Can priming with ascorbic acid or nitric oxide improve the germinability of stored sunflower seeds?

Aline Aparecida Silva Pereira Fernanda Carlota Nery Rafael Agostinho Ferreira Victor Navarro da Silva Mateus Moreira Bernardes Heloisa Oliveira dos Santos Elisa Monteze Bicalho About the authors

Abstract:

Sunflower (Helianthus annuus L.) is a relevant oilseed species used as feed in human and animal nutrition and in multiple industrial applications. However, oilseeds need to deal with the loss of vigor when used as a propagule, due to loss of seed physiological quality (viability and vigor) caused by deterioration events. We have evaluated in the present study the effects of seeds priming techniques (water, ascorbic acid, and sodium nitroprusside) on vigor, germination, antioxidant enzymes and membrane integrity of stored sunflower hybrid HELIO 251 seeds. Germination parameters showed that non-primed seeds (control) delayed germination in almost 72 h while all primed seeds germinated 100% in 24 h. Electrolyte leakage and malondialdehyde levels were higher in non-primed seeds indicating higher degree of membrane damage. An increase in the activity of catalase and ascorbate peroxidase was also observed after 8 h of imbibition of primed seeds compared to non-primed seeds. Regarding superoxide dismutase, there was no significant differences between treatments after 8 h of imbibition, whereas the highest activity was after 24 h of imbibition in non-primed seeds. Therefore, it can be concluded priming with ascorbic acid or sodium nitroprusside exhibited better performance in germination of stored sunflower seeds, probably resulting from lower accumulation of reactive oxygen species and consequent reduced oxidative damage due to an efficient antioxidant enzyme system.

Index terms:
aging; oilseed; oxidative damage; priming; seed vigor

Resumo:

O girassol (Helianthus annuus L.) é uma espécie oleaginosa relevante utilizada na alimentação humana e animal e, em múltiplas aplicações industriais. No entanto, as oleaginosas precisam lidar com a perda de vigor quando utilizadas como propágulos, devido à perda da qualidade fisiológica da semente (viabilidade e vigor) causada por eventos de deterioração. Avaliamos no presente estudo os efeitos de técnicas de condicionamento de sementes (água, ácido ascórbico e nitroprussiato de sódio) no vigor, germinação, enzimas antioxidantes e integridade de membrana de sementes de girassol híbrida HELIO 251 armazenadas. Os parâmetros de germinação mostraram que as sementes não condicionadas (controle) atrasaram a germinação em quase 72 h enquanto todas as sementes condicionadas germinaram 100% em 24 h. O extravasamento de eletrólitos e os níveis de malondialdeído foram maiores nas sementes não condicionadas, indicando maior grau de dano à membrana. Um aumento na atividade da catalase e ascorbato peroxidase também foi observado após 8 h de embebição de sementes condicionadas em comparação com sementes não condicionadas. Em relação à superóxido dismutase, não houve diferenças significativas entre os tratamentos após 8 h de embebição, enquanto a maior atividade foi após 24 h de embebição em sementes não condicionadas. Portanto, pode-se concluir que o priming com ácido ascórbico ou nitroprussiato de sódio apresentou melhor desempenho na germinação de sementes de girassol armazenadas, provavelmente decorrente do menor acúmulo de espécies reativas de oxigênio e consequente redução do dano oxidativo devido a um sistema enzimático antioxidante eficiente.

Termos para indexação:
envelhecimento; oleaginosa; dano oxidativo; priming; vigor da semente

INTRODUCTION

Sunflower (Helianthus annuus L., Asteraceae) is an annual cycle oilseed species (Simões et al., 2020SIMÕES, W.L.; SILVA, J.S.; OLIVEIRA, A.R.; NETO, A.R.; DRUMOND, M.A.; LIMAS, J.A.; NASCIMENTO, B.R. Sunflower cultivation under different irrigation systems and planting spacings in the sub-iddle region of São Francisco Valley. Ciências Agrárias, v.41, n.2, p.2899-2910, 2020. https://doi.org/10.5433/1679-0359.2020v41n6Supl2p2899
https://doi.org/10.5433/1679-0359.2020v4...
) with broad agronomic and feed uses. The high oil content requires improved storage protocols to maintain the quality (germination/vigor) and promote better longevity of stores sunflower seeds (Abreu et al., 2013ABREU, L.A.S.; CARVALHO, M.L.M.; PINTO, C.A.G.; KATAOKA, V.Y.; SILVA, T.T.A. Deterioration of sunflower seeds during storage. Journal of Seed Science, v.35, n.2, p.40-247, 2013. https://doi.org/10.1590/S2317-15372013000200015
https://doi.org/10.1590/S2317-1537201300...
). Seed vigor encompass a set of attributes that ensures the seeds’ potential to germinate faster and with better uniformity (synchronously) in shorter periods (Siadat et al., 2012SIADAT, S.A.; MOOSAVI, A.; ZADEH, M. Effect of seed priming on antioxidant activity and germination characteristics of Maize seeds under different aging treatments. Research Journals of Seed Science, v.5, n.2, p.51-62, 2012. https://doi.org/10.3923/rjss.2012.51.62
https://doi.org/10.3923/rjss.2012.51.62...
). Whereas oil seeds generally show less chemical stability due to the percentage of lipids reserves, the increasing probability of lipid peroxidation and membrane damages intensifying the loss of vigor and ultimately germinability (Rajjou et al., 2012RAJJOU, L.; DUVAL, M.; HALLARDO, K.; CATUSSE, J.; BALLY, J.; JOB, C.; JOB, D. Seed germination and vigor. Annual Review of Plant Biology, v.63, n.1, p.507-533, 2012. https://doi.org/10.1146/annurev-arplant-042811-105550
https://doi.org/10.1146/annurev-arplant-...
).

The reduction of seed vigor and geminability resulting from deterioration of its reserves during storage are associated with changes in cellular and metabolic events resulting in enzymatic inactivation and reduced energy metabolism, beyond overproduction of reactive oxygen species (ROS) (Hu et al., 2012HU, G.; MA, Q.; WANG, J.; YAO, Y.; WANG, H.W. Spatial and temporal nature of reactive oxygen species production and programmed cell death in elm (UlmuspumilaL.) seeds during controlled deterioration. Plant Cell Environment, v.35, n.11, p.2045-2059, 2012. https://doi.org/10.1111/j.1365-3040.2012.02535.x
https://doi.org/10.1111/j.1365-3040.2012...
; Xia et al., 2015XIA, X.J.; ZHOU, Y.H.; SHI, K.; ZHOU, J.; FOYER, C.H.; YU, J.Q. Interplay between reactive oxygen species and hormones in the control of plant development and stress tolerance. Journal of Experimental Botany , v.66, n.10, p.2839-2856, 2015. https://doi.org/10.1093/jxb/erv089
https://doi.org/10.1093/jxb/erv089...
). Under controlled conditions, the levels of ROS in seeds are regulated by superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) which act regulating ROS accumulation and minimizing oxidative damages (Kaur et al., 2019KAUR, N.; KAUR, J.; GREWAL, S.K.; SINGH, I. Effect of heat stress on antioxidative defense system and its amelioration by heat acclimation and salicylic acid pre-treatments in three pigeonpea genotypes. Indian Journal of Agricultural Biochemistry, v.32, n.1, p.106-110, 2019. https://doi.org/10.5958/0974-4479.2019.00014.5
https://doi.org/10.5958/0974-4479.2019.0...
).

Priming is known to recover and/or enhance the physiological quality by recovering from the aging effects that stored seeds usually suffers (Farooq et al., 2009FAROOQ, M.; BASRA, S.M.A.; WAHID, A.; AHMAD, N.; SALEEM, B.A. Improving the drought tolerance in rice (Oryza sativaL.) by exogenous application of salicylic acid. Journal of Agronomy and Crop Science, v.195, n.4, p.237-246, 2009. https://doi.org/10.1111/j.1439-037X.2009.00365.x
https://doi.org/10.1111/j.1439-037X.2009...
). One of the most promisor priming methods to restore seed vigor is with signaling molecules as nitric oxide (NO) and ascorbic acid (AsA). The NO is a gaseous uncharged and lipophilic molecule able to act on some subcellular compartments (Takahashi and Shinozaki, 2019TAKAHASHI, F.; SHINOZAKI, K. Long-distance signaling in plant stress response.Current Opinion in Plant Biology, v.47, n1, p.106-111, 2019. https://doi.org/10.1016/j.pbi.2018.10.006
https://doi.org/10.1016/j.pbi.2018.10.00...
). Besides being the most abundant reactive nitrogen species in plants, NO perform an essential role in several physiological processes such as growth regulation and tolerance to various biotic or abiotic stressful conditions (Sánchez-Vicente et al., 2019SÁNCHEZ-VICENTE, I.; FERNÁNDEZ-ESPINOSA, M.G.; LORENZO, O. Nitric oxide molecular targets: reprogramming plant development upon stress. Journal of Experimental Botany , v.70, n17, p.4441-4460, 2019. https://doi.org/10.1093/jxb/erz339
https://doi.org/10.1093/jxb/erz339...
). While AsA is a non-enzymatic antioxidant molecule with important role in germination as it may protect cells from the cytotoxic effects of ROS (Foyer and Noctor, 2005FOYER, C.; NOCTOR, G. Redox homeostasis and antioxidant signaling: a metabolic interface between stress perception and physiological responses. The Plant Cell, v.17, n.7, p.1866-1875, 2005. https://doi.org/10.1105/tpc.105.033589
https://doi.org/10.1105/tpc.105.033589...
), therefore, reducing oxidative damage (Sharma et al., 2019SHARMA, R.; BHARDWAJ, A.K.; THUKRAL, A.A.; ALHUQAIL, M.H.; SIDDIQUI, P.; AHMAD. Oxidative stress mitigation and initiation of antioxidant and osmoprotectant responses mediated by ascorbic acid in Brassica juncea L. subjected to copper (II) stress. Ecotoxicology and Environmental Safety, v.182, 109436, 2019. https://doi.org/10.1016/j.ecoenv.2019.109436.
https://doi.org/10.1016/j.ecoenv.2019.10...
) and acting on repair of damaged membranes (Tommasi et al., 2011TOMMASI, F.; PACIOLLA, C.; PINTO, M.C.; GARA, L. A comparative study of glutathione an ascorbate metabolism during germination of Pinuspinea L. seeds. Journal of Experimental Botany , v.52, n.361, p.1647-1654, 2011. https://doi.org/10.1093/jexbot/52.361.1647
https://doi.org/10.1093/jexbot/52.361.16...
) resulting from abiotic and biotic stresses.

Besides all efforts to evaluate the physiological responses of primed seeds, and vigor, there is a lack of information regarding this process on long-term stored seeds. Here we hypothesized that long-term stored seeds of the sunflower hybrid, HELIO 251, primed with NO or AsA can exhibit improvements in physiological quality, uniformity, and germination speed. Thereby, this work aimed to evaluate the germinability of stored sunflower hybrid HELIO 251 seeds submitted to priming with NO and AsA.

MATERIAL AND METHODS

Plant material and storage conditions

All experimental assays were performed at the Plant Growth and Development Lab and at the Seeds Central Lab at the Universidade Federal de Lavras (UFLA) located in the state of Minas Gerais. Seeds of the simple sunflower hybrid HELIO 251 were produced in 2016 by Heliagro Agricultura e Pecuária ©. Seeds were harvested, processed, and stored in multilayered Kraft paper inside polyethylene bags and subsequently stored in a cold chamber (10 °C) during 42 months prior to use.

Priming, experimental conditions, and sampling

The priming experiment was carried out in a completely randomized design with 4 treatments and 4 replications each. Treatments were composed by control (non-primed seeds); water (hydropriming with deionized water); NO (priming with 100 mM sodium nitroprusside, a NO donor); and AsA (priming with 100 mM ascorbic acid). The priming processes consisted in imbibing the stored seeds in deionized water or in 100 mM AsA or in 100 mMNO during 4 hours (h) at 25 °C, followed by washing in running tap water and redrying at 35 °C for 12 h. The control treatment was not subjected to priming. Then, these seeds were placed into Petri dishes with germination paper moistened with distilled water, in four replications with 25 seeds each, and incubated in a germination chamber at 12-hour photoperiod and average temperature of 25 °C and 60 % of relative humidity. Germination parameters were evaluated daily (24 h) for four days, by measuring the percentage of seed germination and germination speed index (GSI).

The biochemical analyses were carried on samples collected after 8 and 24 h of imbibition corresponding to phase II and phase III of the germinative process, respectively. Electrical conductivity was performed only with 8 h-imbibed seeds. Biochemical assays were performed with both 8h and 24h-imbibibed seeds and consisted of quantification of hydrogen peroxide (H2O2), malondialdehyde (MDA, an extension of lipid peroxidation) and antioxidant enzymes activities (catalase - CAT, ascorbate peroxidase - APX and superoxide dismutase - SOD) in a 2x4 factorial arrangement (two times of imbibition and three priming treatments plus control).

Germination parameters

Seed germinability was evaluated daily for 4 days. Germinated seed were considered at 2 mm radicle protrusion (Brasil, 2009BRASIL. Ministério da Agricultura, Pecuária e Abastecimento. Regras para Análise de Sementes. Secretaria de Defesa Agropecuária. Brasília: MAPA/ACS, 2009. 399p. https://www.gov.br/agricultura/pt-br/assuntos/insumos-agropecuarios/arquivos-publicacoes-insumos/2946_regras_analise__sementes.pdf
https://www.gov.br/agricultura/pt-br/ass...
). The percentage of seed germination was obtained through quantification of the mean of the daily-germinated seeds during the four days. The germination speed index was performed using the equation proposed by Maguire (1962MAGUIRE, J.D. Speed of germination aid in selection and evaluation for seedling emergence and vigor. Crop Science , v.2, n.2, p.176-77, 1962. https://doi.org/10.2135/cropsci1962.0011183X000200020033
https://doi.org/10.2135/cropsci1962.0011...
), considering the sum of seeds germinated in each day of experiments

Electrical conductivity

The electrical conductivity test consisted in four replications with 25 seeds. Each replication was weighted before imbibition and placed in plastic recipients containing 75 mL of deionized water. Then, they were kept in a germination chamber for 24 h at constant temperature of 25 °C (Vieira and Carvalho, 1994VIEIRA, R.D.; CARVALHO, N.M. Testes de vigor em sementes. Jaboticabal: FUNEP, 1994. 164p.). The samples were shaken, and the electrical conductivity was measured with a conductivity meter (MS TECNOPON®, mCA 150 model) and results expressed in µS.cm.g-1 of seeds.

Biochemical analyzes

Lipid peroxidation and hydrogen peroxide quantification

Lipid peroxidation was determined by malondialdehyde (MDA) quantification (Hodges et al., 1999HODGES, D.M.; DELONG, J.M.; FORNEY, C.F.; PRANGE, R.K. Improving the thiobarbituric acid reactive-substances assay for estimating lipid peroxidation in plant tissues containing anthocyanin and other interfering compounds. Planta, v.207, n.1, p.604-611, 1999. https://doi.org/10.1007/s004250050524
https://doi.org/10.1007/s004250050524...
). Samples of 100 mg of seeds were grinded in liquid nitrogen and homogenized in 1 mL of 80% ethanol, centrifuged, and the supernatant collected. This step was performed three times, totaling 3 mL. The reaction with 1ml of extract and 1 mL of 20% TCA, 0.65% tiobarbituric acid (TBA) and 0.01% butylated hydroxytoluene (BHT) was carried. The estimate lipid peroxidation was calculated usingthe absorbance reading in a spectrophotometer at 440, 532, and 600 nm. Hydrogen peroxide (H2O2) was quantified as described by Velikova et al. (2000VELIKOVA, V.; IORDANOV, I.; EDREVA, A. Oxidative stress and some antioxidant systems in acid rain-treated bean plants: protective role of exogenous polyamines. Plant Science, v.151, n.1, p.59-66, 2000. https://doi.org/10.1016/S0168-9452(99)00197-1
https://doi.org/10.1016/S0168-9452(99)00...
). Samples of 100 mg of seeds were grinded in liquid nitrogen and then homogenized with 1 mL of 0.1% trichloroacetic acid (TCA), and subsequently centrifuged and placed for reaction in 10 mM potassium phosphate (KH2PO4) buffer at pH 7.0 and 1M potassium iodide (KI). Analysis in spectrophotometer occurred by reading at 390 nm and then comparing it to a standard curve for H2O2 quantification.

Antioxidant system enzymes

The enzymatic antioxidant system activity (SOD, CAT, and APX) was evaluated in seeds after priming. The enzymatic extracts were obtained according to Biemelt et al. (1998BIEMELT, S.; KEETMAN, U.; ALBRECHT, G. Re-aeration following hypoxia or anoxia leads to activation of the antioxidative defense system in roots of wheat seedlings. Plant Physiology , v.116, n.2, p.651-658, 1998. https://doi.org/10.1104/pp.116.2.651
https://doi.org/10.1104/pp.116.2.651...
) in which 200 mg of seeds were grinded in liquid nitrogen with PVPP (Polyvinylpyrrolidone) added to 1.5 mL of extraction buffer composed of: 400 mM potassium phosphate (pH 7.8), 10 mM EDTA, and 200 mM ascorbic acid. The solution was centrifuged at 13.000 g for 10 minutes at 4 °C and the supernatant collected to quantify enzymes activities.SOD activity was based on the enzyme capacity of inhibiting the photoreduction of nitrobluetetrazolium (NBT) (Giannopolitis and Ries, 1977GIANNOPOLITIS, C.N.; RIES, S.K. Superoxide dismutase: I. occurrence in higher plants. Plant Physiology , v.59, n.2, p.309-314, 1977. https://doi.org/10.1104/pp.59.2.309
https://doi.org/10.1104/pp.59.2.309...
). Absorbance readings were made at 560 nm. A unity of SOD corresponds to the number of enzymes able to inhibit 50% of NBT photoreduction in the assay conditions. The CAT activity was determined according to Havir and McHale (1987HAVIR, E.A.; MCHALE, N.A. Biochemical and developmental characterization of multiple forms of catalase in tobacco leaves. Plant Physiology , v.84, n.2, p.450-455, 1987. https://doi.org/10.1104/pp.84.2.450
https://doi.org/10.1104/pp.84.2.450...
). This enzyme activity is calculated based on the absorbance decrease at 240 nm, every 15 seconds for 3 minutes, monitored by the hydrogen peroxide consumption. The molar extinction coefficient used was 36 mM-1 .cm-1 (Azevedo et al., 1998AZEVEDO, R.A.; ALAS, R.M.; SMITH, R.J.; LEA, P.J. Response from elevated carbon dioxide to air and ozone fumigation in leaves and roots of wild type and a catalase-deficient mutant of barley. Physiologia Plantarum, v.104, n.2, p.280-292, 1998. https://doi.org/10.1034/j.1399-3054.1998.1040217.x
https://doi.org/10.1034/j.1399-3054.1998...
). APX activity was analyzed as described by Nakano and Asada (1981NAKANO, Y.; ASADA, K. Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant and Cell Physiology , v.22, n.22, p.867-880, 1981. https://doi.org/10.1093/oxfordjournals.pcp.a076232
https://doi.org/10.1093/oxfordjournals.p...
) through ascorbate oxidation at 290 nm with a molar extinction coefficient of 2.8 mM-1 .cm-1.

Statistical analysis

The experiment was carried out in a completely randomized design, considering one-way factor for germination and electrical conductivity and in a two-way 4×2 factorial arrangement for biochemical analysis, consisting of the four priming treatments and two imbibition times, with replications each. Data was subjected to ANOVA and, in case of normal distribution, Scott-Knot means test was selected at 5% significance using the Sisvar 5.6 software package.

RESULTS AND DISCUSSION

Changes in germination only occurred in control non-primed seeds. The cumulative germination (G%) showed that control seeds delayed germination in comparison to primed seeds regardless the priming solution. Primed seeds reached over 90% germination after 24 h imbibition (Figure 1A). The germination speed index (GSI) of control seeds was lower than of primed seeds (Figure 1B). G% and GSI did not show statistical difference among water, NO and AsA seeds.

Figure 1
Cumulative germination (G%) (A) and Germination Speed Index (GSI) (B) of Helianthus annus seeds subjected to priming treatments Water, NO (100mM sodium nitroprusside) and AsA (100mM ascorbate), and the Control. Dots or bars are means ± standard error (n=4). Same letters do not differ each other on Scott-Knott test set at 5% of probability. (*) statistic differences.

All priming methods applied to the stored (aged) sunflower hybrid seeds resulted in better germination and vigor performance (speed and uniformity) in comparison to non-primed. Since germination is one of the most important processes in a plant’s life cycle, its success depends on environmental conditions besides appropriate biochemical and physiological responses (Bewley et al., 2013BEWLEY, J.D.; BRADFORD, K.; HILHORST, H.; NONOGAKI, H. Seeds: Physiology of Development, Germination and Dormancy. New York: Springer, 2013. p.1-405. ). At cellular level, the seed aging is associated with a set of alterations including membrane integrity loss and the reduction of energetic metabolism that can lead to electrolyte leakage and decrease seed germination and vigor (Boniecka et al., 2019 BONIECKA, J.; KOTOWICZ, K.; SKRZYPEK, E.; DZIURKA, K.; REWERS, M.; JEDRZEJCZYK, I.; WILMOWICZ, E.; BERDYCHOWSKA, J.; DĄBROWSKA, G.B. Potential biochemical, genetic and molecular markers of deterioration advancement in seeds of oilseed rape (Brassica napus L.). Industrial Crops and Products, v.130, n.1, p.478-490, 2019. https://doi.org/10.1016/j.indcrop.2018.12.098
https://doi.org/10.1016/j.indcrop.2018.1...
). The GSI and G% results for primed seeds indicate a reestablishment of ideal seed physiological capacity as seeds presented faster and uniform germination, i.e. better vigor.

The seeds’ speed and capacity for repairing damages are directly involved to its performance during the germination and seedling establishment (Long et al., 2015LONG, R.L.; GORECKI, M.J.; RENTON, M.; SCOTT, J.K.; COLVILLE, L.; GOGGIN, D.E.; COMMANDER, L.E.; WESTCOTT, D.A.; CHERRY, H.; FINCH-SAVAGE, W.E. The ecophysiology of seed persistence: a mechanistic view of the journey to germination or demise. Biological Reviews , v.90, n.1, p.31-59, 2015. https://doi.org/10.1111/brv.12095
https://doi.org/10.1111/brv.12095...
). As previously reported, seeds that did not undergo any kind of priming (control) showed lower germination percentage and germination speed index. As practical results of this technique and the treatments established, there is an increase in the germination rate, germination speed, uniformity, and the restructuring of membranes. Once the imbibition process starts, there are a set of repairs that happens at cellular and structural level which are related to delay in germination of aged seeds (Xu et al., 2020XU, L.; XIN, X.; YIN, G.; ZHOU, J.; ZHOU, Y.; LU, X. Timing for antioxidant-priming against rice seed aging: optimal only in non-resistant stage. Scientific Reports , v.10, n.1, p.1-13, 2020. https://doi.org/10.1038/s41598-020-70189-6
https://doi.org/10.1038/s41598-020-70189...
). As seeds go through a post-harvest storage period, they undergo a gradual and continuous deterioration process which leads to reduction of their vigor (Sahu et al., 2017SAHU, B.; SAHU, A.K.; THOMAS, V.; NAITHAN, S.C. Reactive oxygen species, lipid peroxidation, protein oxidation and antioxidative enzymes in dehydrating Karanj (Pongamiapinnata) seeds during storage. South African Journal of Botany, v.112, n.1, p.383-390, 2017. https://doi.org/10.1016/j.sajb.2017.06.030
https://doi.org/10.1016/j.sajb.2017.06.0...
; Boniecka et al., 2019 BONIECKA, J.; KOTOWICZ, K.; SKRZYPEK, E.; DZIURKA, K.; REWERS, M.; JEDRZEJCZYK, I.; WILMOWICZ, E.; BERDYCHOWSKA, J.; DĄBROWSKA, G.B. Potential biochemical, genetic and molecular markers of deterioration advancement in seeds of oilseed rape (Brassica napus L.). Industrial Crops and Products, v.130, n.1, p.478-490, 2019. https://doi.org/10.1016/j.indcrop.2018.12.098
https://doi.org/10.1016/j.indcrop.2018.1...
). As seeds age and lose vigor, more severe damage exceeds the capacity for self-repair (Bewley et al., 2013BEWLEY, J.D.; BRADFORD, K.; HILHORST, H.; NONOGAKI, H. Seeds: Physiology of Development, Germination and Dormancy. New York: Springer, 2013. p.1-405. ).

The levels of hydrogen peroxide (H2O2) (Figure 2A) were not show statistical differences between treatments after 8 h imbibition. However, it was possible to observe that the control non-primed seeds showed the lowest content of H2O2 when compared to any of the primed seeds. At 24 h, a higher concentration of H2O2 was observed in seeds subjected to imbibition in water. NO and AsA treatments did not show statistical difference during the evaluation at 24 h. The content of MDA was higher after 8 h imbibition in control seeds, while it was lower in NO primed seeds (Figure 2B), whereas water and AsA treatments showed similar contents. Similarly, the control seeds had higher content also after 24 h imbibition. Water, NO and AsA treatments did not present statistical differences. Electrical conductivity values were higher in non-primed control seeds, while the seeds subjected to all priming treatments exhibited the lower not significant values (Figure 2C).

Figure 2
Hydrogen peroxide (H2O2) (A), malondialdehyde (MDA) content (B) and electrical conductivity (C) of Helianthus annus seeds subjected to priming treatments Water, NO (100mM sodium nitroprusside) and AsA (100mM ascorbate), and the Control. Bars are means ± standard error (n=4). Uppercase letters compare the levels of H2O2 or MDA between 8 h and 24 h in each treatment. Lowercase letters compare the levels of H2O2 or MDA between treatments of 8 h or 24 h. Equal letters do not differ from each other on Scott-Knott test set at 5% of probability. (*) statistic differences.

The efficiency of seed priming with NO and AsA in improving both percentage and speed of germination correlated with ROS scavenging, thus limiting oxidative stress. The increase in ROS production was identified as one of the most important points that directly affects seed aging by promoting oxidative damage to lipids and proteins, loss of viability and electrolyte leakage (Bailly, 2004BAILLY, C. Active oxygen species and antioxidants in seed biology. Seed Science Research, v.14, n.2, p.93-107, 2004. https://doi.org/10.1079/SSR2004159
https://doi.org/10.1079/SSR2004159...
;Waszczak et al., 2018WASZCZAK, C.; CARMODY, M.; KANGASJÄRVI, J. Reactive oxygen species in plant signaling. Annual Review of Plant Biology , v.69, n.1, p.209-236, 2018. https://doi.org/10.1146/annurev-arplant-042817-040322
https://doi.org/10.1146/annurev-arplant-...
). The imbalance between the level of ROS and the consequent oxidative damage is associated with loss of seed viability (Morscher et al., 2015MORSCHER, F.; KRANNER, I.; ARC, E.; BAILLY, C.; ROACH, T. Glutathione redox state, to cochromanols, fatty acids, antioxidant enzymes and protein carbonylation in sunflower seed embryos associated with after-ripening and aging. Annals of Botany, v.116, n.4, p.669-678, 2015. https://doi.org/10.1093/aob/mcv108
https://doi.org/10.1093/aob/mcv108...
; Sano et al., 2016SANO, N.; RAJJOU, L.; NORTH, H.H.; DEBEAUJON, I.; MARION-POLL, A.; SEO, M. Staying alive: molecular aspects of seed longevity. Plant Cell Physiology, v.57, n.4, p.660-674, 2016. https://doi.org/10.1093/pcp/pcv186
https://doi.org/10.1093/pcp/pcv186...
). Thereby, lipid peroxidation promoted by free radicals, membranes rupture and loss of cellular solutes have been reported as the main damage during deterioration (Fu et al., 2015FU, Y.; AHMED, Z.; DIEDERICHSEN, A.Towards a better monitoring of seed aging under ex situ seed conservation. Conservation Physiology, v.3, n.1, p.1-16, 2015. https://doi.org/10.1093/conphys/cov026
https://doi.org/10.1093/conphys/cov026...
).

In oilseeds, the glyoxysomes produce a greater amount of hydrogen peroxide during the period that seeds mobilize its lipid reserves (Bailly et al., 2008BAILLY, C.; EL-MAAROUF-BOUTEAU, H.; CORBINEAU, F. From intracellular signaling networks to cell death: the dual role of reactive oxygen species in seed physiology. Comptes Rendus Biologies, v.331, n.10, p.806-814, 2008. https://doi.org/10.1016/j.crvi.2008.07.022
https://doi.org/10.1016/j.crvi.2008.07.0...
; Gill and Tuteja, 2010GILL, S.S.; TUTEJA, N. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiology and Biochemistry, v.48, n.12, p.909-930, 2010. https://doi.org/10.1016/j.plaphy.2010.08.016
https://doi.org/10.1016/j.plaphy.2010.08...
). However, the results found demonstrate that, even the treatments being no different statistically regarding the H2O2 content, the control seeds exhibited the higher MDA rate and electrical conductivity after 8 h imbibition when compared to other treatments. The same can be seen after 24 h imbibition, where the non-primed control showed greater MDA content. Although the seeds have several mechanisms of repair and elimination of ROS, they may no longer be viable and may present delays in germination or even not germinate after a prolonged period of storage if the damage is greater than the repair capacity (Xu et al., 2020XU, L.; XIN, X.; YIN, G.; ZHOU, J.; ZHOU, Y.; LU, X. Timing for antioxidant-priming against rice seed aging: optimal only in non-resistant stage. Scientific Reports , v.10, n.1, p.1-13, 2020. https://doi.org/10.1038/s41598-020-70189-6
https://doi.org/10.1038/s41598-020-70189...
).

NO can act both in the production and in the degradation of ROS, presenting a complex feedback regulation between these two signaling molecules. The action of NO in the elimination of ROS can occur directly or in the action of this reactive nitrogen species (RSN) in inducing the action of the antioxidant system (Singh and Bhatla, 2017SINGH, N.; BHATLA, S.C. Signaling through reactive oxygen and nitrogen species is differentially modulated in sunflower seedling root and cotyledon in response to various nitric oxide donors and scavengers. Plant Signaling & Behavior, v.12, n.9, p.1-15, 2017. https://doi.org/10.1080/15592324.2017.1365214
https://doi.org/10.1080/15592324.2017.13...
). Priming with NO or AsA resulted in seeds with greater capacity to repair the damages and consequently presented less oxidative damages, which resulted in the better percentage and speed of germination. As observed in electrical conductivity results, the non-primed control seeds showed the highest rate of electrolyte leakage when compared to the priming seeds. The electrolyte leakage is an indicator of altered cell membrane permeability, consequence of structural modifications induced by lipid peroxidation and damage to the phospholipid layer during seed deterioration and vigor decrease (Mira et al., 2010MIRA, S.; GONZÁLEZ-BENITO, M.E.; HILL, L.M.; WALTERS, C. Characterization of volatile production during storage of lettuce (Lactuca sativa) seed. Journal of Experimental Botany , v.61, n.14, p.3915-3924, 2010. https://doi.org/10.1093/jxb/erq202
https://doi.org/10.1093/jxb/erq202...
; Sahu et al., 2017SAHU, B.; SAHU, A.K.; THOMAS, V.; NAITHAN, S.C. Reactive oxygen species, lipid peroxidation, protein oxidation and antioxidative enzymes in dehydrating Karanj (Pongamiapinnata) seeds during storage. South African Journal of Botany, v.112, n.1, p.383-390, 2017. https://doi.org/10.1016/j.sajb.2017.06.030
https://doi.org/10.1016/j.sajb.2017.06.0...
). The reduction of electrolyte leakage by hydropriming, AsA and NO is strong evidence of the potential of these molecules to induce the retention or restructuration of the cell membrane integrity during germination. In addition, NO can act as a chain-breaking pathway during the lipid peroxidation process, being able to react directly with membranes or with reactive molecules such as superoxide anion, thus reducing damage to the system (Mazid et al., 2011MAZID, M.; KHAN, T.A.; MOHAMMAD, F. Role of Nitric oxide in regulation of H2O2 mediating tolerance of plants to abiotic stress: A synergistic signaling approach. Journal of Stress Physiology & Biochemistry, v.7, n.2, p.34-74, 2011. http://www.jspb.ru/issues/2011/N2/JSPB_2011_2_34-74.pdf
http://www.jspb.ru/issues/2011/N2/JSPB_2...
).

The difference in cell membrane permeability assessed after 8 h imbibition (phase II of germination) suggests changes in the extent of damage caused by ROS to the cell membrane (Nabi et al., 2019NABI, R.B.S.; TAYADE, R.; HUSSAIN, A.; KULKARNI, K.P.; IMRAN, Q.M.; MUN, B.G.; YUN, B.W. Nitric oxide regulates plant response to drought, salinity, and heavy metal stress. Environmental Experimental Botany, v.161, n.1, p.123-133, 2019. https://doi.org/10.1016/j.envexpbot.2019.02.003
https://doi.org/10.1016/j.envexpbot.2019...
). The content of MDA in both treatments is proportional to the activity of the antioxidant systems that act in scavenging of ROS, which affects the integrity of membranes and electrolyte leakage. The proposed priming methods seemed to effectively recover and improve membrane dysfunction while reducing lipid peroxidation. A common fact of aged seeds is the increase of leachates when seeds are immersed in water (López-Fernández et al., 2018LÓPEZ-FERNÁNDEZ, M.P.; MOYANO, L.; CORREA, M.D.; VASILE, F.; BURRIEZA, H.P.; MALDONADO, S. Deterioration of willow seeds during storage. Scientific Reports, v.8, n.1, p.1-11, 2018. https://doi.org/10.1038/s41598-018-35476-3
https://doi.org/10.1038/s41598-018-35476...
). However, the evaluation of MDA as a marker of oxidative stress in plants in this work allowed the observation of a correlation between the evaluated imbibition stages, the H2O2 accumulation and the membrane damage. Variations in the ROS amounts and its production time have also been reported in the work developed by Leymarie et al. (2012LEYMARIE, J.; VITKAUSKAITÉ, G.; HOANG, H.H.; GENDREAU, E.; CHAZOULE, V.; MEIMOUN, P.; CORBINEAU, F.; EL-MAAROUF-BOUTEAU, H.; BAILLY, C. Role of reactive oxygen species in the regulation of Arabidopsis seed dormancy.Plant and Cell Physiology, v.53, n.1, p.96-106, 2012. https://doi.org/10.1093/pcp/pcr129
https://doi.org/10.1093/pcp/pcr129...
), where the authors suggest that ROS assumes different functions during germination.

The values of superoxide dismutase (SOD) activity (Figure 3A) were higher after 8 h imbibition in all treatments, although they were not statistical different during this period. The evaluation at 24 h imbibition showed the highest activity in non-primed control seeds. Lower activity of catalase (CAT) and Ascorbate peroxidase (APX) (Figures 3B and 3C) was only perceived after 8 h imbibition in non-primed control seeds. None of the treatments showed significant differences after 24 h imbibition. Whereas NO and AsA treatments did not present differences after 8- and 24-h imbibition.

Figure 3
Superoxide dismutase (SOD) (A), catalase (CAT) (B), and ascorbate peroxidase (APX) (C) activity of Helianthus annus seeds subjected to priming treatments water, NO (100mM sodium nitroprusside) and AsA (100mM ascorbate), and the Control. Bars are means ± standard error (n=4). Uppercase letters compare the activity of SOD, CAT or APX between 8 h and 24 h in each treatment. Lowercase letters compare the activity of SOD, CAT or APX between treatments of 8 h or 24 h. Equal letters do not differ from each other on Scott-Knott test set at 5% of probability. (*) statistic differences.

Considering the interaction between time factors and types of conditioning factors (priming), it was observed that only SOD and APX presented significant values. Regarding SOD, the activity after 8 h imbibition was greater than after 24 h. In addition, the non-primed control differed from the primed seeds which presented higher average in relation to the other treatments after 8 h. Within 24 h, all treatments, except the control, significantly reduced SOD activity over time. Regarding APX, its activity increased over time in all treatments, being statistically different in control and hydropriming. The non-primed control seeds showed less activity of APX after 8 h imbibition compared to the other treatments. On the other hand, CAT presented greatest activity after 24 h than after 8 h in all treatments. At 8 h, the control treatment differed significantly from the others, presenting the lowest activity of this enzyme in this time interval. Within 24 h, there was no statistical difference between treatments.

It is reported that the reduction in the activity of the antioxidant system, mainly the enzymatic action, may be responsible for the accumulation of ROS, thus leading to loss of seed vigor (Yin et al., 2014YIN, G.; XIN, X.; SONG, C.; CHEN, X.; ZHANG, J.; WU, S.; LI, R.; LIU, X.; LU, X. Activity levels and expression of antioxidant enzymes in the ascorbate-glutathione cycle in artificially aged rice seed. Plant Physiology and Biochemistry, v.80, n.1, p.1-9, 2014. https://doi.org/10.1016/j.plaphy.2014.03.006
https://doi.org/10.1016/j.plaphy.2014.03...
). SOD showed a high activity in the first evaluation, followed by a reduction at 24 h evaluation in all treatments except the control. The activity of CAT, responsible for reducing H2O2 in water (Das and Roychoudhury, 2014DAS, K.; ROYCHOUDHURY, A. Reactive Oxygen Species (ROS) and Response of Antioxidants as ROS-Scavengers during Environmental Stress in Plants. Frontiers in Environmental Science , v.2, n.1, p.1-13, 2014. https://doi.org/10.3389/fenvs.2014.00053
https://doi.org/10.3389/fenvs.2014.00053...
), differed statistically only at the 8 h period in the control treatment, where the lowest activity of this enzyme was observed. Interestingly, the H2O2 level in the 8-hour control treatment was lower than other treatments, although its oxidative damage assessed by the MDA marker was greater. The increase CAT and APX activities over time showed here played a key role in the elimination of ROS and damage decrease. Similar results have been identified in studies with aged soybean seeds (Xin et al., 2014XIN, X.; TIAN, Q.; YIN, G.; CHEN, X.; ZHANG, J.; NQ, S.; LU, X. Reduced mitochondrial and ascorbate-glutathione activity after artificial aging in soybean seed. Journal of Plant Physiology , v.171, n.2, p.140-147, 2014. https://doi.org/10.1016/j.jplph.2013.09.016
https://doi.org/10.1016/j.jplph.2013.09....
). Thus, it can be considered that the membrane damage is the main event that occurs in the aging of seeds (Ratajczak et al., 2019RATAJCZAK, E.; MAŁECKA, A.; CIERESZKO, I.; STASZAK, A.M. Mitochondria are important determinants of the aging of seeds. International Journal of Molecular Sciences, v.20, n.7, p.1-12, 2019. https://doi.org/10.3390/ijms20071568
https://doi.org/10.3390/ijms20071568...
).

The main mechanism of repair of the priming with AsA and NO in naturally aged seeds was the increase of enzymatic activity in relation to control and the reduction of damage by lipid peroxidation. As AsA is water-soluble, it has advantages over other constraints when used in seed pre-treatment, being involved in distinct cell processes, including the cell division, membrane restructuring, and vigor increase (De Gara et al., 2013DE GARA, L. Ascorbate metabolism and plant growth-from germination to cell death. In: ASARD, H.; MAY, J.; SMIRNOFF, N. Vitamin C: its function and biochemistry in animals and plants. Oxford: BIOS Scientific Publishers Ltd, 2013. P.83-95.). The action of AsA on seed germination was observed in studies with sunflower (El-Saidy et al., 2011EL-SAIDY, A.E.; FAROUK, S.; ABD EL-GHANY, H.M. Evaluation of different seed priming on seedling growth, yield and quality components in two sunflower (Hellianthus annus L.) cultivars. Trends in Applied Sciences Research, v.9, n.9, p.977-991, 2011. https://doi.org/10.3923/tasr.2011.977.991
https://doi.org/10.3923/tasr.2011.977.99...
). Regarding NO, there is also a relationship between the involvement of the signaling pathways by ROS and reactive nitrogen species (RNS), where it is assumed that these molecules interact with enzymes self-regulating mechanisms (Begara-Morales et al., 2018BEGARA-MORALES, J.C; CHAKI, M.; VALDERRAMA, R.; SÁNCHEZ-CALVO, B.; MATA-PÉREZ, C.; PADILLA, M.N.; CORPAS, F.J.; BARROSO, J.B. Nitric oxide buffering and conditional nitric oxide release in stress response. Journal of Experimental Botany, v.69, n.14, p.3425-3438. https://doi.org/10.1093/jxb/ery072
https://doi.org/10.1093/jxb/ery072...
). The results of the enzymatic antioxidant activity in the treatment with seeds primed NO show that this molecule acts positively on the germination of aged seeds.

One of the main characteristics of the SNP, a widely used NO donor in the nitrosyl-iron complex category, deals with the formation of the inorganic iron-NO complex, where iron is in its ferrous state (Fe2+) and not bound to this as NO+ (Singh and Bhatla, 2017SINGH, N.; BHATLA, S.C. Signaling through reactive oxygen and nitrogen species is differentially modulated in sunflower seedling root and cotyledon in response to various nitric oxide donors and scavengers. Plant Signaling & Behavior, v.12, n.9, p.1-15, 2017. https://doi.org/10.1080/15592324.2017.1365214
https://doi.org/10.1080/15592324.2017.13...
). This fact may be correlated with the release of Fe2+ by the donor, thus increasing their concentration in the medium and promoting the occurrence of Fenton reactions. However, in response to exogenous SNP application, Keisham et al. (2019KEISHAM, M.; JAIN, P.; SINGH, N.; BHATLA, C.V.C.; LINDERMAYR, C. Deciphering the nitric oxide, cyanide and iron-mediated actions of sodium nitroprusside in cotyledons of salt stressed sunflower seedlings. Nitric Oxide, v.88, n.1, p.10-26, 2019. https://doi.org/10.1016/j.niox.2019.03.008
https://doi.org/10.1016/j.niox.2019.03.0...
) reported an abundance of 117 altered proteins. Among these proteins, functional categories are involved in stress responses, signal translation, proteolysis, among others. Furthermore, these authors also reported that NO plays a protective role during the proteolytic degradation process in oilseeds, thus minimizing the reduction of oxidative damage like was observed.

CONCLUSIONS

It can be concluded that stored sunflower seeds, when subjected to physiological priming with water (hydropriming), 100 mM NO and, 100 mM AsA show better performance in terms of germination speed, cumulative germination, ROS-scavenging system, and their cytotoxic effects, as well as electrolyte leakage and enzymatic antioxidant system. Since the vigor of seeds is essentially dependent on the ability to restructure the damage caused by aging and maintain essential metabolic activities, the NO acted as the primed that maintained the antioxidant enzymatic activity reducing the ROS produced and presented less oxidative damage, that is, favored its repair. Thus, the use of this seed’s technologies is suggested to achieve uniform germination and seedling establishment and to promote the reuse of these seeds, minimizing costs and obtaining quality input.

ACKNOWLEDGMENT

The authors would like to thank FAPEMIG (Fundação de Amparo à Pesquisa do Estado de Minas Gerais) for the scholarship for the first author.

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Publication Dates

  • Publication in this collection
    22 Apr 2022
  • Date of issue
    2022

History

  • Received
    21 Sept 2021
  • Accepted
    24 Mar 2022
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