Watching the days go by: Aging during sunflower seed storage under distinct oxygen availability

Observando o passar dos dias: Envelhecimento de semente de girassol durante o armazenamento sob distinta disponibilidade de oxigênio

Rafael Agostinho Ferreira Alisson Alves de Oliveira Victor Navarro da Silva Aline Aparecida Silva Pereira Mateus Moreira Bernardes Elisa Monteze Bicalho About the authors

ABSTRACT

The maintenance of seed viability is widely studied since preserving the physiological characteristics that will allow efficient germination and adequate field occupation is broadly pursued. However, even under optimal storage conditions, the aging process is inherent to the seed’s life. In order to understand the effects of storage under low and normal oxygen conditions, this work sought to evaluate the physiological responses of two seed lots of two sunflower hybrids stored under different oxygen availability (normoxia and hypoxia) over a 360-day period. Aiming to investigate the effects of storage, the activities of the enzymatic antioxidant metabolism, hydrogen peroxide and MDA content, and the performance of viability, and vigor tests (tetrazolium test and electrolyte leakage) were performed with the stored seeds every 60 days. The hypoxia conditions were not able to keep seed viability over time, probably affecting negatively the embryonic axis. Throughout the evaluations, the viability tests demonstrated that the storage in the two experimental conditions was not able to contain the aging of the seeds. The increased content of H2O2 and MDA, associated with the enhanced electrical conductivity over time, indicate that there were losses by lipid peroxidation and that the aging process was not contained by storage under low oxygen availability.

Index terms:
Oxidative damage; lipid peroxidation; ROS; Helianthus annus; hypoxia.

RESUMO

A manutenção da viabilidade das sementes é amplamente estudada uma vez que se busca preservar as características fisiológicas que permitirão uma germinação eficiente no campo. No entanto, mesmo em condições ideais de armazenamento, o processo de envelhecimento é inerente à vida da semente. Com o objetivo de compreender os efeitos do armazenamento em condições normais e de baixo oxigênio, este trabalho buscou avaliar as respostas fisiológicas de dois lotes de sementes de dois híbridos de girassol armazenados sob diferentes disponibilidades de oxigênio (normoxia e hipóxia) durante um período de 360 dias. Com o objetivo de investigar os efeitos do armazenamento, o metabolismo antioxidante enzimático, teor de peróxido de hidrogênio e MDA, bem como a viabilidade e o vigor (teste de tetrazólio e vazamento de eletrólito) foram realizados com as sementes armazenadas a cada 60 dias. As condições de hipóxia não foram capazes de manter a viabilidade das sementes ao longo do tempo, provavelmente afetando negativamente o eixo embrionário. Ao longo das avaliações, os testes de viabilidade demonstraram que o armazenamento nas duas condições experimentais não foi capaz de conter o envelhecimento das sementes. O aumento do conteúdo de H2O2 e MDA, associado ao aumento da condutividade elétrica ao longo do tempo, indicam que houve perdas por peroxidação lipídica e que o processo de envelhecimento não foi contido pelo armazenamento sob baixa disponibilidade de oxigênio.

Termos para indexação:
Dano oxidativo; peroxidação lipídica; EROs; Helianthus annus; hipóxia.

INTRODUCTION

The loss of vigor in seeds during storage is being studied in recent years since it culminates in agronomiclosses impairing germplasm preservation due tothe aging (Kurek; Plitta-Michalak; Ratajczak, 2019KUREK, K.; PLITTA-MICHALAK, B.; RATAJCZAK, E. Reactive oxygen species as potential drivers of the seed aging process. Plants, 8(6):174, 2019.; Ambarsi et al., 2021AMBARSARI, I. et al. The effects of ozone exposure on aged soybean seeds stored in different packaging. Journal of Southwest Jiaotong University, 56(2):1-11, 2021.; Małecka et al., 2021MAŁECKA, A. et al. Relationship between mitochondrial changes and seed aging as a limitation of viability for the storage of beech seed (Fagus sylvatica L.). PeerJ, 9:e10569, 2021.). Although this process occurs in an irreversible way, the development of technologies that allow long-term storing seeds without the occurrence of losses is constant (Moncaleano-Escandon et al., 2013MONCALEANO-ESCANDON, J. et al. Germination responses of Jatropha curcas L. seeds to storage and aging. Industrial Crops and Products, 44:684-690, 2013.; De Castro Marais, et al., 2021DE CASTRO MORAIS, T. et al. Qualidade fisiológica e ação enzimática antioxidante em sementes de girassol expostas à deterioração. Revista Caatinga, 34(3):570-579, 2021.).

The techniques that exploit changes in the storage conditions aim to control temperature, humidity and oxygen availability to provide suitable conditions for seed metabolism since the variability in these conditions can lead to losses in vigor and viability (Ratajczak et al., 2019RATAJCZAK, E. et al. Mitochondria are important determinants of the aging of seeds. International Journal of Molecular Sciences, 20(7):1568, 2019.; De Vittis et al., 2020DE VITIS, M. et al. Seed storage: maintaining seed viability and vigor for restoration use. Restoration Ecology, 28(S3):S249-S255, 2020.). Considering the storage conditions are controlled, seeds are less susceptible to external oscillations that can lead to increases in reactive oxygen species (ROS) (Biswas et al., 2020BISWAS, N. et al. Vigor difference during storage and germination in Indian mustard explained by reactive oxygen species and antioxidant enzymes. Turkish Journal of Agriculture and Forestry, 44(6): 577-588, 2020.). An important highlight are the oxidative reactions, that result in electrons unpairing of the molecular oxygen, reacting with lipids from bilayer membrane and other cells constituents, inducing lipid peroxidation, protein carbonylation and DNA damage (El-Maarouf-Bouteau; Bailly, 2008EL-MAAROUF-BOUTEAU, H.; BAILLY, C. Oxidative signaling in seed germination and dormancy. Plant Signaling & Behavior, 3(3):175-182, 2008.; Zhang et al., 2021ZHANG, K. et al. Deterioration of orthodox seeds during ageing: Influencing factors, physiological alterations and the role of reactive oxygen species. Plant Physiologyand Biochemistry, 158:475-485, 2021.).

ROS accumulation above the scavenger capacity of antioxidant system can result in metabolic impairments, such as oxidative stress (Bailly, 2019BAILLY, C. The signalling role of ROS in the regulation of seed germination and dormancy. Biochemical Journal, 476(20):3019-3032, 2019.; Sharma; Yadav; Sibi, 2020SHARMA, S.; YADAV, S.; SIBI, G. Seed germination and maturation under the influence of hydrogen peroxide: A review. Journal of Critical Reviews, 7(1):6-10, 2020.). Besides that, when storage conditions are controlled, seeds tend to reduce reserves mobilization for respiratory metabolism and maintain their vigor that, consequently, can prolong seed viability (Chhabra; Singh; Ansari, 2019CHHABRA, R.; SINGH, T; ANSARI, S. Seed aging. storage and deterioration: An irresistible physiological phenomenon. Agricultural Reviews, 40(3):234-238, 2019.; Zhang et al., 2021ZHANG, K. et al. Deterioration of orthodox seeds during ageing: Influencing factors, physiological alterations and the role of reactive oxygen species. Plant Physiologyand Biochemistry, 158:475-485, 2021.). Mostly, the damage that culminates in the loss of vigor by seeds is associated with hydrolytic, oxidative and peroxidative reactions ( Mahjabin; Bilal; Abidi, 2015MAHJABIN; ; BILAL, S ABIDI, A. Physiological and biochemical changes during seed deterioration: A review. International Journal of Recent Scientific Research, 6(4):3416-3422, 2015.). Among these reactions are the enzymatic and non-enzymatic reactions that cause cellular and molecular damage leading to degradation of reserves (Hebelstrup; Moller 2015HEBELSTRUP, K. H.; MØLLER, I. M. Mitochondrial signaling in plants under hypoxia: Use of reactive oxygen species (ROS) and reactive nitrogen species (RNS). In: IGAMBERDIEV, A. V.; GUPTA, K. J. Reactive Oxygen and Nitrogen Species Signaling and Communication in Plants, Springer, p.63-77, 2015.).

The oxidative reactions in dry or stored seeds, especially the Fenton and Maillard reactions, as well as mitochondrial activity, can lead to the formation of ROS, which in turn promote oxidative damage that causes seeds loss of vigor (Chhabra; Shabnam; Singh, 2019CHHABRA, R.; SINGH, T; ANSARI, S. Seed aging. storage and deterioration: An irresistible physiological phenomenon. Agricultural Reviews, 40(3):234-238, 2019.; Önder et al., 2020ÖNDER, S. et al. Biochemical changes stimulated by accelerated aging in safflower seeds (Carthamus tinctorius L.). Journal of Seed Science, 42:e202042015, 2020.). During storage, ROS are originated mainly due to non-enzymatic reactions, such as the oxidation of polyunsaturated fatty acids (PUFA’s) and by Maillard and Fenton reactions, as well as by the natural aging of seeds, thus oxidative stress plays a critical role in seed deterioration (Fotouo et al., 2020FOTOUO-M, H. et al. The effect of natural long-term packaging methods on antioxidant components and malondialdehyde content and seed viability Moringa oleifera oilseed. South African Journal of Botany, 129:17-24, 2020.; Zhang et al., 2021ZHANG, K. et al. Deterioration of orthodox seeds during ageing: Influencing factors, physiological alterations and the role of reactive oxygen species. Plant Physiologyand Biochemistry, 158:475-485, 2021.). This is especially important in oilseed species, in which the lipid reserves are the main target of ROS attack (Whitehouse; Hay; Lusty, 2020WHITEHOUSE, K. J.; HAY, F. R.; LUSTY, C. Why seed physiology is important for genebanking. Plants , 9(5):584, 2020.).

Oxidative stress is the result of the imbalance between the production and elimination of ROS. In general, plant organisms have a network of agents responsible for the elimination of these ROS, including enzymatic and non-enzymatic antioxidant agents, composing a highly regulated line of defense against the action of these reactive molecules (Bailly, 2019BAILLY, C. The signalling role of ROS in the regulation of seed germination and dormancy. Biochemical Journal, 476(20):3019-3032, 2019.). Among the main defense mechanisms, enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and glutathione reductase (GR) and other peroxidases (POX) can be highlighted (Shaban, 2013SHABAN, M. Review on physiological aspects of seed deterioration. International Journal of Agriculture and Crop Sciences, 6(11):627-631, 2013.).

As an inevitable consequence of oxidative damage in seeds, low germination rates are a major problem, since damage to seed reserves compromises the germination and establishment of seedlings, generating agronomic losses by seed aging processes (Tian et al., 2019TIAN, P. et al. Effect of artificial aging on wheat quality deterioration during storage. Journal of Stored Products Research, 80:50-56, 2019.). Thereby, storage techniques are focused in reduce the respiratory metabolism, reducing damages of oxidative stress (Wawrzyniak; Michalak; Chmielarz, 2020WAWRZYNIAK, M.; MICHALAK, M.; CHMIELARZ, P. Effect of different conditions of storage on seed viability and seedling growth of six European wild fruit woody plants. Annals of Forest Science, 77:58, 2020.). This way, storing seeds under low oxygen conditions can promote benefits in reducing the ageing effects. Seeds are related to survive in conditions where can be deathly for plants (Zhang et al., 2021ZHANG, K. et al. Deterioration of orthodox seeds during ageing: Influencing factors, physiological alterations and the role of reactive oxygen species. Plant Physiologyand Biochemistry, 158:475-485, 2021.), so explore low oxygen availability can promote an increase in seed longevity (Pirredda et al., 2020PIRREDDA, M. et al. Genetic and epigenetic stability in rye seeds under different storage conditions: Ageing and oxygen effect. Plants , 9(3):393, 2020. ).

In this work, we hypothesized that during storage under low oxygen availability, sunflower (Helianthus annus)seeds will keep their vigor by reducing the respiratory activity and consequently, avoiding ROS accumulation.We studied here one-year storage of two hybrids of sunflower (Helio 250 and Helio 251) seeds under two distinct oxygen availability. The seed reserves of this species are majority composed of lipids, which could accelerate aging, conducing the seed loss of vigor or even seed death. Therefore, we believe this work brings important contribution to seed storage techniques.

MATERIAL AND METHODS

Plant Material

Seeds from two sunflower hybrids were used in this experiment (Helio 250 and Helio 251 from HeliagroAgricultura e Pecuária©). Both materials were collected from two different periods. One lot, labeled ‘STORED’, consisted in seeds that were stored immediately after its harvesting in 2016 in a cold chamber until the set of the experiment. The other lot, labeled ‘FRESH HARVESTED’, comprised seeds harvested in 2019 which were readily subjected to the experimental conditions.

Experimental conditions

The experiment was carried out in the Laboratory of Plant Growth and Development (LCDP) at the Federal University of Lavras (Lavras-MG, Brazil). The experimental design was entirely randomized, in a 2x7 factorial scheme where two oxygen availability conditions (normoxia and hypoxia), around seven times of evaluation in each hybrid or year of production. The effects of the different storage forms throughout the storage time were compared.The sunflower seeds were packaged in Kraft® paper bags depending on the way of storage (normoxia and hypoxia). The seeds from the hypoxic treatment were placed in a plastic bag and the air removed from the interior using a vacuum pump. The seeds from normoxia treatment were kept in paper bags in normal oxygen conditions, without any type of special storage.The seeds were assessed before of storage, and then every 60 days, for a period of 360 days of storage, totaling seven sampling over time. Each 60 days, the germination and biochemical parameters were carried out. The germination index, germination speed index, viability by tetrazolium test, electrolyte leakage and hydrogen peroxide (H2O2) and malondialdehyde (MDA) contents, as well as enzymatic antioxidant system (SOD and CAT) were performed.

Germination assay

The seeds were placed into paper rolls using four repetitions of 50 sunflower seeds each; 25 seeds were used to measure the germination parameters and 25 seeds were collected for biochemical analysis. To conduct the germination test, plastic bags were used to wrap the sets of paper rolls with the seeds. The paper rolls were moistened with sterile water, in the proportion of 2.5 times the dry weight of the paper. Later the seeds were kept in the BOD, at a 25 ºC temperature with a 12/12 hours photoperiod, where the germination was counted daily over a period of 10 days. The germination assay was based on Seeds Analysis Rules (Brasil, 2009BRASIL. Regras para analises de sementes. Ministério da Agricultura, Pecuária e Abastecimento. Brasília: Mapa/ACS, 2009. 399p.), and Germination speed index as described from Maguire (1962)MAGUIRE, J. D. Speed of germination aid in selection and evaluation for seedling emergence and vigor. Crop Science, 2(2):176-77, 1962.. Samples of 25 seeds used for biochemical analysis were collected after eight hours of imbibition, period that comprehend the phase II of germination, according to imbibition curve previously elaborated.

Electrical conductivity

The electrical conductivity test consisted of four replicates with 25 seeds each. Each replicate sample was placed to soak in a becker containing 75 mL of deionized water. Then, they were kept in a BOD chamber for 24 hours at a constant temperature of 25 °C, according to Vieira (1994)VIEIRA, R. D. Teste de condutividade elétrica. In: VIEIRA, R. D.; CARVALHO, N. M. (Ed.) Testes de vigor em sementes. Jaboticabal: FUNEP, p. 103-132, 1994.. The electrical conductivity was measured with a conductivity meter, being the results expressed in µS.cm-1 g-1 of seeds.

Seeds viability test

For this test, 25 seeds were pre-soaked in distilled water for 24 hours at 25º C. Afterwards, the seed coat was manually removed and a section was made through the tegument and between cotyledons to the center of the seed, then it was immersed in a 0.5% tetrazolium solution (2, 3, 5-triphenyltetrazolium chloride salt), pH 7, for a one-hourperiod at 25° C. The percentage of vigor and viability were calculated according to Silva et al. (2013)SILVA, R. C. et al. Adaptation of the tetrazolium test for assessment of sunflower seed viability and vigor. Pesquisa Agropecuária Brasileira, 48(1):105-113, 2013..

Biochemical analysis

To perform the biochemical analyses, the seeds of each periodof storage were collected after eight hours of soaking on paper rolls. The seeds were frozen in liquid nitrogen and stored at -80 °C until analysis. To obtain an extract that could represent the totality of the seeds, a pool of ten seeds composed each replicate.

Hydrogen peroxide (H2O2)

The levels of H2O2 were quantified by the method of Velikova, Yordanov and Edreva, (2000)VELIKOVA, V.; YORDANOV, I.; EDREVA, A. Oxidative stress and some antioxidant systems in acid rain-treated bean plants: protective role of exogenous polyamines. Plant Science, 151(1): 59-66, 2000.. Four replicates of 100 mg of seeds each were grinded in liquid nitrogen and then homogenized with 1 mL of 0.1% Trichloroacetic Acid (TCA). Samples were centrifuged, and the reactions were carried out with 10 mM potassium phosphate buffer pH 7 and 1M potassium iodide (KI). The samples were read at spectrophotometer at 390 nm and the H2O2 amounts were measured by standard curve.

Lipid peroxidation

Lipid peroxidation estimation were performed by the quantification of Malondialdehyde (MDA) as described by Du and Bramlage (1992)DU, Z.; BRAMLAGE, W. J. Modified thiobarbituric acid assay for measuring lipid oxidation in sugar-rich plant tissue extracts. Journal of Agricultural and Food Chemistry, 40(9):1566-1570, 1992.. 100 mg of seeds weregrinded in liquid nitrogen and then homogenized with 1 mL of 80% ethanol (three times, totaling 3 mL). The reactions were carried out with 1 mL of 20% TCA, 0.65% thiobarbituric acid (TBA) and 0.01% beta-hydroxytoluene (BHT). The estimation of lipid peroxidation was measured in spectrophotometer at 600, 440, and 532 nm.

Antioxidant enzymatic system assays

For the enzymatic extractions of catalase (CAT) and superoxide dismutase (SOD), about 100 mg of fresh material were grindedin liquid nitrogen and polyvinylpyrrolidone (PVPP) and homogenized with 1 mL of phosphate buffer (100 mM pH 7.8) with 100 mM EDTA and 1 mM ascorbic acid. The reaction medium for CAT comprised 67 mM potassium phosphate buffer (pH 7.0), 10 mM H2O2 and an aliquot of the enzyme extract (Biemelt; Keetman; Albrecht, 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 , 116(2):651-658, 1998.). H2O2 consumption was measured through the absorbance at 240 nm as described by Anderson, Prassad and Stewart (1995)ANDERSON, M. D.; PRASAD, T. K.; STEWART, C. R. Changes in isozyme profiles of catalase, peroxidase and glutathione reductase during acclimation to chilling in mesocotyls of maize seedlings. Plant Physiology, 109(4):1247-1257, 1995..

SOD activity was measured using a reaction medium containing 50 mM phosphate buffer (pH 7.8), 13 mM L-methionine, 0.1 mM EDTA, 0.002 mM riboflavin and 0.075 mM NBT, as described by Giannopolitis and Ries (1977)GIANNOPOLITIS, C. N.; RIES, S. K. Superoxide dismutases: Occurrence in higher plants. Plant Physiology , 59(2):309-314, 1977.. The reaction occurred under fluorescent light (15 W) for 7 minutes. The blue formazan compound, derived from the reduction of NBT, was quantified at 575 nm. One unit of SOD was defined as the amount of enzyme required to inhibit NBT reduction by 50%.

Statistical analysis

The statistical analysis was performed with the software Rbio (Bhering, 2017BHERING, L. L. Rbio: A tool for biometric and statistical analysis using the R platform. Crop Breeding and Applied Biotechnology, 17(2):187-190, 2017.). Data was subjected to two-way ANOVA and, in case of normal distribution, Tukey means test was selected at 5% significance. Data without a normal distribution were evaluated by a GLM analysis, assuming normality by observation of the qq-plot graphs and then applying the Tukey test at 5% of significance.

RESULTS AND DISCUSSION

The results showed herepointed out two relevant considerations: 1) the germination impairment in hypoxia condition is due to reductions in seed viability, mainly regarding to embryo axis; and 2) oxidative stress was marked over time of storage independent of the hybrid, oxygen conditions or the lots studied.

Germination percentage (G%) (Figure 1A) decreased in the hypoxia condition after 120 days of the storage, germination showed the lowest percentage of germinated seeds, even in fresh or previously stored seeds, for the two hybrids. These changes indicate that hypoxia was not efficient in promoting physiological conditions for the seeds to remain viable after removal from the storage condition. Seeds from both lots in the normoxia condition showed a germination percentage above 75%, reinforcing that the hypoxia condition was not able to keep viability over time. This is in agreement with observations of Morscher et al. (2015)MORSCHER, F. et al. Glutathione redox state, tocochromanols, fatty acids, antioxidant enzymes and protein carbonylation in sunflower seed embryos associated with after-ripening and ageing. Annals of Botany, 116(4):669-678, 2015. and Bailly (2019)BAILLY, C. The signalling role of ROS in the regulation of seed germination and dormancy. Biochemical Journal, 476(20):3019-3032, 2019..

Figure 1:
Germination percentage (A) and germination speed index (B) of Helianthus annuus hybrids seeds subjected to storage under normoxia (Grey bars) and hypoxia (black bars) during a 360-days period. Bars represent means ± standard error (n=4). Uppercase letters compare the storage period and lowercase letters compare the storage conditions (normoxia or hypoxia) within a certain storage period. Equal letters do not differ statistically at 5% significance level.

Regarding the Germination Speed Index, stored seeds were more sensible over time than non-stored seeds from normoxia conditions. The highest GSI values that result in a higher germination (Figure 1B) could be observed for seeds from both lots under normoxia conditions. Under hypoxia conditions, only at 180 days a higher GSI could be observed for seeds from H250 hybrid, however, after that, this index decreased again showing compromises in germination, that results are similar to results obtained by Sahu et al. (2017)SAHU, B. et al. Reactive oxygen species, lipid peroxidation, protein oxidation and antioxidative enzymes in dehydrating Karanj (Pongamia pinnata) seeds during storage. South African Journal of Botany , 112:383-390, 2017. working with seeds under storage subjected to artificial aging process.

The viability of the seeds gradually decreased in storage conditions under low oxygen availability. At the initial characterization, all hybrids showed values of electrolyte leakage up to 9.03 µS/cm/g and all of the seeds have showed a vivid stained embryo, indicating that the seeds show high viability (Table 1). Although it is possible to observe staining in the cotyledons and in embryonic axis, the embryonic axis did not resist the storage under low oxygen availability (Figure 2). Moreover, the values of electrolyte leakage increased considerably throughout the experimental period; in hypoxic storage conditions the values observed are up to three times higher than those under normal oxygen conditions (Table 1). The increase in electrolyte leakage is associated with a higher incidence of membrane damage, and it is responsible for the low germination of the seeds since there is a compromise of the embryonic tissues, which can lead to embryo death (Liu et al., 2013LIU, R. et al. Changes in physiology and quality of Laiyang pear in long time storage. Scientia Horticulturae, 150:31-36, 2013.).

Figure 2:
Images from the sunflower seeds at 360 days storage subjected to Tetrazolium test: A, B, C, D (hypoxia condition), seeds from hybrids H250 F+V, H251 F+V, H250 PS+V, H251 PS+V, and E, F, G, H (normoxia condition) from hybrids H250 F, H251 F, H250 PS, H251 PS - F+V: Fresh Harvested under hypoxia; PS+V: Pre-stored under hypoxia; F: Fresh harvested kept under normoxia; PS: Pre-stored kept under normoxia.

Table 1:
Electrolyte leakage (µS/cm/g) and results of tetrazolium salt test (percentage of viable, inviable and dead seeds) of Helianthus annuus hybrids seeds subjected to storage under normoxia and hypoxia during a 360-days period. Uppercase letters compare the storage periodand lowercase letters compare thestorage conditions within a certain storage period. Equal letters do not differ statistically at 5% significance level. CV. cultivar; F. fresh seeds; PS. pre-harvested; V. hypoxia.

Clearly, these changes were responsible for inducing redox homeostasis alterations that led to a loss of vigor in the seeds, increasing the mortality. The H2O2 content increased significantly after 120 days of evaluation (Figure 3A), although at low concentrations and under normal conditions tested. This molecule acts directly on signaling in seeds and plants. However, when in conditions that exceeds the “oxidative window” (Bailly; El-Maarouf-Bouteau; Corbineau, 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. Computes Rendus Biologies, 331(10):806-814, 2008.), this molecule reacts with lipids, proteins and nucleic acids.

Figure 3:
H2O2 (A) and MDA content (B) of Helianthus annuus hybrids seeds subjected to storage under normoxia (Grey bars) and hypoxia (black bars) during a 360-days period. Bars represent means ± standard error (n=4).Uppercase letters compare the storage period and lowercase letters compare the storage conditions (normoxia or hypoxia) within a certain storage period. Equal letters do not differ statistically at 5% significance level.

The content of MDA showed a prominent increase after 120 days of storage for all materials in both conditions (Figure 3B). These values indicated an intensification in lipid peroxidation regardless of the storage condition and seed hybrid, that could be associated with a greater increase of ROS, as showed for H2O2 levels. As can be seen, the H2O2 showed an enhancement in its endogenous concentration that accompanied the raise in MDA content, indicating an increased lipid peroxidation, an oxidative damage that contributes to the loss of vigor of seeds (Nagel et al., 2016NAGEL, M. et al. Barley seed aging: genetics behind the dry elevated pressure of oxygen aging and moist controlled deterioration. Frontiers in Plant Science, 7:388, 2016.).

These results indicated that lipid peroxidation might have occurred by the attack of ROS on both cell membranes and these seeds reserves, which is mostly lipid. Throughout the evaluation period, the highest values of MDA content could be observed in the seeds of hybrid H250 at 360 days of evaluation, which indicates that there was a process of aging of the seeds that was accentuated throughout the evaluations.

The oxidative damage and, consequently electrolyte leakage, could be avoided by an efficient antioxidant system to maintain cellular homeostasis, however it was not observed here. During the evaluation period, it was observed that there were oscillations in the activity of SOD (Figure 4A). with a peak of its activity occurring in the first 120 days and then declining in all experimental conditions in all materials. The significant decrease in the activity of this enzyme that occurred after 120 days, is probably related to an imbalance in the capacity of extinction of the superoxide radical. These alterations could be observed in all conditions and for all materials. In order to eliminate the generated ROS, the enzymatic antioxidant metabolism acts by transforming ROS into molecules with less ability to interact with macromolecules and then reducing the possibility of oxidative damage occurrence (Diaz-Vivancos et al., 2013DIAZ-VIVANCOS, P. et al. Ectopic expression of cytosolic superoxide dismutase and ascorbate peroxidase leads to salt stress tolerance in transgenic plums. Journal of Plant Biotechnology, 11(8):976-895, 2013.).

Figure 4:
SOD content (A) CAT content (B) of Helianthus annuus hybrids seeds subjected to storage under normoxia (Grey bars) and hypoxia (black bars) during 360 days period. Bars are means ± standard error (n=4). Upper case letters compare storage period times and lower case letters compare storage conditions (normoxia or hypoxia) within a certain storage time. Equal letters do not differ statistically at 5% significance level.

Trying to avoid the damages associated with ROS, catalase (Figure 4B) is one of the enzymes that act to clean up the H2O2 formed. This enzyme showed a similar activity for the different seed lots in both conditions of oxygen availability until the 120 days of the experiment. There was a decrease in the activity of this enzyme in the evaluations that comprised 180 and 240 days, with an increase in 300 and 360 days. This increase in activity might be seen as an attempt to maintain the redox homeostasis of the seeds,which did not occur efficiently. The H250 hybrid seeds previously stored under low O2 conditions showed higher activity, however it was not possible to observe the efficiency in guaranteeing the scavenging of ROS, which culminated in a low percentage of germination in the seeds under low O2.

Although storing seeds under low oxygen availability is an option to promote the maintenance of desirable physiological characteristics, seeds under storage are inevitably subjected to aging processes that can culminate in the loss of germinability and viability. In order to control this process, it is essential to know the stored material and evaluate the best storage techniques to be adopted. In this work, it was possible to verify that low oxygen conditions were dangerous for embryonic axis of sunflower seeds, which was not able to keep viability over time. It could also be concluded that ageing reduced seed viability by increasing ROS and oxidative damage over time, independently of oxygen conditions. Therefore, this ageing during storage could be related to lipid losses of the cotyledon tissues, which are lipid-rich. Since seeds initiate the aging process after reaching physiological maturity and ROS formation is intrinsic to aerobic metabolism, we can suggest that some techniques such as seed priming or the adoption of molecules that can contain the formation of reactive oxygen species during storage may enable the maintenance of seed viability for long periods. Otherwise, the low oxygen conditions must be deeply investigated to stands out asa viable technique of storing seeds.

CONCLUSIONS

Although storing seeds under low oxygen availability can promote the maintenance of desirable physiological characteristics, this condition can’t stop aging processes that culminate in germinability and viability losses. Knowing the stored material is essential to adopt the best techniques aiming to control aging process. In this work, it was possible to verify that low oxygen conditions were dangerous for embryonic axis of sunflower seeds affecting the viability by increasing ROS and oxidative damage over time, independently of oxygen conditions.

AUTHOR CONTRIBUTION

Conceptual idea: Bicalho, E.M and Ferreira, R. A., Methodology design: Bicalho, E.M and Ferreira, R. A; Data collection: Ferreira, R. A.; Silva, V.N.; Alves, A. O.; Bernardes, M. M.; Pereira, A. A. P. Data analysis and interpretation: Bicalho, E.M; Ferreira, R. A.; Bernardes, M. M. Writing and editing: Ferreira, R. A.; Silva, V.N.; Alves, A. O.; Bernardes, M. M.; Pereira, A. A. P Bicalho, E.M.

ACKNOWLEDGEMENTS

A special thanks to Professor Heloísa Oliveira Araujo for the donation of the seeds and Jober Condé Evangelista for the help in performing statistical analysis, and to UFLA for granting the Scientific Initiation scholarship and to Capes for granting the PhD scholarship to the author.

REFERENCES

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Publication Dates

  • Publication in this collection
    08 Dec 2021
  • Date of issue
    2021

History

  • Received
    10 Sept 2021
  • Accepted
    08 Nov 2021
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