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Evaluation of biochemical and free radical scavengers of Digitaria exilis L. under osmotic stress
J Plant Biotechnol 2019;46:331-337
Published online December 31, 2019
© 2019 Korean Society for Plant Biotechnology.

David Oyinade A. · Osonubi Oluwole · Oyetunji Olusola Jacob

Department of Plant Science and Biotechnology, Federal University Oye-Ekiti, Ekiti State, Nigeria
Botany Department, University of Ibadan, Ibadan, Nigeria
Correspondence to: e-mail: oyinade.dedeke@fuoye.edu.ng
Received June 30, 2019; Revised December 16, 2019; Accepted December 16, 2019.
cc This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract

Digitaria exilis L. is an under-utilized crop with high nutritional and medicinal values. It thrives in and is well-adapted to arid areas with low soil nutrients. Using biochemical markers, this study investigates the mechanisms by which D. exilis responds to osmotic stress. Three accessions Dinat Iburua (DIN), Jakah Iburua (JAK) and Jiw Iburua (JIW) were collected from National Cereal Research Institute, Niger State. Two accessions, NG/11/JD/061 and NG/11/JD/062 were also collected from National Centre for Genetic Resources and Biotechnology, Ibadan. Murashige and Skoog medium of approximately 1.2 L was supplemented with polyethylene glycol 6000 to create osmotic pressures of -9.29, -13.93, -20.13, -26.32, -32.51, and 0 MPa (control). Sterilized seeds were inoculated in the medium and placed in the growth room for 4 weeks. Proline accumulation was significantly high in all JAK plants under osmotic stress. Proline and ascorbate peroxidase (p<0.05) activities were directly correlated, thus reinforcing the survivability of JAK during stress. Catalase (CAT) activity was also significantly induced in JAK under osmotic stress, which synergistically improved its tolerability. As a result, >50% of OH, H2O2, and NO radicals were scavenged. However, other accessions including DIN, NG061, NG062, and JIW showed variations in their responses to different levels of osmotic stress, although not significant. Therefore, JAK possesses a well-equipped free radical quenching system that is protected by the accumulation of the osmolyte proline; therefore, accession JAK is considered osmotolerant. CAT and superoxide dismutase activities were osmostabilized against oxidative stress by proline.

Keywords : free radicals, D. exilis, antioxidant enzymes, proline accumulation, lipid peroxidation
Introduction

Digitaria exilis possesses many nutritional, economic and phytochemical benefits to mankind. Gwete soup is an African delicacy that is locally prepared from D. exilis to treat diabetes1. It yields and survives well in relatively poor climatic conditions such as arid areas. D. exilis is very important among other grains due to its high nutritional composition. D. exilis show generally mineral contents that are in the range of other cereals. However, it contains much more protein that other cereals like millets, maize, sorghum etc. and the protein is mainly concentrated in the grain and not in the husk1. Methionine, which builds up sulphur, is accumulated in D. exilis twice the amount compared to corn or millet and three times compared to rice.

In vitro culture techniques minimize environmental variation due to defined nutrient controlled conditions and homogeneity of stress application. The simplicity of such manipulation enables to study large plant production and stress treatments in a limited space and short period of time. Simulation of drought stress under in vitro conditions during the regeneration process constitutes a convenient way to study the effect of drought stress on the plant. Application of osmotic stress to plants at juvenile stage is an effective method of selecting plants with drought tolerant traits. This will of course make the mature plants cope with the drought stress conditions during growth and reproductive periods. This will confer the potential in screening for drought tolerance. Poly-ethylene glycol 6000 had been used to simulate drought stress in plant as non–penetrating osmotic agent lowering the water potential in a way similar to soil drying. It is frequently assumed that plant water relations are similar whether the plants are growing in soil or in a PEG' solution having an equal water potential. Larger polyethylene glycol molecules such as poly-ethylene glycol 6000 are more useful for simulating soil drying5. The study therefore aimed at the biochemical mechanisms undertaken by D. exilis to survive osmotic stress.

Materials and Methods

Plant material

Five accessions of D. exilis were used in the studies, which were Dinat Iburua (DIN), Jakah Iburua (JAK), Jiw Iburua (JIW), NG/JD/06/11/062 (NG062) and NG/JD/06/11/061 (NG061). Three accessions DID, JAK and JIW were obtained from National Cereal Research Institute, Badeggi, Niger State, Nigeria, while the other two accessions NG061 and NG062 were obtained from National Centre for Genetic Resources and Biotechnology (NACRAB), Moor Plantation Ibadan, Nigeria.

Media preparation

To prepare 1.2 litre of MS (Murashige and Skoog, 1962) media, 6oml of macronutrients, 6 ml stock micro-nutrients, 36 g of sucrose, 0.12 g of inositol, 6 ml of vitamins, 0.04476 g of Sodium (Di) Ethylenediamine Tetraacetate Dihydrate (Na EDTA. 2H2O) and 0.0278 g of Ferrous sulphate were added to 600 ml of deionized water. The mixture was divided equally into 6 sterilized jars. Poly-ethylene glycol PEG 6000 of 30 g/l, 45 g/l, 65 g/l, 85 g/l and 105 g/l and 0 g/l were added to create an osmotic conditions–of -9.29 MPa, -13.93 MPa, -20.13 MPa, -26.32 MPa, -32.51 MPa and 0 MPa (control) to represent A, B, C, D, E and F. Deionized water was added to make up to 200 ml in each jar. The hydrogen ion concentration i.e. pH of 5.7 ± 0.3 was taken using pH meter. About 0.46 g of phytagel (Agar) was added to each jar. All the media were solubilized for 15 minutes in an oven. Five millilitres (5 ml) were dispensed into an autoclaved test-tube. Five sterilized seeds were inoculated on the media inside the laminar airflow, sealed with paraffin and placed inside growth room.

Quantification of chlorophyll contents

Chlorophyll was extracted from the leaves. The extraction of leaf pigments was performed with 75% ethanol, and the absorbance at 663 and 645 nm were measured with a spectrophotometer. The chlorophyll a, chlorophyll b, and total chlorophyll quantities were calculated according to the method of Arnon. The pigment concentrations were expressed as µg/ml. Chlorophyll contents were calculated using the formula stated below.

Chl a=15.65A663-7.340A645Chl b=27.05 A645-11.21 A663

Determination of proline contents

All experiments were performed at 4°C. Leaf samples were homogenized in ice cold 50 mM sodium phosphate buffer (pH 7.8) for the proline extraction. The buffer contained 1 mM disodium EDTA and 2% (w/v) polyvinylipid peroxidationlypyrrolidone (PVPP). Supernatants collected after centrifugation at 13,000 × g for 40 min were used to determine the proline contents. The free proline content was determined according to Bates.

Determination of radical scavenging activity

DPPH (2, 2-diphenyl-1-picrylhydrazyl hydrate) Assay was determined using the stable radical DPPH (2, 2-diphenyl-1-picrylhydrazyl hydrate) as described by Brand-Williams. Nitric oxide radical activity of the extract was carried out according to the method of Green as described by Marcocci. The hydroxyl radical scavenging activity was measured by studying the competition between deoxyribose and the fractions for hydroxyl radicals generated from the Fe3+/ascorbate /EDTA/H2O2 system according to the method of Halliwell. The ability of plant extracts to scavenge hydrogen peroxide was determined according to the method of Ruch.

Determination of antioxidant enzyme

Superoxide dismutase (SOD) was described by Mccord and Fridovich. Catalase (CAT) activity was measured according to the method of Aebi. Ascobate Peroxidase (APX) activity was measured according to the methods of Nakano and Asada.

Determination of lipid peroxidation

Total amount of lipid peroxidation products present in the plant samples was estimated by the thiobarbituric acid (TBA) method which measures the malondialdehyde (MDA) reactive products according to the method of Ohkawa.

Results

Chlorophyll contents of accessions JAK, NG062 and DIN were significantly high at osmotic stress D. Accessions DIN and NG062 had the highest chlorophyll content at osmotic level D. whereas, accessions NG061 and JIW had their highest total chlorophyll contents at osmotic level C. Furthermore, accession JAK had the overall highest Chl A and B at osmotic level D (Table 1). Proline contents in osmotic stressed JAK, DIN and JIW were significantly higher than those without osmotic stress (control) except for accessions NG061 AND NG062 (Table 2). CAT activities of accessions NG061, NG062, JAK and DIN were significantly high at osmotic level A (Table 3). Accession JAK had high APX and CAT at all levels of osmotic when compared to control. Accession JIW had the lowest CAT activities. Though accession JAK had the highest SOD nevertheless, no significant different in the SOD activities was recorded in all levels of osmotic stress and Accessions. APX was significantly reduced in accessions NG061 and DIN but not significant in accession NG062. Highest APX was found in accession JAK with highest level osmotic stress E (Table 3). Percentage inhibition of OH-, H2O2, NO and DPPH radicals during an osmotic stress in accession NG061 was significantly reduced as compared with to control (Table 4). On the contrary, osmotic stressed JAK scavenged above 50% OH-, H2O2 and NO radicals significantly at different osmotic levels. It is important to state that osmotic stressed DIN and NG062 significantly scavenged OH-. Lipid peroxidation of osmotic stressed accession NG061 was not significant with the control (Table 4). Osmotic stressed accessions NG062, DIN and JIW had their lipid peroxidation significantly higher when compared to the control. Accession JAK under all osmotic levels had their lipid peroxidation significantly low compared to control (Table 5). Nitric acid NO was positively correlated to SOD. Hydrogen peroxide was positively correlated to the activities of APX and Proline. Proline was positively correlated to CAT. MDA is negatively correlated to OH, H2O2 and NO (Table 6). Accession JAK had 85% osmotic tolerant level which was higher than the other accessions followed by NG061 (65%), DIN (55%), NG061 (48%) and JIW (47%). Tolerant level of D. exilis to osmotic stress ranged from 85%~47% (Table 7).

Chlorophyll content (µg/mL) of Digitaria exilis accessions under different osmotic potentials+

ACCESSIONSCHL aCHL bTOTAL CHL
NG061A14.93e34.97cd49.90d
B32.91ab77.14b110.04b
C42.12a107.91a150.04a
D19.17d28.27e47.43d
E19.13d29.47de48.6d
F24.24c39.93c64.17c

NG062A20.30b46.06b66.36bc
B20.42b24.16d44.58d
C7.24d13.89e21.13e
D32.82a81.12a113.94a
E16.97c41.40bc58.37c
F30.12a48.35b78.47b

JAKA25.72c78.16b103.88b
B23.84c55.71bc79.54c
C25.79c41.35c67.13d
D87.54a211.07a298.6a
E16.54d17.87e34.41e
F34.20b38.39cd72.6cd

DINA18.86de47.14c65.99c
B29.21c66.8b96.01b
C37.93a78.41ab116.34ab
D33.65b87.89a121.54a
E19.77d20.04d39.8d
F33.44b65.21b98.65b

JIWA24.12ab58.29ab82.41b
B28.34ab42.55b70.89bc
C31.63a78.30a109.94a
D15.38c37.65bc53.03d
E18.13b42.85b60.99c
F34.00a67.82a101.82a

Values with the same letters in each column are not significantly different from each other at Duncan’s multiple range test of P < 0.05. ᴪS = osmotic potential, CHL = chlorophyll


Proline content of Digitaria exilis under different osmotic potentials

ACCESSIONᴪSPROLINE (mg/mL)
NG061A0.044b
B0.038c
C0.043b
D0.034d
E0.038c
F0.050a
NG062A0.016e
B0.036a
C0.018e
D0.030c
E0.020d
F0.036b
JAKA0.052a
B0.019b
C0.013c
D0.014c
E0.015c
F0.009d
DINA0.014d
B0.023ab
C0.025a
D0.015d
E0.020b
F0.017c
JIWA0.014a
B0.013a
C0.014a
D0.009d
E0.010cd
F0.010c
LSD (0.05)0.009

Values with the same letters in each column are not significantly different from each other at Duncan’s multiple range test of P < 0.05. ᴪS = osmotic potential, LSD = least significant difference


Enzyme activities of Digitaria exilis under osmotic potentials

ACCESSIONSAPX (mmol/mL/min)SOD (units/mg protein)CAT (units/mg protein)
NG061A0.010b1.373a2.155a
B0.003f1.129b0.451d
C0.007d1.324a1.937b
D0.008c1.361a0.682c
E0.005eNDND
F0.020a1.341a1.608c
NG062A0.002c1.412a3.301a
B0.004a1.406a0.657f
C0.005a1.119b1.528d
D0.003b0.054e2.005c
E0.004b0.589d1.003e
F0.004b0.765c2.649b
JAKA0.010b1.634a3.628a
B0.005e1.536b1.477d
C0.008c1.560b2.330c
D0.006d1.659a2.344c
E0.016a1.651a2.710b
F0.003f1.648a1.146e
DINA0.002e0.874b2.190a
B0.003d0.723c1.745b
C0.005c0.810b2.341a
D0.014a0.852b1.801b
E0.002e1.615a1.201c
F0.006b1.476a1.745b
JIWA0.002d1.500ab0.801d
B0.003c1.563a0.375e
C0.004b1.556a0.222f
D0.005a1.395ab1.246c
E0.005a1.392b1.948b
F0.002d0.770c2.328a

Values with the same letters in each column are not significantly different from each other at Duncan’s multiple range test of P < 0.05. ᴪS = osmotic potential, ND = not determined, APX = ascorbate peroxidase, SOD = superoxide dismutase, CAT = catalase


Percentage (%) inhibition of radicals under different osmotic potentials

ACCESSIONSOHH2O2NODPPH
NG061A52.61b35.67c17.50e21.99e
B55.02b31.58d27.13c24.81d
C53.61b45.61b21.76d21.75e
D46.59c44.44b25.37c28.63c
E40.36d50.88a33.15b40.07b
F62.27a53.85a57.64a63.59a
NG062A49.60a38.60d38.24b26.11b
B41.77b45.61c34.17c15.99d
C50.20a45.61c42.13ab18.21c
D51.00a43.86c41.20b37.11a
E52.81a50.88b39.35b16.91cd
F42.31b60.07a45.72a27.06b
JAKA63.86bND13.15e19.33d
B65.26b54.39a20.00d20.93d
C46.18d59.65ab12.04f78.29a
D53.61c19.30d50.65a49.13c
E80.22a36.84c29.54c56.07b
F62.25b51.28b43.78b10.17e
DINA59.84a45.61b16.57cd16.91d
B62.45a28.65e13.06e26.89b
C60.04a36.84c17.59c30.28a
D56.63a32 .16d15.83d31.15a
E60.24a36.84c26.39b18.31d
F41.76b62.27a31.81a24.15c
JIWA36.55e31.58c41.02b23.69c
B43.98d50.88a38.61bc14.34d
C56.83b41.52b31.02d29.99a
D46.59c40.35b31.20d26.55b
E40.36d53.22a49.72a31.44a
F62.27a52.38a36.87c26.58b

Values with the same letters in each column are not significantly different from each other at Duncan’s multiple range test of P < 0.05. ᴪS = osmotic potential, ND = not determined, DPPH = 2, 2-diphenyl-1-picrylhydrazyl hydrate


Lipid peroxidation in Digitaria exilis under different osmotic potentials

ACCESSIONᴪSMDA (Molarity M)
NG061A7.84E-07b
B8.99E-07a
C8.39E-07ab
D8.65E-07c
E8.74E-07a
F8.42E-07ab
NG062A5.96E-07b
B5.96E-07b
C5.47E-07c
D6.59E-07a
E6.54E-07a
F4.74E-07d
JAKA5.07E-07c
B4.58E-07e
C3.79E-07d
D6.18E-07b
E5.86E-07b
F7.80E-07a
DINA1.58E-06a
B1.25E-06b
C7.76E-07cd
D7.52E-07d
E8.50E-07c
F6.66E-07e
JIWA1.41E-06c
B9.04E-07d
C2.03E-06a
D1.81E-06b
E6.94E-07e
F5.56E-07f
LSD (0.05)3.98 × 10-7

Values with the same letters in each column are not significantly different from each other at Duncan’s multiple range test of P < 0.05. ᴪS = osmotic potential, LSD = least significant difference, MDA = Malondialdehyde


Correlation among the different assays

APXCATSODPROLINEMDANOOH-H2O2
APX0.150.160.49-0.24-0.050.250.02
CAT0.15-0.070.04-0.37-0.130.27-0.03
SOD0.16-0.07-0.120.040.01-0.04-0.02
PROLINE0.480.04-0.12-0.26-0.06-0.030.05
MDA-0.24-0.380.04-0.25-0.09-0.04-0.29
NO-0.05-0.130.01-0.06-0.09-0.310.18
OH-0.250.27-0.04-0.03-0.04-0.31-0.24
H2O20.02-0.00-0.020.05-0.280.18-0.24

Percentage osmotic tolerant scoring

ACCESSIONCHL aCHL bTotal CHLPROLAPXSODCATMDAOH-H2O2NODPPH% DRGHT TOL
NG06133354324332465
NG06211141143255148
JAK55535455513585
DID44423232421255
JIW22212511144347

PROL = Proline, APX = ascorbate peroxidase, SOD = superoxide dismutase, CAT = catalase, MDA = 2, 2-diphenyl-1-picrylhydrazyl hydrate, DPPH = Malondialdehyde, % DRGHT TOL = Percentage drought tolerant


Discussion

Accession JAK had a significant high level of proline during osmotic stress. It appeared that accumulation of proline protected plants against oxidative stress through stabilization of antioxidant enzymes. High levels of proline enabled the plant to maintain low water potentials. Due to low water potentials, accumulated compatible solutes osmo-regulated the effect of the stress by allowing additional water to be taken up from the environment thus, buffering the immediate effect of water shortages within the organism. With the accumulation of solutes in JAK, the osmotic potential of the cell may have been lowered, which attracts water into the cell hence, provide and support turgor maintenance of the plant tissues. Osmotic adjustment helps to maintain the cell water balance with the active accumulation of solutes in the cytoplasm, thereby minimized the harmful effects of drought stress. The maintenance of turgor despite a decrease in leaf water may have permitted photosynthesis to go on unabated hence; high plant growth was recorded in osmotic-stressed JAK than their unstressed counterparts. Osmotic adjustment is an important trait in delaying dehydration damage in water-limited environments by continued maintenance of cell turgor and physiological processes. The activity of SOD, CAT and APX varies with the level of drought/ osmotic stress. Enzyme SOD was higher in osmotic stressed JAK which played a major role in quenching reactive oxygen. It works as a catalyzer which dismutated singlet O2 − into H2O2 that are later eliminated by CAT and other antioxidant enzymes. Enzyme APX and CAT was high in osmotic stressed JAK than control. Accession JAK had good and consistent high value of APX when subjected to severe osmotic stress. Consequently, the singlet oxygen dismutated by SOD to hydrogen peroxide (H2O2) was later converted to water (H2O) and oxygen (O2) by CAT in accession JAK which actually made it drought tolerant.

Osmotic stressed accession JAK could scavenge above 50% of OH-, H2O2, NO radicals. This could be as a result of positive correlation between APX and proline. Also, high SOD and CAT found in this accession must have directly caused the inhibition. Ascorbate peroxidase (APX), CAT and SOD were practically stabilized by osmo-regulator proline by removal of superoxide ions which was converted to OH- and later to H2O2. Consequently, low lipid peroxidation was observed in osmotic stressed JAK. Lipid peroxidation, in both cellular and organelle membranes, takes place when above-threshold ROS levels are reached, thereby not only directly affecting normal cellular functioning, but also aggravating the oxidative stress through production of lipid-derived radicals. Osmotic stressed DIN, JIW and NG062 had a high lipid peroxidation. It has also been reported that water stress increased the lipid peroxidation, membrane injury index, H2O2 and OH- production in leaves of stressed Phaleolus vulgaris plants.

Also, the positive correlation between APX and proline could have activated the activity of APX. The high accumulations of proline in JAK under osmotic stress could be responsible for high activities of antioxidant enzymes. These results suggested that accession JAK had higher capacity for osmotic adjustment in terms of accumulating proline, which could maintain water absorption under such harsh conditions. Proline stabilized the activities of CAT thus, low lipid peroxidation with high scavenging activities in accessions JAK were recorded. Osmotic tolerant scoring therefore explained that accession JAK is an osmotic tolerant accession, NG061 and DIN are might mild osmotic tolerant accessions and NG062 and JIW are susceptible to osmotic stress.

Conclusion

Osmotic tolerant ability of accession JAK was due to the accumulation of proline which helps to stabilize activities of enzymes CAT and APX consequently, approximately 50% of hydroxyl, hydrogen peroxide and nitric oxide radicals were successfully scavenged during osmotic stress. Hence, lipid peroxidation was drastically reduced during the stress.

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