WANG Peng ,WANG Cheng-dong ,WANG Xiao-lin ,WU Yuan-hua ,ZHANG Yan ,SUN Yan-guoSHI YiMI Guo-hua#

1 Key Laboratory of Tobacco Biology and Processing, Ministry of Agriculture and Rural Affairs, Tobacco Research Institute,Chinese Academy of Agricultural Sciences, Qingdao 266101, P.R.China

2 Department of Plant Nutrition, College of Resources and Environmental Sciences, China Agricultural University, Beijing 100193,P.R.China

Abstract Compared with sole nitrate (NO3–) or sole ammonium (NH4+) supply,mixed nitrogen (N) supply may promote growth of maize seedlings.Previous study suggested that mixed N supply not only increased photosynthesis rate,but also enhanced leaf growth by increasing auxin synthesis to build a large sink for C and N utilization.However,whether this process depends on N absorption is unknown.Here,maize seedlings were grown hydroponically with three N forms (NO3–only,75/25 NO3–/NH4+ and NH4+ only).The study results suggested that maize growth rate and N content of shoots under mixed N supply was little different to that under sole NO3– supply at 0–3 d,but was higher than under sole NO3– supply at 6–9 d.15N influx rate under mixed N supply was greater than under sole NO3– or NH4+ supply at 6–9 d,although NO3–and NH4+ influx under mixed N supply were reduced compared to sole NO3– and NH4+ supply,respectively.qRT-PCR determination suggested that the increased N absorption under mixed N supply may be related to the higher expression of NO3– transporters in roots,such as ZmNRT1.1A,ZmNRT1.1B,ZmNRT1.1C,ZmNRT1.2 and ZmNRT1.3,or NH4+absorption transporters,such as ZmAMT1.1A,especially the latter.Furthermore,plants had higher nitrate reductase (NR)glutamine synthase (GS) activity and amino acid content under mixed N supply than when under sole NO3– supply.The experiments with inhibitors of NR reductase and GS synthase further confirmed that N assimilation ability under mixed N supply was necessary to promote maize growth,especially for the reduction of NO3– by NR reductase.This research suggested that the increased processes of NO3– and NH4+ assimilation by improving N-absorption ability of roots under mixed N supply may be the main driving force to increase maize growth.

Keywords: maize,NO3–/NH4+ ratio,N absorption,N assimilation,plant growth

1.Introduction

Nitrogen (N),a macro-element,is the key component of DNA,RNA,enzymes,chlorophyll and hormones,which also participates multiple important metabolic pathways in plant growth and development (Lea and Morot-Gaudry 2001).N acquisition efficiency of cereals is less than 50% of the N supply (Raun and Johnson 1999;Sylvester-Bradley and Kindred 2009),this value is lower than the N acquisition rate (60%) which is required for maximizing plant growth and yield (Heffer 2013).Currently,improving plant N absorption ability in soil may be a critical means to improve plant N efficiency and plant growth (Garnettet al.2013).Nitrate (NO3–) and ammonium (NH4+) are the main forms of inorganic N in soil for plant N absorption(Marschner 2011).For most plant species,compared to sole NO3–or NH4+supply,plants grow better when both ions are supplied at an appropriate ratio (Liet al.2013).NO3–and NH4+absorption is regulated by NO3–transporters (NRTs) and NH4+transporters (AMTs),respectively.InArabidopsis,the transporter genesAtNTR1.1/NPF6.3andAtNTR1.2/NPF4.6have been reported to have low affinity (LATS) NO3–uptake ability and were mainly responsible for NO3–absorption at high N concentrations (Huanget al.1999).Hoet al.(2009)reported thatAtNRT1.1had both low and high affinity for NO3–absorption,which depended on its phosphorylation status.AtNRT1.1also as an important sensor and can receive nitrate signaling (Hoet al.2009).In addition,AtNRT2.1andAtNRT2.2have high affinity activity and are mainly responsible for NO3–absorption at low N supply (Liet al.2007;Glass and Kotur 2013),AtNRT3.1(AtNAR2.1)andAtNRT3.2(AtNAR2.2) can indirectly affect the activities ofAtNRT2.1andAtNRT2.2(Okamotoet al.2006).In maize,ZmNRT1.1contains four homologous genes,includingZmNRT1.1A,ZmNRT1.1B,ZmNRT1.1CandZmNRT1.1D(Plettet al.2010).ZmNRT3.1contains two homologous genes which areZmNRT3.1AandZmNRT3.1B(Plettet al.2010).For NH4+absorption inArabidopsis,AtAMT1.1andAtAMT1.3as the high affinity NH4+transporters are mainly responsible for NH4+absorption in the root symplast pathway,whileAtAMT1.2is mainly found in the root cortex and endothelial layer,and is mainly responsible for NH4+absorption in the root apoplast pathway (Yuanet al.2007).In maize,ZmAMT1.1AandZmAMT1.3have been identified and characterized for their physiological function in high-affinity NH4+absorption (Guet al.2013).In recent years,studies have suggested that there is an interaction between NO3–and NH4+when they co-exist.NH4+has been reported to inhibit NO3–absorption in barley (Kronzuckeret al.1999a),while NO3–has been reported to promote NH4+absorption in rice (Kronzuckeret al.1999b),oilseed rape (Babourinaet al.2007) andPopuluspopularis (Luoet al.2013).NO3–and NH4+have different assimilation pathways.In general,most of the NO3–in plants is transported to the shoots in the xylem and is assimilated in chloroplasts with the reducing power coming mainly from photosynthesis,while most of the NH4+is assimilated in the root with the reducing power coming mainly from the carbon transport from the shoot to the roots (Lea and Morot-Gaudry 2001).NO3–reductase and glutamine synthase are the key enzymes in the process of NO3–and NH4+assimilation(Marschner 2011).It is not known whether N absorption and assimilation are necessary conditions for improving plant growth under mixed N supply compared NO3–alone supply.In this study,we aimed to clarify the relationship between N absorption and assimilation under mixed N supply compared with sole NO3–supply,and finally to explore if N absorption and assimilation under mixed N supply is a critical factor to determine the increasing growth of maize.

2.Materials and methods

2.1.Experimental procedures

In the present study,hydroponic experiments were carried out in a growth room with a constant light intensity of 400 μmol m–2s–1,14 h light/10 h dark,day and night temperature of 28 and 22°C,and relative humidity of 60%.Maize hybrid ZD958 was used in the experiments.The seeds were sterilized with 10% (v/v) H2O2for 30 min and then transfered to saturated CaSO4solution for 6 h.The seeds were then germinated on filter paper in a dark environment until the length of the primary roots were 1.5 cm.The seeds were transferred to culture in rolled papers for 6–7 days until the seedlings had one expanded leaf.After removal of the kernels the seedlings were transferred to a container (55 cm×45 cm×35 cm)which could contain 90 L of solution for the hydroponic experiments.Images of the experimental equipment and seedling cultivation are given in Wanget al.(2019b).We used the modified Hoagland nutrient solution containing 0.5 mmol L–1K2SO4,0.6 mmol L–1MgSO4·7H2O,0.3 mmol L–1KH2PO4,0.5 mmol L–1CaCl2·2H2O,1 μmol L–1H3BO3,0.5 μmol L–1MnSO4·H2O,0.5 μmol L–1ZnSO4·7H2O,0.2 μmol L–1CuSO4·5H2O,0.07 μmol L–1Na2MoO4·2H2O,and 0.1 mmol L–1Na-Fe-EDTA (Guet al.2013).We choose the optimal NO3–/NH4+ratio for maize growth as 75%/25% based on our previous work (Wanget al.2019a,b).In the present study,N was supplied at a concentration of 1 mmol L–1with three different NO3–/NH4+ratios (NO3–only,75/25NO3–/NH4+,and NH4+only) using KNO3and/or (NH4)2SO4.MgSO4and K2SO4were added to balance the potassium concentrations in the solutions.Solution pH was adjusted to 5.8 every 6–12 h.The containers were randomly placed in the growth room and their places were changed every three days.The nutrient solution was aerated continuously and renewed every three days.

For the growth experiments related to inhibitors,we used the same nutrient solution treatments as above.We used a 10-L gray box for maize seedling culture,where the concentrations of nitrate reductase (NR) inhibitor sodium tungstate (Na2WO4),glutamine synthase (GS) inhibitor methionine sulfoximine (MSO) and Gln were 500 μmol L–1(Gorskaet al.2008),1 μmol L–1(Wanget al.2016) and 1 mmol L–1,respectively (Sivasankaret al.1997).

2.2.Plant growth and N concentration

Four seedlings from each treatment were sampled at 1,3,6,and 9 d after treatments were applied.Shoots and roots were dried to determine plant biomass.Average growth rate per plant was calculated as the change in biomass per plant in one day.Milled dry shoot and root samples (80 mg) were used to measure N concentration using an N/C analyzer (vario MACRO cube,ELEMENTAR).There were four biological replicates for each treatment.

2.3.15N influx rate

The method to measure15N influx rate of maize roots is given in Guet al.(2013).Maize seedlings under different NO3–and NH4+ion ratios and grown for 1,3,6 and 9 d were used in the experiment.After 5 h light exposure,seedling roots were washed softly with dd-H2O and transferred to 1 mmol L–1CaSO4solution for 1 min,and then the seedlings were transferred to nutrient solution(pH 5.8) of15N-labeled NO3–(10.16 atom%15N) or NH4+(99.14 atom%15N) for 5 min.After rinsing roots in 1 mmol L–1CaSO4solution for 1 min to exchange the tracer from the apoplast,roots were harvested and freeze dried.A 1.5-mg aliquot of ground sample was used for15N analysis by isotope ratio mass spectrometry (DELTAplus XP,Thermo-Finnigan,Germany).There were four biological replicates for each treatment.

2.4.qRT-PCR analysis

Maize roots were harvested in 1,3,6 and 9 d.Whole roots were excised and snap-frozen in liquid N2and stored at–80°C.Total RNA was extracted as described in a former study (Guet al.2013).A 7500 Real-Time PCR System (Applied Biosystems,USA) was used to carry out a two-step PCR procedure.The maizeZmUbiquitingene was used as an internal control for normalizing gene expression in maize.The primers used in the quantitative PCR analyses are listed in Appendix A.Partial NO3–and NH4+absorption transporter gene primers are referenced in previous studies (Garnettet al.2013;Guet al.2013).

2.5.Free nitrate and ammonium

For free NO3–,2 g fresh shoot or root material was sampled at 1,3,6 and 9 d and put in a centrifuge tube to which 8 mL dd-H2O was added.The tubes were placed in a water bath at 95°C for 30 min.After centrifuging at 4 000 r min–1for 15 min at 4°C,the supernatant solution was filtered into a 25-mL volumetric flask.A total of 5 mL dd-H2O was added to the residue,and then the procedure was repeated as above.The specific experimental steps follow Cataldoet al.(1975).KNO3was used as a standard for NO3–.There were four biological replicates for each treatment.

For free NH4+,100 mg of fresh shoot or root material sampled at 1,3,6 and 9 d were put in a centrifuge tube to which 1 mL pre-cooled 10 mmol L–1formic acid was added,and the tubes were then centrifuged at 14 000 r min–1for 15 min.A total of 24 μL of supernatant was taken and mixed with 400 μL OPA buffer (50 mL,pH=6.8,100 mmol L–1KH2PO4/K2HPO4buffer with 0.201 g OPA and 35.2 μL β-mercaptoethanol).A column-free Agilent 1260 HPLC with a fluorescence detector was used to measure NH4+at an excitation wavelength of 410 nm and a wavelength of 470 nm.

2.6.Total free amino acid concentration

To determine free amino acid concentration,100 mg fresh shoot or root material sampled at 1,3,6 and 9 d were put in a centrifuge tube to which 900 μL of 0.01 mol L–1pH=7.3 PBS buffer was added.The mixture was centrifuged at 3 000 r min–1for 20 min.The supernatant was used to determine total free amino acid concentration using an ELISA kit (Crowther 1995).ELISA kit corresponding substance antibody was provided by Shanghai Runfu Biotechnology Co.,Ltd.,China.

2.7.Nitrate reductase (NR) and glutamine synthase(GS) enzyme activity

To determine NR and GS activity,100 mg fresh shoots or roots sampled at 1,3,6 and 9 d were put in the centrifuge tube.After 900 μL of 0.01 mol L–1pH=7.3 PBS buffer was added,the mixture was centrifuged at 1 500× g for 20 min.The supernatant was used to determine NR and GS activity.NR activity was determined by using an ELISA kit(Crowther 1995).Supernatant solution (10 μL) and diluent solution (40 μL) were transferred to an enzyme labeled plate with NR-antibody,then the plate was incubated at 37°C for 30 min and the liquid was discarded.After the enzyme labeled plate was dry,we washed the plate with washing solution for 30 s and repeated this for 5 times.We added 50 μL enzyme labeled reagent before incubating for 10 min and then washed the plate again for 5 times.Finally,we added 50 μL color reagent A and 50 μL color reagent B to the plate,respectively.After the plate was incubated at 37°C in the dark for 10 min,we added 50 μL termination solution and determined enzyme activity at 450 nm wavelength using an enzyme labeling instrument.ELISA kit corresponding substance antibody was provided by Shanghai Run Yu Biotechnology Co.,Ltd.GS activity was determined by reference to Xue(1985).A total of 100 μL of the crude enzyme extract was added to 160 μL assay mixture (60 μL of 0.25 mol L–1imidazole–HCl buffer,40 μL of 0.30 mol L–1sodium hydrogen glutamate,40 μL of 30 mol L–1ATP-Na and 20 μL of 0.5 mol L–1MgSO4).After the mixture was incubated for 5 min at 25°C,20 μL of hydroxylamine hydrochloride (a mixture of 1 mol L–1hydroxylamine hydrochloride and 1 mol L–1HCl,1:1) was added and left standing for 15 min.GS enzyme activity was determined in the supernatants at 540 nm using a GS kit bought from Nanjing Construction Engineering,China.Four biological replicates were used for each treatment.

2.8.Statistical analysis

Data means and standard errors (SE) were calculated using Microsoft Excel 2010.Data were subjected to variance analysis using the one-way ANOVA (Tukey-test)procedure implemented in SPSS Statistics 19.0 (SPSS,Inc.,Chicage,IL,USA).Differences between treatment means were compared using the least significant difference (LSD) test at 0.05 level of probability.

3.Results

3.1.Average growth rate and 15N influx

There was little difference between the N forms in average plant growth rate at 0–3 d.Average growth rate under mixed N supply was significantly higher,by 1.7 folds,than under sole NO3–supply at 3–6 d (Fig.1-A).Average growth rate under mixed N supply at 6–9 d was 1.3 folds that under sole NO3–supply (Fig.1-A).

Fig.1 Average growth rate per plant (A) and 15N influx rate in roots (B) under different forms of N supply.NO NH4+ only respectively represent 100/0,75/25 and 0/100 NO3–/NH4+ ratios.15N-mixed N-NO3– represents NO33– influx rate under– only,mixed N and mixed N supply and 15N-mixed N-NH4+ represents NH4+ influx rate under mixed N supply respectively.Values are mean±SE (n=4).On the same day,significant differences at P<0.05 are shown with different letters;ns,no significant difference.

At 1 and 3 d,15N influx rate under mixed N supply and sole NH4+supply was higher,by 1.3–2.1 and 1.7–2.1 folds than under sole NO3–supply,respectively,and there was no significant difference between mixed N supply and sole NH4+supply.NO3–influx rate under mixed N supply was 43.0–71.9% of that under sole NO3–supply,while NH4+influx rate under mixed N supply was 41.3–79.4% of that under sole NH4+supply.At 6 and 9 d,15N influx rate under mixed N supply was significantly higher than under sole NO3–or NH4+supply,being 3.3–3.4 and 1.3 folds greater,respectively.NO3–influx rate under mixed N supply was 80.8–91.0% of NO3–influx rate under sole NO3–supply,while NH4+influx rate under mixed N supply was 91.2–95.1% of NH4+influx rate under sole NH4+supply (Fig.1-B).

3.2.Shoot and root N content

There was no significant difference between mixed N supply and NO3–supply in shoot and root N content at 1 and 3 d,but N content in mixed N supply was 1.2–1.3 and 1.2 folds that of sole NO3–supply in shoots and roots respectively at 6–9 d (Fig.2-A and B).In addition,in shoots,there was no significant difference in N content between NH4+supply and NO3–supply at 1–9 d (Fig.2-A).In roots,there was no significant difference between sole NH4+and NO3–supply at 1,3 and 9 d,but N content in mixed supply was 1.1 folds that of sole NO3–supply at 6 d(Fig.2-C).

Fig.2 N content of shoots (A) and roots (B) under different forms of N supply at 1–9 d after treatment.NO3– only,mixed N and NH4+only respectively represent 100/0,75/25 and 0/100 NO3–/NH4+ ratios.Values are mean±SE (n=4).On the same day,significant differences at P<0.05 are shown with different letters;ns,no significant difference.

3.3.Gene expression of nitrate and ammonium absorption related transporters

Compared to the expression of NO3–transporters under sole NO3–supply,at 1 d,the expression ofZmNRT1.1Bunder mixed N supply was up-regulated,the expression ofZmNRT1.1D,ZmNRT1.2,ZmNRT1.3andZmNRT3.1Bunder mixed N supply were unchanged,and the expression ofZmNRT1.1A,ZmNRT1.1C,ZmNRT2.1,ZmNRT2.2,ZmNTR3.1AandZmNRT3.2under mixed N supply were down-regulated (Fig.3).At 3 d,the expression ofZmNRT1.1DandZmNRT1.2were upregulated,the expression ofZmNRT1.1AZmNRT1.1B,ZmNRT1.1C,ZmNRT1.1D,ZmNRT1.3andZmNRT3.1Bunder mixed N supply were unchanged,and the expression ofZmNRT2.1,ZmNRT2.2,ZmNTR3.1AandZmNRT3.2under mixed N supply were down-regulated.At 6 d,the expression ofZmNRT1.1C,ZmNRT1.3andZmNRT3.2under mixed N supply were up-regulated,the expression ofZmNRT1.1A,ZmNRT1.1B,ZmNRT1.2andZmNRT3.1Bunder mixed N supply were unchanged,and the expression ofZmNRT1.1D,ZmNTR2.1,ZmNRT2.2andZmNRT3.1Aunder mixed N supply were downregulated.At 9 d,the expression ofZmNRT1.1A,ZmNRT1.1B,ZmNRT1.1C,ZmNRT1.2,ZmNRT1.3andZmNRT3.1Bunder mixed N supply were up-regulated,the expression ofZmNRT1.1D,ZmNRT3.1AandZmNRT3.2under mixed N supply were unchanged,and the expression ofZmNTR2.1andZmNRT2.2under mixed N supply were down-regulated (Fig.3).Compared to the expression of NH4+transporters under sole NH4+supply,at 1–3 d,the expression ofZmAMT1.3under mixed N supply was unchanged,and the expression ofZmAMT1.1AandZmAMT1.1Bwere down-regulated.At 6–9 d,the expression ofZmAMT1.1Aunder mixed N supply was up-regulated,the expression ofZmAMT1.3under mixed N supply was unchanged,and the expression ofZmAMT1.1Bwas down-regulated (Fig.4).

Fig.3 The expression of nitrate absorption transporters under different forms of N supply at 1–9 d after treatment.NO3– only,mixed N and NH4+ only respectively represent 100/0,75/25 and 0/100 NO3–/NH4+ ratios.Values are mean±SE (n=4).On the same day,significant differences at P<0.05 are shown with different letters;ns,no significant difference.

Fig.4 The expression of ammonium absorption transporters under different forms of N supply at 1–9 d after treatment.NO3– only,mixed N and NH4+ only respectively represent 100/0,75/25 and 0/100 NO3–/NH4+ ratios.Values are mean±SE (n=4).On the same day,significant differences at P<0.05 are shown with different letters;ns,no significant difference.

3.4.The changes of free nitrate,ammonium and total amino acid

Compared to sole NO3–supply,with increasing NH4+supply as a proportion of total N supply,free NO3–concentrations in the shoots were decreased significantly each day (Fig.5-A).However,free NH4+concentrations in shoots under each N form were low each day,which illustrates that most of the NH4+was assimilated (Fig.5-B).NO3–concentrations in the roots showed similar trends to those in the shoots each day (Fig.5-C).In roots,free NH4+concentrations under mixed N supply and NH4+supply were all higher than under NO3–supply at 0–9 d,and respectively were 1.2–2.5 and 1.9–2.0 folds greater than under sole NO3–supply (Fig.5-D).

Fig.5 Nitrate and ammonium concentration of shoots (A and B) and roots (C and D) under different forms of N supply at 1–9 d after treatment.NO3– only,mixed N and NH4+ only respectively represent 100/0,75/25 and 0/100 NO3–/NH4+ ratios.Values are mean±SE (n=4).On the same plant day,significant differences at P<0.05 are shown with different letters;ns,no significant difference.

There was no difference in total amino acid concentration in shoots under different N supply forms supply at 1–3 d.At 6–9 d,total amino acid concentrations of shoots under mixed N supply and sole NH4+supply was 1.3–1.5 and 1.3–1.5 folds higher,respectively,than under sole NO3–supply (Fig.6-A).In roots,with increasing NH4+supply as a proportion of total N supply,total amino acid concentration increased significantly at 1–9 d.Total amino acid concentrations under mixed N supply were 1.2–1.3 folds greater than under sole NO3–supply,while under sole NH4+supply they were 1.2–1.5 folds greater than under sole NO3–supply at 0–9 d (Fig.6-C).

Fig.6 Amino acid concentration and content of shoots (A and B) and roots (C and D) under different forms of N supply at 1–9 d after treatment.NO3– only,mixed N and NH4+ only respectively represent 100/0,75/25 and 0/100 NO3–/NH4+ ratios.Values are mean±SE (n=4).On the same plant day,significant differences at P<0.05 are shown with different letters;ns,no significant difference.

There was no difference between treatments in total amino acid content in shoots at 0–3 d.At 6–9 d,total amino acid content in shoots was the highest under mixed N supply.Values under mixed N supply were 1.6–2.0 and 1.2–1.3 folds greater than under sole NO3–supply and sole NH4+supply,respectively (Fig.6-B).In roots,with the increase in NH4+supply as a proportion of total N supply,total amino acid contents increased significantly at 1–6 d.At 9 d,there was no significant difference in amino acid content of roots under mixed N supply and sole NH4+supply,but contents were 1.5 and 1.4 folds greater than that of the sole NO3–supply treatment (Fig.6-D).

3.5.Nitrate reductase and glutamine synthase activities

There was no significant difference in NR activity in shoots between the mixed N and sole NO3–supply treatments at 1–9 d,but both had significantly higher NR activity in shoots than under the sole NH4+supply treatment.In addition,there was no significant difference between the three treatments in root NR activity at 1–3 d,however the mixed N supply treatment had the highest NR activity in roots,which was 1.3–2.0 folds greater than under sole NO3–supply at 6–9 d (Fig.7-A and C).There was no significant difference in GS activity of shoots between mixed N supply and sole NH4+supply at 0–9 d,but values in both were significantly higher than under sole NO3–supply at 3–9 d,when they were 1.2–1.5 and 1.3–1.5 folds greater than under sole NO3–supply.In addition,the GS activity in roots under mixed N supply and sole NH4+supply was significantly higher than that under sole NO3–supply at 0–9 d,but in the 6–9 d period,the activity of GS enzyme under mixed N supply was significantly lower than under sole NH4+supply (Fig.7-B and D).

Fig.7 Nitrate reductase (NR) and glutamine synthase (GS) activity of shoots (A and B) and roots (C and D) under different forms of N supply at 1–9 d after treatment.NO3– only,mixed N and NH4+ only respectively represent 100/0,75/25 and 0/100 NO3–/NH4+ratios.Values are mean±SE (n=4).On the same day,significant differences at P<0.05 are shown with different letters;ns,no significant difference.

3.6.Plant growth after exogenous addition of NR and GS inhibitor and Gln

After addition of NR inhibitor,we found that shoot biomass was reduced under the three N supply forms.The difference in shoot biomass between the mixed N supply and sole NO3–supply treatment was significant,shoot biomass under mixed N supply being 1.3 folds greater than under sole NO3–supply.Shoot biomass under mixed N supply and sole NH4+supply was not significantly different.After addiditon of GS inhibitor,the shoot biomass showed a significant down trend with the increased NH4+supply.Shoot biomass under mixed N supply and sole NH4+supply were 0.7 and 0.5 folds of that under sole NO3–supply.However,after exogenous addition of Gln,we found that there was no significant difference in shoot biomass under three N forms supply.

4.Discussion

N absorption capacity of the majority of plants species is extremely variable across the life cycle.Studies have shown that there is a close link between plant growth and N absorption capacity (Clementet al.1978;Clarksonet al.1986;Garnettet al.2013).In the present study,compared to sole NO3–supply,mixed N supply promoted plant growth,and this has been reported in a variety of crops (Guoet al.2007;Liet al.2013),including maize(Georgeet al.2016;Wanget al.2019a,b).Our previous studies found that mixed N supply promoted carbon and N metabolism in maize,thereby promoting plant growth(Wanget al.2019a,b).However,whether the enhanced N metabolism under mixed N supply was related to plant N uptake rate was still unknown.In the present research,we found that,compared with sole NO3–supply or sole NH4+supply,mixed N supply could promote15N influx rate,and this promotion effect was more significant at 6–9 d(Fig.1-B).Studies have shown that there is an interactive effect on their uptake when NO3–and NH4+co-exist(Hachiya and Sakakibara 2017).For example,in barley,the addition of NH4+to NO3–supply not only inhibited NO3–influx rate to the roots,but also promoted NO3–efflux rate from the roots.InArabidopsis,the expression of geneAtNRT2.1may be inhibited when NH4+is present(Cerezoet al.2001;Wirthet al.2007).In the present experiment,although nitrate concentration in the mixed N supply treatment was only 75% of that in the nitrate alone treatment,the NO3–influx rate under mixed N supply only decreased slightly (81–91% at 6–9 d of that under sole NO3–supply;Fig.1-B).In addition,an13N experiment in rice showed that the addition of NO3–can promote NH4+influx and reduce NH4+efflux from roots,and similar results have also been found in oilseed rape andPopuluspopularis(Kronzuckeret al.1999b;Babourinaet al.2007;Luoet al.2013).In the present experiment,we also found a similar phenomenon.Although there was only 25% of the NH4+in the mixed N supply treatment as in the sole NH4+supply treatment,the NH4+influx rate under mixed N supply was 91–95% of that under sole NH4+supply at 6–9 d.Based on the results above,we infer that the enhancement of N absorption capacity under mixed N supply may be the main driving force for promoting maize growth,and a small amount of NH4+supply may be the main reason for the improvement in N absorption.Furthermore,we found that,compared with sole NO3–supply,although the instantaneous absorption capacity under sole NH4+was increased,plant growth was not increased significantly,this may be due to the different changes of N solution concentration between NH4+absorption rate in the longterm and instantaneous absorption rate in the short term.

The uptake of NO3–and NH4+in maize is usually regulated by related transporters.When the N requirement of maize plants was low,ZmNPF6.3(ZmNRT1.1) may have been mainly responsible for N absorption,however when plant N requirement was high,ZmNRT1.1may not have been able to fully meet plant N uptake requirements,and absorption of NO3–may have been regulated by a highaffinity transport system,such asZmNRT2.1,ZmNRT2.2andZmNRT3.1Aunder sole NO3–supply (Sabermaneshet al.2017).In the present experiment,ZmNRT2.1andZmNRT2.2had lower expression values at 0–9 d under the mixed N supply treatment compared with sole NO3–supply(Fig.3).It has been reported that NH4+inhibits high-affinity transporter gene expression in barley (Kronzuckeret al.1999a) andArabidopsis(Cerezoet al.2001).Higher free NH4+or amino acid concentrations in roots may be the main reason for inhibition of the expression ofAtNRT2.1(Hachiya and Sakakibara 2017),which may be the main reason for down-regulation in the three genes in this experiment.In addition,an interesting phenomenon is that NO3–influx rate under mixed N supply in the longer term was slightly lower or not much different to that under sole NO3–supply.Compared with sole NO3–supply,mixed N supply may have promoted the up-regulation ofZmNRT1.1A,ZmNRT1.1B,ZmNRT1.1C,ZmNRT1.2andZmNRT1.3in roots under long-term conditions (Fig.3).Therefore,we speculate that the expression of some lowaffinity transporter genes may be significantly enhanced in the long term to ensure sufficient NO3–absorption under mixed N supply,but this phenomenon may not be obvious in the short term.In this study,the expression ofZmNRT1.1Bunder mixed N supply was higher at 1 and 9 d than under the other two treatments,but there was no significant difference at other time points.We believe that the up-regulation ofZmNRT1.1Bunder mixed N supply in the short term (1 d) may have been a response to nitrate signaling,and the lack of significant changes later may have been due to the lack of significant phenotype changes.However,the up-regulation ofZmNRT1.1Bat 9 d may be related to the phenotypic promotion effect.Previous studies have shown thatZmAMT1.1AandZmAMT1.3are the main genes responsible for NH4+absorption in maize (Guet al.2013).In this study,we found that the expression ofZmAMT1.1Aunder mixed N supply was significantly higher than that under sole NH4+supply in the long term (Fig.4).In the longer term,the NH4+infulx rate to roots under mixed N supply was closer to that under sole NH4+supply,even though the concentration of NH4+in the mixed N supply treatment was only 25% of that in the sole NH4+supply treatment.Therefore,we infer that the greater expression ofZmAMT1.1Ain the mixed N supply treatment had a critical role in NH4+absorption.The enhancement of ammonium absorption by nitrate supply under mixed N supply may be the main reason for the upregulation ofZmAMT1.1A.

Previous studies have reported that NO3–is mainly assimilated in shoots,while NH4+is mainly assimilated in the roots (Marschner 2011).In present study,NR activity in shoots under mixed N supply was the same as that under sole NO3–supply,while the GS activity in the roots under mixed N supply was significantly greater than that under sole NO3–supply,thus greatly improving the N assimilation ability of plants (Fig.7).Thus,the N metabolism in plant was promoted,and the total amount of amino acids in the plant was increased.The inhibitor experiment further verified this hypothesis,compared with the control treatment,there was a greater difference in the shoot biomass between mixed N supply and single NO3–supply after added NR inhibitor.With the increase in NH4+supply,shoot biomass was decreased significantly after addition of GS inhibitor.Therefore,based on the above results,we further speculate that mixed N supply can not only ensure a similar NO3–assimilation level compared to that for sole NO3–supply conditions,but can also further promote N assimilation due to the small amount of added NH4+,which may the main reason for plant growth promotion where N is supplied as a mixture of NH4+and NO3–(Fig.8).

Fig.8 Maize growth after treatment with nitrate reductase (NR) and glutamine synthase (GS) inhibitors and Gln combined with different forms of N supply 9 days after treatment.100/0,75/25 and 0/100 respectively represent 100/0,75/25 and 0/100 NO3–/NH4+ ratios.Sodium tungstate (Na2WO4) and methionine sulfoximine (MSO) respectively represent nitrate reductase and glutamine synthetase inhibitors.Values are mean±SE (n=3).For the same treatment,significant differences at P<0.05 are shown with different letters.

5.Conclusion

In the long term,compared to sole NO3–or NH4+supply,mixed N supply promoted N absorption in maize plants,and this process may be regulated by the high transcriptional levels of the genes ofZmNRT1.1A,ZmNRT1.1B,ZmNRT1.1C,ZmNRT1.2,ZmNRT1.3andZmAMT1.1A.NR and GS activity in shoots and roots was greater under mixed N supply than under sole NO3–or NH4+supply,which promoted the assimilation of NO3–and NH4+as well as related N metabolism pathways in plants,and in turn the growth of plants was promoted.This process may enhance the utilization of N and C in plants.Furthermore,the enhanced utilization of C and N may have a feedback promoting effect on N assimilation and N absorption (Fig.9).

Fig.9 A model explaining the promoting effect of NO3– and NH4+ uptake under mixed N supply in the long term.NR,nitrate reductase;GS,glutamine synthase.The thick arrows represent the promoting effect;the dotted arrows represent positive feedback regulation.

Acknowledgements

The study was supported by the National Basic Research Program of China (2015CB150402),the National Natural Science Foundation of China (31672221 and 31421092)and the Science Foundation for Young Scholars of Tobacco Research Institute of Chinese Academy of Agricultural Sciences (2022C03 and 20211302).

Declaration of competing interest

The authors declare that they have no conflict of interest.

Appendixassociated with this paper is available on https://doi.org/10.1016/j.jia.2023.04.037