The Effect of Phosphinothricin (Glufosinate) on Photosynthesis
I.
Inhibition of Photosynthesis and Accumulation of Ammonia
Aloysius Wild, H eidrun Sauer, and Wolfgang Rühle
Institut für A llgem eine Botanik der Johannes G utenberg-U niversität, Saarstraße 21,
D-6500 Mainz, Bundesrepublik D eutschland
Z. Naturforsch.
42c, 263 —269 (1987); received Septem ber 12, 1986
Am m onia A ccum ulation, G lufosinate, G lutam ine Synthetase, Phosphinothricin, Photorespira­
tion, Photosynthesis
Phosphinothricin (glufosinate) is an irreversible inhibitor o f glutam ine synthetase in plants. E n­
zym e inhibition results in substantial light-dependent accumulation of ammonia, inhibition of
photosynthesis and ultim ately the death o f the plants. First o f all the m etabolic processes in which
N H 3 is liberated were established. Com parative investigations under photorespiratory and nonphotorespiratory conditions with Sinapis alba plants revealed that about 60% of the accumulated
N H 3 derives from photorespiration. Trials with PPT and K N 0 3 show ed that nitrate assimilation is
insignificant as a source o f N H 3, and so the remaining 40% is accum ulated in catabolic or anabolic
processes. Under photorespiratory conditions C 0 2 fixation after treatment with 1 mM PPT finally
fell below the com pensation point, and this was independent o f the addition o f nitrate. In nonphotorespiratory conditions, by contrast, photosynthesis continued at a level o f over 80% for
several hours in entire plants and in severed leaves, although at the same time substantial amounts
of ammonia had accumulated.
Introduction
Phosphinothricin, a phosphinic acid analogue of
glutam ate, inhibits GS in bacteria [1], algae [2] and
higher plants [2, 3]. As shown recently by means of
an isolated enzyme of wheat and m ustard, this is an
irreversible inhibitor [4]. GS, the key enzyme in the
now generally recognized G S/G O G A T pathway,
plays a crucial role —at least in higher plants — in the
(re)assimilation of NH 3 [5,6]. Thus in earlier investi­
gations with MSO, a structural analogue of PPT that
is similarly familiar as a GS inhibitor [7], elimination
of N H 3 was observed in bacteria and aquatic plants
[8 , 9], whereas accumulation was noted in higher
plants [10, 11]. Various authors have been able to
show that this accumulation of ammonia is in fact the
direct result of GS inhibition by MSO [12—14].
The primary aim of this paper is to quantify the
inhibition of photosynthesis of higher plants that oc­
curs as a result of the application of PPT. The second
point investigated is the metabolic processes in which
the accumulated ammonia occurs. There are three
m ajor potential sources: the assimilation of inorganic
A bbreviations: G D H , glutamate dehydrogenase; G O G A T ,
glutamine: 2-oxoglutarate aminotransferase; G S, glutamine
synthetase; M SO , m ethionine sulphoxim ine; PPT, phos­
phinothricin.
Reprint requests to Prof. Dr. A . Wild.
Verlag der Zeitschrift für Naturforschung, D -7400 Tübingen
0341 - 0382/87/0300 - 0263 $ 0 1 .3 0 /0
nitrogen, photorespiration, and catabolic or anabolic
processes. Previous studies of the effect of MSO
have produced contradictory results. A part from the
groundwork done in our paper [15] no results involv­
ing PPT are yet available. Therefore firstly the influ­
ence of nitrate assimilation and secondly the role of
photorespiration as a cause of ammonia accumula­
tion will be clarified.
Material and Methods
Plant material and chemicals
16- to 18-day-old plants of Sinapis alba (white mus­
tard) were used for investigating the toxic effect of
the herbicide; for the growing conditions see [15].
The active ingredient PPT was supplied by
Hoechst A G (Frankfurt/M ain, West Germany) (code
no. Hoe 35956). This is a phosphinic acid analogue
of glutam ate. Chemical name (IUPA C): d l homoalanin-4yl (methyl)phosphinic acid. The N H j
salt of this com pound (Code No. Hoe 039866, com­
mon name: glufosinate-ammonium) was described as
a new non selective herbicide [16, 17].
M easurement o f photosynthesis
Photosynthesis was measured as the C 0 2 fixation
rate by means of an infrared gas analyser, as de­
scribed in [18]. The experiments were conducted on
entire plants and on severed primary leaves. The
latter could be fed with PPT via the petiole.
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264
A . Wild et al. ■Effect o f Phosphinothricin on Photosynthesis, I
The experim ents with individual leaves involve the
risk of im pairm ent of the transpiration stream when
the leaves are severed: the resultant closure of the
stom ata leads to inhibition of photosynthesis.
W hether this was the case or w hether the toxic effect
of the PPT caused the rate of photosynthesis to drop
had to be proved. This could be achieved by tracing
the resistance to water vapour via a humidity sensor
(Nova Sina, M ünchen) as PPT showed no effect on
the behaviour of stomata. Experim ents in which the
stom atal resistance increased were discarded.
To clarify the question of nitrate assimilation as a
cause of N H 3 accumulation the leaves were treated
via the petiole with the pure herbicide in a concentra­
tion of 1 mM as well as with the same substance plus
50 mM K N 0 3. In order to clarify the role of photo­
respiration, experiments under normal atmospheric,
i.e. photorespiratory, conditions were compared
with those under non-photorespiratory conditions.
M easurement o f ammonia
The ammonia was determ ined by the Berthelot
detection m ethod, in which the blue dye complex
phenylquinone monimine is formed. The application
m ethod is based on investigations by W eatherburn
[19].
Results
Fig. 1 shows the rates of photosynthesis of entire
plants that were sprayed with a PPT dose equivalent
to 1 kg/ha and then exposed to photorespiratory or
non-photorespiratory conditions. To create nonphotorespiratory conditions the 0 2 partial pressure
was reduced to 2 % and in addition the carbon
dioxide level was raised from 400 to 1000 ppm.
100-
2
%0
2
1000 ppm CO2
50-
21 °/o0 2
400 ppm CO2
200
400
It is evident that under normal C 0 2 and 0 2 condi­
tions the initial rate of photosynthesis fell by 50%
after only about 30 min, whereas it remained fairly
constant for 7.5 h with 2% 0 2. The controls which
were sprayed only with wetting agent, are om itted in
Fig. 1 for the sake of clarity. They showed that C 0 2
fixation was fairly constant and that photosynthesis
even rose slightly during the trial period (from 1 0 0 %
to 1 1 1 % under photorespiratory conditions and from
1 0 0 % to
128% under non-photorespiratory condi­
tions).
Table I illustrates the effect of PPT on the rates of
photosynthesis of severed leaves. The absolute initial
Table I. Tim e course o f photosynthetic rates (in % of a H 20 control) o f severed mustard primary
leaves under photorespiratory and non-photorespiratory conditions after petiolar feeding with
1 m M PPT, 50 m M K N O 3 , and both. M eans from 10 experim ents. Standard deviations were
usually about 4%. Further conditions in the assimilation cham ber as in Fig. 1.
Photosynthetic rate
Treatm ent
Conditions
PPT PPT + K N O ,
- KNO,
photorespiratory:
21 % O , +
0.04% C 0 2
PPT PPT + K N O ,
- KNO,
2% 0 2
0. 1 % c o 2
non-photorespiratory:
Time [min]
Fig. 1. Photosynthetic activity of mustard primary leaves
with and without photorespiration after the application of
1 kg/ha PPT. PPT was sprayed 16 h before exposure to
light. During the light period the leaf (still attached to the
plant) was in an assimilation chamber at 25 °C, 56% rel­
ative humidity, 190 W m -2 photosynthetically active radia­
tion (P h A R ), with different com position o f the air flowing
through the leaf cuvette: O = 400 ppm C 0 2, 21 % 0 2; □ =
1000 ppm C 0 2, 2% 0 2.
10
20
30
40
60
90
120
100
100
99
99
98
98
80
95
96
38
91
94
8
71
89
0
17
89
-2
-2
91
93
93
88
94
97
95
92
100
102
88
100
103
84
95
85
89
93
79
84
92
99
min
A. Wild et al. ■Effect o f Phosphinothricin on Photosynthesis, I
265
values for C 0 2 fixation under photorespiratory con­
ditions were 46 ± 8 mmol C 0 2 m - 2 h _ 1 (treated) and
47 ± 6 mmol C 0 2 m - 2 h - 1 (control); under nonphotorespiratory conditions they were 72 ± 4 mmol
C 0 2 m -: h _ 1 (treated) and 74 ± 5 mmol C 0 2 m - 2 h _ 1
(control). This difference is attributable firstly to the
Warburg effect and secondly to the fact that the
limiting factor is eliminated by raising the C 0 2 con­
centration from 400 to 1000 ppm. W hen m easure­
ments are carried out with 2 1 % 0 2 the rate of photo­
synthesis falls to zero over a period of about 80 min.
At 2% 0 2 however it falls by only about 25% after
more than two hours.
In order to determ ine the influence of nitrate as­
similation on N H 3 accumulation the plants were
treated in a further series of trials with K N 0 3 in a
concentration of 50 mM as well as with PPT (Table
I). As a result the rate of photosynthesis of the con­
trols falls slightly (Fig. 2 A). This is undoubtedly
attributable to the unnaturally high nitrate content,
which clearly involves osmotic stress for the plants.
A t a concentration of 5 mM K N 0 3, which is in any
case closer to the normal natural conditions in the
plant [2 0 ], photosynthesis in fact remains constant
over a period of 100 min. In previous work with
MSO and nitrate the concentrations normally used
were between 3 and 21 m M , though concentrations
up to 100 mM were also em ployed [14, 20—22]. A
nitrate concentration of 50 mM was chosen despite
the unnatural conditions so as to ensure that the N O J
content was high enough for nitrate assimilation to
be investigated as a source of N H 3.
In these experiments the absolute initial rates
under photorespiratory conditions were 48 ± 10
mmol C 0 2 m - 2 h - 1 (treated) and 44 ± 2 mmol C 0 2
m ~ 2 h - 1 (control) and under non-photorespiratory
conditions 78 ± 8 mmol C 0 2 m - 2 h - 1 (treated) and
77 ± 4 mmol C 0 2 m - 2 h _ 1 (control).
The rate of photosynthesis of the P P T /K N 0 3 -treated plants was similarly completely reduced under
normal atmospheric conditions — it finally fell below
the C 0 2 compensation point to the respiratory val­
ues. However, inhibition of C 0 2 fixation was longer
in setting in than in the case of plants that received
only PPT (Table I). Incidentally, there was no m ajor
difference between the experim ents with PPT and
those with P P T /K N 0 3 at 2% 0 2.
Reduction in photosynthesis is accompanied by ac­
cumulation of ammonia when plants are treated with
Time [min]
Fig. 2. Photosynthesis and ammonia accumulation of m us­
tard primary leaves after petiolar feeding with 1 mM PPT
and 50 m M K N 0 3 under photorespiratory conditions
(400 ppm C 0 2, 21% 0 2). A , photosynthesis: • = treated,
© = control; B , ammonia content: ■ = treated, □ =
control.
phosphinothricin. Fig. 2 illustrates this for a series of
trials on leaves to which P P T /K N 0 3 was applied.
Table II shows the average N H 3 accumulation
rates. Contrary to our expectation even less N H 3 was
accumulated in plants that received nitrate in addi­
tion to the herbicide. Comparison of the experiments
under photorespiratory conditions with those under
non-photorespiratory conditions reveals however
that with 2 % 0 2 the amount of ammonia formed was
only about 40% of that formed under normal atm os­
pheric conditions.
The ammonia accumulation that takes place some­
what more slowly in the experiments with P P T /K N 0 3
266
A. Wild et al. ■Effect o f Phosphinothricin on Photosynthesis. I
Table II. A m m onia accum ulation o f mustard primary leaves provided with PPT or PPT/KNO3,
respectively, treated under conditions with or without photorespiration.
Treatm ent
Conditions
A m m onia accumulation rate
(^imol N H 3 g -1 fr wt hr-1)
PPT
Control (FLO)
PPT
Control (PLO)
PPT /K N O ^
Control (K N O ,)
p p t/k n o 3
Control (K N O ,)
2 1 % o 2, 400 ppm c o 2
2 % o 2, 1000 ppm c o 2
2 1 % o 2, 400 ppm c o 2
2 % o 2, 1000 ppm c o 2
31.1 ±6.3 (13)1
4.9±0.52 (4)
11.6 ±2.7 (12)
3.5±0.7 (4)
21.7±5.7 (15)
5.5±0.9 (4)
9.6±4.3 (11)
4.7±0.9 (4)
% o f am monia at
photorespiratory
conditions
100.0
37.3
100.0
44.2
1 Num bers in parentheses represent the number o f experim ents.
2 A m m onia content o f the controls did not alter during the course o f the experim ents. This is the
reason, why the control rates are given in fxmol N H 3 g ' 1 fr wt.
does correlate with the delayed inhibition of C 0 2
fixation of these plants.
The rates of photosynthesis in relation to the am­
monia content thus prove to be basically identical in
the leaves that were treated only with PPT and those
that received K N 0 3 in addition. U nder photores­
piratory conditions C 0 2 exchange decreases at about
35 mM N H 3 to the respiratory value (Fig. 3). The
trials with 2 % 0 2 were conducted for a considerably
longer time than those with 2 1 % 0 2, and so in these
conditions enorm ous quantities of ammonia were ac­
cum ulated in the course of time (Fig. 4). The plants
treated with PPT and P P T /K N 0 3 displayed the same
characteristics.
N H 3 accumulation is, furtherm ore, light-depend­
ent. This became clear in trials with PPT-sprayed
plants, in which only one primary leaf was exposed
whilst the other was kept in the dark. It proved that
the ammonia content in the exposed leaf was many
times higher than that in the leaf kept in the dark
(data not shown). Com pared to the controls, how­
ever, even in the dark some N H 3 had been accumu­
lated. Consequently, light-dependent processes are
responsible largely though not exclusively for am­
monia accumulation.
100-
-
50-
• ------- • 50
N h V [ ( j m o l g ' 1 f r. w t ]
Fig. 3. Photosynthesis as a function
of am m onia content o f mustard
primary leaves provided with PPT
or P P T /K N 0 3, respectively, under
photorespiratory conditions. A m ­
monia content of the controls with
K N 0 3 is given in Table II. O =
PPT. • = PPT/K N O ,, © = control
(H 20 ) .
A. Wild et al. ■Effect o f Phosphinothricin on P hotosynthesis, I
100
267
-
50-
10
20
30
40
NH4+[ pmol g -1 fr. wt.]
Discussion
A t normal atmospheric conditions, PPT treatm ent
results in inhibition of photosynthesis. This is shown
in the trials in which the herbicide is sprayed direct
onto the leaves and in the experim ents with the ac­
tive ingredient applied via the petiole.
PPT at a dose equivalent to 1 kg/ha was used for
spraying entire plants; the C 0 2 fixation rate fell to
zero within about 80 min (Fig. 1).
Photosynthesis was also inhibited completely in
the case of plants with severed leaves. It was recently
confirmed that the PPT concentration of 1 mM ap­
plied to the petiole was sufficient to inhibit GS com ­
pletely [23]. On adding 0.25 mM PPT the GS of ex­
tracts of leaves had a residual activity of only about
20% after only 2 min [4],
The photosynthesis of leaves to which PPT/K N O 3
was applied fell below the com pensation point within
2 h. By adding both nitrate and PPT the C 0 2 fixation
is however reduced more slowly than when the herbi­
cide is used on its own (Table I). This was also ob­
served in experiments with MSO and K N 0 3 [22],
Completely different results can be observed
under non-photorespiratory conditions. In entire
plants and in severed leaves photosynthesis con­
tinues for several hours at more than 75% of the
initial rate. This behaviour is independent of any
addition of NO^. In previous work with the GS in­
hibitor MSO there are contrasting data on this point,
though a num ber of authors were able to observe
similar findings with MSO [21, 24—26]. Evidently the
Fig. 4. Photosynthesis as a function o f am­
monia content o f mustard primary leaves
provided with PPT or PPT/K N O 3, respective­
ly, under non-photorespiratory conditions
(1000 ppm C 0 2, 2% 0 2). A m m onia content
o f the controls with K N O , is given in
Table II. □ = PPT, ■ = P P T /K N 0 3; □ =
control (H 20 ) .
interruption of photorespiration in any way is crucial
to the inhibition of photosynthesis.
PPT treatm ent of plants causes high ammonia ac­
cumulation in the tissues [15]. There may be three
m ajor potential sources for the ammonia. In the C 2
pathway of photorespiration the glycine decarboxyl­
ase reaction produces not only C 0 2 but also an
equivalent quantity of ammonia. A nother metabolic
process forming ammonia is the evolution of N H 3 in
the course of nitrate assimilation by reduction of the
externally absorbed NOT. Thirdly, ammonia also oc­
curs in catabolic and anabolic processes, such as in
the breakdown of proteins or nucleotides and in the
oxidative deam ination of phenylalanine or tyrosine.
In each case the N H 3 liberated is reassimilated via
the glutamine synthetase. Inhibition of the G O G A T
cycle at any point therefore results in accumulation
of ammonia.
Table II shows that the total amount of ammonia
liberated under normal atmospheric conditions is
31 (amol N H 3 h - 1 g _ 1 fr wt. Assuming that 334 [imol
C 0 2 h - 1 g - 1 fr wt are reduced at an average rate of
photosynthesis, this value at 9% of the apparent C 0 2
fixation is quite low. However, one possible assump­
tion is that the internally produced and reassimilated
am ount of N H 3 is higher [27, 28]. If so, the plants
have developed detoxification mechanisms by em ­
ploying the enzymes G D H and aspartate-synthetase.
With the aid of these mechanisms they are able to
elim inate not only the normally occurring concentra­
tions of N H 3 but also much higher ones. In any case
268
other authors have shown in more recent work that
the amm onia concentration accumulated in the pres­
ence of a GS inhibitor can not be used as a measure
of the normal intensity of photorespiration [22, 29].
If the main cause of ammonia accumulation lies in
photorespiration, it should be reduced accordingly
by inhibiting this process. In fact the N H 3 concentra­
tion at 2% 0 2 and 1 0 0 0 ppm C 0 2 was only 37% of
the value at 21% 0 2 and 400 ppm C 0 2. The num er­
ous results published so far with MSO as the source
of the ammonia differ for one and the same plant
species: some authors found that the majority of the
accumulated N H 3 in wheat and spinach is formed
under non-photorespiratory conditions [22, 30];
other findings, similarly on wheat [2 1 , 26] but also on
oats [20], pepper [24, 25] and barley [21] point to
photorespiration as the primary source of N H 3: in
these cases the maximum amount of ammonia ac­
cumulated under non-photorespiratory conditions is
40% of that accumulated under photorespiratory
conditions. C ontradictory results were also obtained
in comparing C 3 and C 4 plants: some authors ob­
served in maize and sorghum only about 1/4 of the
amm onia content of C 3 plants [21, 31], whereas
others observed equivalent N H 3 concentrations [2,
32]. The effect of 0 2 and C 0 2 on N H 3 accumulation
in the green algae A nkistrodesm us and Chlamydom onas and the effect of isonicotine hydrazide and
MSO on Chlam ydom onas and on higher plants illus­
trate clearly the role of photorespiration in this con­
nection [33, 34]. According to our results, too, 63%
of the accumulated ammonia is derived from the
glycolate pathway.
Investigation of nitrate as a possible source of am ­
monia in PPT-treated plants produced some surpris­
ing results. If N O J is assumed to be the source of
ammonia nitrogen, then a plant ought to accumulate
more N H 3 if fed with additional nitrate. In our exper­
A. Wild et al. • Effect o f Phosphinothricin on Photosynthesis, I
im ents however the exact opposite was observed: the
P P T /K N 0 3-treated plants contained about 30% less
am m onia than the plants to which only PPT was ap­
plied. In each case it is clear that at least within the
trial period of two hours — the early effects of PPT
action were therefore observed here — more am­
m onia does not occur as a result of reducing the
nitrate. Similar results were obtained with MSO on
various higher plants [21, 30].
With regard to the source of nitrogen for the
accum ulated ammonia it can therefore be said that
nitrate reduction is unim portant at an early stage of
photosynthesis inhibition, that photorespiration is
responsible for 63% of the N H 3 formed and that
therefore 37% has to originate from catabolic or
anabolic processes.
These findings tally well with the results of the
experim ents on the light dependence of N H 3 ac­
cumulation. Confirmation was gained here of what
other authors have already observed with MSO [35,
36], namely that after the application of PPT the
m ajority of the ammonia is formed in the light —this
would correspond to the ammonia from the lightdependent glycolate pathway; but as this paper has
shown, part of the N H 3 is liberated in the dark- this
would be attributable to the light-independent
catabolic and anabolic processes.
The correlation between photosynthesis and am­
monia concentration always shows similar values re­
gardless of the addition of nitrate. A t first glance it
could appear that ammonia accumulation is responsi­
ble for inhibiting photosynthesis, as many authors
have so far postulated for MSO. On the other hand,
C 0 2 fixation under non-photorespiratory conditions
remains largely intact despite high N H 3 concentra­
tions. The cause of photosynthesis inhibition by the
influence of PPT will therefore investigated at great­
er length in a second part of this paper [37].
A . Wild et al. ■Effect of Phosphinothricin on Photosynthesis, I
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