Indian Journal of Experimental Biology Vol. 37, July 1999, pp. 701-705

Ammonia volatilization during moisture stress from wheat (Triticum aestivum L.) plant canopy

Ajay Arora l & Jitender Mohan2

ID ivision of Plant Physiology, Indian Agricultural Research Institute, New Delhi 1100 12, India.

2Department of Botany, JV College, Baraut, Meerut, India

Received 14 August 1998; revised 2 March 1999

The physiological and biochemical factors that govern the exchange of NH) between plants and air were studied together with method for measuring the gas exchange. The investigations were conducted with Uniculm 'Gigas ' culti var of wheat with soil moisture was maintained at two different levels of moisture stress. It was clear from the data, that water stressed plants lost more nitrogen in the form of NH) than the control, irrigated plants. The neutral protease acti vity wat> found to be significantly higher in water stressed plants at all the growth stages after anthesis and max imum at 2 1 days after anthesis as compared to the control plants. eutral protease acti vity and an1monia loss were well corrt:lated wi th each other. No positive relationship was observed between ammoni a loss, transpiration rate and carbon diox ide exchange rate. The result of prescnt study confi rmed that senescent and moisture stressed ti ssues achieved relati vely high Nil ) vo latili.zati on rates as compared to the control plants.

Ammonia volatilization directly from the shoots of agricultural plant spec ies has been observed under both fi e ld conditions l

•2 and under laboratory

conditi ons3-6 . Natura l vegetation also may til some cases emit NH3 to the atmosphere7

.

Vo latilization of NH3 results when NH/ -generating processes (e.g. , protein degradation) exceed assimilation reaction s, primarily those catalyzed by g lutamate synthase (GOGA T) and g lutamine synthetase (GS)6. Therefore, evolution of N H3 is o ften greatest when N remobilization rates are hi gh, such 3S d uring reproducti on and senescence, or WhUl N availab ility is high7

•8

.

A number of workers have reported that the magnitude of losses is s ignificant3,6,9. However, their magnitude v is-a-vis an effect on the overall balance sheet is a subject wh ich needs further investigation 10.

A great deal of indirect ev idences points to a loss of nitrogen from the crops between heading and maturity. A lo~s of 45 kg N ha· 1 was estimated from the fo liage of wheat during the growth stages between anthes is and maturit/ I. Stud ies with the main shoot offield grown wheat l2 and barle/ 3 have shown that a cons iderable amount of N is lost from the shoot before N accumulation sta rted in the grain .

Response of vo latilizati on to water stress have been studied very little . The reports of volatilization available at present under stress are controversial. According to one groupl 4, an increase in ammonia

loss was observed under water stress , wh i Ie the other group5 observed the vo latili zatio n decresed in mild water stressed wheat

The objective o f the present experiment "vas to assess the magnitude of gaseous ammonia losses during flowerin g to maturity under water stress and to e luc idate relationships between protease enzyme and ammonia loss.

Materials and Methods The experiment was carri ed o ut in the pot c ulture

of the Divi s ion of Pl ant Phys io logy, Indi an Agricultural Research Institute, New De lhi . Uni culm ' Gigas' cultivar (with s in g le culm o nl y) of wheat (Triticum aestivum L. ) served as exper imenta l materia I for th is study. The seed mate ri a I \Va obtained from the Wheat Directo rate, Indi an Agricultural Researh In stitute, New Delhi.

The experiment was conducted under natura l condition in 12x 12 inches pots containing 10 kg of soil. Seven seeds of culti va r ' Gi gas' were sown on 2nd November, 1996, in pots containing a m ix ture o f sandy loam so i I and fa rm ya rd manure. After germination the emerged plants were thinned to four per pot at the two leaf stage.

The first split dose of nitrogen in the form of urea (having 46% N ), v iz. @ 60 kg ha· 1 was g iven a long with the basal dressing of phosphorus and potass ium at the time of sowing eaeh as superphosphate and

702 INDIAN 1. EXP. BIOL., JULY 1999

muriate of potash, respect ively. Second dose of nitrogen viz. @ 60 kg ha-I were given at 25 days after sow lllg.

The pots were watered uniformly with measured amount of water upto anthesis, and thereafter, soil moisture was maintained at two different level s of mo isture stress by providing two litres of water per pot in the case of irrigated soil while the amount of water used for unirrigated plants was hal f litre per pot. Water stress treatment was given one week before anthesis by reducing the amount of water to one half of the pots . Plants were sampled at one week interval started from anthesis to maturity.

Ammonia volatilization measurements The measurements took place in a closed enclosure

chamber, in which there .. was no contact between ambient air and air inside the chamber during the experiment. The measurement started from anthesis onwards on potted wheat plants by placing a cylindrical, ptexiglass box over the plants. The surface of the soil in the portion of the pot covered by the plexiglass was sealed with a flexible but impervious silicon seal so that gas exchange from the soil dose not interfere with the measurements5

.

Pressurized, filtered NHJ- - free air (by using boric acid 5%) with a relative humidity of 60% was let into the chamber @ 30 L min-I . The temperature in the plant enclosure chamber during the measurements was 24°C and the light intensity approximately 300 Ilmol m-2s-l. The air inside the chamber had a slightly overpressure in order to avoid intrusion of outside atmospheric air. A parallel control with no plant was also employed. No attempt was made to measure the evolution of oxides of nitrogen. The released ammonia was trapped in 2% solution of boric ~cid. The remaining NHJ adsorbed to the chamber was flushed after removing the shoot. Then the chamber were wiped with moistened paper. The paper were digested in acid and analysed for N-content. No additional N was recovered from the paper indicates that flushing "the chamber wall was sufficient to removed any adsorbed NHJ. Ammonia was estimated following the procedure of Novozamsky el at. 15. The volatilization measurements were made for four plants per pot for 2 hr. and later it is converted and presented on the basis of per plant per hour.

Neutral protease enzyme estimation-Samples were homogenized in 10 ml of extraction medium. (PH 7.5). The homogenate was centrifuged at 25000

rpm for 20 min . Neutral protease content was assayed by the method of Frith et at. 16 and the amino acid analysis was done following the method of Lee and Takahashi l7 . The absorbance was read at 570 nm.

Photosynthetic measurements--A closed flow portable photosythesis system Model LI-6200 (L1-COR) was used for measuring the photosynthet ic rate . Data were collected between 10 and I 1:30 hrs.

Transpiration measurements - Transpiration rate was determined by using steady state porometer (L1COR, LI-1600).

Water potential measurements- The topmost fully expanded leaf was used to determine the leaf water potential using plant water status console (Model-3005, Soil moisture equipment corp. , Ca lifo rnia, USA), following the procedure of Scholander et at. 18

Statistical analysis-.Standard p. rrors of the experimental results were calculated on the basi s of the squre root mean errors obtained in the analysis of variance.

Results Leaf water potential and transpiration rate- The

flag leaf water potential varied from - 0.9 MPa at

athesis to - 1.6 MPa at maturity in control plants. 1 n

treated (water stressed) plants it varied from - 1.3 MPa at anthesis to -2.4 MPa at maturity. The transpiration rate varied from 3.21 Ilg cm-2s- 1 from

anthesis to 4.29 Ilg cm-2s- 1 at 7 days after anthesis (DAA) in control plants. Thereafter, it declined till maturity. In treated plants, transpiration rate was

maximum at '7 DAA (3.82 Ilg cm-2s- I), thereafter, it declined till maturity (Table 4).

Neutral protease activity (NPA)- The NPA (p H

7.5) increased from 53.2 (Ilmol g lycine equivalent h-Ig-Idw) at anthesis to 92.5 (Ilmol glycine equivalent h-Ig-Idw) at 21 DAA, but, thereafter, it s lowly declined in control plants. In treated plants, the activity of the enzyme was higher than the control plants from 64.4 at anthesis to 162 .2 at 21 DAA (Table I).

Ammonia loss (nmole NH3 plant' H')--Ammonia losses were low at anthesis (10.33 nmoles NHJ planr l h- I) and maxnmum at 21 DAA (35.20 nmoles NH J

planrl h- I). Thereafter, it declined. In the treated plants, it was more than the control plants and it varied from 12.38 to 48.41 nmoles NH, planrl h-I, 21 DAA (Table 2).

ARORA & MOHAN: AMMONIA VOLA TIUZA TION DURING MOISTURE STRESS 703

Carbon-dioxide exchange rate (CER)--The flag leaf blade photosynthetic rate declined from anth~s' (21.69 Ilmol m·2s· l) to maturity (5.36 )lffiol m·2sil in control plants. In treated plants, it varied from 7.79 Jlmol m·2s·1 at anthesis to 3.19 at maturity (28 DAA). The photosynthetic rate in the treated plants was less than the control plants at all the growth stages studied (Table 3).

Discussion The earlier studies on whel\t, from our laboratories,

on the basis of indirect evidence, it was shown that considerable amount of N is lost from flowering to maturityl9. Accordingly, the present experiment was carried out to study the magnitude of gaseous ammonia losses during this period under water stress. Since the measurements were run over a period of only two hours no influence of plant age could be

Table I-Neutral protease enzyme activity (Ilmol glycine equiv . h·1 g. ldw) of control and water stressed wheat plants at different

growth stages after anthesis [Values are mean ±SE]

Treatment Da;ts after anthesis 0 7 14 21 28

Control 53 .20 58.45 65.52 92.47 64. 11 ±6.24 ±7.35 ±4.67 ±12.37 ±0.77

Water stressed 64.36 58.43 95.30 162.1 j' 81.08 ±3 .01 ± 1.64 ±3 .34 ± 14.29 ±6.30

Table 2-AmmQJl.ia loss (nmol NHJ planrlh· l) from wheat plant canopy at different leaf water potential (MPa)

[Values are mean ±SE]

Treatment Da;ts after anthesis 0 7 14 21 28

Control 10.33 13.57 14.54 35 .20 15.58 ±1.32 ±1.28 ±4.65 ±4.96 ±0.28 (-0.9) (- 1.0) (- 1.1 ) (-1.6) (- 1.6)

Water stressed 12.38 15 .57 17.74 48.41 15.19 ±3 .34 ±3 .02 ±3 .23 ±3 .26 ±0.77 (- 1.3) (- 1.4) (- 1.7) (- 2.1 ) (- 2.4)

'rable ~arbon dioxide exchange rate (')..lmol CO2 m·2 S· I) of control and water stressed wheat plants at different growth stages

.after anthesis [Values are mean ±SE]

Treatment Da;ts after anthesi s 0 7 14 21 28

Control 21.69 12.32 10.48 6.29 5.36 ±L29 ±2.07 ±2.36 ±0.77 ±1.59

Water stressed 17.79 11.52 8.42 3.54 3. 19 ±0.69 ±0.29 ±0.96 +1.74 +0.13

Table 4--Transpiration rate (Ilg cm·2 s· ljof control ana-water stressed wheat plants at different growth stages aft~r antlutsis

[Values are mean ±SE]

Treatment Da;ts after anthesis 0 7 14 21 28

Control 3.21 4.29 3.31 2.80 2.43 ±O.II ±0.36 ±0.21 ±0.21 ±0.02

Water stressed 2.49 3.82 2.80 2.17 1.75 ±0.06 ±0.05 ±0. 14 +0.1 7 +0. 11

seen on NH3 emission. Experiments run over longer periods have shown an increase in NH3 emission in reproductive stages of growth relative to vegetative stages2.5, . During reproductive growth stages NH3 generation can be very different from that in young plants due to senescence induced protein degradation and utilization of retranslocated nitrogen com­pounds20,2 I.

It is clear from our data as well as of others I4,22

that, water stressed .plants lose more N in the form of NH3 than the control, irrigated plants. Under water stress, protein breakdown by enzyme proteinases and specifically neutral proteases, led to high free NH4 +

levels as comJ1ared to the control plants. The neutral protease activity (PH 7.5) in our experiment was found to be significantly higher in water stressed plants at all the growth stages after anthesis and maximum at 21 days after anthesis (162.15 Jlmol glycine equivalent h·1g.1 dw at a water potential of -2. 1 MPa) as compared to the control plants (92.47 Jlmol glycine equivalent h·1g.1 dw at a water potential of -1.6 MPa) 21 days after anthesis (Table 1). These facts supported the observed higher loss of N from the water stressed plants.

Increase in the neutral protease activity in the flag leaf blades of both water stressed and control plants in our experiment was observed 7 days after anthesis and maximum peak was observed at 21 days after anthesis, might results in failure of the reassimilation to cope with the production of free NH/. This will lead to build up of free NH/ and the result would be a steep concentration gradient between the ambient

atmosphere and the tissues allowing the release of NH3 more in water stressed treatment than the control plants. Other workers also observed the strong relationship between N loss and proteinase activity but the/ suggested that the bimodal response of N H3 volatilization may be due to a double burst of acid proteinase activity23. We observed on Iy one burst of

704 INDI AN 1. EX P. SIOL. , l UL Y 1999

neutral protease activity and that was at 2 1 days after anthes is (F ig. I). Others5 also coul d not found th e biomodal response and suspect th at it is not always expressed. Periods of increased NH, vo lati lization from anthes is to maturi ty re fl ect a change in the balance between NH4 + releasing reactions (deamina­ti on reactions, nitrate reducti on, senescence-induced proteo lys is) and NH/ - uptake reactions (N tra nsport and NH/ ass imilation via glutamine synthetase that shi fts in favour of NH/ release, resu lting in the establishment of new, higher steady-state ti ssues [NH/ ].

Both photosynthes is rate ana NH, vo lati !iza ti on appeared to be strongly influenced by plant leaf age, although considerable temporal variability in gas exchange was also observed when measurements were expressed on a leaf area bas is. No relationship between NH3 vo latili zati on and photosynthesis or transpiration was fo und in the present experiment. Correlation between photosynthesis and NHl vo lat ili­zation have been described for spring wheat' but the influence seemed to be relative ly small. Us ing single encased leaves of soybean') fo und a correlat ion between transpiration and NH1' vo latili zation. Others24 suggested that transpiration dependence may be overriden by vari ations wi th temperature.

Ammonia emiss ion from plant leaves occurs when the atmospheric concentration of NH.,' is below the NH3' compensation point, i.e. the concentration of gaseous NHl above the water film present in the mesophyll cell walls (apoplast) . In contrast, ammoni a is absorbed if the atmospheric NH, concentrati ons is higher than the NH,' compensation point. The NH, compensation point in growing plants seems in most cases to be below 3 Ilg NHJ m·J

7,20 . In the present study, the equilibrium concentration of NH,' in the plant enclosure was lower as those typica lly measured above agricultural cropland2

,7.25. The emiss ions reported by many workers mostly fa ll in the range of 5-50 g NHr N ha" dai' with the majori ty of the measured lossess below 35 g NH3-N ha" dai' . As emissions vary with conditions of temperature, water status and plant development stage, daily emiss ions can not be directly converted to yearly lossess. However, where yearly lossess are reported they mostly are in the range of 1-2 kg N ha" . When conditions are unfavourable for high crop yields, emiss ions can be larger which is in agreement with the present study (1 .5 kg ha" month" )

15o r---------------~------------~ 250 -- Am mo n,a IO S8 ( e ) SE M (:!: )

~ - A mmo n ia 103 8 ( W S ) -<- N P A (e)

:i --- N P A ( W S )

~ :::I: 1 00 z 0 E E-O> ..

-2 .. 5 0 'c 0 E E

""

2 0 0 ""i" " ,F

1 50 ~ .~

0> 100 (5

E .3. ;t

5 0 Z

oL-----------------------------~o o 5 10 1 5 20 25 30

D a y s afte,. anthesl s

Fig. I-Relationship between tOlal ammon ia loss and neutral protease enzyme acti vity (NPA) of control and water stressed wheat pl ants at di ffe rent growth stages alkr anthesis

Acknowledgement The author acknowledge the fi nancial ass istance

from CS IR, New Delhi , in the form of senior research fe llowship. Authors al so thankfu l to the Head, Division of Plant Phys iology, IARI fo r prov iding necessary facilit ies.

References I Harper L A, Sharpe R R. Langdale G W & Giddens J E.

;/gron J, 79 ( 1987) 965 2 Schjoerring J K. Ky lli ngsbaek A. Mortensen J V &

l3yskov-Nielsen S, Plant Cell Environ, 16 ( 1993) 16 1 3 Mo rgan J A & Parton W J. Crop Sci, 29 ( 1989) 726 4 O' Deen W A & Porter L K. Agron J, 78 ( 1986) 746 5 Parton W J, Morgan J A, Attenhofen J M & Harper L A,

Agron J. 80 ( 1988) 4 19 6 Schjoerring .ll K. in Trace gas emissions by plants, edi ted by T

D Sharkey. E A Holl and & H A Mooney (Academic Pn:ss Inc, New York) 267

7 Sutton M A, Schjoerring 1 K & Wyers P. Phil Trans R Soc Lond A, 35 1 ( 1995) 26 1

8 Rabe E. J Horl Sci, 65 ( 1990) 23 I 9 Stutte C A Weil and R T & I3lem A R. Agrol1 J, 7 1 ( 1979) 95

10 Chatterj ee S R & Abrol Y P, Fertilizer Ne ws, ( 1990) 13 I I Daigger LA. Sanders 0 II & Paterson G A, Agron J, 68

( 1977) 85 1 12 Abrol Y P. Kumar P A & Nair T V R. Adv Cereal Sci & Tech.

6 ( 1984) I 13 Chatte ~i ee S R. Pokhriya l T C & Ahrol Y P. J bp BOi. 33

( 1982) 876 14 We ilandR T&StLllteCA,CropSci, 19 (1979)545 15 Novozamsky I, Van Eek R. Van Schouwenburg J Ch &

Wallinger L, Neth J IIgric Sci, 22 ~ 1974) 3 16 Frith G J T, Bruce D G & Dalling M J. Plant Cell Physiol. 16

(1 975) 108 5 17 Lee Y R & Takahashi T, Analy t Biochem, 14 ( 1966) 7 1 18 Scholander P F, Hammel H T. Hemm ingsen E A & Bradstreet

E 0, Proc Nat Acad Sci, USA 52 ( 1964) 11 9 ' 19 Abrol Y p, Kaim M S & Nai r T V R, in Fundamental

ecological & agricultural aspects of nitrogen metabolism in higher p lants, edited by H. Lambers J J. Neeteson & I. Stu len

ARORA & MOHAN : AMMONIA VOLATILIZATION DURING MOISTURE STRESS 705

(Martinus Nijihoff Publishers, Dordrcht) 1986, 399 20 Berger M G, Woo K C, Wong S C & Fock H P, Plant

Physiol, 78 (1985) 779 21 Kamachi K, Yamaya T, Mac T & Ojima K, Plant Physiol, 96

(1991 ) 411 22 Heckathorn S A & De Lucia E H, Decologia. 10 1(3) (1 995)

36 1 23 Dalling M J, Boland G & Wilson J H. Aust J Plant Physiol, 3

( 1976) 721 24 Stune C A & Wei land R T, Crop Sci. 2 1 ( 1978) 596 25 Sutton M A, Fowler D, Moncri efI' J B & Storeton-West R L.

Q J R Metor Soc. 11 9 ( 1993) 1047