HUANG Hui-ying,LI Huan,XIANG Dan,LIU Qing,LI Fei,LIANG Bin

College of Resources and Environmental Sciences,Qingdao Agricultural University,Qingdao 266109,P.R.China

Abstract In order to completely evaluate ammonia emission from greenhouse vegetable fields,crop canopy absorption should not be neglected.The foliar uptake of NH3 applied at two growth stages and the subsequent 15N-labeled N translocation to other plant components were investigated under greenhouse conditions using chambers covered with the soil of a tomato field.Treatments comprised three NH3-N application rates (70,140,and 210 mg/plot)using 15N-labeled ammonium sulfate.Plants were harvested immediately after exposure for 24 h,and the total N concentrations and 15N/14N ratios were determined.With increased NH3 concentration,total 15NH3-N absorption increased considerably,whereas the applied 15NH3-N uptake decreased gradually.The tomato plants absorbed 33-38% and 24-31% of the 15NH3-N generated at the anthesis and fruit growth stages,respectively.A total of 71-80% of the recovered NH3 was observed in the leaves and 20-30% of the recovered NH3 was remobilized to other components.Among them,an average of 10% of the absorbed 15NH3-N was transferred into the tomato fruits.All these results indicated the potential of the tested tomatoes for the foliar uptake of atmospheric 15NH3 and the distribution of 15N-labeled vegetative N among different plant components.The results are of great importance for the complete evaluation of nitrogen use efficiency in the greenhouse tomato fields.

Keywords:greenhouse tomato,15N,foliar ammonia absorption,NH3,canopy

1.Introduction

Greenhouse production has become economically important,especially in northern China (Heet al.2007).In the major vegetable production regions of China,N fertilizer application rates are approximately 1 100-1 500 kg N ha?1in the north (Heet al.2007; Juet al.2009)and 900-1 300 kg N ha?1in the south (Huanet al.2007;Shiet al.2009; Minet al.2011).Excessive N input leads to low N use efficiency,because the losses of N,through nitrification,denitrification,immobilization/mineralization,leaching,and volatilization,increase as the N input increases(Boarettoet al.2013).Among these pathways,ammonia volatilization is one of the important components of soil N loss in vegetable land.Studies have demonstrated that ammonia volatilization can deplete up to 24% of the total N applied in vegetable field soil (Gonget al.2013).If urea is co-applied with composted manure,the high urease activity of compost may stimulate urea hydrolysis,thus leading to more ammonia volatilization loss (Matsushimaet al.2009).So,the amount of N loss through NH3volatilization cannot be ignored.

With the occurrence of NH3diffusion from the soil surface to the atmosphere,part of the “l(fā)ost” NH3can be taken up by the leaves of crops (Hutchinsonet al.1972;Schoningeret al.2018).The foliar uptake of NH3is determined by the NH3compensation point of the plant(Farquharet al.1980)and the NH3concentration in the atmosphere (Gessleret al.2002).The NH3compensation point is defined as the NH3concentration in the interior of the leaves where gains and losses of the gas are equal.The leaf absorption of NH3occurs when the vapor pressure of NH3at the canopy level after N fertilization is above the leaf compensation point (Sparks 2009).The exchanges between the canopy and atmosphere are also affected by the amount of fertilizer application,environmental conditions,and crop growth characteristics of the plants (Krupa 2003).Several researchers have measured NH3uptake using the15N isotopic dilution technique in crops,such as wheat(Pinget al.2000),rice (Ashrafet al.2003),coffee (Fenilliet al.2007),Tanzania grass (Martha Júnioret al.2009),orange trees (Boarettoet al.2013),and maize (Schoningeret al.2018).However,the NH3uptake by tomatoes in the greenhouse remains unknown.In addition,the canopy absorption of volatilized ammonia has mainly concentrated on field crops.However,studies on greenhouse vegetables are rarely reported (Caoet al.2006).

Different from the crops grown in low-N applied agricultural systems and open-air environments,greenhouse tomato plants usually have higher levels of applied fertilizers and are grown in semi-enclosed conditions.On one hand,high rates of nitrogen fertilizer (several times the amount used in wheat or maize)are applied in greenhouse vegetable fields to maximize production (Ju and Christie 2011).However,after the application of side-dress urea,NH3concentrations in the atmosphere next to the emission source (i.e.,urea on the soil surface)can be elevated,favoring foliar NH3uptake(Schoningeret al.2018).Furthermore,the greenhouse is a semi-closed environment; thus,volatilized ammonia cannot immediately spread to the outside air environment (Salazaret al.2015),which might provide greater residence time of volatilized NH3in the air in the vicinity of plant foliage (Fenilliet al.2007).Therefore,the NH3-N canopy uptake in such intensive vegetable production remains to be explored.

The objective of this paper is to study the foliar uptake of15NH3under three ammonia concentrations at two growth stages (anthesis and fruit growth stages)and determine the distribution/fate of the15N-labeled vegetative N to different plant components by using15N-labeling.

2.Materials and methods

2.1.Experimental site and design

The greenhouse study was carried out during the growing season of tomatoes from December 2017 to May 2018 at the Shouguang Experimental Station (36°9′N,118°7′E)of China Agricultural University located in Shandong Province on the North China Plain,which is one of the intensive greenhouse tomato production bases.The region has a typical continental monsoon climate featuring warm and rainy summers and dry,cold winters.The average annual temperature is 12.4°C and the precipitation is 558 mm.One typical non-heated greenhouse (100 m×11.5 m),which is constructed of clay walls and covered with polyethylene film,was randomly selected for this study.The experiment was arranged in a continuous greenhouse tomato system which had been cropped since 2014,with a typical double cropping system per year.The experimental site has a loamy sand soil containing 1.8 g of total N,13 g of organic C,77.2 mg of available P (Olsen-P),and 133.6 mg of available K (NH4OAc-K).The pH (H2O:soil,2.5:1,v/v)is 7.14.In the growing season,the total amount of nitrogen fertilizer was 750 kg N ha?1as urea and potassium nitrate.In addition,45 kg ha?1P as single superphosphate and 90 kg ha?1K as K2SO4were broadcasted and incorporated before planting.Tomato was planted on December 10,2017,with stand density of 32 500 plants ha?1.

The experiment was conducted in the chambers under the greenhouse at the Shouguang Experimental Station.The experiment used a randomized complete block design with three replicates.Chambers with tomato plants separated by at least 5 m were selected from the experimental site.Six uniform sets of tomato plants were exposed to15NH3at anthesis stage (60 days after transplanting)and two uniform sets of tomato plants were exposed at fruit growth stage(115 days after transplanting),respectively.The exposure was carried out in an airtight transparent polyethylene chamber(1.4 m×1.0 m×0.7 m,length×width×height),equipped with a circulating fan,thermometer and hygrometer.To avoid the diffusion of gaseous15N into the soil and absorption by plant roots,plastic mulching was used to cover the soil surface to ensure that the NH3gas could only be taken up by the foliar surfaces.During the exposure period for plants at the anthesis stage,the temperature and relative humidity inside the chamber were 22-30°C and 76-80%,respectively,whereas the corresponding values were 24-32°C and 81-83%,respectively,during exposure at the fruit growth stage.At each stage,plants were exposed to three15NH3concentrations (70,140,and 210 mg of15NH3-N (99.12 atom%15N excess))in the closed chambers for 24 h.15NH3was produced by the reaction of (15NH4)2SO4with sodium in 1 mol L-1NaOH solution.During each15NH3exposure,one set of control plants was also kept under similar culturing conditions.

2.2.Sampling and analysis

All plants in each chamber were harvested immediately after exposure,and the plants were fractionated into different components.Tomato shoots were harvested by cutting at the soil surface and fractionating into the three stem,leaf,and fruit components.Roots were washed out of the soil using a 2-mm mesh.The plant leaves,stems,fruits and roots were washed carefully by distilled water before the analyses.Shoots and roots were oven-dried at 65°C for 48 h and weighed.Total N concentrations and15N signatures were determined by a coupled system comprising an elemental analyzer (Model NA2500,CE Instruments,Milan,Italy)and an isotopic ratio mass spectrometer using combustion gases (Delta Plus; Thermo Electron Corp.,San Jose,CA).

2.3.Statistics and data analyses

For the tomato components,the percentage of N derived from fertilizer (Ndff)was calculated using the isotopic dilution equation described by Boaretto (2013)as follows:

Ndff (%)=[(AT%15Nsamp?AT%15Nuft)/(AT%15Nfert?AT%15Nuft)]×100

Ndff (mg/plant)=[(AT%15Nsamp?AT%15Nuft)/(AT%15Nfert?AT%15Nuft)]×N content in plant components

where Ndff (%)is the percentage of N derived from fertilizer,AT%15Nsampis the atom% of15N in the sample,AT%15Nuftdenotes the atom% of15N in unfertilized tissues,and AT%15Nfertis the atom% of15N in the15NH3.The total amount of N recovered from fertilizer by tomato was calculated using the determinations of dry mass,total N,and Ndff (%)of the components.

Statistical analysis of the data was tested by ANOVA and significant difference (LSR)at the 5% level using the SPSS software package version 19.0 (SPSS Institute,Inc.,Cary,NC).

3.Results

Increased NH3concentrations increased the Ndff (%)values of all components of the plants at the two growth stages considerably (P＜0.05).Furthermore,the relationships among the Ndff (%)values of the different components were as follows: leaf＞stem＞root at antithesis stage (Fig.1-A),and leaf＞stem＞fruit＞root at fruit growth stage (Fig.1-B).Comparing the two growth stages,the Ndff (%)values of leaves were substantially increased whereas those of roots were considerably reduced; while stems were not different at anthesis stage from those at fruit growth stage.

The quantity of15N derived from15NH3(Ndff values)depended on the atmospheric concentrations and this tendency was similar for the Ndff (%)values at the anthesis and fruit growth stages.Comparing the two growth stages,the Ndff values of leaves were substantially increased;values of stems were not different at the two growth stages;and the values of fruits were significantly increased at the fruit growth stage (Fig.2).

Fig.1 Ndff (%)in different parts of tomato under three ammonia concentrations at anthesis (A)and fruit growth stages (B).Ndff (%)is the percentage of N derived from NH3 absorbed by the tomato of each experimental plot.N1,N2 and N3 stand for three 15NH3 concentrations (70,140,and 210 mg of 15NH3-N)in the closed chambers,respectively.Different letters in three ammonia concentrations are significantly different at P＜0.05.Bars represent mean±SD (n=3).

Fig.2 Ndff (mg/plant)in different parts of tomato under three ammonia concentrations at anthesis (A)and fruit growth (B)stages.Ndff (mg/plant)is the quantity of N derived from NH3 absorbed by the tomato of each experimental plot.N1,N2 and N3 stand for three 15NH3 concentrations (70,140,and 210 mg of 15NH3-N)in the closed chambers,respectively.Different letters in three ammonia concentrations are significantly different at P＜0.05.Bars represent mean±SD (n=3).

Fig.3 15N accumulation of the whole plant (leaf+stem+root+fruit)under three ammonia concentrations at two growth periods.N1,N2 and N3 stand for three 15NH3 concentrations (70,140,and 210 mg of 15NH3-N)in the closed chambers,respectively.Different letters in three ammonia concentrations are significantly different at P＜0.05.Bars represent mean±SD(n=3).

With increased15NH3concentration,total15NH3-N absorption (mg/plant)increased.Furthermore,total15NH3-N absorption (mg/plant)increased considerably at the fruit growth stage compared with that at the anthesis stage(Fig.3).However,with increased15NH3concentration,the uptake of the applied15NH3-N decreased considerably(exposure of plants to 140 mg and 210 mg NH3-N application was insignificantly different at anthesis stage).Uptake of the applied15NH3-N decreased considerably at fruit growth stage compared with that at anthesis stage (Fig.4).The tomato plants absorbed 33-38% and 24-31% of the15NH3-N generated in the canopy under different ammonia concentrations at the anthesis and fruit growth stages,respectively.The absorption rate was approximately 1.4 times higher when plants were exposed to15NH3at anthesis stage compared with that at fruit growth stage (Fig.4).

The15NH3absorbed by plants was recovered in the leaves(75.9-77.8%),stems (20.1-22.2%),and roots (1.9-2.1%)at the anthesis stage and in the leaves (71.5-79.9%),stems(12.3-15.0%),roots (0.3-0.7%),and fruits (7.4-12.8%)at the fruit growth stage (Table 1).These distributions indicate that a total of 71-80% of the recovered NH3was observed in the leaves and 20-30% of the recovered NH3was remobilized to other components.Among them,an average of 10% of the absorbed15NH3-N was transferred into the tomato fruits.

Fig.4 The absorption of 15NH3-N applied in the foliar under three ammonia concentrations at two growth periods.N1,N2 and N3 stand for three 15NH3 concentrations (70,140,and 210 mg of 15NH3-N)in the closed chambers,respectively.Different letters in three ammonia concentrations are significantly different atP＜0.05.Bars represent mean±SD (n=3).

4.Discussion

Due to frequent irrigation,long-term moist soil and higher soil temperatures in greenhouse production,soil evaporation and plant transpiration were more pronounced than in open-air cultivation.By adding large amounts of fertilizer,ammonia volatilization occurs readily,and even a large amount of ammonia volatilization loss may be caused by open air in the greenhouse during daytime (Heet al.2005).Although NH3volatilization was not the major pathway of N losses from the greenhouse due to several reasons(lowered pH,high soil moisture level,and a closed system),assessing the fate of NH3emitted from the soil surface is necessary to make a closed nitrogen cycle (Gallowayet al.2004).

Table 1 Recovery of the 15NH3-N absorbed by foliage under three ammonia concentrations at two growth periods

Earlier studies have shown that plants can absorb substantial amounts of NH3through leaves (Hutchinsonet al.1972); therefore,part of the volatilized fertilizer N can again be reused by plants during its convective diffusion to the atmosphere (Fenilliet al.2007).Evidence of NH3absorption from the atmosphere and N assimilation by leaves has been obtained by monitoring the NH3decrease within a plant chamber or using the15N tracer technique(Castroet al.2006; Fenilliet al.2007; Boarettoet al.2013;Schoningeret al.2018),which demonstrated the NH3uptake from the volatilized N up to 7-43%.Although variable,the estimates of foliar uptake suggest that it is a consequential pathway for N entry into agroecosystems (Sparks 2009).In the experiments conducted here,the tomato plants absorbed 33-38% and 24-31% of the15NH3-N generated in the canopy at the two growth stages,and these results are the first report of ammonia volatilization in vegetable fields.Some similar studies reported the uptake of15NH3volatilized from labeled urea for wheat (3.3%,Sommeret al.1993;11%,Pinget al.2000),maize (14.8%,Schoningeret al.2018),orange tree (7%,Boarettoet al.2013)and Tanzania grass (16%,Martha Júnioret al.2009),at levels much lower than our findings.One of the possible reasons was that the tomato plants grown in chambers were protected from the wind,which provided longer residence time of volatilized NH3on the vicinity of plant canopy.

Absorption was affected considerably by growth stages.Following the anthesis stage,an average of 35%of volatilized N was recovered in the tomato plants,while 28% of volatilized N was recovered following the second stage.Lower recoveries following the second stage were probably due to less physiological activity of the tomato plants at this time.Part of the leaves are under the shade of the upper part of the canopy; thus,low light incidence and low photosynthetic activity may be occurring,consequently leading to a restriction in stomatal opening (Taiz and Zeiger 2010).

Suttonet al.(2001)reported that plant NH3uptake may be responsible for the N-nutrient status of the vegetation.NH3stimulation requires a corresponding increase in nutrient acquisition to maintain the plant N metabolism and nutrient balance (Chenet al.2010).Nitrogen uptake is repressed at high internal N levels and becomes depressed when N supplies are limited (Glass and Siddiqi 1995).Moreover,plants growing on low soil N take up more leaf-derived N than those fertilized with high N (Chenet al.2010).However,in this study,the Ndff (%)and Ndff values of all components of the plants increased with the NH3concentrations immediately after NH3exposure at both growth stages (Figs.1 and 2),which indicated that enriched atmospheric NH3may have direct “fertilizer” effects on plant nutrient uptake.The probable reasons for such findings are as follows: (1)A considerable NH3uptake by the tomato canopy at different growth stages is in agreement with earlier studies with maize(Porteret al.1972)and Italian ryegrass (Wollenweber and Raven 1993)and may be attributed to a linear NH3influx gradient between the atmosphere and inside the plant(Holtan-Hartwig and Bockman 1994).(2)Although plants normally take up N by the roots (Glasset al.2002),when excessive N application inhibits root growth and nutrient uptake (N fertilizer application rates are approximately 1 100-1 500 kg N ha?1in intensive production),plant shoots can absorb NH3from the atmosphere and partially replace the root system as an additional N source (Pérez-Sobaet al.1994).(3)A high demand for carbon skeletons due to NH3assimilation was responsible for increased CO2fixation and stomatal conductance,which are regulated by internal CO2concentrations (Van Hoveet al.1987).NH3uptake may cause an autocatalytic increase in additional NH3flux into the leaves by inducing stomatal openingviathe internal CO2level as long as the photon flux density is sufficient for equivalent photosynthesis (Van der Eerden and Pérez-Soba 1992).

Grundmanet al.(1993)demonstrated two possible modes of atmospheric NH3-N absorption and assimilation in plants: a fast uptake with immediate assimilation and a fast storage with progressive metabolism.The NH3is rapidly assimilated into organic compounds in the shoot to avoid the physiological damage caused by toxic endogenous levels of free ammonium in the leaf cells (Pearson and Stewart 1993; Tamaki and Mercier 2001),and it is then redistributed to the growing organs and reserves (Yoneyamaet al.2003).In the present study,71-80% of the recovered NH3was observed in the leaves,indicating that the leaves were the main NH3absorption organ.Furthermore,20-30% of the recovered NH3was observed in stems,roots,and fruits,which suggested that absorbed15NH3was remobilized to other components when plants were immediately harvested after exposure.Similar with our experimental results,Ashrafet al.(2003)found that soon after NH3exposure at different rice growth stages,recovery of a small portion of15N-labelled N in the roots indicated an immediate assimilation and translocation of the absorbed NH3towards other plant parts.This phenomenon occurs because the NH3absorbed through the leaves is a rapidly utilizable N form for the plant (Zhanget al.2011)provided that carbon skeletons are sufficient to incorporate these N into organic forms (Adriaenssenset al.2012).

To date,a few studies on the impact of foliar NH3uptake and translocation to grain have been reported.Ashrafet al.(2003)showed that approximately 15% of the absorbed15NH3-N was recovered in the rice grain.The15NH3absorbed by orchards trees that was recovered in the fruits was approximately 6% (Boarettoet al.2013).In the present study,foliar uptake of applied15NH3and the subsequent distribution of15N-labeled N to other components were different at the two growth stages.An average of 10%of the absorbed15NH3-N was transferred into the tomato fruits (Table 1).This finding suggests that atmospheric NH3can be absorbed as an additional N source by the plant and can affect fruitage N status.The absorbed foliar NH3was probably incorporated into simple or complex organic compounds,such as amino acids and amides (Ashrafet al.2003).Therefore,further investigations are necessary to understand the relationship between foliar NH3uptake and fruit quality.

5.Conclusion

Our results showed for the first time that a considerable amount of foliar NH3uptake by tomato occurs when plants were exposed to high15NH3for a limited period.Therefore,not all N fertilizer emitted as NH3from the soil can be considered lost from the plant-soil system.In addition,the absorption was strongly affected by different growth stages.Moreover,atmospheric NH3can be absorbed as an additional N source by plants and can affect fruitage N status.The factors controlling NH3absorption and the direction of NH3absorbed by the crop canopy are still largely unknown and would be worthy of further investigation.

Acknowledgements

This research was funded by the National Key Research and Development Program of China (2017YFD0200106).We are grateful to Prof.Chen Qing from China Agricultural University for valuable comments on an earlier version of this manuscript.We are also thankful for the constructive comments received from anonymous reviewers and the editors.