Introduction
Cold stress or low temperature stress is major
environmental factor affecting plant growth and yield and resulting
considerable loss to crops (Farooq et al. 2009; Sanghera et al. 2011; Ahmad et al. 2014). Low temperature has a lethal effect on germination that
results in reduced photosynthetic rate along with reduced mass of above ground
organs. Cold stress (low temperature) causes numerous alterations in
biochemical and physiological processes of plants (Zhao et al. 2009; Farooq et al. 2017). Generally, photosynthesis and many other physiological processes are sensitive
to chilling stress, which result in the decline of growth and yield in plants.
By understanding the responses of plants towards stress, crops can be made
stress tolerant (Zhao et al. 2009). Among crops, wheat (Triticum aestivum L.) is an important food crop for more than one
third of the world population and its yield is being influenced because of global climate
change and low temperature stress in the environment (Farooq et al. 2008; Chaves and Oliveira 2004). Wheat often experiences cold stressed conditions
during its life cycle so it is necessary to understand the natural genetic
variation in characters related to stress tolerance (Loggini et al. 1999).
Oxidative
stress is induced in the cell because of higher electron leakage towards O2
during respiratory and photosynthetic processes leading to augmentation
in generation of reactive oxygen species (ROS) which can damage to membrane
proteins, DNA and lipids leading to cell death (Mittler
2002; Simova-Stoilova et al. 2008). During optimal conditions,
balance is tightly controlled between ROS formation and its consumption by
antioxidant defense system of the plant. Catalase (CAT), Superoxide dismutase
(SOD) and peroxidase (POD) are key antioxidants that play a crucial role in
plant defense against ROS (Noctor and Foyer
1998; Simova-Stoilova et al. 2008).
Many studies have revealed that
increasing antioxidant defense resulted in stress tolerance to temperature
extremes (Almeselmani et al. 2006).
Zhao et al. (2009) reported that
chilling tolerance in tomato varieties could be designated by higher activities
of SOD, CAT, POX and APX enzymes. Studies suggest that nitric oxide (NO) has the potential
to induce tolerance in plants against different environmental stresses. These
investigations suggest that NO has antioxidant properties and may act as a
signal to activate ROS scavenging enzymes under abiotic stress. It plays an
imperative role in resistance to heavy metal, UV-B, drought, salt as well as to
low and high temperature stress (Nabi et al. 2019). It has been accepted that NO
plays a crucial role in the varied physiological functions of plants (Libourel et al.
2006; Zheng et al. 2009).
Differential antioxidant defense reactions of susceptible versus resistant
wheat genotypes to stress-induced oxidative stress at a specific growth stage
and in controlled conditions have been reported (Loggini et al. 1999; Lascano et al. 2001).
The
effect of stress on plant species depends on variety, duration and intensity of
the stress in addition to developmental stage (Simova-Stoilova et
al. 2008). There is probability that genotypes could respond
variously under varied growth conditions and priming at similar growth stage.
So, true chilling acclimation potential of wheat genotypes could differ under
changed growth conditions and priming (Afzal et al. 2008). Any genotype can be more competent and specific due to better
adoptive changes in metabolic and anti-oxidative processes due to hormonal (Sgherri et al.
2000) biochemical grounds. Therefore, present study was carried out to
provide insights in the relationship of SNP priming and chilling stress
tolerance of seven wheat genotypes by affecting changes in antioxidant enzymes
and subsequent growth of the seedlings.
Materials and Methods
Wheat seeds of seven genotypes, viz. NARC-2011, AAS-2011,
Punjab-2011, Faisalabad-2008, Uqab-2002, Chakwal-50 and Lasani were taken from
the Agriculture Department Muzaffarabad, Azad Jammu & Kashmir, Pakistan.
Seeds were soaked in aerated solution of 10-4 and
10-5 molar sodium nitroprusside (SNP) in 100 mL glass beakers for 6
h at 25°C. After SNP priming, seeds were washed with distilled water (Bradford 1986) then dried back on dry filter
papers. Three replicates of seeds (90) of each variety for each treatment were
placed in Petri dishes on blotting paper at 25°C as a control and 04°C as a
stress for 15 days. About 5 mL of distilled water was used to moisturize each
Petri dish. Germination was counted on daily basis. After termination of
germination, seedlings were measured for following parameters and then
harvested for biochemical analysis.
Germination
speed was measured by using equation
of Rajabi and Poustini (2005). Germination percentage was determined by using
the procedure and formula of AOSA (1983). Length of the shoot was taken from
five randomly selected seedlings. Number of leaves was determined by counting
the number of leaves from five randomly selected seedlings in each pot. Number
of roots was determined by counting the number of leaves from all the studied
samples by randomly selected five seedlings of each pot.
Antioxidant enzymes activity
Supernatant was
used to estimate the activity of SOD by recording the decrease in absorbance of
superoxide-nitro blue tetrazolium complex by the enzyme. About 3 mL of reaction
mixture was taken in test tubes in duplicate from each enzyme sample. Two tubes
without enzyme extract were taken as control. The reaction was started by
adding 0.1 mL riboflavin (60 µM) and
placing the tubes below a light source of two 15 W florescent lamps for 15 min.
Reaction was stopped by switching off the light and covering the tubes with
black cloth. Absorbance was recorded at 560 nm and one unit of enzyme activity
was taken as the quantity of enzyme which reduced the absorbance reading of the
samples to 50 percent in comparison with tubes lacking enzymes.
The activity
of indole acetic acid oxidase was determined according to the method of Omran
(1980); Talwar (et al. 1985) with
some modifications. The enzyme extract was incubated for 60 min with 0.2 M sodium phosphate citrate buffer having
pH 5.6, 200 µg mL-1 of IAA
solution in 0.5 mM MnCl2
and 0.1 mM of 2,4 dichlorophenol.
Salkowski’s reagent was used to stop the incubation process in addition of its
reaction with unoxidized IAA. The absorbance of the sample was measured
spectrophotometrically at 540 nm.
The activity of Ascorbate peroxidase
was determined according to Bartoli et al. (1999) from the extract prepared for PPO activity. The activity
of enzyme was analyzed by following the decrease in the absorbance (265 nm) of
the reaction mixture having 50 µM
ascorbate, 50 µM of potassium
phosphate buffer, pH 6.5 and 90 µM of
hydrogen peroxide.
Guaiacol peroxidase activity was
determined following the method of Plewa et
al. (1991) from the extract. Reaction mixture contained 2.77 mL of 50 mM Phosphate buffer (pH 7), 25 µL of enzyme extract, 0.1 mL of 1% H2O2
and 0.1 mL of 4% guaiacol. Increase in absorbance due to guaiacol
oxidation was recorded at 470 nm.
Statistical analysis of data
Significance of the
data was tested by analysis of variance and Duncan’s Multiple Range Test at P < 0.05 and where applicable at P < 0.01 and P < 0.03 using MSTAT
software. Standard
error was performed to determine
random error in the data
Results
Interaction of
wheat varieties with SNP concentrations showed that same variety after various
seed priming responded differently as varied germination speed was obtained
after different SNP treatments (Fig. 1). Maximum germination speed was recorded
in AAS-2011, NARC-2011 and Lasani seeds primed with 10-4 M SNP in normal growth condition.
Minimum germination speed was recorded from 10-4 M SNP primed Punjab-2011 and
Faisalabad-2008 under chilling stress. All SNP priming treatments improved
%20919-926_files/image002.png)
Fig. 1: Germination
speed of wheat varieties as affected by SNP priming under normal and chilling
conditions
%20919-926_files/image004.png)
Fig. 2: Final
germination of wheat varieties as affected by SNP priming under normal and
chilling conditions
final germination in all varieties
except Faisalabad-2008 in controlled conditions while 10-4 M SNP priming showed maximum final
germination in most of cultivars under chilling stress (Fig. 2).
Comparison of wheat varieties with
varied SNP priming and growth conditions showed very highly significant
variations in shoot length (Fig. 3). Maximum shoot length was recorded from 10-5
M SNP primed Uqab-2002 and
NARC-2011 in normal conditions and the minimum from Chakwal-50 with 0 M SNP treatment in chilled conditions.
Leaf numbers were determined from all the studied samples and non-significant
variation was observed by wheat varieties, SNP priming and growth conditions
(Fig. 4). Wheat varieties under different growth conditions and SNP
concentrations revealed very highly significant variations for root numbers
(Fig. 5). Unprimed Lasani gave more number of roots as compared to SNP priming
treatments under chilling stress. However, SNP priming improved root number in
AAS-2011 during low temperature conditions.
The activity of superoxide dismutase (SOD) significantly
varied among wheat varieties under different growth conditions and SNP priming
(Fig. 6). SNP priming significantly improved SOD activity in Punjab-2011 under
controlled conditions whereas maximum SOD activity was recorded in seedlings
raised from unprimed seeds of NARC and Lasani under chilling stress. Regarding
indole acetic acid, maximum activity was recorded by Faisalabad-2008 with 10-5
M SNP priming in chilled growth condition
(Fig. 7). Ascorbate peroxidase activity was not significantly improved in all
varieties under both normal and chilling stress conditions (Fig. 8). The
response of SNP priming and wheat varieties for Guaiacol peroxidase was found
quite variable, however, activity was more pronounced in unprimed seeds of
NARC, AAS-2011 and Uqab-2002 (Fig. 9).
%20919-926_files/image006.png)
Fig. 3: Variations
in shoot length of wheat varieties as affected by SNP priming and chilling
%20919-926_files/image008.png)
Fig. 4: Variations
in leaf number of wheat varieties as affected by SNP priming and chilling
Discussion
Present study revealed positive effect
of NO priming and negative effect of chilling on germination traits of wheat
varieties (Fig. 1–2). Negative effect of chilling is due to fact that chilling
caused reduction in germination by detaining metabolic reactions and reducing
water potential in germinating seeds (Bibi et
al. 2017). Chilling stress
restrains different metabolic reactions by preventing the expression of total
genetic potential of the plants (Chinnusamy et
al. 2007). Data from literature provide evidence in favor of our
findings that nitric oxide regulates the response of plants towards stress
(Bibi et al. 2017). Ansari et
al. (2012) and Sharafizad et
al. (2012) reported negative effect
of stress and positive effect of priming on germination traits because priming
has the ability to induce some metabolic changes in seed that ultimately
enhance its germination speed and percentage as well. During priming,
uniform and rapid stimulation of some physiological changes occur in seeds that
boost their speed of germination (Bradford 1986). Nitric
Oxide has been implicated in promotion of seed germination in many species
either by reducing seed dormancy, or by reducing the effects of adverse
environmental conditions (Krasuska et al. 2017).
SNP has been found to promote seed germination by inducing the activity of
β-amylase during early stages of seed germination in many plant species
such as Mouse-air cress, wheat and alfalfa (Duan et al. 2007;
Maurice et al. 2016).
%20919-926_files/image010.png)
Fig. 5: Variations
in root number of wheat varieties as affected by SNP priming and chilling
%20919-926_files/image012.png)
Fig. 6: Variations
in superoxide dismutase of wheat varieties as affected by SNP priming and
chilling
Different varieties have different
genetic potential of growth and response; hence show variations in their
morphology (Aghamolki et al. 2014). Chilling stress prevents expression of
total genetic potential in plants leading to negative effects on plants’ growth
and morphology due to blockage of different metabolic reactions. The increase in
plant growth i.e. shoot length, root length is linked with the SNP-mediated
mitigation of chilling-induced over-production of reactive oxygen species
possibly by up-regulating the antioxidative defense mechanisms. Exogenous SNP
as seed priming is effective in maintaining the plant water relations by
up-regulating the synthesis of Proline (Chinnusamy et al. 2007). Our results are in accordance with
other researchers (Aghamolki et al.
2014; Lianopoulou and Bosabalidis 2014) who
reported negative change in morphology and differences among varieties due to
temperature stress. Bibi et al.
(2018) reported positive effect of NO priming on morphological attributes in
wheat varieties and adverse effects of chilling stress. Although they found non-significant
variations in total number of leaves despite varied conditions.
Ali et al. (2019) reported that
priming exposure time also affected antioxidative defense mechanism variously
in various varieties. If priming duration couldn’t stimulate antioxidant enzyme activities in any plant
species, then it stimulates other defense mechanisms like, proline, soluble
sugars etc. for defense purpose. Nejadalimoradi et al. (2014) reported that SNP application could raise the
activity of antioxidant enzymes in SNP treated plants compared to untreated
one. In present study, many variations in antioxidant enzyme activities were
observed from different wheat varieties under same treatment. This might be due
to their varied tolerance to stress due to varied genetic makeup. Sairam et
al. (2011) observed inconsistent antioxidant defense response in genotypes
and could not notice any pattern vis-ŕ-vis
tolerant and susceptible genotypes. Shi et
al. (2007) reported that NO enhanced the activity of CAT, APX and SOD in
cucumber roots, and apoplastic H2O2 in NO-induced
antioxidant defence. Increased SOD activity could enhance the ability of
tissues to eliminate H2O2, explaining the lower level of
H2O2 that was observed in NO-treated fruit. Treatment
with nitric oxide significantly increased the activities of CAT, POD, SOD and
APX under chilling stress (Ghorbani et al. 2018).
%20919-926_files/image014.png)
Fig. 7: Variations
in indole acetic acid oxidase of wheat varieties as affected by SNP priming and
chilling
%20919-926_files/image016.png)
Fig. 8: Variations
in ascorbate peroxidase of wheat varieties as affected by SNP priming and
chilling
Conclusion
Genotypic differences in chilling
stress tolerance were chiefly accredited to the capacity of wheat varieties to
activate antioxidant defense. Capability of varieties to persuade the
antioxidant response differs by different priming and growth conditions. Wheat
varieties with better chilling tolerance than others sustained higher
antioxidant enzyme activities that result in decreased oxidative damage and
increased growth. However, SNP priming affected its antioxidant enzyme
production via changing its metabolic
reactions. Consequently, resistance against chilling induced oxidative stress
was primarily dependent on the genetic potential (superior antioxidant defense
system) of wheat varieties. Germination speed, germination percentage, seedling
growth and antioxidant status might be used as indices of chilling tolerance in
wheat.
%20919-926_files/image018.png)
Fig. 9:
Variations in guaiacol peroxidase of wheat
varieties as affected by SNP priming and chilling
References
Afzal I, SMA Basra, M Shahid, M
Saleem (2008). Priming enhances germination of spring maize (Zea mays L.) under cool conditions. Seed
Sci Technol 36:497‒503
Ahmad I,
SMA Basra, A Wahid (2014). Exogenous application of ascorbic acid, salicylic
acid and hydrogen peroxide improves the productivity of hybrid maize under at
low temperature stress. Intl J Agric Biol
16:825‒830
Ali M, S Hayat, H Ahmad, MI
Ghani, B Amin, MJ Atif, Z Cheng (2019). Priming of Solanum melongena L. seeds enhances germination, alters antioxidant
enzymes, modulates ROS, and improves early seedling growth; Indicating aqueous
garlic extract as seed-priming bio-stimulant for eggplant production. Appl
Sci 9:2203‒2220
Ansari O, H Chogazardi, F
Sharifzadeh, H Nazarli (2012). Seed reserve utilization and seedling growth of
treated seeds of mountain rye (Secale montanum) as affected by drought stress. Cer Agron
Moldova 45:43–48
AOSA (1983). Seed Vigor Testing Handbook. In
Contribution No. 32 to the Handbook on Seed Testing, Bassersdorf, Switzerland
Bartoli CG, M Simontacchi, E
Tambussi, J Beltrano, E Montaldi, S Puntarulo (1999). Drought and
watering-dependent oxidative stress: effect on antioxidant content in Triticum aestivum L. leaves. J Exp Bot 50:375–383
Bibi A, SA Majid, A Munir, A
Ulfat, G Javed, S Khatoon, N Azhar, S Ashraf, S Aziz, N Mumtaz (2018). Chilling effects after priming by nitric oxide
applications on amelioration of leaf growth and photosynthetic pigments.
Phyton 87:178‒182
Bibi A, SA Majid,
A Ulfat, S Khatoon, A Munir, G Javed (2017). Effect of nitric oxide seed priming on chilling induced water related
physiological attributes in germinating wheat. J Anim Plant Sci 27:186‒191
Chinnusamy V, J Zhu, JK Zhu (2007). Cold stress
regulation of gene expression in plants. Trends Plant Sci 12:444‒451
Duan P, F Ding,
F Wang, BS Wang (2007). Priming of seeds with nitric oxide donor sodium
nitroprusside (SNP) alleviates the inhibition on wheat seed germination by salt
stress. J Plant Physiol Mol Biol
33:244‒250
Farooq M, M
Hussain, A Nawaz, D-J Lee, SS Alghamdi, KHM Siddique (2017) Seed priming
improves chilling tolerance in chickpea by modulating germination metabolism,
trehalose accumulation and carbon assimilation. Plant Physiol Biochem
111:274–283
Farooq M, T
Aziz, A Wahid, D-J Lee, KHM Siddique (2009) Chilling tolerance in maize:
agronomic and physiological applications. Crop Pasture Sci 60:501–516
Farooq M, SMA
Basra, H Rehman, BA Saleem (2008) Seed priming enhances the performance of late
sown wheat (Triticum aestivum L.) by improving the chilling tolerance.
J Agron Crop Sci 194:55–60
Ghorbani B, Z Pakkish,
M Khezri (2018). Nitric oxide increases antioxidant enzyme activity and reduces
chilling injury in orange fruit during storage. NZ J Crop Hortic Sci 46:101‒116
Krasuska U, K
Ciacka, A Gniazdowska (2017). Nitric oxide-polyamines cross-talk during
dormancy release and germination of apple embryos. Nitric Oxide 68:38‒50
Loggini B, A
Scartazza, E Brugnoli, F Navari-Izzo (1999). Antioxidative defense system, pigment composition,
and photosynthetic efficiency in two wheat cultivars subjected to drought. Plant Physiol 119:1091‒1100
Maurice N, CY
Ping, Q Miaomiao, U Constantine, Y Bo, KY Qi (2016). Effects of exogenous
nitric oxide on germination and carbohydrates mobilization in alfalfa seedlings
under cadmium stress. Intl J Environ Sci Technol 4:2337‒2350
Mittler R (2002).
Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci 7:405‒410
Nabi RBS, R Tayade,
A Hussain, KP Kulkarni, QM Imran, BG Mun, BW Yun (2019). Nitric oxide regulates
plant responses to drought, salinity, and heavy metal stress. Environ Exp Bot
161:120‒133
Nejadalimoradi HA, FA Nasibi, KM
Kalantari, RO Zanganeh (2014). Effect of seed priming with L-arginine and
sodium nitroprusside on some physiological parameters and antioxidant enzymes
of sunflower plants exposed to salt stress. Agric
Commun 2:23‒30
Omran, RG(1980). Peroxide levels
and activity of catalase, peroxidase and IAA oxidase during and after chilling
cucumber seedlings. Plant Physiol 65:
407–408
Plewa MJ (1991) The Mechanisms and Effects of the Plant Activation
of Chemicals in the Environment. University of Urbana, Chamapign, Illinois,
USA
Rajabi R, K
Poustini (2005) Effects of NaCl salinity on seed germination of 30 wheat (Triticum aestivum L.) cultivars. Sci J Agric 28: 29‒44
Sairam RK, K Dharmar, S Lekshmy,
V Chinnusamy (2011). Expression of antioxidant defense genes in mung bean (Vigna radiata L.) roots under
water-logging is associated with hypoxia tolerance. Acta Physiol Plantarum 33:735‒744
Sharafizad M, A Naderi, SA
Siadat, T Sakinejad, S Lak (2012). Effect of salicylic acid pretreatment on
yield, its components and remobilization of stored material of wheat under
drought stress. J Agric Sci 4:115–126
Simova-Stoilova L, K Demirevska, T Petrova, N Tsenov, U Feller (2008).
Antioxidative protection in wheat varieties under severe recoverable drought at
seedling stage. Plant Soil Environ 54:529‒536
Talwar G, JPS
Dendsay,VK Gupta (1985). Kinetic properties of IAA oxidase from mung bean
cotyledons. Phytochemistry 24:673–676
Zhao MG, L Chen, LL Zhang, WH Zhang (2009). Nitric reductase-dependent
nitric oxide production is involved in cold acclimation and freezing tolerance
in Arabidopsis. Plant Physiol
151:755‒767