Revista
de la
Universidad
del Zulia
Fundada en 1947
por el Dr. Jesús Enrique Lossada
75
ANIVERSARIO
DEPÓSITO LEGAL ZU2020000153
ISSN 0041-8811
E-ISSN 2665-0428
Ciencias
Exactas,
Naturales
y de la Salud
Año 13 N° 37
Mayo - Agosto 2022
Tercera Época
Maracaibo-Venezuela
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DOI: http://dx.doi.org/10.46925//rdluz.37.23
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Features of changes in prooxidant-antioxidant balance of tissues
during activation of seed germination
Mariia Bobrova *
Olena Holodaieva **
Svitlana Koval ***
Olha Tsviakh ****
Olena Kucher *****
ABSTRACT
Aim of the research: to identify changes in the value of indicators of the state of the prooxidant-
antioxidant system (PAS) in seed tissues at rest and the initiation of its germination processes. The
subject of the research is the role of individual components of the PAS in ensuring the activation of
seeds before germination. Methodology. Quantitative determination of indicators of the state of PAS
was performed on tissue samples of seeds of the following plants: Glycine max L., Helianthus annuus L.,
Fagopyrum esculentum L., Linum usitatissimum L., Sinapis alba L., Chenopodium quinoa L., Panicum miliaceum L.,
Oryza sativa L., Avena sativa L., Zea mays L., Hordeum vulgare L., Triticum durum Desf. The concentration of
superoxide anion radical (•O2-), TBA-active products, cytochrome oxidase activity, superoxide
dismutase activity, catalase, the concentration of ascorbic acid, glutathione was determined. The
results of the research show that for the tissues of seeds of experimental Magnoliopsida plants at
rest, both links of PAS are more powerful than in Liliopsida, the level of free radical peroxidation
(FRPO) is lower, which is achieved by both enzymatic and low molecular weight antioxidants (AO).
Germination activation enhances both links of PAS in all experimental groups of plants, however, in
Magnoliopsida, we observe the stronger generation of •O2-, and the predominance of protection by
enzymatic AO, and in Liliopsida - low molecular weight.
KEYWORDS: antioxidants, germination, glutathione, ascorbic acid, catalase, superoxide anion
radical, superoxide dismutase, cytochrome oxidase.
*Ph.D in Biology, Associate Professor of the Department of Physics, Biology and Methods of Teaching of the
Volodymyr Vynnychenko Central Ukrainian State Pedagogical University. Kropyvnytskyi, Ukraine. ORCID ID:
0000-0001-7703-651X. Tel.: +380957473989. E-mail: kazna4eeva@gmail.com
**PhD in Chemistry, Associate Professor, Head of the Department of Chemistry and Pharmacognosy of Kyiv
International University. Kyiv, Ukraine. ORCID ID 0000-0002-4922-7033. E-mail: elena.gologaeva@gmail.com
***Senior Lecturer of the Department of the Fundamental Disciplines of the International European University.
Kyiv, Ukraine. ORCID ID: 0000-0002-4907-177X. E-mail: kovalsyu@gmail.com
****PhD in Biology, Senior Lecturer of the Department of Chemistry of the V.O. Sukhomlynskyi Mykolaiv
National University. Mykolaiv, Ukraine. ORCID ID: 0000-0002-1119-2170. E-mail: tsvyakho@gmail.com
***** PhD in Agricultural science, Senior Lecturer of the Department of Chemistry of the V.O. Sukhomlynskyi
Mykolaiv National University. Mykolaiv, Ukraine. ORCID ID: 0000-0002-9963-6855. E-mail:
hrizantema84.84@gmail.com
Recibido: 09/02/2022 Aceptado: 06/04/2022
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Características de los cambios en el equilibrio prooxidante-
antioxidante de los tejidos durante la activación de la germinación
de semillas
RESUMEN
Objetivo de la investigación: identificar cambios en el valor de indicadores del estado del
sistema prooxidante-antioxidante (SPA) en tejidos de semillas en reposo y el inicio de sus
procesos de germinación. El tema de la investigación es el papel de los componentes
individuales del SPA para garantizar la activación de las semillas antes de la germinación.
Metodología. La determinación cuantitativa de indicadores del estado de SPA se realizó en
muestras de tejido de semillas de las siguientes plantas: Glycine max L., Helianthus annuus
L., Fagopyrum esculentum L., Linum usitatissimum L., Sinapis alba L., Chenopodium quinoa
L. , Panicum miliaceum L., Oryza sativa L., Avena sativa L., Zea mays L., Hordeum vulgare L.,
Triticum durum Desf. Se determinó la concentración de radical anión superóxido (•O2-),
productos activos de TBA, actividad de citocromo oxidasa, actividad de superóxido
dismutasa, catalasa, concentración de ácido ascórbico, glutatión. Los resultados de la
investigación muestran que para los tejidos de semillas de plantas experimentales de
Magnoliopsida en reposo, ambos enlaces de SPA son más potentes que en Liliopsida, el nivel
de peroxidación de radicales libres (PRL) es menor, lo que se logra tanto por enzimática como
por bajo antioxidantes de peso molecular (AO). La activación de la germinación potencia
ambos enlaces de SPA en todos los grupos experimentales de plantas. Sin embargo, en
Magnoliopsida observamos la mayor generación de •O2-, y el predominio de la protección
por AO enzimático, y en Liliopsida -de bajo peso molecular.
PALABRAS CLAVE: antioxidantes, germinación, glutatión, ácido ascórbico, catalasa, radical
anión superóxido, superóxido dismutasa, citocromo oxidasa.
Introduction
The transition of a plant from a latent state to a pregenerative one is the initial stage
of its ontogenesis. The starting and the most responsible process is the germination process.
Induction, maintenance, and exit of seeds from dormancy are controlled by complex
physiological and biochemical mechanisms, which are influenced by a wide range of
endogenous and exogenous factors. The study of changes in the components of the
prooxidant-antioxidant system (PAS) that initiate the process of seed germination opens the
possibility of regulating and correcting this stage of plant ontogenesis, increasing
germination, and friendliness of crops is particularly relevant and economically justified in
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terms of crop intensification. The use of human processes of seed germination initiation in
everyday life deserves special attention. Thus, among supporters of a healthy lifestyle and a
balanced diet, attention to cereals and so-called "live cereals" has increased significantly, in
the preparation of which pre-soaking and minimal heat treatment is recommended to
preserve the maximum amount of biologically active substances in food. Given the growing
number of supporters of this issue among physicians, nutritionists, and scientists (Baiano &
del Nobile, 2015; Xu et al., 2017; Pacheco et al., 2018), the study of biochemical changes that
accompany the processes of seed germination is of great practical importance.
Aim of the research: to investigate the change in prooxidant-antioxidant homeostasis
in seed tissues at rest and the initiation of its germination processes.
To achieve this aim, the following tasks were identified:
1. To investigate the state of PAS in the tissues of seeds of experimental plants at rest.
2. To investigate the changes in the values of PAS in the tissues of seeds of experimental
plants in the initiation of the germination process.
3. To compare how the initiation of sampling affects the prooxidant (PO) and
antioxidant (AO) links in the tissues of experimental Magnoliopsida and Liliopsida plants.
4. To determine species-specific features of PAS change of tissues of seeds of
experimental plants of different systematic categories.
1. Literature review
The seeds of wild and cultivated plants are characterized by a state of forced
dormancy. In this case, the ability of seeds to be at rest provides plants with the opportunity
to experience unfavorable for their existence periods of the year. However, with increasing
hydration in the seeds, the main metabolic processes are activated, and respiration increases
to the maximum level, which characterizes their growth and development. The period of seed
germination is divided into three stages: 1) activation of metabolism (stage of physical
swelling of seeds); 2) preparation for the beginning of growth by stretching (gluing of seeds
due to transition to stretching of cells of axial organs of a germ); 3) the actual growth of the
organs of the seedling (Nonogaki, 2017; Wolny et al., 2018). In wet seeds, there is an active
consumption of oxygen, which can cause oxidative tissue damage (Oracz & Karpinski, 2016).
Reactive oxygen species (ROS) play a role in the development of oxidative stress: •О2-, Н2О2,
НО-, НОС1, etc. (Apel & Hirt, 2004; Halliwell, 2006)
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Reactive oxygen species (ROS) are currently considered "dual agents" (Dat et al., 2000;
Mittler, 2017). They either directly initiate intense oxidative stress, accompanied by damage
or death of cells and the body, or act as signaling molecules that induce physiological and
biochemical reactions that increase the body's resistance (Vranova et al., 2002; Jaspers &
Kangasjarvi, 2010; Kolupaev & Karpets, 2010; Kreslavski et al., 2012).
Recent studies have shown that ROS are involved in the regulation of the most
important physiological processes, including growth and development, response to biotic
and abiotic stresses, and programmed cell death (Kreslavski et al., 2012; Suzuki et al., 2012;
Silvina et al., 2016; Gautam et al., 2017). The greatest progress in understanding the role of
ROS in growth and development has been achieved in the study of the early embryogenesis
of Fucus and the formation of Arabidopsis root hairs (Silvina et al., 2016). Using similar
approaches, it was previously shown that growth processes in plant somatic cells also
depend on the level of extracellular ROS (Gautam et al., 2017). Two mechanisms have been
proposed to explain this phenomenon. According to the first of them, some ROS can act as
signaling factors; according to the second, ROS are involved in the chemical modification of
the polymer matrix of the cell wall and thereby affect its mechanical properties (Mittler et
al., 2004).
ROS generation in plants occurs in cell walls, plasma membrane, chloroplasts,
mitochondria, peroxisomes, and, possibly, in other compartments (Foyer & Noctor, 2009).
The greatest contribution to the formation of ROS during seed germination is made by
mitochondria, which is associated with the activation of cellular respiration, as well as the
cell wall of actively dividing cells (Kolupaev et al., 2019). Complexes I and III are considered
to be the main sites of electron leakage in plant mitochondria (Rhoads et al., 2006;
Cvetkovska & Vanlerberghe, 2013). In plants, the relationship between electron transport,
oxidative phosphorylation, and ROS generation is complicated by the presence of an
alternative oxidase that catalyzes the oxidation of ubiquinone and the reduction of molecular
oxygen to water. In this case, the likelihood of the formation of •О2- due to the leakage of an
electron from complex III is prevented. Along with this, the transport of electrons bypassing
complex III, cytochrome c, and complex IV reduces mitochondrial re-reduction, their
membrane potential, and, as a consequence, the likelihood of ROS formation (Moller, 2001;
Kolupaev & Karpets, 2014).
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During the germination process, the plant cell is exposed to exogenous environmental
factors. ROS accompany the formation of the metabolic reaction of plants to the first
influence of abiotic factors, take part in the formation of the adaptation reaction, as well as
the response to stress changes (Gill & Tuteja, 2010; Hasanuzzaman et al., 2019; Kolupaev &
Karpets, 2019; Pacheco et al., 2018). Thus, using hypothetical sensors (Los et al., 2010;
Hirayama & Shinozaki, 2010), the cell surface perceives signals of hyperthermia, osmotic
action, or salinity. This leads to the activation of transmembrane NADPH-oxidase, apoplastic
forms of peroxidase and increased generation of •О2-. The latter, with the help of apoplastic
forms of superoxide dismutase (SOD), can be converted into H2O2, which freely penetrates
the cytoplasm through the plasma membrane. In addition, under certain conditions •О2- can
be converted to hydroperoxyl and pass through membranes (Sagi & Fluhr, 2006).
Simultaneously, under the action of stressors, an increase in the stochastic formation of ROS
in chloroplasts and mitochondria and activation of photorespiration can occur (Foyer &
Noctor, 2009). An increase in the concentration of H2O2 in cells leads to the modification of
intracellular protein redox sensors. Ultimately, it is likely that the ROS signal leads to a
change in the state of transcript factors that control the genes of antioxidant enzymes and
enzymes involved in proline synthesis, and other protective reactions (Suzuki et al., 2012).
ROS signals are also closely integrated with the signals of phytohormones, in
particular, ethylene, abscisic, salicylic and jasmonic acids, brassinosteroids (Galiba et al.,
2013; Bartoli et al., 2013), regulating cell metabolism during germination and rest (Kolupaev
& Karpets, 2014; Oracz & Karpinski, 2016).
However, the accumulation of ROS in cells leads to disruption of the course of
transcription and replication processes, changing the composition of membrane lipids. •О2-
modify proteins, disrupt the structure of DNA, destroy hormones and other functionally
active substances. Therefore, the study of the manifestation of compensatory mechanisms in
seeds on the action of ROS is a general biological task [5. The control over the content of ROS
in seeds is carried out by antioxidants (AO) (Apel & Hirt, 2004; Kumar et al., 2011).
The key low molecular weight AO is ascorbic acid (AA) and glutathione (GSH), and
the enzymatic ones are SOD, catalase, and peroxidase (Polesskaja, 2007; Kolupaev et al.,
2019). Recently, the relevance of publications on a significant increase in the amount of
antioxidants in the tissues of germinated seeds, but comprehensive information on the
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consistency of the components of prooxidant and antioxidant systems in the initiation of
germination is absent or fragmentary, which necessitates comprehensive research and
systematization.
2. Research methodology
Quantitative determination of PAS was performed on seed tissue samples of the
following plants: Glycine max L., Heliаnthus аnnuus L., Fagopyrum esculentum L., Linum usitatissimum
L., Sinapis alba L., Chenopodium quinoa L., Panicum miliaceum L., Oryza sativa L., Avena sativa L., Zea mays
L., Hordeum vulgare L., Triticum durum Desf. Seed analysis was performed on objects that were in
a state of physiological rest. In parallel, the seeds were examined during the initiation of
germination, which was carried out by the previous 12-hour soaking in water. Each
experimental group included 10 samples, so the experiment analyzed 1680 samples.
NBT test (spectrophotometric variant) was used to determine the concentration of
superoxidanion radical: 0.1 g of tissue was homogenized in 0.9 cm3 of phosphate buffer (pH =
7.4). The obtained homogenate and buffer solution (0,05 cm3 each) were mixed in equal
proportions, shaken (time – 2 min), 0,05 cm3 NBT was added. After 30 minutes incubation
of the resulting solution (at 240C) was added 2 cm3 of a solution of dimethyl sulfoxide-
chloroform (2:1). The extraction was performed for 1 min, сentrifuged for 5 min (аt 1500 rpm).
The supernatant of the cetrifugate solution was photometered against the control at 540 nm
(cuvette 1 cm3, thickness 0,5 cm).
Distilled water was used instead of the homogenate to prepare the control sample.
Superoxide production was calculated according to the calibration graph, for the
construction of which the extinction of the samples was determined from 0,01, 0,02, 0,05,
0,07, 0,1, 0,2 cm3 NBT (w = 0.2%), 0,1 cm3 1 М KOH and 0,1 cm3 AK solution (18 mg/10 cm3).
To determine the increase in the level of TBA-active products (Δ TBAap), their baseline
level (TBAap0) and stimulated (TBAap1,5) level were determined. The final value of the
indicator (in μmol/kg) was calculated by the formula:
Δ TBAap = | TBAap1,5 – TBAap0 | / TBAap0 ∙ 100%
To determine TBAap0 0,5 g of the test object was homogenized in 4,5 cm3 of tris-buffer
solution (pH = 7,4). 2 cm3 of homogenate were taken, mixed with trichloroacetic acid (w =
30%) and centrifuged (conditions: 30 min, at 3000 rpm).
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The 2 cm3 centrifuge supernatant was mixed with 3 cm3 of thiobarbituric acid solution
(w = 0,338%, ex tempore preparation). The resulting trimethine complex was photometered
at 540 nm against a control that did not contain homogenate (reagent control composition:
1,2 cm3 of buffer solution, 0,7 cm3 of trichloroacetic acid, 0,1 cm3 of water and 3 cm3 of TBA
reagent). Determination of TBAap1,5 was performed by a similar method, with pre-incubation
of the sample in prooxidant iron-ascorbate buffer for 90 min. The concentration of TBAap
was calculated by the formula:
C = E ∙ 240,4
where C is the concentration of TBAap in μmol/kg; E – extinction; 240.4 – coefficient
taking into account molar extinction and dilution.
Superoxide dismutase (SOD) activity was determined in the following sequence: 0,5 g of
test tissue was homogenized in 0,5 cm3 of water. To precipitate pigments after 10 minutes
was added 2 cm3 of a mixture of ethanol chloroform (5:3), incubated at -40C. After 24 hours,
stirred and centrifuged for 15 min at 3000 rpm. 0,1 cm3 of the centrifuge was mixed in a
cuvette (1 cm) with a prepared solution containing 4,4 cm3 of carbonate buffer (pH = 10,2)
and 0,5 cm3 of a solution of adrenaline (C = 0,01 mol/dm3) in lemon acid (C = 0,01 mol/dm3).
Extinction was determined every minute until it stopped increasing. 0,1 cm3 of distilled water
was introduced into the control sample instead of the centrifugate. The calculation of SOD
activity was carried out according to the formula:
T = (E1– E2) ∙ 100 / E1
where T is the percentage of inhibition of oxidation of •O2- adrenaline to
adrenochrome (%); E1 – average extinction control for 1 min (E/t); E2 – average extinction of
the experiment for 1 min; 100 – the maximum percentage (%) of inhibition.
SOD activity was expressed in conventional units (OD):
OD = T / (100 – t)
where 1 OD corresponds to the inhibition of the reaction rate by 50%.
Catalase activity was determined by the following method: to a flask with 7 cm3 distilled
water was added 1 cm3 of aqueous tissue homogenate of the test object (0,1 g in 20 cm3 of
H2O) and 2 cm3 of H2O2 (w = 1%), shaken every 10 minutes. After 30 minutes 3 cm3 of H2SO4
solution (w = 10%) was added and titrated with 0,1 M KMnO4 solution to a pale pink color
that did not disappear for 30 seconds.
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The calculation of catalase number (А) (in μmol of substrate per unit time per unit
mass of protein) was carried out by the formula:
A = (Vcontrol – Vexperimental) ∙ 1.7/t ∙ M (H2O2)
where Vcontrol and Vexperimental is the volume of KMnO4 solution spent on titration of the
control and test samples, respectively, cm3 (boiled homogenate was used in the control
sample); 1,7 – amount of H2O2 (mg), which corresponds to 1 cm3 of KMnO4 solution; t –
incubation time of the sample (30 s); M(H2O2) – molar mass of H2O2 (34 g/mol).
When determining the concentration of GSH, the homogenate was prepared on
trichloroacetic acid (0,1 g of tissue with 2,4 cm3 of trichloroacetic acid) and left for 10
minutes. Subsequently, the samples were centrifuged for 15 min at 3000 rpm. 0,2 cm3 of
centrifugate was mixed with 0,05 cm3 of NaOH solution (w = 20%) and 5 cm3 of tris-buffer
(pH = 8,05) and 0,1 cm3 of Elman's reagent (99 mg DTNBK in 25 cm3 ethanol). After 20
minutes keeping the samples in the dark, performed their photometry at 412 nm in a cuvette
per 1 cm against control of reagents that did not contain homogenate. GSH concentration
was determined according to a standard calibration schedule.
When determining the concentration of AА used acid homogenate (1 g of tissue in 9 cm3
of HCl solution (w = 2%)), which after 10 minutes filtered. 3 cm3 of the filtrate was titrated
with a solution of 2,6-dichlorophenolindophenol (0,001 mol/dm3) to a stable pink color.
Boiled filtrate in the presence of 3 drops of 3% H2O2 was used in the control sample. The
concentration of AА (in mmol/kg) was calculated by the formula:
С = Q ∙ (Aexp - Acontr) ∙ V0 / (V1 ∙ a)
where Q is the amount of AА corresponding to 1 cm3 of a solution of 2,6-
dichlorophenolindophenol (0,088 mg); V0 – total amount of extract, cm3; V1 - volume of
extract taken for titration, cm3; a – weight of the sample, g; Acontr, Aexp – the volume of a
solution of 2,6-dichlorophenolindophenol spent on titration of the control and experimental
sample, cm3.
To determine the activity of cytochrome oxidase, a tissue homogenate of the test object
was prepared in phosphate buffer (ratio 0,5 g in 4,5 cm3) at pH 7,6. To 1 cm3 of homogenate
was added 1 cm3 of the reaction mixture containing 0,25 cm3 of α-naphthol in 50 cm3 of
ethanol (w = 22%), 0,35 cm3 of aqueous solution of N,N-dimethyl-para-phenylenediamine
hydrochloride (w = 0,1%), 0,25 cm3 of phosphate buffer, 0,15 cm3 of cytochrome c solution (w
= 0,02%). After 5 minutes diethyl ether and ethanol were added in a volume ratio of 9:1 (V =
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10 cm3), kept at 40C for 30 minutes, shaking periodically. The extract was photometered at
540 nm against the control, which instead of the homogenate contained 1 cm3 of diluted
buffer solution. The calculations were performed according to the formula:
А = Eexp ∙ 10 / Est ∙ 5 = 2 Eexp / Est
A – cytochrome oxidase activity in indophenolic units per gram of tissue per minute;
Eexp – extinction of the test sample;
Est – extinction of the standard, calculated from the calibration graph at a dose of 100
mg/cm3 of α-naphthol;
10 – breeding; 5 – incubation time.
All the results of the determination of biochemical indicators of the state of the
prooxidant-antioxidant system were processed statistically according to generally accepted
methods.
3. Results and discussion
The results of determining the indicators of the PO link of PAS and FRPO tissues of
dormant seeds are shown in table 1, AO link of PAS - in table 2. The effect of activation of
germination processes on PO link of PAS and FRPO is shown in table 3, the effect on AO link
- in table 4.
Table 1. The results of determining the prooxidant activity and the level of FRPO in
seed tissues at rest
Experimental plants
Indicators of prooxidant activity
Cytochrome oxidase
activity, OD
Δ TBAap, %
Magnoliopsida
Glycine max L.
66,15 ± 5,01
0,314 ±0,019
Heliаnthus аnnuus L.
42,23 ± 1,06
0,204±0,003
Fagopyrum esculentum L.
99,22 ± 4,11
0,183±0,005
Linum usitatissimum L.
29,88 ± 1,44
0,262±0,009
Sinapis alba L.
35,18 ± 1,22
0,240±0,004
Chenopodium quinoa L.
85,14 ± 3,67
0,436 ±0,011
Liliopsida
Panicum miliaceum L.
136,49 ± 6,22
0,118±0,006
Oryza sativa L.
21,63 ± 1,10
0,398 ±0,006
Avena sativa L.
11,27 ± 2,01
0,418 ±0,009
Zea mays L.
111,83 ± 5,19
0,159±0,008
Hordeum vulgare L.
128,45 ± 18,35
0,276±0,005
Triticum durum Desf.
27,86 ± 4,11
0,346 ±0,001
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Table 2. The results of determining the antioxidant activity in the tissues of seeds at rest
Experimental plants
Enzymatic antioxidants
Low molecular weight
antioxidants
Catalase
activity, 𝜇𝑚𝑜𝑙
𝑘𝑔∗𝑚𝑖𝑛
SOD activity,
OD
Concentration
of AA, 𝑚𝑚𝑜𝑙
𝑘𝑔
Concentratio
n of GSH,
𝑚𝑚𝑜𝑙
𝑘𝑔
Magnoliopsida
Glycine max L.
0,48 ± 0,02
0,53 ± 0,02
0,293 ± 0,03
59,32 ± 0,95
Heliаnthus аnnuus L.
0,19 ± 0,01
0,28 ± 0,01
0,096 ± 0,01
39,11 ± 0,72
Fagopyrum esculentum L.
0,31 ± 0,02
0,28 ± 0,02
0,141 ± 0,02
43,22 ± 0,96
Linum usitatissimum L.
0,11 ± 0,01
0,30 ± 0,01
0,135 ± 0,02
46,79 ± 0,48
Sinapis alba L.
0,25 ± 0,01
0,36 ± 0,03
0,110 ± 0,01
41,01 ± 0,63
Chenopodium quinoa L.
0,36 ± 0,01
0,44 ± 0,02
0,120 ± 0,02
51,67 ± 0,11
Liliopsida
Avena sativa L.
0,39 ± 0,03
0,46 ± 0,02
0,111 ± 0,03
54,19 ± 0,34
Oryza sativa L.
0,31 ± 0,01
0,42 ± 0,01
0,092 ± 0,01
45,18 ± 0,78
Hordeum vulgare L.
0,23 ± 0,02
0,29 ± 0,01
0,076±0,01
48,05 ± 0,10
Triticum durum Desf.
0,09 ± 0,02
0,22 ± 0,01
0,057 ± 0,01
40,79 ± 0,25
Zea mays L.
0,09 ± 0,01
0,19 ± 0,01
0,085 ± 0,02
37,16 ± 0,99
Panicum miliaceum L.
0,07 ± 0,01
0,16 ± 0,01
0,037 ± 0,01
43,14 ± 0,67
Among the researched Magnoliopsida, the lowest level of •О2- generation has the
seeds of Glycine max L., the highest is characteristic of the tissues of Helianthus annuus L., Linum
usitatissimum L., and Sinapis alba L. It is noteworthy that the reserve substances in the last three
experimental plant species are lipids, while in Glycine max L. – proteins. Moreover, the level
of •О2- generation is higher the lower the content of polyunsaturated fatty acids (PUFA)
among lipid inclusions. The lowest values of ΔTBAap are also characteristic of plants that
specialize in lipid storage, within this group we also observe a decrease in ΔTBAap with
increasing PUFA content. The largest difference in ΔTBAap is characteristic of the tissues of
Fagopyrum esculentum L. and Chenopodium quinoa L., whose reserve substance is carbohydrates.
However, according to the literature, among these two plant species, the seeds of Chenopodium
quinoa L., in addition to carbohydrates, have a significantly higher percentage of protein in the
reserve than Fagopyrum esculentum L., and according to the results - a lower level of generation
•О2- and ΔTBAap. All of the above suggests that the accumulation of tissue tissues of
experimental plants of protein inclusions, or PUFA helps to reduce the generation of •О2-
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and ΔTBAap. The degree of FRPO can be judged by the activity of cytochrome oxidase. Thus,
in the tissues of Glycine max L. and Chenopodium quinoa L. it is the highest, and in the tissues of
Helianthus annuus L., Linum usitatissimum L., and Sinapis alba L. we again observe a decrease in
enzyme activity following a decrease in PUFA content in lipid inclusions.
The activity of enzymatic antioxidants in the tissues of experimental Magnoliopsida
is 1,35 times higher than that of Liliopsida (the predominance of the average activity of
catalase is 1.44 times, SOD – 1,26 times). The increase in the activity of enzymes within the
experimental group is also manifested against the background of increasing protein content
in the reserve components of seeds. The highest activity of both SOD and catalase was found
in the tissues of Glycine max L. and Chenopodium quinoa L., which confirms the fact that SOD
supplies a substrate for catalase. A parallel decrease in the activity of these two enzymes is
observed in a number: Sinapis alba L., Helianthus annuus L., and Linum usitatissimum L. (dependence
on PUFA content was not detected). In the tissues of Fagopyrum esculentum L., catalase activity
remains high, while the activity of SOD decreases, which may be explained by the
participation of catalase in the inactivation of PO, the source of which is not SOD.
Table 3. The results of determining the prooxidant activity and the level of FRPO in
seed tissues, with the activation of germination processes
Experimental plants
Indicators of prooxidant activity
Cytochrome
oxidase activity,
OD
NBT test (base level),
nmol •О2-/(g*s)
Δ TBAap, %
Magnoliopsida
Glycine max L.
0,111 ±0,06
30,41 ± 1,26
0,465 ±0,011
Heliаnthus аnnuus L.
2,444 ± 0,003
106,84 ± 1,88
0,220±0,006
Fagopyrum esculentum L.
0,364 ± 0,006
58,15 ± 2,01
0,230±0,009
Linum usitatissimum L.
1,809 ± 0,022
61,29 ± 1,55
0,299±0,008
Sinapis alba L.
1,464 ± 0,018
80,80 ± 2,16
0,263±0,004
Chenopodium quinoa L.
0,197 ± 0,011
52,94 ± 1,43
0,572 ±0,020
Liliopsida
Panicum miliaceum L.
1,506±0,021
154,07 ± 8,02
0,126±0,004
Oryza sativa L.
0,826±0,022
33,38 ± 2,65
0,644 ±0,016
Avena sativa L.
0,062±0,003
17,36 ± 1,28
0,610 ±0,023
Zea mays L.
1,809±0,026
129,14 ± 3,89
0,177±0,005
Hordeum vulgare L.
0,124±0,005
144,29 ± 4,07
0,337±0,009
Triticum durum Desf.
0,112 ±0,004
53,97 ± 1,88
0,364 ±0,008
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Table 4. The results of determining the antioxidant activity in seed tissues, with the
activation of germination processes
Experimental plants
Enzymatic antioxidants
Low molecular weight
antioxidants
Catalase
activity, 𝜇𝑚𝑜𝑙
𝑘𝑔∗𝑚𝑖𝑛
SOD activity,
OD
Concentration
of AA, 𝑚𝑚𝑜𝑙
𝑘𝑔
Concentratio
n of GSH,
𝑚𝑚𝑜𝑙
𝑘𝑔
Magnoliopsida
Glycine max L.
1,21 ± 0,07
0,88 ± 0,05
2,063 ± 0,03
94,43 ± 3,44
Heliаnthus аnnuus L.
0,27 ± 0,01
0,34 ± 0,01
0,137 ± 0,02
41,07 ± 0,18
Fagopyrum esculentum L.
0,64 ± 0,02
0,42 ± 0,02
0,845 ± 0,02
51,44 ± 2,18
Linum usitatissimum L.
0,15 ± 0,01
0,38 ± 0,03
0,469 ± 0,01
50,96 ± 0,83
Sinapis alba L. горчица
0,39 ± 0,01
0,41 ± 0,01
0,318 ± 0,01
46,16 ± 0,99
Chenopodium quinoa L.
0,79 ± 0,04
0,70 ± 0,03
0,614 ± 0,02
70,42 ± 1,76
Liliopsida
Avena sativa L.
0,69 ± 0,04
0,86 ± 0,03
1,703 ± 0,09
62,88 ± 0,22
Oryza sativa L.
0,64 ± 0,02
0,80 ± 0,02
0,481 ± 0,02
50,49 ± 0,93
Hordeum vulgare L.
0,33 ± 0,03
0,40 ± 0,02
0,749 ± 0,08
54,91 ± 0,19
Triticum durum Desf.
0,12 ± 0,02
0,29 ± 0,02
0,459 ± 0,04
45,34 ± 0,21
Zea mays L.
0,12 ± 0,01
0,23 ± 0,01
0,690 ± 0,03
42,17 ± 1,04
Panicum miliaceum L.
0,09 ± 0,01
0,18 ± 0,01
0,101 ± 0,01
46,51 ± 0,34
Initiation of germination causes an increase in the level of generation •О2- in all
experimental plants. Thus, in the tissues of Glycine max L. this figure increased by 54,17%, in
the tissues of Helianthus annuus L. – by 115,52%, Fagopyrum esculentum L. – 24,46%, Linum
usitatissimum L. – 79,82%, Sinapis alba L. – 88,17%, Chenopodium quinoa L. – 61,47%. Moreover,
among Magnoliopsida, the largest increase in the level of TBA-active products was found in
the tissues of Helianthus annuus L., Linum usitatissimum L., and Sinapis alba L., where we again
observe a connection with the content of PUFA. However, in the tissues of these three plant
species, cytochrome oxidase activity does not decrease and even increases by a small
percentage (7,82% for Helianthus annuus L., 14,18% for Linum usitatissimum L. and 9,56% for
Sinapis alba L.) at a slight increase in the activity of enzymatic AO and the content of low
molecular weight. A possible explanation for such results is that with the strengthening of
the link, the level of non-structural FRPO increases, namely reserve lipids, which are used to
meet the energy needs of the body, which is especially important in the transition from rest
to active growth. The largest increase •О2- with the smallest increase in TBAap level and the
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highest increase in cytochrome oxidase activity (48,15%) was found in the tissues of Glycine
max L. A similar pattern is observed for Chenopodium quinoa L. but with slightly lower values
(increase in cytochrome oxidase 31,24%). In the tissues of Fagopyrum esculentum L., cytochrome
oxidase activity increases by 26,03% and occupies an intermediate position between the
results described above.
Analyzing the effect of germination activation on the AO link in the tissues of the
researched Magnoliopsida, we also observe the largest increase in enzyme AO in the tissues
of Glycine max L. (66,04% for SOD and 152,08% for catalase). A significant increase in the value
of AO enzymes is observed in the tissues of Fagopyrum esculentum L. (51,28% for SOD and
107,01% for catalase) and Chenopodium quinoa L. (58,22% for SOD and 119,16% for catalase).
The increase in the activity of antioxidant enzymes in Magnoliopsida′s tissues, that
specializing in lipid inclusions is insignificant, depending on the amount of PUFA was not
detected. Thus, for Helianthus annuus L., the increase in catalase activity was 40,22%, SOD –
28,01%, for Linum usitatissimum L. – 33,79% and 28,28%, respectively, for Sinapis alba L. – 54,13%
and 14,93%
Analyzing the results of determination of low molecular weight AO, it can be stated
that the highest base level of AA has Glycine max L., which is characteristic of plants of the
Fabaceae, and more than 2 times higher than ascorbate content in all other experimental
plants. Helianthus annuus L. seed tissues have the lowest AA content, which also has the
smallest increase in AA content when germination is activated (1,43 times). Activation of
germination processes increases the amount of ascorbate in the tissues of Fagopyrum esculentum
L. by 5,99 times, Chenopodium quinoa L. – by 5,12, Linum usitatissimum L. – by 3,48 times, Sinapis
alba L. – by 2,89 times. Glycine max L. has the largest increase in the concentration of AA, which
is 7,04 times.
Seed tissues of Glycine max L. have the highest content of GSH, both at rest and the
largest increase in germination (59,18%), which is naturally due to the presence of a large
number of amino acids L-cysteine, L-glutamic acid, and glycine required for its synthesis.
Helianthus annuus L. seed tissue, which specializes in lipid inclusions, has the lowest level of
GSH and has the lowest protein content among all experimental plants. Activation of
germination processes increases the amount of GSH in the tissues of Chenopodium quinoa L. by
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36,28%, Fagopyrum esculentum L. – by 19,01%, Sinapis alba L. - by 12,55%, Linum usitatissimum L. –
8,92%. Helianthus annuus L. has the smallest increase in GSH concentration, which is 5,02%.
The average GSH growth rate for experimental Magnoliopsida is 26.11%.
Summarizing the results obtained on experimental Magnoliopsida, we can say that in
the tissues of seeds at rest, the average level of generation •О2- is 0,567 nmol •О2-/(g*s),
ΔTBAap – 59,63%, SOD – 0,37 OD, catalase – 0,28 μmol/(kg*min), AА – 0,149 mmol/kg, GSH
– 46.85 mmol/kg, cytochrome oxidase – 0,273 OD. When activating the germination
processes, the average level of generation •О2- was 1,065 nmol •О2-/(g*s), ΔTBAap - 65.07%,
SOD – 0,52 OD, catalase – 0,58 μmol/(kg*min), AА – 0,741 mmol/kg, GSH – 59,08 mmol/kg,
cytochrome oxidase – 0,342 OD. Thus, the activation of germination caused an increase in
the value of the average PAS as follows: for the generation of •О2- growth is 87,83%, for
ΔTBAap – 9,12%, for SOD – 40,54%, catalase – 107,14% (2.07 times), AА – 397,32% (4,97
times), GSH – 26,11%, cytochrome oxidase – 25,28%.
Analyzing the state of prooxidant activity of seed tissues at rest, we can say that
among the experimental Liliopsida the highest base level of •О2- generation have Zea mays L.
and Panicum miliaceum L. The same plants have the highest ΔTBAap and rather low values of
cytochrome oxidase activity, indicating a high degree of FRPO macromolecules in their cells.
This assumption is natural, because, according to the results, both in the tissues of Zea mays
L. and Panicum miliaceum L. found the lowest level of activity of enzymatic antioxidants and
close to the lowest content of AA and GSH. A similar prooxidant pattern is observed in the
tissues of Hordeum vulgare L., however, increased generation of low molecular weight AO in
combination with increased SOD and catalase activity suggests a lower level of FRPO and is
confirmed by significantly higher cytochrome oxidase activity compared to the previous two
experiments. Avena sativa L. tissues have the most powerful link in AO protection, cytochrome
oxidase activity is also the highest, TBAap value is the lowest, but the base level of •О2- is
also low. The peculiarity found in the tissues of Oryza sativa L.: so at a fairly high level of
generation •О2- we observe one of the lowest levels of ΔTBAap and the level of cytochrome
oxidase activity, which does not differ significantly from the value set for Avena sativa L. Thus,
the AO link of PAS in the tissues of Oryza sativa L. is more powerful than for Avena sativa L.,
which is achieved both by SOD and catalase and several other AOs.
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Analyzing the results of the research of PAS of Liliopsida, it can be argued that the
initiation of germination causes an increase in the level of •О2- generation in the tissues of
Panicum miliaceum L. by 38,67%, in the tissues of Oryza sativa L. – by 88,97%, Zea mays L. –
42,14%, Avena sativa L. – 72,14%, Hordeum vulgare L. – 36,36%, Triticum durum Desf. – by 24,39%.
The largest increase in the level of ΔTBAap is characteristic of Triticum durum Desf. (26,11%),
the least - for Avena sativa L. (6,09%). Germination initiation increases the activity of
cytochrome oxidase in the tissues of Avena sativa L. by 46,02%, Panicum miliaceum L. by 6,99%,
in the tissues of Oryza sativa L. – by 61,80%, Zea mays L. – 11,42%, Hordeum vulgare L. – 22,16%,
Triticum durum Desf. – at 5,18%. Catalase activity is enhanced in experimental plants by the
following values: Avena sativa L. by 75,79%, Panicum miliaceum L. by 21,04%, in tissues of Oryza
sativa L. – by 105,13%, Zea mays L. – 29,99%, Hordeum vulgare L. – 42,62%, Triticum durum Desf. –
at 33,91%. By increasing the growth of SOD experimental plants can be placed in the
following order: Panicum miliaceum L. (15,11%), Zea mays L. (23,20%), Triticum durum Desf.
(30,40%), Hordeum vulgare L. (38,46%), Avena sativa L. (86,03%), Oryza sativa L. (91,07%).
Analyzing the content of low molecular weight antioxidants, it should be noted that the
concentration of ascorbate in the seed tissues of experimental Liliopsida plants is on average
1,96 times lower than in Magnoliopsida. Oats Avena sativa L., Oryza sativa L., and Zea mays L. have
the highest background level of AA, Panicum miliaceum L. has the lowest.
Activation of germination processes increases the amount of ascorbate in the tissues
of Avena sativa L. by 15,34 times, Hordeum vulgare L. by 9,86, Zea mays L. – by 8,15 times, Triticum
durum Desf. – 8,06 times, Oryza sativa L. – 5,23 times. The smallest increase in the concentration
of AA has Panicum miliaceum L., which is 2,74 times. The content of GSH has a similar tendency,
so the average concentration of GSH in the tissues of experimental Magnoliopsida is 1,49
times higher than in Liliopsida. The maximum value of the indicator for dormant seeds
recorded for Avena sativa L., the minimum – for Zea mays L. Activation of germination processes
increases the amount of GSH in the tissues of Avena sativa L. by 16,04%, Hordeum vulgare L. – by
14,29%, Zea mays L. – by 13,48%, Triticum durum Desf. – by 11,23%, Oryza sativa L. – 11,76%. The
smallest increase in the concentration of GSH has Panicum miliaceum L., which is 7,81%. As a
result of the analysis of changes in the amount of low molecular weight AO, it can be assumed
that when activating the processes of germination of AA seeds has a more protective value
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compared to GSH. The increase in the concentration of AA is more species-specific, while for
GSH is more uniform.
Summarizing the results obtained on experimental Liliopsida, we can say that in the
tissues of seeds at rest, the average level of generation •О2- is 0,502 nmol •О2-/(g*s), ΔTBAap
– 72,79%, SOD – 0,29 OD, catalase – 0,20 μmol/(kg*min), AА – 0,076 mmol/kg, GSH – 44,75
mmol/kg, cytochrome oxidase – 0,241 OD. When activating the germination processes, the
average level of •О2- generation is 0,740 nmol •О2-/(g*s), ΔTBAap – 88,70%, SOD – 0,46 OD,
catalase – 0,33 μmol/(kg*min), AА – 0,697 mmol/kg, GSH – 50,38 mmol/kg, cytochrome
oxidase – 0,376 OD.
Thus, the activation of germination caused an increase in the value of the experimental
average PAS as follows: for the generation of •О2- growth is 41,47%, for ΔTBAap – 21,64%,
for SOD – 58,62%, catalase – 68,37%, AA – 817.11% (9,17 times), GSH – 12,58%, cytochrome
oxidase – 56,02%.
Comparison of the average values of the obtained results makes it possible to identify
the following patterns:
At rest in experimental Magnoliopsida compared to Liliopsida, the level of
generation •О2- is higher by 12,96%, the level of ΔTBAap is lower by 13,29%, cytochrome
oxidase activity is higher by 13,29%. The activity of SOD is higher by 27,59%, catalase - by
40%, the concentration of AA - by 96,06%, GSH - by 4.69%.
When germination is activated, we have the following predominance of
indicators: the level of •О2- generation is higher by 43,92%, the level of ΔTBAap is lower by
33,63%, the activity of cytochrome oxidase is lower by 9.94%. The activity of SOD is higher
by 13,04%, catalase – by 75,76%, the concentration of AA – by 6,31%, GSH – by 17,27%.
Thus, for the tissues of seeds of experimental Magnoliopsida, which are at rest,
both links of PAS are more powerful than in Liliopsida, the level of FRPO is lower, which is
achieved due to both enzymatic and low molecular weight antioxidants. Germination
activation enhances both levels of PAS in all experimental groups of plants, but in
Magnoliopsida, we observe the stronger generation of •О2-, and the predominance of
protection by enzymatic AO, and in Liliopsida - low molecular weight
If we characterize the effect of germination activation on individual
components of the AO protection system, we can see that germination activation enhances
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SOD activity in Liliopsida more intensely than in Magnoliopsida, which in turn show
increased catalase activity. Among low molecular weight AO, we also observe a cross pattern:
a significant increase in the generation of AA Liliopsida and the concentration of GSH
Magnoliopsida.
When comparing changes in the values of the indicators of the PAS of different species
of plants, the following assumptions can be made:
The level of AO protection during germination activation depends on the
quantitative and qualitative composition of reserve inclusions in the composition of seed
tissues. For example, an increase in the content of protein inclusions promotes the increased
synthesis of enzyme AO and GSH, and an increase in the content of PUFA among lipid
inclusions reduces the intensity of FRPO.
According to the growth of AO content during germination activation,
experimental plants can be placed in the following order: Glycine max L., Chenopodium
quinoa L., Avena sativa L., Fagopyrum esculentum L., Oryza sativa L., Hordeum vulgare L.,
Linum usitatissimum L ., Sinapis alba L., Panicum miliaceum L., Zea mays L., Helianthus
annuus L. This is a biochemical basis for practical recommendations on the feasibility and
benefits of pre-soaking cereals in their culinary processing.
Conclusions
1. In the tissues of seeds of experimental Magnoliopsida at rest, the average level of
generation •О2- is 0,567 nmol •О2-/(g*s), ΔTBAap – 59,63%, SOD – 0,37 OD, catalase
– 0,28 μmol/(kg*min), AА – 0,149 mmol/kg, GSH – 46.85 mmol/kg, cytochrome
oxidase – 0,273 OD. For Liliopsida: the level of generation •О2- is 0,502 nmol •О2-
/(g*s), ΔTBAap – 72.92%, SOD – 0,29 OD, catalase – 0,20 μmol/(kg*min), AА – 0,076
mmol/kg, GSH – 44.75 mmol/kg, cytochrome oxidase – 0,241 OD.
2. Activation of germination caused an increase in the value of the average PAS in the
tissues of experimental Magnoliopsida as follows: for generation •О2- growth is
87,83%, for ΔTBAap – 9,12%, for SOD – 40,54%, catalase – 107,14% (2.07 times), AА –
397,32% (4,97 times), GSH – 26,11%, cytochrome oxidase – 25,28%. For Liliopsida: for
generation •О2- growth is 41,47%, for ΔTBAap – 21,64%, for SOD – 58,62%, catalase –
68,37%, AА – 817,11% (9,17 times), GSH – 12,58%, cytochrome oxidase – 56,02%.
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3. For seed tissues of experimental Magnoliopsida, which are at rest, both links of PAS
are more powerful than in Liliopsida, the level of FRPO is lower, which is achieved
due to both enzymatic and low molecular weight AO. Germination activation
enhances both links of PAS in all experimental groups of plants, however, in
Magnoliopsida, we observe the stronger generation of •О2-, and the predominance of
protection by enzymatic AO, and in Liliopsida – low molecular weight. Activation of
germination enhances the activity of SOD in Liliopsida more intensely, compared with
Magnoliopsida, which in turn revealed enhanced catalase activity. Among low
molecular weight AO, we also observe a cross pattern: a significant increase in the
generation of AA in Liliopsida and the concentration of GSH in Magnoliopsida.
4. As the growth rate of generation •О2- increases during germination activation, the
experimental plants form the following series: Triticum durum Desf., Fagopyrum
esculentum L., Hordeum vulgare L., Panicum miliaceum L., Zea mays L., Glycine max L.,
Chenopodium quinoa L., Avena sativa L., Linum usitatissimum L., Sinapis alba L., Oryza sativa L.,
Helianthus annuus L. According to the growth of AO content during germination
activation, experimental plants can be placed in the following order: Helianthus annuus
L., Zea mays L., Panicum miliaceum L., Sinapis alba L., Linum usitatissimum L., Oryza sativa L.,
Hordeum vulgare L., Fagopyrum esculentum L., Avena sativa L., Chenopodium quinoa L., Glycine
max L. This is a biochemical basis for practical recommendations on the feasibility and
benefits of pre-soaking cereals in their culinary processing. The results of the study
also indicate the need for amendments to the methods of biochemical analysis of seeds
to differentiate dry homogenization and its pre-soaking during sample preparation.
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