LIU Yang,LI Yu-xiang,LI Yi-xiangTIAN Zhong-weiHU Jin-lingSteve ADKINS,DAI Ting-bo

1 Key Laboratory of Crop Physiology and Ecology in Southern China/College of Agriculture,Nanjing Agricultural University,Nanjing 210095,P.R.China

2 College of Agriculture,Shihezi University,Shihezi 823003,P.R.China

3 School of Agriculture and Food Sciences,the University of Queensland,Gatton QLD 4343,Australia

Abstract To elucidate the response of oxidative metabolism,triggered by elevated ammonium (NH4+) concentrations,on root growth of wheat seedlings,Yumai 49 (NH4+-tolerant) and Lumai 15 (NH4+-sensitive) cultivars were supplied with either 5.0 mmol L–1 NH4+-N (EAC) or 5.0 mmol L–1 NO3–-N (CON) under hydroponic conditions.Root growth in both cultivars was significantly reduced under EAC,and the negative effect was greater in Lumai 15.EAC enhanced the activities of monodehydroascorbate reductase and dehydroascorbate reductase in the roots of both cultivars,while it decreased ascorbic acid (ASA) content and GDP-mannose pyrophosphorylase (GMPase) activity at the 12th day after treatment in Lumai 15 by 62.0 and 71.4%;and in Yumai 49 by 38.8 and 62.2%,respectively,indicating that the regeneration of ASA was increased,but the biosynthesis of ASA was reduced under EAC treatment.Moreover,EAC increased DHA/ASA,reactive oxygen species (ROS),and malondialdehyde contents,as well as antioxidant enzyme activities in the roots of both cultivars.Relatively greater increases in ROS and soluble sugar,and lower antioxidant enzyme activities in Lumai 15 indicate severe disruption of oxidative metabolism when compared to Yumai 49.Results reveal that the reduction of ASA biosynthesis via decreased GMPase activity under the EAC condition probably acts as a trigger for accumulated ROS and imbalanced redox status,resulting in root growth inhibition during wheat seedling growth stage.Yumai 49,being an NH4+ -tolerant cultivar,had the stronger capacity to protect itself from oxidative stress,which allowed it to retain a lower DHA to ASA ratio by maintaining a better redox homeostasis than could be maintained in the NH4+ -sensitive cultivar Lumai 15.

Keywords:elevated ammonium concentrations,oxidative metabolism,redox homeostasis,root morphology,wheat

1.Introduction

Ammonium (NH4+),as a major nitrogen source,is important for plant growth,while excessive NH4+can be toxic to cellular functions and plant growth (Liet al.2013;Liuet al.2013).NH4+-induced growth inhibition is now a common phenomenon in certain Chinese intensive agriculture systems.This has come about through the exclusive and excessive application of NH4+fertilizer,the use of dry or wet deposition of ammonia and the use of nitrification inhibitors,which all cause high NH4+concentrations to form in the soil profile (Liet al.2010).Several studies have shown that ion and plant growth regulator imbalances,and toxicity symptoms can be observed when plants are exposed to high NH4+concentrations (Britto and Kronzucker 2002;Bittsánszkyet al.2015;Wanget al.2019).Moreover,the increased production of reactive oxygen species (ROS),in the form of superoxide (O2-·) and hydrogen peroxide(H2O2),have been also observed in common duckweed and grape under high NH4+levels (Skopelitiset al.2006;Wang Get al.2016).

Antioxidant enzymes,such as superoxide dismutase(SOD,EC 1.15.1.1) and peroxidase (POD,EC 1.11.1.x),are ROS scavengers and play important roles in protecting plants from cell membrane structural damage caused by lipid peroxidation.Studies on duckweed,sunflower and maize revealed that the oxidative damage and programmed cell death were induced by elevated concentrations of NH4+(EAC) (Wang Get al.2016).However,research on hydrilla(Hydrilla verticillataL.f) has shown the opposite trend,with enhanced activities of antioxidant enzymes and a decreased content of non-enzymatic antioxidants occurring when exposed to EAC (Wanget al.2010).

In addition to antioxidant enzymes,ascorbic acid (ASA) is a natural,low molecular weight non-enzymatic antioxidant,which plays a crucial role in the shielding aspects of plant growth and oxidative metabolism (Fotopoulos and Kanellis 2013).TheArabidopsismutantvtc1,which lacks ASA biosynthesis (about 30% of wild-type levels),was more sensitive to ozone damage than the wild type (Conklinet al.1997).Further,exogenous application of ASA has been shown to improve plant responses to abiotic stresses,which indicates that sufficient ASA biosynthesis could provide an approach for an improved tolerance to abiotic stresses(Zhanget al.2011).In the process of ASA biosynthesis,GDP-mannose pyrophosphorylase (GMPase) plays an important role and GMPase may also influence plant sensitivity to NH4+conditions (Conklinet al.1999).Research has shown that overexpression of the GMPase gene in potato (Solanum tuberosumL.) and tomato (Solanum lycopersicumL.) leaf tissue significantly increases ASA levels and delays their senescence under abiotic stress(Linet al.2011;Wanget al.2012).Furthermore,GMPase activity may also influenceArabidopsissensitivity to NH4+by modulating the N-glycosylation of proteins,which in turn triggers protein folding,cellulose synthesis,and decreases in cell wall stability and cell viability,all of which can result in root growth inhibition (Qinet al.2008;Tanakaet al.2015).However,whether wheat growth sensitivity towards to NH4+is related to overproduction of ROS by GMPase activity or ASA biosynthesis still unclear.

The content of ASA in plants not only depends on the level of biosynthesis,but also the levels of degradation and regeneration.Regeneration of ASA is ensured by the enzymes monodehydroascorbate reductase (MDHAR)and dehydroascorbate reductase (DHAR).MDHAR is an NDAPH-dependent enzyme which can reduce MDHA into ASA,but it can also reduce phenoxyl radicals (Sakihamaet al.2002).DHAR uses glutathione as electron donor to reduce DHA into ASA.ASA recycling ensures a relatively high turnover rate of the ASA pool.The recycling pathway controls the redox state of the ASA pool and is important during stress responses.Plants lacking the ability to synthesize ASA or to balance the redox state level showed no development beyond the seedling stage (Dowdleet al.2007).Wang (2010) revealed that EAC decreases the content of ASA and the ASA/DHA ratio in aquatic plants,and can trigger oxidative stress,leading to inhibition of plant growth.However,there is little information as to whether inhibited root growth in wheat,under EAC conditions,is related to a reduction in ASA production.

Wheat is a NO3–preferring plant and is highly sensitive to high NH4+concentrations (Liet al.2017).This sensitivity has been shown to increase where NH4+-based nitrogen fertilizers have been applied as a basal crop growth simulant,especially in China (Liuet al.2013).Seed germination and early seedling growth of wheat is severely reduced under EAC conditions (Wang Fet al.2016;Liuet al.2017).Previous studies have shown that enhanced production of ROS and altered antioxidant enzyme activities occur under EAC conditions in the early stages of wheat growth(Polesskayaet al.2004);however,no further research has been conducted to explore the reasons for this enhanced ROS production or to explore the relationship between ASA redox status and ROS accumulation under EAC conditions in wheat seedlings.

The objective of this study was to observe root growth responses of wheat to EAC during early seedling growth,with an emphasis on how oxidative metabolism and the redox status are affected by EAC.The expected results will help in elucidating the response mechanisms in EACtolerant cultivars,which in turn will help identify techniques to alleviate NH4+stress in other wheat cultivars.

2.Materials and methods

2.1.Plant materials and experimental design

Seeds from two wheat cultivars,Yumai 49 (NH4+-tolerant)and Lumai 15 (NH4+-sensitive) were surface-sterilized with 20% (v/v) hydrogen peroxide for 15 min,rinsed thoroughly with sterile distilled water,then germinated in a hydroponic system.They were supplied with either a 5.0-mmol L–1NH4+-N (EAC) treatment or a 5.0-mmol L–1NO3–-N (CON)treatment as the control,as previously described (Liuet al.2017).When radicle lengths of 2.0 mm were reached,seedlings were transferred to specially-designed plastic containers (31 cm×21 cm×11 cm;l/w/h) with a gauze layer inserted 5.0 cm above the container base.The containers were then filled with 850 mL of two modified Hoagland’s nutrient solutions so that the seedlings sat on the gauze and in direct contact with the solution.The containers were placed in a dark growth chamber set at 80% relative humidity,and a 24°C/16°C temperature regime set with a 10 h/14 h thermoperiod.The Hoagland’s nutrient solutions used to grow the seedlings were prepared as previously described (Liuet al.2017),and contained 5.0 mmol L–1N as 1.5 mmol L–1Ca(NO3)2with 2.0 mmol L–1KNO3or 2.5 mmol L–1(NH4)2SO4,3.0 mmol L–1K as 1.0 mmol L–1KH2PO4and 2.0 mmol L–1KNO3or 1.0 mmol L–1K2SO4,1.5 mmol L–1Ca as 1.5 mmol L–1Ca(NO3)2or 0.5 mmol L–1CaCl2and CaSO4,1.0 mmol L–1Mg as 1.0 mmol L–1MgSO4·7H2O,1.0 mmol L–1P as 1.0 mmol L–1KH2PO4,0.5 mmol L–1Na as 0.5 mmol L–1NaCl,0.5 μmol L–1Fe-EDTA,0.55 μmol L–1MnSO4·H2O,0.0385 μmol L–1ZnSO4·7H2O,0.0165 μmol L–1Cu as CuSO4·5H2O,2.35 μmol L–1H3BO3,and 0.0065 μmol L–1H2MoO4.To match the 5.0 mmol L–1NH4+treatment (EAC) in terms of Ca and K concentrations,the Hoagland’s solution was modified by adding CaCl2and CaSO4to replace the calcium normally supplied as Ca(NO3)2,and K2SO4was added to replace the potassium normally supplied as KNO3,compared to 5.0 mmol L–1NO3–control treatment (CON).To ensure stability of the pH,the two Hoagland’s solutions were replenished every second day and the pH was adjusted to 6.8–7.0 with daily drop-wise additions of 1.0 mol L–1NaOH.The experiment was undertaken in a completely randomized block design with three replications for each treatment.

The whole roots were sampled from the containers at 5,7,9,and 12 days after treatment (DAT),then frozen in liquid nitrogen and stored at–40°C.In addition,from other seedlings undergoing the same treatments,shoot and root samples were taken after 7 and 12 DAT,and dried at 75°C to constant weight.These dry and further fresh samples were used for the analysis of growth.

2.2.Morphological measurements

Root length was determined using a flatbed scanner (Epson 1680,Jakarta,Indonesia) running WinRHIZO Software (Pro Vision 5.0,Canada).

2.3.GDP-mannose pyrophosphorylase (GMPase),monodehydroascorbate reductase (MDHAR),and dehydroascorbate reductase (DHAR) activity assays

For GMPase activity,fresh root samples (0.5 g) were homogenized in a chilled mortar and pestle with 4.0 mL 100 mmol L–1Tris-HCl buffer (pH 7.6) containing 1%PVP,5.0 mmol L–1DTT and 1.0 mmol L–1EDTA.The homogenate was centrifuged at 10 000×g for 10 min to produce a supernatant.All operations were carried out at 4°C.A mixture of 50 mmol L–1triethylamine hydrochloride(pH 7.0),containing 0.1 mmol L–1EDTA,2.5 mmol L–1MgCl2,0.1% BSA,0.5 mmol L–1NADP+,1.0 mmol L–1β-mercaptoethanol,1.0 mmol L–1glucose,1.0 mol L–1ADP,1.0 mmol L–1sodium pyrophosphate,0.5 mmol L–1GDPmannose,0.5 U mL–1of nucleoside diphosphate kinase(Sigma Chemical Co.,Beijing),1.0 U mL–1of hexokinase(Sigma Chemical Co.,Beijing) and 2.0 U mL–1glucose-6-phosphate dehydrogenase (Roche Chemical Co.,Beijing)was added to 100 μL of extract supernatant,and after 1 min the GMPase activity was determined at an absorbance of 340 nm (Szumiloet al.1993).

For MDHAR and DHAR activities,fresh root samples(0.5 g) were homogenized in a chilled mortar and pestle with 5.0 mL of 50 mmol L–1Tris-HCl buffer (pH 7.2) containing 2%PVP,1.0 mmol L–1EDTA,0.1% BSA,0.05% cysteine,and 0.3 mol L–1mannose.The homogenate was centrifuged at 10 000×g for 30 min to produce a supernatant.All operations were carried out at 4°C.MDHAR activity was assayed following the method of Hossain (1984).Briefly,3.0 mL of 100 mmol L–1Tris-HCl (pH 6.3) containing 0.2 m L–1NADH,1.0 mmol L–1ASA and 0.2 mL AAO (0.25 U mL–1) was added to 1.0 mL of extract supernatant,and the MDHAR activity was determined at an absorbance of 340 nm.DHAR activity was assayed following the method of Douliset al.(1997).A total of 3.0 mL of 100 mmol L–1potassium phosphate buffer (pH 7.0) containing 1.0 mmol L–1DHA,2.0 mmol L–1GSH,and 0.1 mmol L–1EDTA was added to 0.1 mL extract supernatant.DHAR activity was determined at an absorbance of 256 nm.

2.4.Ascorbic acid and dehydroascorbic acid content assays

The protocols for ASA and DHA measurements followed the method of Zhang and Kirkham (1996).Briefly,fresh root samples (0.5 g) were homogenized with 3.0 mL of 5.0% trichloroacetic acid (TCA) and the homogenate was centrifuged at 10 000×g for 15 min to produce a supernatant.To measure total ASA content,a mixture of 0.75 mL phosphate buffer (150 mmol L–1,pH 7.4) and 0.15 mL of 10 mmol L–1dithiothreitol (DTT) was added to 0.3 mL of extract and incubated at room temperature for 10 min.Thereafter,0.15 mL of 0.5% N-ethylmaleimide was added to remove all remaining DTT.To this was added 0.6 mL of 10% TCA,0.6 mL of 44% phosphate buffer,0.6 mL of 4%2,2´-bipyridine ethanol solution and 0.15 mL of 0.3% FeCl3and the mixture was incubated at 40°C for 40 min.The total ASA content was determined from the absorbance at 525 nm.For the ASA content,the assay was repeated with the DTT and N-ethylmaleimide replaced by 0.3 mL of distilled water,with all other steps in the assay the same.The DHA content was determined as the difference between the total ASA and ASA contents.

2.5.Antioxidant enzyme activity assays,and malondialdehyde,hydrogen peroxide,and superoxide anion content assays

Fresh root samples (0.5 g) taken directly from the seedlings in hydroponic culture were ground and homogenized in a chilled mortar and pestle with 5.0 mL of extraction buffer containing 50 mmol L–1potassium phosphate buffer(PPB;pH 7.0) and 0.4% polyvinylpoly pyrrolidone.The homogenate was centrifuged at 10 000×g for 30 min and the supernatant was collected and stored as the extract.All operations and storage were undertaken at 4°C.

The measurements of POD and CAT activities,as well as malondialdehyde (MDA) content of roots,were carried out as described by Tanet al.(2008).Briefly,20% trichloroacetic acid containing 0.5% 2-thiobarturic acid was added to 0.2 mL of the supernatant and heated at 95°C for 25 min,then the mixture was cooled down in a water bath to 25°C,and centrifuged at 3 000×g for 5 min.MDA concentration was determined from the absorbances of the extract at 532 and 450 nm using a spectrophotometer (UV-2401,Shimadzu Corporation,Japan) and corrected by subtracting the absorbance at 600 nm.

For POD activity assessment,transparent tubes with 3.0 mL reaction mixture containing 1.9 mL of 200 mmol L–1PBS (pH 6.0),1.0 mL of 29% H2O2,guaiacol and 10 μL of extract were mixed,then after 3 min,absorbance was determined at 470 nm.

For CAT activity assessment,transparent tubes with 3.0 mL reaction mixture containing 1.9 mL of 100 mmol L–1PPB (pH 7.0),1.0 mL of 0.075% H2O2and 10 μL of extract,were mixed,then after 1 min,absorbance was determined at 240 nm.

APX activity was measured according to the methodology of Polesskayaet al.(2004).Briefly,transparent tubes with 3.0 mL of reaction mixture containing 100 mmol L–1PPB(pH 7.0),0.5 mmol L–1ASA,0.1 mmol L–1EDTA,and 50 μL extract were set up.H2O2(0.1 mmol L–1) was added to initiate the reaction,then after 1 min,activity was determined by the absorbance of the solution at 240 nm.

The O2−· content was determined following the method of Wang (2006).A mixture of 0.5 mL of 50 mmol L–1PBS(pH 7.8) and 1.0 mL of 10 mmol L–1hydroxyl ammonium chloride was added to 1.0 mL of extract,and the mixture was incubated at 25°C for 60 min.After that time,1.0 mL of 17 mmol L–1p-aminobenzene sulfonic acid and 1.0 mL of 7.0 mmol L–1α-naphthylamine were added prior to a further 20 min incubation at 25°C.O2−· content was determined by the absorbance at 530 nm.Tissue H2O2content was determined by using a H2O2analysis kit (A064) purchased from the Chengjian Bioengineering Institute (Nanjing,China) with the generated products measured at 405 nm.The protein content was measured following the method of Bradford (1976).

2.6.Ammonium content assay

Fresh root samples (0.5 g) were homogenized with 5.0 mL Tris-HCl buffer (25 mmol L–1pH 7.6) in a pre-chilled mortar and pestle.The extract was stored at 45°C for 1 h,followed by centrifugation of the homogenate at 15 000×g for 20 min.The measurement of NH4+content followed the method of Leleuet al.(2004) with NH4Cl being used as the standard.

2.7.Free amino acid and soluble sugar content assays

Dried samples were pulverized using a ball mill.For each sample,0.1 g powder was weighed and extracted three times with 80% (v/v) ethanol at 80°C and then centrifuged at 3 000×g.The supernatant was transferred to a glass tube and the final volume was made up to 20 mL with 80%ethanol.Total soluble sugar content was measured using the anthrone method (Fales 1951),and the content of free amino acids was measured according to the ninhydrin reagent method (Moore and Stein 1954).

2.8.Statistical analysis

Means from three replicate samples,from all studies,were subjected to an analysis of variance (ANOVA) test to determine significant differences between treatments,using Duncan’s multiple range test at the 0.05 level of significance.The ANOVA analysis was performed using SPSS predictive analytics software (ver.16.0;SPSS Inc.,Chicago,IL,USA).

3.Results

3.1.General growth responses

Morphological growth responses of both Yumai 49 and Lumai 15 cultivars,under EAC and CON conditions,were recorded at 7 and 12 DAT (Table 1).Significant decreases in biomass accumulation and root length were observed under EAC treatment for both cultivars.When compared to CON treatment,the decreases in root length and plant biomass accumulation in Yumai 49 at 7 DAT were 24.0 and 9.6%;while they were 32.7 and 17.7% in Lumai 15.In addition,at 12 DAT,the decreases in root length and plant biomass accumulation were 33.4 and 21.9% for Yumai 49,while they were 41.5 and 30.6% in Lumai 15.The adverse growth effects caused by EAC as seen in Yumai 49 were significantly less than those seen in Lumai 15.

3.2.Activities of GMPase,MDHAR and DHAR

In general,the activity of GMPase decreased in the roots of both cultivars over time.More specifically,as a result of the EAC treatment,the GMPase activity significantly decreased in both cultivars and the differences between EAC and CON after 5,7,9,and 12 DAT were respectively 61.5,51.2,55.4,and 62.2% for Yumai 49 and 54.7,51.9,60.7,and 71.4%for Lumai 15 (Fig.1).

The activities of MDHAR and DHAR in the EAC-treated roots increased along with time (Fig.2),and the significant differences in Yumai 49 were observed at the 9th d for DHAR activity and the 12th d for MDHAR activity,respectively.In contrast,the significant differences in Lumai 15 were found at the 12th d for DHAR activity,and 9th d for MDHAR activity,respectively.

3.3.Contents of ASA and DHA

The content of ASA increased with time in the roots of both cultivars,except after 7 DAT in the EAC-treated roots of Lumai 15.The rate of increase in ASA was lower in Lumai 15 than in Yumai 49,while the rate of decrease after 7 DAT was higher in the EAC-treated roots of Lumai 15 than in Yumai 49 (Fig.3-A).Similarly,the content of DHA and the DHA/ASA ratio increased in both cultivars and under both treatments.The increases in DHA content underEAC (as compared to the respective CON treatments)after 9 and 12 DAT were 213.6 and 93.8% in the roots of Lumai 15,and 21.9 and 64.5% in the roots of Yumai 49,respectively.The increases in the DHA/ASA ratio under EAC (as compared to the respective CON treatments) after 9 and 12 DAT were 5.0 and 4.9 times higher in the roots of Lumai 15,and 55.9% and 1.0 times higher in the roots of Yumai 49,respectively (Fig.3-B and C).

Table 1 Root length and plant biomass as affected by elevated ammonium concentrations after seed emergence in two wheat cultivars1)

Fig.1 The activity of GDP-mannose pyrophosphorylase in the roots of Yumai 49 and Lumai 15 under elevated ammonium concentrations during early wheat seedling growth stage.CON,control;EAC,elevated ammonium concentrations.Data represent means of three replicates for each genotype.Bars mean SD (n=3).* within the same day refers to a significant difference (P<0.05).

Fig.2 The activities of monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR) in the roots of Yumai 49 and Lumai 15 under elevated ammonium concentrations during early wheat seedling growth stage.Data represent means of three replicates for each genotype.Bars mean SD (n=3).* within the same day refers to a significant difference (P<0.05).

3.4.Activity of antioxidant enzymes

The activity of the antioxidant enzymes in the roots of both Yumai 49 and Lumai 15 increased under the EAC treatment when compared to the respective CON treatment.The rate of increase in activity of antioxidants was higher in the roots of Yumai 49 than in the roots of Lumai 15 under both CON and EAC.At 7,9 and 12 DAT,the differences between EAC and CON in POD activity were respectively 42.1,57.8 and 71.3% in Yumai 49 whereas they were 21.5,25.1 and 95.3%in Lumai 15 (Fig.4-A).The increase in the activity of CAT under EAC in the roots of Yumai 49 was significantly higher than in CON at 7 DAT,while in Lumai 15 it was only high at 9 DAT (Fig.4-B).The increase in the activity of APX in EAC-treated roots of both cultivars was most prominent after 9 DAT.When compared to the respective CON treatments at 12 DAT,APX activity was 4.2 times higher in Yumai 49 and 4.2 times higher in Lumai 15 (Fig.4-C).

3.5.Production rate of O2-·,contents of H2O2and MDA

In the EAC treatment,the increase in the production rate of O2-· was statistically significant from 7 DAT onwards in Lumai 15 roots,while in Yumai 49 roots there was no significant difference in the production rate over time.Furthermore,the increase in the production rate of O2-·as a result of EAC treatment and at 9 DAT was 2.0 times greater in Lumai 15 than in Yumai 49 when compared to their respective CON treatments (Fig.5-A).The increases in the contents of H2O2and MDA as a result of the EAC were greater for both cultivars when compared to CON.Notably,the content of H2O2in the roots of Yumai 49 was significantly greater than in the respective CON treatment,but only after 7 DAT.For Lumai 15,it was significantly higher under the EAC treatment than in CON,even after 5 DAT (Fig.5-B).In addition,the difference in MDA content in both EAC and CON treatments was significantly greater in the roots of Lumai 15 than in Yumai 49 (Fig.5-C).

3.6.Contents of ammonium,free amino acid and soluble sugar

Fig.3 Ascorbic acid (ASA) content,dehydroascorbic acid (DHA) content and DHA to ASA ratio in the roots of Yumai 49 and Lumai 15 under elevated ammonium concentrations during early wheat seedling growth stage.CON,control;EAC,elevated ammonium concentrations.Data represent means of three replicates for each genotype.Bars mean SD (n=3).* within the same day refers to a significant difference (P<0.05).

The NH4+content under the EAC treatment increased in the roots of both cultivars as compared to CON,however the rate of increase over time was higher in Lumai 15 than in Yumai 49 (Fig.6).At the 7th d of EAC treatment,the increases of NH4+content in the roots of Lumai 15 were 94.6 and 58.9% in Yumai 49 respectively when compared to their respective CON.

The free amino acid and soluble sugar contents were increased in the EAC-treated roots in both cultivars at 7 DAT,the increase in free amino acid of Yumai 49 was more obvious than that in Lumai 15,and the increase in soluble sugar of Lumai 15 was greater than that in Yumai 49(Fig.7-A and C).Further,the free amino acid and soluble sugar contents were increased in the EAC-treated roots in both cultivars at 12 DAT,and the increases in free amino acid and soluble sugar were more significant in Yumai 49 and Lumai 15,respectively (Fig.7-B and D).The ratio of soluble sugar to free amino acid was increased in both cultivars under EAC treatment at 7 DAT,and the increase in Lumai 15 was greater than that in Yumai 49 (Fig.7-E).Moreover,the ratio of soluble sugar to free amino acid was increased in EAC-treated root of Yumai 49 but decreased in Lumai 15 at 12 DAT (Fig.7-F).

4.Discussion

Fig.4 The activities of antioxidant enzymes in the roots of Yumai 49 and Lumai 15 under elevated ammonium concentrations during early wheat seedling growth stage.CON,control;EAC,elevated ammonium concentrations.Data represent means of three replicates for each genotype.Bars mean SD (n=3).* within the same day refers to a significant difference (P<0.05).

Ammonium is the preferred nitrogen source for many plants,however,when it is applied as the sole nitrogen source to crops,high concentrations can cause growth inhibition and significant yield losses (Liet al.2014).In a previous study,the higher ammonium concentrations suppressed wheat seed germination and early seedling growth through reduced reserve mobilization and ATP production in seeds,and decreased sugar utilization capability in radicles (Liuet al.2017).The root growth inhibition and the higher sensitivity of root growth,as compared to shoot growth,in two cultivars of wheat under EAC treatment (Table 1),were identical to those seen in previous reports involving both aquatic and terrestrial plants (Polesskayaet al.2004;Wanget al.2010).This growth inhibition was attributed to oxidative stressviaalterations to the redox balance,through the stimulation of ROS production,as well as by affecting the activity of certain antioxidant enzymes (Polesskayaet al.2004;Wanget al.2010).It has been found that a higher NH4+tolerance in rice was achieved both by the activation of antioxidant defense signaling and by the modulation of ROS homeostasis,thereby neutralizing excess oxygen species that are produced in response to NH4+stress (Xieet al.2014).However,the underlying mechanism of how EAC affected the redox balance and the inhibition of root growth during the early seedling growth stage of wheat has not yet been determined.

Redox balance is determined by the balance between ROS production and scavenging.Overproduction of ROS or a low level of ROS scavenging may cause oxidative stress and lead to redox imbalance.Links between ASA redox homoeostasis and oxidative stress have previously been reported under EAC treatment (Wanget al.2010).In the ASA biosynthesis,GDP-mannose pyrophosphorylase is one of the important key enzymes involved in this process in higher plants (Conklinet al.1997,1999),and Qin (2008)found that GMPase activity decreased under high NH4+concentrations inArabidopsis,owing to an increased medium pH and internal alkalinization.In the present results,GMPase activity and ASA content were decreased in the roots of both cultivars under EAC treatment (Figs.1 and 3-A).Further,the ASA recycling pathway also controls the redox state in plants,and is important during stress responses.Two key enzymes,MDHAR and DHAR,are involved in the ASA recycling pathway,and the reduction of MDHAR and DHAR activities suppresses growth and yield of plants (Truffaultet al.2016).In the present study,MDHAR and DHAR activities were increased along with time under the EAC condition,indicating the ability to regenerate ASA was not inhibited by EAC,and EAC might affect ASA biosynthesis or catabolismviadecreased GMPase activity in the roots of wheat seedlings.

Fig.5 The production rate of superoxide anions,content of hydrogen peroxide and content of malondialdehyde in the roots of Yumai 49 and Lumai 15 under elevated ammonium concentrations during early wheat seedling growth stage.CON,control;EAC,elevated ammonium concentrations.Data represent means of three replicates for each genotype.Bars mean SD (n=3).* within the same day refers to a significant difference (P<0.05).

Fig.6 Ammonium content in the roots of Yumai 49 and Lumai 15 under elevated ammonium concentrations during early wheat seedling growth stage.CON,control;EAC,elevated ammonium concentrations.Data represent means of three replicates for each genotype.Bars mean SD (n=3).* within the same day refers to a significant difference (P<0.05).

Fig.7 Free amino acid content,soluble sugar content and ratio of soluble sugar to free amino acid in the roots of Yumai 49 and Lumai 15 under elevated ammonium concentrations during early wheat seedling growth stage.CON,control;EAC,elevated ammonium concentrations.Bars mean SD (n=3).Data represent means of three replicates for each genotype.Small letters within the same graph refer to a significant difference (P<0.05).

As root growth is related to cell division and cell expansion,several studies have mentioned that ASA metabolism is closely linked to these processes:ASA stimulates mitosisviathe G1 to S phase transition (Pintoet al.1999),while DHA is an inhibitor of the G1 phase (Potterset al.2004).The ASA/DHA ratio could be a signal controlling the path from cell division to cell expansion,and also an important indicator of redox status of the cell (Fotopoulos and Kanellis 2013).In the present study,ASA content decreased after 7 DAT under EAC treatment,while the DHA content,DHA/ASA ratio and soluble sugar content in roots increased(Figs.3 and 7-B),which indicates oxidative stress might be triggered by EAC treatment during the early seedling growth stage of wheat,and soluble sugar is used to adjust the endogenous osmotic pressure to protect cell membranes from the damage attributed to EAC.Although a previous study on spinach and pea (Pisum sativumL.) revealed that the inhibition of plant growth by EAC treatment was due to application of NH4+as a sole nitrogen source and not from oxidative stress (Domínguez-Valdiviaet al.2008).However,there are some studies on the responses of aquatic plants andArabidopsisthat have found oxidative stress to be elicited by NH4+(Pattersonet al.2010;Bittsánszkyet al.2015).The differences among these results might be due to the fact that different plant growth stages,stress durations and species were involved.

Ascorbic acid,as a non-enzymatic antioxidant,was affected by EAC treatment,which may be attributed to over accumulated ROS production.Production of ROS can activate a cascade system in plants that creates a response to oxidative stress,which includes an enhancement or suppression in the activities of antioxidant enzymes.The increased activities of POD and CAT seen in both wheat cultivars,as well as the greater activities of both in EACtreated roots of Yumai 49 than in Lumai 15 (Fig.4-A and B),indicates that the increases in antioxidant enzyme activities in early seedling growth of wheat are used to help resist the NH4+stress and there is a stronger possibility of antioxidant protection in the tolerant cultivar as compared to the sensitive cultivar.Additionally,the APX activity was increased significantly in the later stages of seedling growth as compared to the activities in the previous days(Fig.4-C),implying that APX,CAT and POD collectively were able to remove the accumulated H2O2from the root,while APX played an important role in scavenging ROS,using ASA as a substrate,during long-term stress conditions(Barrameda-Medinaet al.2014).In our study,an increased O2-·production rate and H2O2content (Fig.5-A and B) were found in the roots of both cultivars under EAC treatment,which may have resulted from the lower content of ASA and ratio of ASA to DHA leading to a greater possibility of redox imbalance.The increased MDA content (Fig.5-C) was probably associated with the EAC treatment stimulation of ROS which caused oxidative damage.Previously,it has been shown that NH4+(or any of its assimilation products such as glutamine or glutamate) serves as a stress signal in sunflower,maize and spinach (Spinacia oleraceaL.)to trigger excessive ROS production (Barrameda-Medinaet al.2014).

The ASA to DHA ratio could be a signal affecting plant growth,and the activity of GMPase also could be a trigger for reduced ASA production and a lower ASA/DHA state,which results in growth inhibition (Barthet al.2010;Raquelet al.2016).Therefore,future studies which determine whether elevated NH4+concentrations cause oxidative metabolism changes depending on GMPase activity or ASA itself during wheat seedling growth stage will be required to improve our knowledge of plant production and adaptation to abiotic stress.

5.Conclusion

The process of ASA regeneration was enhanced (increased activities of MDHAR and DHAR),and enzymes in the enzymatic system were increased by EAC,while the ROS was overproduced and root growth was inhibited.Thus,the reduction of ASA biosynthesisviadecreased GMPase activity,probably a trigger for accumulated ROS and imbalanced redox status,resulting in root growth inhibition during wheat seedling growth stage.Yumai 49,being an NH4+-tolerant cultivar,had the capacity to protect itself from the antioxidant action which allowed it to retain a lower DHA to ASA ratio by maintaining a better redox homeostasis than could be maintained in the NH4+-sensitive cultivar Lumai 15.

Acknowledgements

This study was funded by a project of the National Natural Science Foundation of China (31471443),the Jiangsu Collaborative Innovation Center for Modern Crop Production,China (JCIC-MCP) and the Priority Academic Program Development of Jiangsu Higher Education Institutions,China (PAPD).

Declaration of competing interest

The authors declare that they have no conflict of interest.