Nitrogen dioxide fumigation alters the glucosinolate and nitrate levels in pak choy (Brassica campestris ssp. chinensis)

Scientia Horticulturae, 56 (1993) 87-100

87

Elsevier Science Publishers B.V., Amsterdam

Nitrogen dioxide fumigation alters the glucosinolate and nitrate levels in pak choy ( Brassica campestris ssp. chinensis ) V.I. Shattuck*, W. Wang Department of Horticultural Science, University of Guelph, Guelph, Ont. N I G 2 W1, Canada (Accepted 30 June 1993)

Abstract

The influence of continuous short-term nitrogen dioxide fumigation on glucosinolate and nitratenitrogen levels in pal( choy was investigated. Hydroponically grown seedling and commercially mature plants were exposed to increasing concentrations (0.6, 1.2, and 1.8 pl 1-1) of nitrogen dioxide for 1, 3 and 5 days. Visual symptoms of NO2 fumigation included increased green leaf color and leaf distortion. The treatments altered the dry matter accumulation, total glucosinolate (GS) concentration and composition, and nitrate levels in plants. These changes depended on the stage of plant and tissue development, nitrogen dioxide concentration and exposure duration. Seven predominant glucosinolates were identified in pak choy with 1-methoxy-3-indolylmethyl-GS, 4-methoxy-3-indolylmethyl-GS, and 3-indolylmethyl-GSreported for the first time. In both seedlings and older plants, the proportion of indolyl-glucosinolatesincreased in response to increasing nitrogen dioxide concentration. The nitrogen dioxide treatments increased nitrate levels up to 21% in seedlings and 89% in older plant tissues. Variation in the total glucosinolate concentration and nitrate levels were not associated over the treatments. The implications of these findings on pak choy quality are discussed. Key words: Air pollution; Brassica campestris; Glucosinolate; Nitrate; Nitrogen dioxide Abbreviations: GS = glucosinolate (s)

Introduction

Pak choy (Brassica campestris ssp. chinensis) is a popular vegetable grown worldwide for its attractive glossy green leaves and blanched hearts. At times this vegetable is exposed to gaseous air pollutants when grown commercially around large industrial cities or in CO2-enriched greenhouses. The most troublesome air pollutants for plants include ozone, peroxyacetyl nitrate, nitrogen oxides, sulfur dioxide, and fluoride (Ormrod, 1978). Information is lacking on the effects of these air pollutants on pak choy metabolism. Nitrogen dioxide (NO2) is a widespread air pollutant that is generated when *Corresponding author.

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ILl. Shattuck, W Wang / Scientia Horticulturae 56 (1993) 87-100

nitrogen monoxide emissions from the combustion of fossil fuels are oxidized by atmospheric molecular oxygen or ozone (Wellburn, 1990). High concentrations of NO2 can cause acute damage or kill plants outright (Taylor and Eaton, 1966; Li Li and Shimazaki, 1988 ), but at low concentrations and short exposure times, can stimulate overall plant growth (Sandhu and Gupta, 1989 ). Unfortunately, plant responses to NO2 exposure are far from understood. One area needing more attention is the influence of NO2 on the quality of horticultural products. Nitrogen dioxide induced shifts in plant metabolism could adversely affect the quality of horticultural products without changing biomass. Glucosinolate (s) (GS) and nitrate-nitrogen are compounds that affect the quality of brassica plants. Glucosinolate degradation products contribute to the flavors ofbrassica vegetables while the excessive accumulation of nitrate in horticultural food products is undesirable. Glucosinolate and nitrate levels are also closely associated with the nitrogen metabolism of plants; GS are derived from amino acids (Fenwick et al., 1983). A common plant response to NO2 fumigation is a change in nitrogen metabolism (Troiano and Leone, 1977; Takeuchi et al., 1985; Ito et al., 1986). Nitrogen dioxide induced alterations in the nitrogen metabolism of pak choy could cause changes in the GS and nitrate levels, thereby influencing the quality of this vegetable. With this in mind, we studied the effects of continuous short-term NO2 exposure on GS and nitrate levels in hydroponically grown pak choy plants. Materials and methods

F~ hybrid pak choy plants, cultivar 'Mei Qin Choi', were germinated from seed in fiats containing vermiculite. Ten days after seeding, representative plants were transplanted to 25-1 plastic trays and grown hydroponically. Each hydroponic tray contained a removable black polyethylene inner lining and a tight fitting styrofoam lid with holes for the plants and an air intake hose. The plants were supported on the lids with a rockwool collar that inhibited light penetration into the solution. Each tray held 9-12 plants. Air was discharged at the bottom of each tray by an electric air pump to provide aeration. The hydroponic solution contained (rag 1-1 ): N, 98; P, 31; K, 176; Ca, 60; Mg, 49; S, 96; Fe, 1.1; Mn, 0.1; Zn, 0.05; Cu, 0.02; B, 0.5; Mo, 0.02; C1, 0.15. The nutrient solution pH was 6.0 and the ratio of NO3- to NH4 + was 3.7: 1. Our preliminary studies showed that pak choy plants developed well in this nutrient culture. The solution was maintained at 3 cm below the top of the container, and the temperature was 25 ° C. The solution was changed every 3 days which allowed us to maintain a constant pH and electrical conductivity ( 1.2 mS cm-~). Plants were grown in the Horticultural Science Department greenhouse prior to NO2 fumigation. High-pressure sodium lamps provided

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400 pmol s- 1 m - 2 of light at the top of plants during a photoperiod of 16 h. The greenhouse was maintained at 23/18 °C (day/night). Plants were exposed to NO2 at the four and 11 leaf stages, which corresponded to 25 days and 38 days after seeding, respectively. These represented two distinct developmental stages of pak choy, i.e. seedling plants and plants at an acceptable size for commercial harvest. The hydroponic flats were randomly assigned to four continuously stirred tank reactors (Heck et al., 1978). The treatments, comprising three to six plants each, consisted of an air control (0 ~11-1 NO2) and NO/concentrations of 0.6, 1.2, and 1.8 #11-1. Plants were removed from the chambers and evaluated after 1, 3, and 5 days of NO2 exposure. The NO/was provided from a pressurized cylinder ( 5000 ~11-1 ) and injected into the air flowing through the chambers at a rate of 1.5 m s- 1. The concentration of N O / i n each chamber was monitored seven times per hour by a Thermo Electron 14BE ChemiluminescentNO-NO/-NOx analyzer. The pH of the hydroponic solution was maintained between 5.8 and 6.2 during the experiment. Growth conditions for the plants during the NO/fumigation were similar to the greenhouse, with the exception of the light intensity, which was lower (200 pmol s- 1 m - 2). During and after the treatments, the plants were examined for visible NO/exposure symptoms. The plant portion above the styrofoam lid was harvested and flesh weight determined. Seedling plants were evaluated intact, but for older plants, the leaves including the stems were separated from each plant and placed into three developmental classes (mature, mid-development,young) for evaluation. After harvest, the plant tissues were quickly frozen in liquid nitrogen and stored at - 20 oC for 1 week. The frozen samples were freeze-dried, dry tissue weights obtained, then ground to a fine powder using an electric coffee grinder prior to sampling. Glucosinolates were extracted from 100 mg of ground tissue using 5 ml of boiling high-performance liquid chromatography (HPLC) grade methanol maintained in a water bath at 60°C for l0 min. A known quantity ofbenzylGS was added to each sample during the extraction as an internal standard. The tissue was re-extracted and the combined extracts were evaporated to dryness at 4°C on a Speed Vac Concentrator (Model SVC200H-115; Savant Instruments, Hicksville, NY). The residue was defatted by redissolving in 1 ml ofhexane then in 1 ml of water; the solution was vortexed, centrifuged and the upper hexane layer was discarded. To the lower layer containing the water soluble GS, 50 ~tl of a 0.5 M lead and barium acetate solution was added to remove proteins. The solution was vortexed then centrifuged. The supernatant was loaded onto prepared ion-exchange mini-columns and the GS desulfated using procedures described previously (Daun and McGregor, 1981; Sang et al., 1984). The desulfoglucosinolate solution was filtered through an aqueous 0.45 ~tm ACRO LC3A filter, then analyzed by gradient system HPLC (Waters Associates, Milford, MA). The HPLC system consisted of a Model

90

~
481 gradient programmer and Model 45 pumps. Separation was performed using a C 18 ( 5/~m ) reverse phase column (25 cm × 4.6 mm ) (Alltech, Avondale, PA) with a linear gradient (0-30%) acetonitrile/H20 over 70 min at a flow rate of 1 ml min- 1. Values of the various GS were adjusted according to their response factors (Buchner, 1987). The identity of individual GS were further confirmed using LC plasma spray mass spectrometry. For the determination of nitrate nitrogen, 250 mg tissue samples were mixed with water in glass vials, and shaken at a speed of 250 rev min- ~for 30 min. The mixture was filtered and the extracted nitrate was determined using a TRAACS 800 autoanalyzer (Tel and Heseltine, 1990). The data were evaluated using ANOVA and regression analysis. Where appropriate, the means were separated using Dunnett's test (Steel and Torrie, 1980). Results

When pak choy plants were fumigated with high NO2 concentrations (more than 5 #l 1-1 ) in preliminary experiments, acute foliar injury was evident during the first 24 h of exposure. Injury symptoms included necrotic flecking of the intraveinal tissues of both mature and immature leaves and/or numerous small irregularly shaped collapsed tissue areas on the upper leaf surface. With the NO2 concentrations and exposure durations used herein, acute foliar damage or leaf senescence did not occur. However, seedling and older plants exposed to 1.2 and 1.8 #1 1-~ NO2 had darker green leaves than the control by the second day of gas exposure, suggesting that the leaf chlorophyll content increased (Sandhu and Gupta, 1989). Plants exposed to 1.8/tl 1-1 NO2 for 5 days also had mature and immature leaves that cupped downward. The dry matter accumulation of plants after NO2 exposure for 1 and 5 days is shown in Table I. Linear and quadratic increases in plant dry weight occurred for plants over the 5 day exposure period (data not shown). The NO2 treatments had little effect on the average dry weight of seedlings (Table 1 ). Older plants exposed to 1.8/zl 1-~ NO2 for 1 day had greater dry weight and percentage dry matter than the control (Table 1). However, older plants continuously exposed to 1.8/~l l- 1NO2 beyond 1 day did not readily accumulate dry matter, and by Day 5 were significantly lower in dry weight than the control. Seedling and older plants exposed to 1.2 and 1.8/~1 l-1 NO2 for 5 days both increased in percentage dry matter above the control. In all cases, seedling GS concentration decreased over the 5 days of treatment (Table 1 ); the GS concentration in control seedlings decreased by 62%. The primary control seedling GS at Day 1 included 3-butenyl-GS and 2-phenylethyl-GS, which comprised 57% of the total GS concentration (Fig. 1). When seedlings were exposed to 1.2 #l 1-1 and 1.8 pl 1-~ NO 2 for 1 day the glucosinolate concentrations increased 36% and 19%, respectively above the

EL Shattuck, W. Wang/Scientia Horticulturae56 (1993) 87-100

91

Table 1 Influence of NO2concentrations and exposure duration on the dry weight (DW), dry matter percentage (DM%) and glucosinolate concentration (GS) of 'Mei Qin Choi' pak choy plants at different developmental stages

NO2

Seedling

concentration (#11 -t )

11

Commercial maturity 5

1

5

0.36 0.26 NS 0.35 NS 0.30 NS

2.93 3.09 NS 3.00 NS 3.86*

4.52 4.23 NS 4.53 NS 3.79*

7.8 7.8 NS 8.6* 8.5*

4.7 4.9 NS 5.4* 5.7*

5.9 5.6 NS 6.6* 6.9*

1.00 1.10 NS 0.51" 0.67*

2.03 2.53* 4.03* 3,90*

4.30 3.66* 3,73* 3.14"

D W (g)

0 0.6 1.2 1.8

0.15 0. l0 NS 0.13 NS 0.08 NS

DM%

0 0.6 1.2 1.8

7.4 8.8* 10.1" 8.1 NS

GS (#mol g -1 DW)

0 0.6 1.2 1.8

2.52 2.33 NS 3.44* 3.01"

Days of NO2 exposure. Values are expressed on a per plant basis and are non-significant (NS) or significantly different (*) from the control at the P< 0.05% level according to Dunnett's test.

control. This increase in GS concentration was accompanied by a change in the percentage GS composition and an increase in the indolyl-GS, such as 4methoxy-3-indolylmethyl-GS (Fig. 1 ). The GS concentration decreased and the percentage GS composition was altered in seedlings exposed to 1.2 and 1.8 gl 1-~ NO2 for 5 days. A linear increase in the indolyl-GS was accompanied by a linear decrease in alkyl-GS in seedlings exposed to increasing NO2 concentrations at 1 and 5 days (Fig. 1 ). The mean GS concentration increased in older control plants and in plants exposed to 0.6/zl 1-1 NO2 over the 5 day treatment period (Table 1 ). However, the mean GS concentration was lower on Day 5 than on Day 1 in plants exposed to 1.2 and 1.8% NO2. The primary GS on Day 1 in older control plants included 1-methoxy-3-indolylmethyl-GS and 2-phenylethyl-GS (Fig. 2), which comprised 57%, 54%, and 47% of the total GS concentration of the mature, mid-developed and young leaves, respectively. Overall, the GS concentration was highest in the young and lowest in the mature leaves of older plants. On Day 1, the GS concentration was higher in the mature leaves exposed to 1.2 gl 1- ~ NO2 and in the mid-developed leaves exposed to 1.2/tl 1- ~ NO2 than in control tissues (Fig. 2). A linear increase in GS concentration from

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141. Shattuck, W. Wang / Scientia Horticulturae 56 (1993) 87-1 O0

% Indolyl GS

A

25

29

33

39

-3

E

o

05

@ C' O0

0 6

12

NO 2 c o n c e n t r a t i o n

E

18

( #1 L - l )

3 bu[

[]~

1 methind

4 methind

[EEE~ 4 p e n t

2 pe

~

others

Fig. 1. Glucosinolate (GS) concentration and composition of 'Mei Qin Choi' pak choy seedlings at the four leaf stage following exposure to varying concentrations of NO2 for 1 (A) and 5 (B) days. The primary GS are presented in the staked bars in the same order as the symbol legend and includes: 3-butenyl-GS (3 but); 4-methoxy-3-indolylmethyl-GS (4 methind); 2phenylethyl-GS (2 pe ); 1-methoxy-3-indolylmethyl-GS ( 1 methind); 4-pentenyl-GS (4 pent ); and 3-indolylmethyl-GS and 2-hydroxy-3-butenyl-GS (others). Total indolyl-GS are expressed as a percentage o f the total detected.

4.3 to 9.1 g m o l g-1 dry weight ( D W ) in response to increasing NO2 concentration was noted in young leaf tissues. Alterations in the ratio o f alkyl-GS to the indolyl-GS occurred for the treatments and depended on the leaf developmental stage and the NO2 exposure concentration (Fig. 2). In mature leaves, 2-phenylethyl-GS decreased and 4-methoxy-3-indolylmethyl-GS increased as the NO2 concentration was raised. The percentage GS composition

93

El. Shattuck, W. Wang/Scientia Horticulturae 56 (1993) 87--100 Indolyl-GS A

60 43 51

75 44 50

64 47 50

78 54 4~

B6 40 30

77 32

~* I n d o l y l - GS 29 BO 48 41

B3 4~ 44

12

I0

fl

8

'i 2

0 12

10

8

6

2

o

o6 NO

3

but

2

12

concentration

18

(/~l L - t )

[]]]]m 1

methir~d

[~

4 methind

~

4 pent

m

2 pe

~

others

Fig. 2. Glucosinolate (GS) concentration and composition of'Mei Qin Choi' pak choy plants at the 11 leaf stage following exposure to varying concentrations of NO2 for 1 (A) and 5 (B) days. The primary GS in the mature (O), mid-developed (M) and young (Y) leaves are presented in the staked bars in the same order as the symbol legend and includes: 3-butenyl-GS (3 but ); 4-methoxy-3-indolylmethyl-GS (4 methind); 2-phenylethyl-GS (2 pc); 1-methoxy-3-indolylmethyl-GS ( 1 methind); 4-pentenyl-GS (4 pent); 3-indolylmethyl-GS and 2-hydroxy-3butenyl-GS (others). Total indolyl-GS are expressed as a percentage of the total detected. in young tissues remained relatively unchanged by the NO2 treatments at Day 1 as the GS levels in these tissues markedly increased. The GS concentration in the mature leaves o f plants exposed to increasing NO2 concentrations for 5 days were similar (Fig. 2 ). However, a striking linear increase in the indolyl-GS to increasing NO2 concentration along with changes in the percentage GS composition were evident in these leaves. The

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~ I. Shauuck, ~E. Wang / Scientia Itorticulturae 56 (1993) 87-1 O0

decrease in GS concentration from the control in plants exposed to 1.2 and 1.8/~l 1-1 N O 2 for 5 days (Table l ) was due to reductions in the GS concentrations in the mid-developed and young leaves (Fig. 2). The percentage GS composition in the different leaf tissues was affected by 5 days of N O 2 exposure; in some instances these alterations were striking. For example, 4-methoxy-3-indolyl-methyl-GS markedly increased in concentration above the control in mature (86%) and young (450%) leaves exposed to 1.2 #l 1-~ NO2 (Fig. 2). Foliar nitrate-nitrogen levels in pak choy ranged from 1.33 to 1.92% dry weight in seedlings and 0.86 to 3.85% in the tissues of older plants (Figs. 3 and 4). Nitrate levels in the control seedlings increased linearly over the 5 day treatment period. When seedlings were exposed to 0.6/zl l-~ NO2 for 1 day, 3 days, and 5 days, the nitrate levels were higher by 20%, 16% and 21%, respectively, compared with control plants (Fig. 3 ). Seedlings exposed continuously for 5 days to 1.8 #l l-1 NO2 were 12% higher in nitrate levels than the control plants. Mature leaves of control plants had the highest (:~= 3.26%) nitrate levels followed by the mid-developed (~=2.37%) and young (~= 1.14%) leaves (Fig. 4). Nitrate levels in the foliar tissues of older plants varied and depended on the N O 2 concentration and exposure period. Plants exposed to 0.6 #l l- 1 N O 2 showed nitrate accumulation in old and mid-developed leaves that was evident by the first day of treatment (Fig. 4). Nitrate accumulation by Day 1 also occurred in the mid-developed leaves of plants exposed to 1.8 #l 1-1 NO2. Plants fumigated with 0.6 #l l- ~ and 1.2 #l l- ~ N O 2 for 5 days had mid-developed leaves with 24% and 19% higher nitrate levels, E.1

v

1.9

o

k3 .~

1.7

o o Z

1.5

1.3 Z 1

3

5

Days

Fig. 3. Effect of NO2 concentration and exposure time on the nitrate-nitrogen concentration in 'Mei Qin Choi' pak choy seedlings. Seedlings were exposed to 0 (O), 0.6 ( • ) , 1.2 ( I ) , and 1.8 ( • )/~l 1-1 NO 2 for l, 3, and 5 days. Vertical bars represent the standard error of the mean. Error bars are smaller than the data symbols where not visible.

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V.I. Shattuck, W. Wang/Scientia Horticuhurae 56 (1993) 87-100

1.8

A

1.6

T

1.4 ~--~

1.2

1.0

;<

0.8 l

a.~ © •~

-

I

l

I

I

B

3.0

2.8

©

~-)

2,6 2. 4

© C)

Z

2.2

2.0 I

(1.)

I 4.1

C T

3.9 .,..~

3.7

Z

3.5 3.3 3.1 2.9 2.7 I

I

I

i

3

5

Days Fig. 4, Effect of NO2 concentration and exposure time on the nitrate-nitrogen concentration of young (A), mid-developed (B), and mature (C) 'Mei Qin Choi' pak choy leaves. Leaves were exposed to 0 (©), 0.6 (D), 1.2 (m), and 1.8 ( • ) H1l- l NO2 for I, 3, and 5 days. Vertical bars represent the standard error of the mean. Error bars are smaller than the data symbols where not visible.

respectively, than control plants. Plants exposed to 1.8 H11-1 N O 2 had significantly higher (15%) and lower (31%) nitrate levels in mature and young leaves, respectively, than the control. Changes in foliar nitrate and GS levels were not associated over the treatments. Discussion The short and continuous NO2 treatments (Table 1 ) caused alterations in the dry matter accumulation in pak choy which reflected disruptions in the

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v.L Shattuck, W. Wang / Scientia Horticulturae 56 (1993) 87-100

metabolism of plants by this air pollutant. These results are in accordance with previous studies (Taylor and Eaton, 1966; Ito et al., 1985; Sandhu and Gupta, 1989) showing that NO2 effects on plants were rapid, and either stimulated or inhibited dry matter production. Plants exposed to increasing NO2 concentration and treatment period generally suffer decreased dry matter production (Ito et al., 1985 ). A comparison of foliar biomass accumulation in seedlings and older plants after 5 days exposure to 1.8 #11- l NO2 indicated that seedlings were less sensitive to this concentration than older plants (Table 1 ). This is not that surprising since plant sensitivity to air pollutants is associated with the developmental stage of tissues, with greatest sensitivity typically occurring in older tissues (Ormrod, 1978 ). Seven predominant GS were identified in pak choy and included 3-butenylGS, 4-pentenyl-GS, 1-methoxy-3-indolylmethyl-GS, 2-phenylethyl-GS, 4methoxy-3-indolylmethyl-GS, 3-indolylmethyl-GS, and 2-hydroxy-3-butenyl-GS (Figs. 1 and 2). For the first time, 1-methoxy-3-indolylmethyl-GS, 4-methoxy-3-indolylmethyl-GS, and 3-indolylmethyl-GS are reported in pak choy. An earlier study (Cole, 1976) reported the presence of isopropyl-GS, 2-propenyl-GS, and 3-methylthiopropyl-GS in pak choy; these GS were not detected in our study. The differences in GS profiles between that paper and ours could be due to the use of different cultivars and plant growth conditions. It is well recognized that plant GS profiles are greatly influenced by the genotype and environment (Fenwick et al., 1983; Shattuck et al., 1991a). Glucosinolates are important because their breakdown products contribute to the characteristic odor and flavor of brassica crops (Fenwick et al., 1983) and may be goitrogenic and cytotoxic (Nishie and Daxenbichler, 1980; Uda et al., 1992). Consumer preference for brassica plant products could be affected by changes in the GS concentration and profile of these products. Marked differences in the total GS concentration and GS profiles were detected between control seedlings and older control plants (Table 1; Figs. 1 and 2). For example, 3-butenyl-GS predominated in seedling tissues, while in older plants l-methoxy-3-indolyl-methyl-GS was overall the most abundant GS. Moreover, changes in the GS concentration and profile occurred during the 5 day treatment period. Thus, the developmental stage determined the GS concentration and composition in pak choy foliage. Our findings are consistent with previous studies that showed that GS concentration and profiles varied in different plant tissues and fluctuated in these tissues during normal plant development (Clossais-Besnard and Larher, 1991 ). The total GS concentration and profiles in seedlings and older plants were altered and the concentration and relative proportion of indolyl-GS increased in response to the NO2 treatments (Table 1; Figs. 1 and 2). Changes in the GS concentration and profiles can occur when plants are subjected to stress during development. These changes can occur without visual symptoms of plant injury being apparent. In certain instances indolyl-GS have been ob-

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served to accumulate in tissues from plant stress, such as injury (Koritsas et al., 1989; Bodnaryk, 1992 ), disease infection (Rausch et al., 1983 ), nutrient deficiency (Shelp et al., 1992), and low temperature exposure (Shattuck et al., 1991b). Amino acids are the precursors of GS. Two-phenylethyl-GS and the GS containing the 3-indolylmethyl ring structure are derived from phenylalanine and tryptophan, respectively, with the remaining GS originating from methionine (Haughn et al., 1991 ). Our data suggested that the NO: treatments increased the activities of the pentose phosphate and shikimic acid pathways, causing an increased production of tryptophan and ultimately an accumulation of indolyl-GS (Shelp et al., 1992). Nitrogen nutrition affects GS changes within and among plant tissues. It is well recognized that the GS content of brassicas generally increases when nitrogen fertilization rates are elevated (Gustine and Jung, 1985 ). Unfortunately, the physiological regulation of nitrogen induced GS production in plants is not known at this time. The biological importance of stress induced GS changes also remains to be established. The NO2 flux into plant leaves is complex and depends on many factors, including stomatal and cuticle conductance, mesophyll resistance, the nitrogen status and nitrogen metabolism of plants, the light intensity during NO2 exposure, and the NO2 movement and concentration over leaf surfaces (Yoneyama et al., 1980; Rowland-Bamford and Drew, 1988; Wellburn, 1990). Gaseous NO2 reacts with extracellular water or the cytoplasm to form nitrate or nitrite ions (Wellburn, 1990), which are rapidly converted to the ammonium ion by the activities of nitrate and nitrite reductase. The resulting ammonium ion is rapidly converted to amino acids and proteins through the normal pathway of nitrogen metabolism (Ito et al., 1986; Wellburn, 1990). We hypothesized that pak choy plants grown with sufficient nitrate-nitrogen might accumulate foliar nitrate following NO2 treatment if nitrate accumulation exceeded nitrate assimilation. Under the conditions of our experiment, the foliar nitrate concentrations of the older pak choy controls were relatively high, but not unusual. For example, the nitrate levels in the edible parts of certain vegetables can frequently exceed 2500 mg kg-1 fresh weight; this is especially true of products from greenhouse environments (Corr6 and Breimer, 1979). When seedling and older plants were fumigated with NO2, nitrates rapidly accumulated in the foliage. Seedling and certain tissues of older plants contained consistently higher nitrate levels following the 0.6 pl 1-1 NO2 treatment, while in the other treatments, the foliar nitrate levels varied with respect to the stage of plant and leaf development, the NO2 concentration, and the treatment duration. In our study, we did not discriminate if these elevated foliar nitrate levels were from the nutrient culture or the NO2 treatment. Nevertheless, these higher nitrate levels suggest that the NO2 treatments affected the nitrate content in plants.

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Recent consumer demand for horticultural products of high quality has prompted interest in the nitrate-nitrogen levels in vegetables. The accumulation of nitrates in edible plant parts is a concern in human and livestock health, and working standards for nitrate levels in water and foods have been suggested in certain European countries (Corr6 and Breimer, 1979). Although the NO2 concentrations examined herein are seldom found outdoors, it is possible that levels approaching 0.6 ]tl l-1 NO2 could accumulate in domestic and commercial glasshouse environments where hydrocarbon burners are used to provide CO2 enrichment (Capron and Mansfield, 1975, 1976; Ashenden et al., 1977 ). Under these conditions, pak choy plants grown with a sufficient supply of nitrate-nitrogen might be prone to accumulating nitrates and altered GS content in foliar tissues. Alternatively, the NO2 could respond additively or synergistically with other air pollutants, such as SO2 arising during CO2 enrichment, and produce growth reductions and/or plant injury (Hogsett et al., 1984). Not surprisingly, these data indicated that chemical rather than biomass changes were the first plant signal of N O 2 induced stress in pak choy. Thus, attention should be given to both the deviations in normal growth and chemical composition of pak choy when assessing the effects of this air pollutant on the quality of this vegetable. References Ashenden, T.W., Mansfield, T.A. and Harrison, R.M., 1977. Generation of air pollutants from kerosene combustion in commercial and domestic glasshouses. Environ. Pollut., 14: 93-100. Bodnaryk, R.P., 1992. Effects of wounding on glucosinolates in the cotyledons of oilseed rape and mustard. Phytochemistry, 31: 2671-2677. Buchner, R., 1987. Response factors. In: J.P. Wathelet (Editor), Glucosinolates in Rapeseed: Analytical Aspects. Martinus Nijhoff, Boston, pp. 50-58. Capron, T.M. and Mansfield, T.A., 1975. Generation of nitrogen oxide pollutants during CO2 enrichment of glasshouse atmospheres. J. Hortic. Sci., 50: 233-238. Capron, T.M. and Mansfield, T.A., 1976. Inhibition of net photosynthesis in tomato in air polluted with NO and NO2. J. Exp. Bot., 27:1181-1186. Clossais-Besnard, N. and Larher, F., 1991. Physiological role of glucosinolates in Brassica napus. Concentration and distribution pattern of glucosinolates among plant organs during a complete life cycle. J. Sci. Food Agric., 56: 25-38. Cole, R., 1976. Isothiocyanates, nitriles and thiocyanates as products of autolysis of glucosinolates in Cruciferae. Phytochemistry, 15:759-762. Corr6, W.J. and Breimer, T., 1979. Nitrate and nitrite in vegetables. Lit. Surv. 39, Department of Soil Fertility, Agricultural University, Wageningen, Netherlands, 83 pp. (Unpublished.) Daun, J.K. and McGregor, D.I., 1981. Glucosinolate analysis of rapeseed (Canola). Canadian Grain Commission, Winnipeg, 27 pp. Fenwick, G.R., Heaney, R.K. and Mullin, W.J., 1983. Glucosinolates and their breakdown products in food and food plants. CRC Crit. Rev. Food Sci. Nutr., 18:123-201. Gustine, D.L. and Jung, G.A., 1985. Influence of some management parameters on glucosinolate levels in Brassica forage. Agron. J., 77: 593-597.

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