both ion tolerance and restrictions on water availability (Liu et al., 2022). As salinity increases, there is a super production of reactive oxygen species (ROS), which induces damage to the cell membrane, structural proteins, enzymes, and nucleic acids and consequently may result in metabolic and structural damage to the cell. Superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) are some of the enzymes that are part of the antioxidant system that work to eliminate or reduce the amount of ROS produced (García-Caparrós et al., 2020). ROS are naturally produced under natural conditions, however, under stressful conditions, such as the presence of salt and heavy metals, the amount of ROS produced increases. Therefore, the system has difficulty balancing ROS production and ROS elimination, leading to oxidative stress.

Seeds are essential for genetic variability and the application of new technology, helping to adapt to biotic or abiotic stresses. The study of the physiological of plants to these stresses has been fundamental to understand their tolerance and adaptation (Barbieri et al., 2019). Among the improvement techniques, priming stands out, which consists of the controlled imbibition of the seeds to activate your metabolism without radicle protrusion. This process favors uniform germination and increases the resistance of the seeds to different stresses (Bonome et al., 2017).

In combination with priming technique, the use of compounds such as indoleacetic acid (IAA), melatonin, chitosan, and sodium nitroprusside (SNP) will allow these molecules to act as signal inductors that will protect the plants against abiotic stress (Carmo et al., 2024). These molecules are associated with plant defense mechanisms acting as signaling molecules by increasing the efficiency of seed antioxidant system defense (Wang et al., 2019). Therefore, this research was performed to evaluate the effects of priming on seed germination, seedling development, and antioxidant system activation of upland rice under salt and cadmium stress.

MATERIAL AND METHODS

The research was performed at the Universidade Federal de Lavras using two rice cultivars: Douradão (National Cultivar Registry nº 00630) and BRS-Soberana (National Cultivar Registry nº 04360). Priming technique was performed on both cultivars separately, immersing the seeds in aerated priming solutions in a biochemical oxygen demand (B.O.D.; Photoperiod Climate Controlled Chambers, Model EL202, Eletrolab, Brazil) at 25 °C in the dark for 20 hours. The amount of 40g of seeds from each treatment was placed inside an erlenmeyer with 400 mL of each priming solution in the following concentrations: 0.018 g.L^-1 of IAA (Lecube et al., 2014), 0.099 g.L^-1 of chitosan (Yang et al., 2010), 0.232 g.L^-1 of melatonin (Ye et al., 2016), 8.6 mL.L^-1 of hydrogen peroxide (H₂O₂) (Santhy et al., 2014) and sodium nitroprusside (SNP) at 0.0298 g.L^-1 (Faraji et al., 2018). As a control treatment, unprimed seeds without any germination stress conditions were used. After priming, seeds were washed in running water for 10 minutes and after that, seeds were placed in a single layer inside a tray, and dried in an oven with forced air circulation for 48 hours at 25 °C. To keep uniform water content of all treatments, the control treatment was also dried in the same condition. Water content was measured immediately after priming and after the drying process using two replications (3 g of seeds for each replication) from each treatment, following the Brasil (2009) protocol (oven at 105 °C for 24 hours), and results expressed in percentage.

For germination test, seeds from both cultivars and all priming solutions were placed to germinate under three conditions, no-stress condition (water), stressed by salty, and stressed by cadmium. For cadmium stress, the cadmium was diluted in 250 mL of distilled water to obtain a solution with concentration of 250 πM of cadmium (0.0458 g.L^-1) (Pereira et al., 2017). For saline condition, NaCl was diluted in 250 mL of water to obtain a concentration solution of 100 mM of NaCl (Ma et al., 2018). The tests were performed separately, with each stress condition using four replications with 25 seeds each placed on a germination paper (sterilized, neutral pH, 28x38 cm, porosity: 3 µ, grammage: 65g.m^-2) roll moistened with 250 mL of distilled water, or with 250 mL of NaCl solution (Ma et al., 2018), or with 250 mL of cadmium solution (Pereira et al., 2017). The germination test was conducted in a B.O.D. at 25 °C and evaluation was performed 5 days after (first count) and 14 days (final count) after sowing. Five and fourteen days after sowing the germination rolls were opened and counted the number of normal seedlings (seedlings with all healthy structures of leaves, stem, and radicle) on each replication, and results expressed in percentage of normal seedlings (Brasil, 2009). Based on germination test performed on control seeds, by seedling structures comparation, it is possible to identify whether cadmium contamination affects germination or not, and if priming techniques will provide any support to avoid damage from cadmium contamination.

To obtain the results of the seedling growth, root and shoot lengths were measured using the image analysis system GroundEye® version S800. Seedlings from each germination test after 5 days were placed inside the equipment drawer and the reading was performed through software reading, providing measurements of the seedlings. Contamination may affect seedling development; therefore, seedling images are essential to evaluate the contamination effects on seedling growth.

To perform the biochemical analysis, a sample of seedlings 14 days after sowing from each treatment was stored in a deep freezer at -80 °C (VIP Series MDF-U53VA Ultra Low Temperature Freezer, Sanyo, Japan) for 7 days until biochemical analyses. For enzyme extraction, 200 mg of seedling sample were macerated in liquid nitrogen and 0.01g of polyvinylpolypyrrolidone (PVPP) and homogenized in 465 µL of extraction buffer (400 mmol.L^-1 of potassium phosphate (54.4 g.L^-1 of monobasic phosphate and 69.7 g.L^-1 of dibasic phosphate) at 7.8 pH, 3.7 g.L^-1 of EDTA, and 35.2 g.L^-1of ascorbic acid). After that, the mixture of sample and buffer was centrifuged at 12.000 rpm for 10 minutes at 4 °C (Micro High Speed Refrigerated Centrifuge VS-15000 CFNII, Vision, Korea), and the supernatant was collected for enzymatic analysis (Biemelt et al., 1998). SOD activity was determined by the enzyme’s ability to inhibit the photochemical reduction of nitrobluetetrazolium (NBT) according to Giannopolitis and Ries (1977) protocol. Therefore, 10 µL of the extract (supernatant) was added to 160 µL of buffer (100 mmol.L^-1 of potassium phosphate (13.6 g.L^-1 of monobasic phosphate and 17.4 g.L^-1 of dibasic phosphate) at pH 7.0, 10.4 g.L^-1 of methionine, 3.7 g.L^-1 of EDTA, 0.8 g.L^-1 of NBT, and 0.08 g.L^-1 of riboflavin). The mixture (extract + buffer) was kept under fluorescent light (20W) for 7 minutes, and readings were performed at 560 nm at a spectrophotometer (96-well plates, Eon™ Microplate Spectrophotometer, BioTek Instruments, United States). A unit of SOD was determined by the amount of enzyme that inhibits 50% of the NBT reduction ratio.

CAT activity was determined according to Havir and McHale (1987) protocol using 9 µL of the extract mixed with 90 µL of buffer (200 mmol.L^-1 of potassium phosphate (27.2 g.L^-1 of monobasic phosphate and 34,8 g.L^-1 of dibasic phosphate) at pH 7.0). To this mixture, was added 9 µL of 21.54 mL.L^-1 H₂O₂ solution, and the readings were performed immediately after that, monitoring the consumption of 21.54 mL.L^-1 of H₂O₂ at a spectrophotometer (96-well plates, Eon™ Microplate Spectrophotometer, BioTek Instruments, United States). CAT activity was determined by the amount of enzyme necessary to decompose 1 µmol·min^-1 of H₂O₂.

APX activity was measured according to Nakano and Asada (1981) protocol, adding 9 µL of enzyme extract to 99 µL of buffer (200 mmol.L^-1 of potassium phosphate (27.2 g.L^-1 of monobasic phosphate and 34.8 g.L^-1 of dibasic phosphate) at pH 7.0, and 1.8 g.L^-1 of ascorbic acid) at 30 °C preheated in a water bath for 15 minutes (Small digital water bath, model CT-245, Cientec Laboratory equipment Ltda., Brazil). Along with this mixture, 9 µL of 0.17 mL.L^-1 of H₂O₂ was added, and readings were performed immediately. An APX unit was expressed by the amount of enzyme that oxidizes 1 µmol·min^-1 ascorbic acid.

For hydrogen peroxide and lipid peroxidation analysis, 200 mg of seedling sample was macerated in liquid nitrogen and 0.01 g of PVPP and homogenized in 1.5 mL of 0.1% trichloroacetic acid solution (TCA-0.1%; 1 g.L^-1). The material was centrifuged at 12000 rpm for 15 minutes at 4 °C (Vision Micro High Speed Refrigerated Centrifuge VS-15000 CFNII, Korea), and the supernatant was collected (Buege and Aust, 1978). Lipid peroxidation was analyzed through malondialdehyde content (MDA) produced by reaction with thiobarbituric acid (TBA) according to the TBARS method (Buege and Aust 1978), where the quantity of 125 µL of extract was added to 250 µL of buffer (0.5 % TBA (5 g.L^-1) + 10% TCA (100 g.L^-1)). The mixture was heated at 95 °C for 30 minutes and quickly cooled on ice. Reading was performed at a spectrophotometer at 535 and 600 nm (96-well plates, Eon™ Microplate Spectrophotometer, BioTek Instruments, United States), and the MDA content was calculated using an extinction coefficient of 155 mM¹·cm^-1 (Baryla et al., 2000). To analyze hydrogen peroxide content, 45 μL of extract was added to 125 μL of buffer (100 mmol/L^-1 of potassium phosphate (13.6 g.L^-1 of monobasic phosphate and 17.4 g.L^-1 of dibasic phosphate) at pH 7.0, and 166 g.L^-1 of potassium iodide). Reading was performed at a spectrophotometer at 390 nm (96-well plates, Eon™ Microplate Spectrophotometer, BioTek Instruments, United States), and the results were compared to a standard H₂O₂ dilution curve. The H₂O₂ content was expressed in µmol H₂O₂·g^-1 of fresh mass (Velikova et al., 2000).

All experiments were established on a completely randomized design with 2 (cultivars) x 3 (germination conditions) x 5 (priming solutions) + 1 (control). Data were analyzed on a triple factorial scheme with 1 additional treatment through ANOVA. Treatments were compared by Tukey’s test at 5% probability. Treatments were compared with the control by Dunnett’s test, in R software for Windows 4.1.1.

RESULTS AND DISCUSSION

After priming, seeds from both cultivars absorbed, on average, twice the value of the initial water content. Seeds from ‘Douradão’ and ‘BRS Soberana’ cultivars showed similar water content among the priming solutions (Figure 1). Priming allows necessary water for reactivation of physiological processes for metabolism at a balanced ratio, and seed water potential becomes equal to the priming solution (Almeida et al., 2016). Post-priming drying is necessary for seeds to reach a low water content allowing storage of the seeds for some periods without losing the benefits of the technique or its physiological quality (Ribeiro et al., 2019). Under low water content, seed metabolism is reduced, with water imbibition being essential for its reactivation and germination to proceed (Almeida et al., 2016). The responses of the seeds to priming and germination conditions were different according to the cultivar analyzed (Figure 2). Under cadmium conditions, Douradão and Soberana do not show significant differences between the priming solutions. Under water germination, IAA improved germination at first count for both cultivars (Figures 2A and 2B). The results of the first count were higher when the seeds were primed with IAA and SNP for Douradão (Figure 2A) and SNP and chitosan for Soberana (Figure 2B), under saline stress.

Figure 1
Water content of two different cultivars of rice seeds (Douradão and BRS Soberana) after priming with different solutions and after drying. For each cultivar, equal letters, uppercase comparing priming solutions on each drying process (after priming and after drying), and lower cases comparing the drying process on each priming solution, indicate no significant differences by Tukey’s test (p≥ 0.05).

Figure 2
The percentage of first (A and B) and final count (C and D) germination under three germination conditions (water, salinity (NaCl), and cadmium) from two different cultivars of rice seeds, Douradão (A and C) and Soberana (B and D), primed with different solutions For each cultivar, equal letters, uppercase comparing priming solutions on each germination conditions, and lower cases comparing each priming solution among the germination conditions, indicate no significant differences by Tukey’s test (p≥ 0.05). The symbol “*” indicates a significant difference between the treatment and control for each cultivar.

For the final germination percentage (Figures 2C and 2D), no differences were observed between the solutions for both cultivar and germination conditions. The IAA for Douradão seeds showed higher values in the water condition (Figure 2C), while SNP and chitosan were higher in BRS Soberana under cadmium condition (Figure 2D). SNP was the priming solution that improved the first and final germination counts of BRS Soberana seeds, indicating that not only germination but also seed vigor was improved under high salinity conditions (Figures 2B and 2D). SNP, a nitric oxide donor, promotes plant tolerance to salt stress by reducing the transport of Na⁺ and Cl^- to the leaves and allowing the plant to accumulate these ions in vacuoles to prevent their accumulation in the cytoplasm or cell walls, thereby avoiding salt toxicity (Silva et al., 2019).

The efficiency of SNP in increasing seed stress tolerance has been reported in several species such as wheat (Triticum aestivum) (Ali et al., 2017), Urochloa brizantha (Oliveira et al., 2021) and cotton (Gossypium arboreum L.) (Guaraldo et al., 2023). In studies with other species, such as Urochloa ruzizienses (Oliveira et al., 2022) the potential of SNP as a priming agent was highlighted.

On the other hand, for Douradão seeds under cadmium stress, none of the solutions were efficient in improving seed performance, since all solutions showed significantly lower first count values ​​when compared to the control (Figure 2A). However, despite the difference in germination speed among germination conditions, the results on final germination values ​​were the same in both water conditions and compared to control seeds (Figure 2C). This may indicate that seed performance and germination are affected by priming in different ways depending on the cultivar, highlighting the importance of understanding the effect of priming in each species and even across different cultivars.

Overall, the roots of all treatments and both cultivars developed twice as much as the shoots of all treatments (Figure 3). Under stressful conditions (cadmium and saline), priming with H₂O₂ resulted in greater shoot lengths in Douradão seedlings. When placed to germinate in water, the greatest shoot length of Douradão seedlings was observed with the use of melatonin (Figure 3A). In BRS Soberana seedlings, compared to the control, priming was not efficient in increasing shoot length in any germination condition (Figure 3B). Higher shoot lengths were observed in BRS Soberana seedlings treated with chitosan under cadmium stress, with IAA under salt stress and with IAA and H₂O₂ when germinated in water (Figure 3A). Regarding root length, priming with H₂O₂ resulted in greater root lengths compared to control seedlings when Douradão seeds were germinated under cadmium conditions. The same was observed with the use of chitosan in seedlings germinated under saline stress and IAA and H₂O₂ under normal germination conditions (Figure 3C). The physiological priming technique had a great effect on the root length of BRS Soberana seedlings in all germination conditions compared to control seedlings (Figure 3D). Under saline stress, priming with SNP and H₂O₂ resulted in greater root lengths in BRS Soberana seedlings. Additionally, H₂O₂ improved the root length of ‘BRS Soberana’ seedlings under cadmium conditions. Among the priming solutions, H₂O₂ presented the best results for root length under cadmium conditions in both cultivars (Figures 3C and 3D).

Figure 3
Seedling shoot length (A and B) and root length (C and D) under three germination conditions (water, salinity (NaCl), and cadmium) from two different cultivars of rice seeds, Douradão (A and C) and Soberana (B and D), primed with different solutions For each cultivar, equal letters, uppercase comparing priming solutions on each germination conditions, and lower cases comparing each priming solution among the germination conditions, indicate no significant differences by Tukey’s test (p≥ 0.05). The symbol “*” indicates a significant difference in the treatment from control for each cultivar.

In plants, Cd usually accumulates in the roots, preventing its transport to other tissues. As a result, this organ may suffer further damage due to excessive concentrations of cadmium (Abedi, 2021). Thus, the use of stress tolerance induction molecules that enable greater root length under cadmium stress conditions is of great importance. In our work, H₂O₂ presented the best results for root length under cadmium conditions (Figures 3C and 3D), validating the importance of hydrogen peroxide as a stress tolerance induction molecule for rice. Based on the seedling growth results (Figure 3), the treatments with IAA, H₂O₂, chitosan and SNP stand out as the best treatments for rice priming, especially under cadmium and salinity stress conditions, in both cultivars. The agronomic efficacy of chitosan in increasing plant tolerance to stress has already been reported, involving several metabolic pathways and promoting better plant development (Rabêlo et al., 2019). Similarly, melatonin is widely recognized for improving stress tolerance, mainly by increasing the activity of the antioxidant system and the chelation of heavy metals (Gu et al., 2021). Although its excessive accumulation in cells is harmful, H₂O₂ is also considered an interesting molecule in increasing plant tolerance to stress, if it is in low and controlled doses, acting to activate the antioxidant system (Dikilitas et al., 2020).

Reduced plant growth due to the negative effects of stresses such as exposure to cadmium and salinity has been reported in other studies (García-Caparrós et al., 2020; Gu et al., 2021). These effects are related to increased accumulation of reactive oxygen species (ROS) and damage to the photosynthetic machinery (Khan et al., 2020). Senna macranthera seeds germinated under saline stress induced by NaCl at -0.3 and -0.4 MP, showed reduction in root protrusion, shoot and root length, shoot and root dry mass (Silva et al., 2019). The accumulation of cadmium in plants also reduces seedling growth, as it affects DNA replication and consequently cell replication (Huybrechts et al., 2019). Therefore, it is important to use an effective priming agent to stimulate the enzymatic activity of the antioxidant system.

When ROS levels are higher than what can be controlled by defense mechanisms, the cell experiences oxidative stress. Increased ROS production during periods of stress poses a threat to cells due to lipid peroxidation (Khan et al., 2020). Malondialdehyde (MDA) is one of the products of this process, therefore, an increase in MDA content is highly linked to the negative effects of stress conditions (Schwember and Bradford, 2010), such as the presence of heavy metals and salinity. To evaluate the ROS activity in seedlings germinated under different conditions, the MDA and H₂O₂ contents in each priming solution were evaluated, and the results can be seen in Figure 4. For BRS Soberana seedlings, melatonin and SNP were the solutions that reduced malondialdehyde production under stressful conditions (Figure 4B). For Douradão seedlings, the lowest MDA values were with the use of melatonin, SNP, H₂O₂ and IAA for stressful conditions. All solutions used for the priming technique in the Douradão cultivar showed MDA values lower than the control seeds, indicating that priming is a good option to avoid lipid peroxidation in this cultivar (Figure 4A).

Figure 4
MDA content (A and B) and H₂O₂ content (C and D) from seedlings under three germination conditions (water, salinity (NaCl), and cadmium) of two different cultivars of rice seeds, Douradão (A and C) and Soberana (B and D), primed with different solutions For each cultivar, equal letters, uppercase comparing priming solutions on each germination conditions, and lower cases comparing each priming solution among the germination conditions, indicate no significant differences by Tukey’s test (p≥ 0.05). The symbol “*” indicates a significant difference in the treatment from control for each cultivar.

The results found in this research indicate that IAA, melatonin, SNP and H₂O₂ are promising priming solutions to increase tolerance to cadmium and salt stress in seeds by reducing the MDA content. Reductions in MDA content due to priming have been reported before. Carmo et al. (2024), when working with primed Urochloa ruzizienses seeds, they observed a significant reduction in lipid peroxidation with the application of chitosan under conditions of stress caused by Cd. Similarly, wheat seeds primed with SNP had improved salt tolerance by reducing the plant’s MDA content (Ali et al., 2017). Rice seeds, in turn, primed with different concentrations of H₂O₂ showed lower MDA values under drought conditions compared to unprimed seeds (Jira-Anunkul and Pattanagul, 2020). IAA also significantly reduced MDA content and increased cell membrane stability in wheat seedlings under low temperature conditions (Kanjevac et al., 2023). According to Nawaz et al. (2012), the lower MDA concentration may be attributed to the reorganization of cell membranes that enhances membrane repair and antioxidant systems after priming, which synergistically eliminates oxidative damage.

In small amounts, hydrogen peroxide, a reactive oxygen species, can act as a signaling molecule, improving the plant’s response to stress. However, in higher concentrations, this molecule is toxic, posing a strong risk of oxidative stress in the plant, as it can cross all cellular compartments (Waszczak et al., 2018). Therefore, molecules that make it possible to reduce the levels of this reactive oxygen species under stressful conditions are of great importance. For seedlings germinated under cadmium conditions, lower H₂O₂ was observed in Douradão seedlings primed with chitosan (Figure 4C) and in Soberana seedlings primed with IAA and melatonin (Figure 4D). For Douradão, H₂O₂ also had positive effects on seedlings germinated under salt stress conditions (Figure 4C). For Douradão, seedlings primed with IAA, melatonin and SNP presented lower H₂O₂ values regardless of germination conditions. Reduction in H₂O₂ levels under stressful conditions has been reported before (Ali et al., 2017) However, actions on ROS may depend on the solution and concentration used to prepare the seeds (Sen and Puthur, 2020). Therefore, the results in Figures 4C and 4D indicate that these priming solutions reduced or prevented H₂O₂ formation under stressful conditions compared to the other solutions and to normal germination conditions (water).

Enzymes of the antioxidant system of plants can act directly in response to stress, preventing the formation and accumulation of reactive oxygen species (ROS) in cells. The action of the antioxidant system associated with the use of priming agents, such as molecules that stimulate responses to stress in seeds and seedlings, has been highlighted in several species, including Brachiaria spp. (Oliveira et al., 2021; Oliveira et al., 2022; Carmo et al., 2024), coffee (Coffea arabica) (Frota et al., 2024), cotton (Guaraldo et al., 2023), and rice (Hussain et al., 2015). Among these enzymes, ascorbate peroxidase (APX), catalase (CAT) and superoxide dismutase (SOD) have been widely investigated and related due to their importance in stress tolerance in plants. Peroxidases, such as APX, are enzymes that can oxidize any substrate in the presence of H₂O₂ or any organic hydroperoxides. This enzyme uses ascorbate as a reducing agent to reduce H₂O₂ to H₂O, preventing the accumulation of H₂O₂ (Huchzermeyer et al., 2022).

Overall, physiological priming increased APX activity in all germination conditions for Douradão (Figure 5A). Under stressful conditions (cadmium and saline stress), higher APX activity was observed in Soberana seedlings primed with H₂O₂ (Figure 5B). Under saline stress, Douradão seedlings primed with IAA showed higher enzymatic activity. Similar results were observed in Douradão seedlings treated with SNP and chitosan under cadmium stress conditions (Figure 5A). While APX preferentially captures H₂O₂ in the cytosol and chloroplast, CAT fulfills a similar role in peroxisomes and mitochondria. During stress, catalases are responsible for eliminating excess H₂O₂ from cells, converting it into water and molecular oxygen (García-Caparrós et al., 2020; Huchzermeyer et al., 2022). In our research, priming increased CAT activity in all germination conditions for Douradão. Under saline stress, Douradão seedlings primed with melatonin, H₂O₂ and chitosan showed higher CAT activity (Figure 5C). Hydrogen peroxide also increased CAT activity in Douradão seedlings under cadmium stress and in BRS Soberana seedlings under saline stress (Figures 5C and 5D). Likewise, similar results were observed in BRS Soberana seedlings treated with melatonin under cadmium stress conditions (Figure 5D).

Figure 5
Ascorbate peroxidase (APX) (A and B), catalase (CAT) (B and C), and superoxide dismutase (SOD) (E and F) content in seedlings under three germination conditions (water, salinity (NaCl), and cadmium) of two different cultivars of rice seeds, Douradão (A) and Soberana (B), primed with different solutions For each cultivar, equal letters, uppercase comparing priming solutions on each germination conditions, and lower cases comparing each priming solution among the germination conditions, indicate no significant differences by Tukey’s test (p≥ 0.05). The symbol “*” indicates a significant difference in the treatment from control for each cultivar.

Since both APX and CAT function as H₂O₂ reducers or scavengers, looking at all the results together, we observed high APX and CAT activity, as well as low H₂O₂ content using chitosan under cadmium stress in Douradão and H₂O₂ under saline stress in Douradão and BRS Soberana, indicating that these priming solutions are efficient in activating the antioxidant system, thus reducing the oxidation process. On the other hand, H₂O₂ as a priming solution, for BRS Soberana under cadmium stress condition, showed high APX and CAT activity, and high H₂O₂ content. This suggests that the antioxidant system, even with high activity, is not efficient in preventing H₂O₂ accumulation and, consequently, preventing oxidative stress. Therefore, H₂O₂ is not indicated to increase tolerance to cadmium stress in the BRS Soberana cultivar.

Superoxide dismutase (SOD) activity has been used as an additional tool to analyze the physiological quality of seeds of several crops, such as corn (Zea mays), soybean (Glycine max L.) and pepper (Capsicum annuum) (García-Caparrós et al., 2020). SOD is part of the group of metalloenzymes, presenting three isoforms, depending on the metal present in the active site (copper, iron or manganese). This enzyme acts in cellular protection, converting superoxide into H₂O₂ and O₂ (Huchzermeyer et al., 2022), being considered the first line of defense against ROS poisoning (García-Caparrós et al., 2020). SOD activity under stressful conditions, primed with different priming solutions, can be observed in Figure 5. In Douradão seedlings, no priming solution was efficient in increasing SOD activity compared to the control, in any germination condition (Figure 5E). Under cadmium conditions, the priming solutions did not differ from each other for Douradão, while for Soberana, the highest SOD activity was in seedlings treated with H₂O₂ and chitosan. Under saline stress, the highest activity was observed in Douradão seedlings primed with chitosan and in BRS Soberana seedlings treated with SNP (Figures 5E and 5F).

As the first defense barrier against ROS, higher SOD activity indicates greater cellular protection. Thus, under stress conditions, increased SOD activity indicates that the plant can tolerate this condition, while reduced levels of this enzyme can result in oxidative damage to cells (García-Caparrós et al., 2020). SOD activity has been positively correlated with priming in several species, including rice, cotton, coffee and brachiaria (Sen and Puthur, 2020; Guaraldo et al., 2023; Carmo et al., 2024; Frota et al., 2024). The benefits of chitosan in increasing SOD activity have already been reported in the literature. Cauliflower (Brassica oleracea) and sorghum (Sorghum bicolor L.) seeds primed with H₂O₂ also increased SOD activity under stressful conditions (Guo et al., 2022). All these studies corroborate the results found in this research. Although the values ​​of the antioxidant system enzymes are presented separately here, their actions all occur at the same time, and it is necessary to understand the results as a group. Since the main objective of SOD is to reduce ^O2- into H₂O₂ and O₂, the higher H₂O₂ values ​​found in seedlings primed with chitosan and H₂O₂ (Figure 4B) can be explained because of the action of SOD induced by the effect of these two priming solutions. However, for the antioxidant system to be efficient, the APX and CAT enzymes will need to eliminate all the H₂O₂ generated by SOD to prevent oxidation (Habib et al., 2021).

When primed with H₂O₂, stressed BRS Soberana seedlings increased APX activity compared to other priming solutions but had low activity in unstressed seedlings (Figure 5B). SNP and chitosan increased APX activity in Douradão seedlings stressed by cadmium, while under saline stress the highest activity was in seedlings treated with IAA (Figure 5A). For the CAT enzyme, it had good activity with H₂O₂ in Douradão seedlings and melatonin in BRS Soberana under cadmium stress, while for saline stress, the best activity was observed in Douradão seedlings treated with melatonin, H₂O₂ and chitosan in BRS Soberana seedlings primed with H₂O₂ (Figures 5C and 5D). These results indicate that chitosan and H₂O₂ are efficient in SOD activity, but H₂O₂ was not as efficient in maximizing the induction of APX and CAT enzymes especially, resulting in insufficient ROS scavenging. Priming is a technique with high potential to increase plant stress tolerance, and the benefits of the molecules tested in this research have been reported in other species (Sadak et al., 2022; Guaraldo et al., 2023; Carmo et al., 2024; Frota et al., 2024). Despite the great potential, no universal priming agent can be mentioned, since each species and even cultivars may respond differently to the chosen molecules and the priming technique applied. Furthermore, some priming solutions will act more on improving germination and vigor pathways, while others will act more on improving the antioxidant system pathways.

The results were directly influenced by the cultivar in question and the germination condition applied. In general, SNP, H₂O₂, and IAA are molecules with greater potential to induce tolerance to saline and cadmium stress, through the priming technique. Analyzing the performance of the priming solutions based on the cultivars studied, H₂O₂ stands out for the Douradão cultivar and SNP for the BRS Soberana cultivar. This research contributes to sustainable agricultural practices by offering strategies to mitigate the impact of abiotic stresses on rice production.

CONCLUSIONS

The priming of rice seeds improves seed performance and response to stress conditions and the best priming agent varies according to the cultivar and stress condition.

Hydrogen peroxide improved seed germination and root growth under salinity stress, while melatonin and H₂O₂ were more effective under cadmium stress. The activity of antioxidant enzymes is influenced by priming treatments and stress conditions.

Among the solutions used in priming, H₂O₂, SNP and IAA stand out to increase rice tolerance to cadmium and salinity stresses.

ACKNOWLEDGMENTS

The authors thank the research funding agencies Coordination for the Improvement of Higher Education Personnel (CAPES - Brazil, Process 88887.511203/2020-00), the Foundation for Research Support of the State of Minas Gerais (FAPEMIG) and the National Council for Scientific and (CNPq- Process 426309/2018-9). HOS is a CNPq productivity fell. Cadmium stress in rice plants: The effect of cadmium on seed germination and seedling growth of rice plant (Oryza sativa L.).

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Edited by

• Editor:
  Denise Cunha Fernandes dos Santos Dias

Publication Dates

• Publication in this collection
  04 Apr 2025
• Date of issue
  2025

History

• Received
  11 July 2024
• Accepted
  17 Feb 2025