PESTICIDE BIOCHEMISTRY AND PHYSIOLOGY 12, 156- 162 (1979)

Effects of Ametryn [2-(ethylamino)-4-(isopropylamino)-6-(methylthio)-

s-triazine] on Nitrate Reductase Activity and Nitrite Content of
Wheat (Triticum aestiwum L.)’
KATHLEEN CHURCHILL AND LOWELL KLEPPER"
Department of Agronomy, University of Nebraska. Lincoln. Nebrasha 68583
Received December 14, 1978; accepted June 27. 1979
The objectives of this study were to show that: (a) a herbicide, such as ametryn, which interferes
with the photosynthetic electron transport system, causes nitrite to accumulate in illuminated
leaves and (b) that nitrite is toxic and contributes to the herbicidal damage and death of the plant.
Tests were conducted on wheat seedlings grown on 5 mM nitrate, 5 mM ammonia, and zero
nitrogen. Ametryn treatment decreased in viva and in vitro nitrate reductase activity (NRA) within a
26-hr period. In virv NRA decreased more rapidly than in vitro NRA. Compared with control
tissue, only 3% in klo NRA remained at the end of 26 hr. The in rive assay conducted in light
confirmed the inhibition of photosynthetic electron flow by ametryn within the leaf tissue. Nitrate-
grown, ametryn-treated plants accumulated nitrite and, after 10 days were the only plants that were
completely desiccated and dead. Ammonia- and zero-nitrogen. ametryn-treated plants did not
accumulate nitrite. were only partially chlorotic after the IO-day period, and were still living. Low
levels of NO,,, (NO, and/or NO) emissions were demonstrated by nitrate-grown ametryn-treated
plants.

INTRODUCTION providing carbohydrate for glycolysis and

Most plants take up nitrogen from the soil
as nitrate but must reduce it to ammonia
before it can be utilized. Nitrate reductase
(NR) is the first enzyme in the reductive
process, mediating the reduction of nitrate
to nitrite. It is located cytoplasmically (1),
has a rapid turn-over rate (2), is nitrate-
inducible (3), exhibits diurnal variation (4),
and is subject to environmental control (5).
The electron donor for NR is reduced
nicotinamide adenine dinucleotide (NADH)
derived from cytoplasmic oxidation of
glyceraldehyde-3-phosphate (6) or malate
(7). Photosynthesis indirectly supplies
energy necessary for nitrate reduction by
* Cooperative investigations of the Nebraska Ag-
ricultural Experiment Station and the Agricultural Re-
search Service, USDA, Lincoln, Nebr. under Con-
tracts AIDicsd-1208 and AID/cm/ta-C-73-71 with the
Agency for International Development, Department of
State. Published as paper 5685, Journal Series,
Nebraska Agricultural Experiment Station.
Z Now at Department of Botany. University of Kan-
sas, Lawrence, Kans., and University of Nebraska,
Lincoln, Nebr.
156
0048-3575/79/060156-07$02.00/O
Copyright @ 1979 by Academic Press. Inc.
Ail rights of reproduction in any form reserved.
Kreb’s cycle operation. Photosynthetically
derived energy is also necessary for con-
tinued induction (8) and resynthesis of NR
(9). Nitrate reduction is not thought to
occur in darkness under a normal, aerobic
environment (5, 10).
Nitrite, the product of nitrate reduction is
further reduced by nitrite reductase (NiR)
to ammonia. NiR is located in the chloro-
plast (1) and receives its electrons from
photosynthetically reduced ferredoxin (9,
11). Nitrite, a potent toxin, is only barely
detectable in intact, healthy plants and is
thought to occur in an “enzyme-bound”
form (11).
Klepper (12, 13) demonstrated nitrite ac-
cumulation in green leaf tissue when photo-
synthetic inhibitor herbicides were included
in the in viva nitrate reductase medium and
the assay was conducted in light. He con-
cluded that nitrite accumulation was due to
interference in photosynthetic electron flow
which was necessary for nitrite reduction.
Rates of nitrate reduction were not af-
fected. Several researchers (14- 16) have
 EFFECTS OF AMETRYN ON NITROGEN METABOLISM OF WHEAT 157

confirmed the inhibition of nitrite reduction Ametryn Trecrtmerlt

by photosynthetic inhibitor herbicides. Be- The leaves (8-10 cm) of 7- to lo-day-old
cause of its known toxicity and reactivity, seedlings were dipped into an aqueous so-
nitrite was proposed as a phytotoxin re- lution composed of 1 mg/ml (4.4 mM ame-
sponsible for rapid injury and death of tryn), 5% ethanol, and 0.15% Tergitol 15-S-7
plants treated with photosynthetic inhibitor (a nonionic surfactant). Excess solution
herbicides (12). was immediately shaken off to prevent ac-
It is well established that plants treated cumulation of the solution at the base of the
with photosynthetic inhibitor herbicides do plant. The plants were replaced into growth
not slowly die of starvation. Visible chambers after a 30-min drying period.
phytotoxic symptoms often appear rapidly Control plants were treated in an identical
and do not resemble the appearance of manner except that ametryn was not in-
plants merely kept in darkness. It has been cluded in the treatment solution. All plants
suggested that due to an interaction be- were exposed to 2-3 hr light prior to ame-
tween the herbicide and light, some free tryn treatment.
radical is formed and is responsible for the
Sampling tmd Assays
rapid injury symptoms (17).
To assure representative samples, 15
Experiments were designed to: (a) ob-
leaves were harvested from each of four
serve the effect of ametryn on nitrogen
different pots for each treatment. The
metabolism as reflected in the in Go and in
leaves were cornposited and then cut into 7-
vitro nitrate reductase assays, (b) examine
the phytotoxicity of ametryn to wheat to lo-mm sections and then divided into
duplicate samples for analyses of in ~*i~*rt
(Triticum arstivtrm L.) seedlings grown on
NRA, in vitro NRA, and nitrite.
different sources of nitrogen and, (c) inter-
The in \*itro NRA analysis has been pre-
pret the results toward obtaining a better
viously described by Eilrich and Hageman
understanding of the interrelationship be-
( 19). The in \,i\~j assay in darkness and light
tween the mode of action of a photosynthet-
was basically that described by Klepper
ic inhibitor herbicide such as ametryn and
(13) except that 0.15% Tergitol 15-S-7 was
nitrogen metabolism.
included in the reaction medium.
For nitrite determinations, 0.25 g of leaf
MATERIALS AND METHODS
sections were homogenized by a Polytron
Plant Culturr homogenizer in a 4:l solution of wa-
Wheat (var. Atlas 66) was grown in ter:methylene chloride (25). After centrifu-
growth chambers in a vermiculite base in gation (lO,OOOg), 2 ml of the clear, aqueous
180-ml Styrofoam pots. Light intensity was portion was analyzed for nitrite as previ-
1500 peinsteins/sec/m? for a 14.5-hr period. ously described (12).
Temperature was maintained at 20°C during The method for NO,\-, measurements has
the light period and 10°C during the dark. been previously described ( 19). All experi-
Seedlings were watered daily with 100 ml of ments were conducted at least twice. Stan-
a modified Hoagland’s solution (18). The dard errors of treatment means (SE) are in-
nutrient solution was modified by supplying cluded in each figure legend. Treatment
nitrogen as 5 mM NO;, 5 mM NH:, or zero means ? standard deviations (SD) are re-
nitrogen. Additional CaCl, and KC1 were ported in tables.
supplied to maintain proper ionic balance.
N-Serve [2-chloro-6-(trichloromethyl)pyri- RESULTS AND DISCUSSION
dine] (0.1 ppm) was included in all nutrient Control wheat leaves from plants raised
solutions to prevent bacterial nitrification of on 5-M nitrate exhibited typical diurnal
ammonia. variation of in \,i\*o NRA (Fig. 1). Little in
 CHURCHILL AND KLEPPER

thetic inhibitor, ametryn, appeared to chem-

ically mimic the effect of manual depriva-
tion of CO, or light. The low levels of in \>itro
NRA in the ammonia-grown plants also
decreased.
Diurnal variation of in \yi~~j NRA was also
observed in nitrate-grown control wheat
leaves (Fig. 2). Ammonia-grown plants
contained very low levels of in L+IW NRA.
In \li\~ NRA of nitrate-grown, ametryn-
treated plants began a steady decline after
treatment, similar to that of in ritro activity
(Fig. I) but at a faster rate. In r,i\~) NR not
only reflects the presence of NR but also
the nitrate reduction potential of tissue
under accelerated glycolytic conditions (6)
(induced by anaerobiosis). A decrease in in
\~~LYINRA could indicate a decrease in NR,
carbohydrate supply, cofactor availability,

HOURS AFTER TREATMENT

FIG. 1. In lho nitrate rcdrrc~tase uc’tivify oj’cwntrol
( -J- or umetryn (----rreuted bvheat seedlings
raised on 5-mM nitrate ( 0) or ammonia (0). Shaded
area repwsrnts dark prriod. (SE c~fc~r,ntn)/-arnmonitr~ni~7
means. 0.07: of c,ontrol-r7itrrrtr merrns. 0.78: 0.j
Irn7efr?n-clmmorlia ~7ea~7.s. 0.09: c7nd o,f rrmcIfr.vn-
,7itrrltc n7eat7s. 0.34.)

~?tro NRA was found in ammonia-raised

seedlings. This was expected because NR is
a substrate-inducible enzyme. The fact that
some NRA was present indicated either
that the ammonia-grown plants were not
entirely nitrate-free or presence of a low
level of a “constitutive” enzyme.
Since neither nitrate nor NADH was lim-
iting in the in vitro assays, they represented
a measure of the enzyme activity under op-
timal conditions. Thus, fluctuations of in
~itvo activity reflected changes in the rela-
tive rate of degradation and synthesis or in-
activation and activation of the enzyme.
The in \litro NRA of the nitrate-grown,
ametryn-treated tissue began a steady de-
cline shortly after treatment. Numerous re-
searchers have shown that plant tissues de-
prived of light or CO, exhibit a steady de-
cline in NR (5, 10, 21, 22). The photosyn-
 EFFECTS OF AMETRYN ON NITROGEN METABOLISM OF WHEAT 159

or endogenous nitrate supply. A portion of IO’

the rapid decrease of ilz viva NRA can be 9.

attributed to the decrease in in vitro NRA
(Fig. 1). After 26 hr, approximately 30% of
8-
the in i*itro NRA remained in the nitrate-
grown ametryn-treated plants compared to r-
control plants while only 3% of the in rho
NRA remained. This indicated that al- 6-
though an appreciable level of NR was still
present, probably little nitrate reduction
was occurring.
It is well known that CO, fixation is
quickly inhibited in plants treated with a
photosynthetic inhibitor herbicide such as
ametryn. Any reduction in carbohydrate
supply due to herbicide treatment should be
reflected in rates of irz ~~ivo NRA since
NADH generation for nitrate reduction is
closely linked to glycolytic oxidation of
carbohydrate (6). This effect also agrees
2 6 IO 14 I8 22 26
well with a study by Aslam and Huffaker HOURS AFTER TREATMENT
(231 which demonstrated that although NR FIG. 3. In vi\v nitrute rrductase crctirnity. conducted
was present, the photosynthetic inhibitors in light. of control and umetryn-treated, nitrate-grobvn
diuron [3-(3,4-dichlorophenyl) - 1,1- u,heat srrdlings. Shaded area represents dark period.
dimethylurea], atrazine [2-chloro-4- (SE c;f control means. 0.10: of arnetcn means. 0.22.~
(ethylaminol-6-(isopropylaminol-s-tri-
azine] , and simazine [2-chloro-4,6-bis present in the leaf tissue and was actively
(ethylamino-s-triazine] indirectly stopped inhibiting photosynthetic electron flow
the flow of electrons necessary for ni- necessary for nitrite reduction.
trate reduction. In a companion study over Even though photosynthetic electron
several days, analyses revealed that this flow was severely inhibited by ametryn,
decrease of in \~i~~~and in tlitro NRA caused thus limiting carbohydrate synthesis neces-
by ametryn treatment was irreversible (data sary for nitrate reduction, nitrate reduction
not shown). could have continued for a period utilizing
The in rvit~j NR assay is normally tested reserve carbohydrates as it does in the
in the dark but can also be conducted in anaerobic in ~vi\~ assay (no carbohydrate
light if a photosynthetic inhibitor is present substrate supplied). Any reduction of ni-
in the leaf tissue. This provides a measure trate under these circumstances would re-
of the amount of inhibition of nitrite reduc- sult in nitrite accumulation.
tion by the herbicide (12, 13). Only the Abnormally high concentrations of free
nitrate-grown, ametryn-treated plants ac- nitrite were found in the nitrate-grown,
cumulated any significant amount of nitrite ametryn-treated seedlings shortly after
(Fig. 3). Large amounts of nitrite accumu- treatment (Fig. 4). Nitrite concentration of
lated in this light assay immediately after 0.64 pmol/g fresh wt for the nitrate-grown,
treatment with ametryn, and declined ametryn-treated wheat 6 hr after treatment
throughout the day, mirroring the decline in is more than 30 times higher than that found
the in \lilw NRA assay conducted in dark- in control plants (0.005-0.020 pmol/g fresh
ness (Fig. 2). When conducted in light, this wt) .
assay provides evidence that ametryn was For nitrogen metabolism studies. wheat
160 CHURCHILL AND KLEPPER

TABLE 1

Fresh trnd Dry Weight of Wheat Seedlings Gro,t,n on

Different Nitrogen Sources IO Days ufter
Trecttment \l,ith 1000 ppm Ametryn (Mean + SD
c?f‘ Three Replic,ates)

Wheat

Nutrient Fresh wt Dry wt

medium Treatment 02) (!a

Zero N Control 5.96 * 0.02 0.93 t 0.01

Ametryn 2.95 i- 0.19 0.49 -c 0.06
Control IV) 49.5 53.1
5lIlMN Control 6.80 2 0.49 1.17 k 0.09
as NH: Ametryn 2.56 + 0.65 0.52 i 0.03
Control (%I 31.7 44.4
5mMN Control 9.91 k 0.41 1.50 +- 0.09
as NO, Ametryn 0.50 2 0.01 0.47 + 0. I I
Control (%) 5.1 31.3

ametryn-treated seedlings were smaller

than their controls and contained partially
chlorotic areas but appeared relatively
healthy. The ametryn-treated zero-nitrogen
0 2 6 IO 14 16 22 26 plants closely resembled the treated
HOURS AFTER TREATMENT
ammonia-grown plants. The nitrate-grown,
FIG. 4. Endogenorrs nitrite concentration of control
( -)- or ametryn (---)-treated w,heat seedlings ametryn-treated plants were dead (com-
raised on 5-mM nitrate ( 0) or ammonia (0). Shaded pletely chlorotic and desiccated). A com-
area represents dark period. (SE of control-crmmonia panion study using oats as the test plant
means. 0.00: of c.ontrol-nitrate means. 0.01: c!f produced identical results (data not shown).
ametyvn~mmonia means. 0.00: and of ametryn+i-
trate means, 0.04.)
In a similar study, Fedtke (15) grew oats
on 5-M nitrate or ammonia and treated
them with diuron. He reported neither free
is commonly grown on higher nitrate nutri- nitrite in the leaf tissue nor visible herbici-
ent media than was used in this study (12, da1 effects within 24 hr. However, if plants
13, 24). However, in preliminary experi- were subjected to anaerobic conditions,
ments, visible NH’, toxicity was observed both nitrate- and ammonia-grown plants
in wheat whenever concentrations greater died (only the nitrate-grown plants ac-
than 5 mM NH: were used. Nitrate levels cumulated nitrite during this treatment). He
had to be lowered in order to obtain valid concluded that there was no relationship
comparisons between treatments. In other between herbicidal effect on nitrogen
experiments with identical ametryn treat- metabolism and physical injury symptoms.
ments, but with wheat grown on 15 mM In comparison with this study, his in rsi\~
NO; nutrient solutions, nitrite concentra- NRA values were very low, possibly due to
tions up to 1.5 pmol/g fresh wt were mea- a low light environment or that his mea-
sured. surements were not made until approxi-
Within 24 hr, only the nitrate-grown, mately 24 hr after treatment. In our study, a
ametryn-treated seedlings began to wilt. large portion of metabolic damage had oc-
The study was terminated 10 days after curred within 24 hr but no visible injury
treatment. Fresh and dry weights of the other than wilting was observed. In his ex-
treated plants were determined (Table I). A periments, an anaerobic environment and
zero nitrogen treatment was included for a blockage of photosynthetic electron flow by
further comparison. The ammonia-grown, diuron should have impaired ammonia in-
 EFFECTS OF AMETRYN
corporation. Further plant uptake of NH+,
could have produced toxic effects. The
method he reported for nitrite measurement
is not as sensitive as the nitrite extraction
technique used in this study (25). According
to our data (Fig. 3), nitrite accumulated to
its maximum 6 hr after treatment and was
very low 24 hr after treatment.
Nitrite is an extremely reactive com-
pound, particularly under the mildly acid
conditions within the plant cell. If it had
reacted with substances with which it is ca-
pable (amines, amino acids, proteins,
phenols, tannins, hemes, indole acetic acid)
( 12) it would no longer have been detectable
but would have disappeared. By reacting
with certain vital components of the cell,
and inhibiting the functions of certain me-
tabolites, nitrite could have appeared and
disappeared long before gross morphologi-
cal changes could be observed in the plant.
Air pollution researchers have defined this
problem by describing two forms of injury,
first, metabolic injury, and later, visible in-
jury (26).
Klepper (20, 27, 28) has demonstrated
emission of considerable quantities of the
free radicals, nitric oxide (NO) and nitrogen
dioxide (NO,) gases, from herbicide-treated
soybeans. These plants contained relatively
high levels of endogenous nitrite and the
gases were evolved hours before any visible
symptoms were observed.
In a separate study, with 5-M nitrate-
grown plants and ametryn treatments iden-
tical to those in other studies, intact plants
were tested for NO,,, emissions (Table 2).
Three hours after treatment, NO,,, was de-
tected. Highest levels were recorded after 6
Treatment 0 3 hr
Control 0 0
+ Ametryn 0 0.54 (k0.19)
” Each pot contained approximately 3 g fresh
ON NITROGEN METABOLISM OF WHEAT 161
hr with decreasing emission rates after 12 hr.
No NOCs, was detected after 24 hr. These
NO,,Y, values correspond in time very close-
ly to the nitrite accumulation pattern (Fig.
4). As previously proposed (20, 27, 28)
NOCs, is thought to be a reaction product be-
tween nitrite and certain plant metabolites.
The amounts emitted by wheat are small in
comparison to those reported in soybeans
(pg quantities/min) (20, 28). However, the
data establish the formation of NO,s, within
the leaf in response to herbicide treatment.
There are several explanations to account
for such small levels of NO,,,: (a) Only very
low levels of NOW, were formed; (b) the
internal chemistry of the wheat plant may
be such that the free radicals of NO,\-, could
react to form an NO- or NO,-organic de-
rivative which would not have been de-
tected; (c) the cuticle or stomata1 aperture
of wheat may have prohibited emission of
the gases; (d) other nitrogen gases (N,O,
N2) could have been formed (12). Presently,
there is no technique available to measure
internal leaf NO,,, concentration. Only that
portion that escapes into the atmosphere
can be detected.
In recent experiments in this laboratory,
much higher levels of NOts, have been de-
tected from wheat grown on lo- and 15-r&2
nitrate and treated with different herbicides
(data not shown).
It was concluded that seedlings which
contained NR and were actively reducing
nitrate when foliarly treated with the photo-
synthetic inhibitor, ametryn, were more
susceptible to herbicide injury and death
than plants with little or no NRA. The
drastic difference in response to ametryn
TABLE 2
pg NO,,, evolvedihr”
6 hr 12 hr 24 hr
0 0 0
0.90 (k0.33) 0.39 (20.53) 0
wt of tissue. Values are means of four replications (&SD)
 162 CHURCHILL AND KLEPPER

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grown, and zero nitrogen plants adds evi- Inhibition of the normal process of nitrite re-
duction, Nrbr. Res. Bull 259, 1 (1974).
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