Waterlogging effects on some antioxidant enzymes activities and yield of three wheat promising lines

Waterlogging is one of the most important environmental stresses that have negative effects on wheat growth and yield. The purpose of this study was to investigate the effect of waterlogging (0, 7, 14 and 21 d) at tillering (ZG21) and stem elongation (ZG31) stages on the content of photosynthetic pigments, proline, malondialdehyde (MDA), antioxidant enzymes, grain yield and yield components of three wheat promising lines (N-93-19, N-93-9 and N-92-9). Increasing waterlogging stress reduce the photosynthetic pigments contents and the activity of catalase enzyme while increase the proline content, MDA, superoxide dismutase and peroxidase enzymes in three wheat genotypes in both tillering and stem elongation stages. Waterlogging also reduced yield and yield components in three wheat genotypes. The results showed that N-92-9 genotype had better response than other two genotypes in all studied traits under waterlogging conditions.


INTRODUCTION
Waterlogging, which is due to poor drainage of the soil under inappropriate irrigation practices or heavy rainfall, is one of the abiotic stresses and considered as one of the main limitation factors for the growth and yield of crops.Therefore, excess water around the plant's root causes a decrease in oxygen concentration and the plant encounters oxygen deficiency, which is known as hypoxia (Dennis et al., 2000).Plants need to exchange atmospheric gases to maintain their natural growth and photosynthesis and respiration processes (Suzuki and Mittler, 2006).Therefore, under flooding conditions, excess water around the root reduces gas diffusion, which directly affects the absorption of nutrients, plant growth and yield (Fukao and Bailey-Serres, 2004).
The tolerance to flooding conditions varies in crops that depend on biochemical and anatomical adaptations (Liu et al., 2005).The closure of the stomata to prevent water loss along with the adjustment of photosynthetic apparatus is one of the main responses of the plant under waterlogging stress (Arbona et al., 2008).In addition, under environmental stresses, antioxidant defense systems play an important role in plant tolerance to stress.Reactive oxygen species (ROS) damage the living tissues through the oxidation of biological macromolecules such as lipids, proteins, and nucleic acid (Mittler et al., 2004).By converting ROS to harmless compounds by enzymatic and non-enzymatic antioxidants, the toxic effect of ROS is reduced.The first cellular defense line is superoxide dismutase (SOD) followed by catalase (CAT) and peroxidase (POD) (Edreva, 2005).In various studies, increased activity of antioxidant enzymes has been reported under various environmental stresses (Kumutha et al., 2009;Amador et al., 2012;Gerami et al., 2018).Van Toai and Bolles (1991) showed that high SOD activity with superoxide detoxification could help tolerate the plant under flood stress.
Wheat (Triticum aestivum L.) is one of the most important crops that is widely cultivated under different climatic conditions.Wheat is grown in areas with different moisture conditions where rainfall ranges from 250 to 1800 millimeters.However, the most areas of wheat cultivation receive an average annual rainfall of 380 and 880 millimeters (Herzog et al., 2016).Wheat cultivation requires adequate moisture during the growing season; however, excessive irrigation or precipitation causes waterlogging.According to estimates, more than 10 to 15 million ha of wheat fields are under waterlogging threat (Sayre et al., 1994).Olgun et al. (2008) reported that wheat yield reduced under waterlogging stress.In other study, Collaku and Harrison (2002) examined the effect of different levels of waterlogging (0, 10, 20 and 30 d) on nine wheat cultivars, indicating that waterlogging stress reduced the yield of nine wheat cultivars from 35 to 60 %.
The purpose of this study was to evaluate the effects of waterlogging on biochemical traits such as content of chlorophyll a and b, proline and antioxidant enzymes activity of three wheat-promising lines in two tillering and stem elongation stages.In addition, at physiological maturity, grain yield, number of seeds per spike, grain mass per spike and number of spikes per square meter were investigated.

MATERIALS AND METHODS
Seeds (20 seeds per pot) of three wheat-promising lines (N-93-19, N-93-9 and N-92-9) were sown in plastic pots (19 cm depth, 23 cm diameter).The pots were filled with 4 kg of sterilized field soil amended with 2.4 g K 2 O, 3.2 g P 2 O 5 and 4 g (NH 4 ) 2 SO 4 per pot (240 pots).After germination, plants were thinned to 10 plants per pot.Waterlogging treatments included 0, 7, 14 and 21 days in tillering (ZG21) and stem elongation (ZG31) stages.In the stem elongation stage, plants of all three genotypes after 21 days of waterlogging were completely died, which were not sampled.Waterlogging stress was applied by placing the pots in larger pots (30 cm depth, 30 cm diameter) filled with water to 2 cm above the surface of the soil.The control plants were watered as much as needed to avoid waterlogging stress and drought stress.At the end of the waterlogging treatments, half of the pots (5 pots) were harvested to measure the biochemical traits, while the remaining pots were grown until physiological maturity.Then seeds/spike, seeds mass/spike, spikes/m 2 and grain yield were measured according to Ceylan (1994).
To measure chlorophyll (a and b) and carotenoids, extracts of fresh leaves with 80 % acetone according to method of Lichtenthaler and Wellburm (1983) were used.For proline measurement, fresh leaves were extracted with methanol (40 %).Then 1 ml of methanolic extract was mixed with 25 ml of ninhydrin and 1 ml of the mixture of orthophosphoric acid (6M) and glacial acetic acid (2.3 v/v).The tubes were incubated at 100 °C and after cooling, toluene (5 ml) was added.By reading the absorbance of supernatant at 528 nm using spectrophotometer (Varian, Carry 300, California, USA), free proline content was calculated according to Bates et al. (1973) method.In order to measure lipid peroxidation, malondialdehyde (MDA) content was determined using thiobarbituric acid method according to Heath and Packer (1968) and an extinction coefficient of 155 mM -1 cm -1 .
The superoxide dismutase (SOD) activity was measured using photo-reduction of NBT (nitrobluetetrazolium).The amount of protein used to inhibit 50 % of the NBT photo-reduction at 560 nm was considered as one unit of activity of SOD enzyme.The protocol of Luck (1971) has been applied for determination of catalase (CAT) activity by using the rate of decrease in absorbance at 240 nm for two minutes.Meahly and Chance (1954) method was used to measure the peroxidase (POD) activity based on the oxidation of guaiacol in the presence of hydrogen peroxide (H 2 O 2 ).All spectrophotometric experiments were performed using a Varian, Carry 300, California, USA spectrophotometer.Two-way ANOVA has been used for the analysis of the results by with SAS 9.1.3software and means were compared with the LSD test (P < 0.05).

Photosynthetic pigments
In tillering and stem elongation stages, waterlogging treatments reduced the content of chlorophyll a and b in all three wheat genotypes.The highest reduction of chlorophylls was observed during 14 and 21 d waterlogging in tillering and stem elongation stages, respectively.In all treatments, N-93-19 and N-92-9 genotypes had the highest and lowest reductions, respectively, as compared to the corresponding control (Fig 1 and 2

DISCUSSION
High chlorophyll content under waterlogging conditions can be the most appropriate way for achieving high yield under waterlogging conditions (Gardner et al., 1993).Our results indicated that waterlogging treatments in tillering and stem elongation stages significantly reduced photosynthetic pigments (chl a, b and carotenoids) in all three wheat genotypes, however, N-92-9 genotype had higher photosynthetic pigments than other genotypes under all waterlogging duration.Pang et al. (2004) stated that waterlogging stress reduced the content of chlorophyll and consequently reduced the rate of CO 2 assimilation.In another report, Smethurst and Shabala, (2003) indicated that the content of chlorophyll a and b reduced in genotypes under waterlogging treatments.Screening of the effect of waterlogging duration on chlorophyll and carotenoids content presented that by increasing the duration of waterlogging, the content of chlorophyll and carotenoids more decreased, which is according to the results of Olgun et al. (2008).
The maintaining of water balance of the plant under environmental stresses is very important for plant tolerance, which proline is one of the most compatible solutes (Claussen, 2005;Ghorbani et al., 2018).Our results showed that proline contents under waterlogging stress increased in tillering and stem elongation stages.The increase of proline contents in N-92-9 genotype was significantly higher than the other two varieties under same waterlogging duration.Increasing of waterlogging duration significantly increased the contents of proline, which is in accordance to results of Olgun et al. (2008).Increasing proline contents in plants under waterlogging treatments can be due to oxidation inhibition, loss of inhibition of feedback and protein synthesis impairment.Videmšek et al. (2006) reported that wheat genotypes have different ability to synthesize and accumulate proline, which affects their tolerance to waterlogging.
The stomatal closure and the reduction of CO 2 availability resulting from waterlogging conditions increase the production of reactive oxygen species (ROS) and thus induce oxidative stress (Gossett et al., 1994).Our results showed that waterlogging stress increased MDA production, which indicating the degree of damage to membrane lipids.However, there was a significant difference between wheat genotypes, which indicates their different defensive capacity against waterlogging-induced ROS.Increasing the activity of antioxidant enzymes such as SOD, CAT and POD can play an important role in plant tolerance to waterlogging stress.The results of this study showed a continuous increase in the activity of SOD and a decrease in the activity of POD in all three wheat genotypes under waterlogging stress.However, CAT activity under waterlogging treatments in N-92-9 genotype showed a continuous increase while there was decline in CAT activity in two genotypes of N-93-19 and N-93-9.Different researchers have suggested that the activity of antioxidant enzymes under waterlogging conditions increases to take care of hypoxia-induced oxidative stress (Arbona et al., 2008;Kumutha et al., 2009).Our results are in accordance with the results of Blokhina et al. ( 2001) who found more oxidative stress under waterlogging and increased antioxidant enzymes could reduce ROS production.It is clear from the results that wheat genotypes suffered from ROS under waterlogging stress that intensified with increasing the waterlogging duration.Therefore, the rapid increase in the activity of antioxidant enzymes in plants under waterlogging stress can protect the plant against the oxidative stress caused by waterlogging.Many researchers have been previously reported increased activity of CAT (Ushimaru et al., 1997), POD (Meloni et al., 2003) and SOD (VanToai andBolles, 1991, Biemelt et al., 2000) enzymes under waterlogging conditions.Kernels mass/spike is one of the most important components of wheat yield.Our results showed that waterlogging treatments reduced the kernels/spike and kernels mass/spike traits of all three studied wheat genotypes in both tillering and stem elongation stages, however, the lowest decrease was observed in N-92-9 genotype.These findings are consistent with the results of Collaku and Harrison (2002) who stated that increasing the duration of waterlogging reduce the kernels/spike.They also showed that tolerant genotypes had higher kernels/spike compared to other genotypes.In another report, Saqib et al. (2004) showed that the kernels mass/spike in wheat genotypes under waterlogging stress was reduced by 50-80 % as compared to control treatments.The results related to yield and spikes/m 2 showed that these traits significantly decreased with increasing waterlogging duration in all three genotypes and the highest decrease was observed in N-93-19 genotype.Reducing the yield of wheat genotypes under waterlogging according to Barret-Lennard (2003) and Setter et al. (2001) who showed that increasing waterlogging duration reduced wheat yield from 10 to 80 %.Collaku and Harrison (2002) also reported that waterlogging stress reduced the spikes/m 2 in winter wheat.

CONCLUSION
In general, the results showed that waterlogging stress caused oxidative stress in wheat genotypes that enhanced with duration of exposure.N-92-9 genotype with higher activity of antioxidant enzymes (CAT, SOD and POD) which resulted in higher grain yield and yield components under waterlogging conditions was more tolerant to waterlogging stress.