Plaint Physiol. (1967) 42, 1419-1422
Inhibitory Effect of Peroxyacetyl Nitrate on Cyclic Photophosphorylation
by Chloroplasts from Black Valentine Bean Leaves'
Jane Koukol, W. M. Dugger, Jr., and R. L. Palmer
Statewide Air Pollution Research Center and Departments of Life Sciences and Horticulture,
University of California, Riverside, California 92502
Received June 16, 1967.
Summiiary. The inhibitory effect of peroxyacetyl nitrate on the cyclic photophosphorylation of chloroplasts isolated ifrom Black Valentine variety bean leaves (Phaseolis
vulgaris L.) has been studied. Peroxyacetyl nitrate caused inhibition to photophosphorylation, in either the dark or the ltight, by afifecting the chloroplast Evidence is
presented which suggests that peroxyacetyl nitrate could oxidize sullfhydryl groups on
enzymes necessary for -photophosphoryllation. The inhibition to photophosphorylation
caused by peroxyacetyl nitrate cannot be reversed by glutathione, even when added in
large amounts, whereas the inhibition to photophosphorylation caused by para-chloromercuriphenylsuifonic acid is easily reversed by small quantities of glutathione. This
suggests that if ,peroxyacetyl nitrate is oxidizing sulf'hydryl groups necessary for photophosphorylation, this oxidation is proceeding beyond the disulifide stage.
Peroxyacetyl nitrate (PAN), a component of
photochemical smog, is known; to cause severe damage
to plants ( 10). Light is necessary ibefore, during,
and after the fumigation of intact bean plants with
PAN in order for damage to occur (12). In vitro
studies have shown that PAN can inhibit photosynthetic reactions (3,4) but can also inactivate isol-ated
enzymes which are not involved in photosynthesis
(9). Thus, it appears that PAN could cause damage
by intee-fering in both the photosynthetic and nonphotosynthetic processes of the plant. This report
describes studies on the inhibitory effect of PAN
on cyclic photophosphorylation by chloroplasts obtained from Black Valentine variety bean leaves.
The purpose of this investigation was to study the
inhibitory effect of iPAN on a given photosynthetic
reaction in a more detailed manner than had previously
been done.
Materials and Methods
Chloroplasts were prepared from primary leaves
of 8 to 12 day old Black Valentine variety bean
plants (Phaseolis vulgaris L.) by a procedure adapted
from Margulies and Jagendorf (6) and were washed
once. Chlorophyll content was determined according
to Arnon (1).
Photophosphorylation was measured by deternmning the decrease in Pi which occurred when the
1 This work was supported by a grant (AP-40) from
the National Center for Air Pollution Control, United
States Public Health Service.
chloroplasts were incubated in the reaction mixture
for 10 minutes at 150 in the light (4000 ift-c) with
shaking. The reaction mixture contained 100 umoles
tris buffer (pH 8.0), 10 p,moles MgC12, 10 ,umoles
KH,PO4, 10 umoles ADP, and 0.1 ,mole phenazine
methosulfate (PMS), and water in a final volume
of 2.0 ml -in a 25 ml Erlenmeyer flask. The chlorophyll concentration was 0.150 mg per flask. The
reaction mixture was denatured by trichlioroacetic acid.
Pi was determined according to Fiske and Subba
Row (5).
A manifold arrangement over the bath pennitted
the simultaneous gassing of 11 flasks with PAN and
11 'flasks with N2 or the gassing of 22 flasks with
N9. Flow rates were controlled by flowmeters. All
N2 gassing was at the rate of 100 ml per minute per
flask. The uniform distribution of the PAN into
the flasks was achieved by using glass tubing of small
and uniform inside diameter (0.5 mm) for the gas
outlet. The entire PAN gassing system was first
flushed with PAiN before the PAN gassing of the
experiment was carried out. The uniformity of PAN
gassing was determined by the oxidation of KI by
PAN to I2, which could then be determined spectrophotometrically.
PAN was synthesized, purified chromatographically, diluted with N2 and the concentration was
determined according to the methods of Stephens
et al. (11).
For the dark inhibition studies, the complete reaction mixture including the chloroplasts was incubated at 150 in darkness and gassed with PAN for
10 minutes. Control, flasks were gassed with N2.
At the end' of the 10-minute dark period, the flasks
1419
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1 420
PLANT PHYSIOLOGY
were incubated in the light for 10 minutes with N2
gassing. The PAN gassing during the dark incubation was at the rate of 0.089 umole per minute per
flask but only 0.025 ,umole per minute was recovered
in the reaction mixture.
For the light inhibition studies, the react-on flasks
were gassed with PAN during the photophosphorylation reaction period itself. Nitrogen gassed control
flasks 'were always run simultaneously. The PAN
gassing for the light inhibition studies was at the rate
of 0.27 zumole per minute per flask but only 0.11
,mole per minute was recovered in the reaction mix-
ture.
Results
Inhibition of Cyclic Photophosphorylation by PAN.
The inhibition of cyclic photophosphorylation by PAN
increased progressively when the flow rate was increased in both kinds of inhibition. Inhilbition was
also progressive with time in the case of the dark
inhibition (;fig 1A). However, when PAN was
introduced only when the light was turned on, it was
several minutes before inhibition to photophosphorylation was observed (fig 1B).
The Effect of PAN on the Chloroplasts and on
PMS. Table I shows that gassing with PAN in the
dark affected the chloroplasts and not the PMS.
Flushing the reaction mixture with nitrogen after the
chloroplasts had 'been gassed with PAN did not serve
to reverse the inhibition (see legend, table I). This
indicated that the damage caused by the PAN is
irreversilble. However, since PAN is known to yield
inorganic nitrite (8), under certain conditions, it is
possible that the inhibition could ihave been due to
this product which would remain after flushing. The
vt
0
0
0
E
0
w
E
w
y
Y
a-
ED
a.
1 2 3 4 5 6 7 8 9 10 11 12 13
T IM E (minutes)
1
2 3 4 5 6 7 8 9 10 11 12 13
TI ME (minutes)
FIG. 1. The time course of the inhibition of cyclic photophosphorylation by PAN. A (left) Dark inhibition.
The components of the reaction mixture were as usual except for the Pi and ADP (Pi = ADP), which were
as follows: 5 jAmoles for 10, 8 and 6 minute flasks, 10 ,umoles for 4 and 2 minute flasks. Flasks were run in
duplicate. B (right) Light inhibition. The components of the reaction mixture were as usual except for the Pi
and ADP (Pi = ADP), which were as follows: For both No (0) and PAN (0) flasks; 10 /imoles for 12 and 9
minute flasks, and 5 ,umoles for 6 and 3 minute flasks. Both N2 and PAN gassed flasks were run in duplicate.
Table I. The Effect of PAN on Bean Chloroplasts and PMS Catalyzed Photophosphorylation in the Dark
Flasks were gassed as indicated during the first 10 minute dark period. All flasks were then gassed with N2
for 10 minutes in the dark to remove any PAN remaining. Then, either PMS or the chloroplasts was added and
the flasks were assayed in the light under N2. The flasks were run in triplicate.
Conditions during first dark
10 min gassing
Chloroplasts absent, PMS present
Chloroplasts absent, PMS present
Chloroplasts present, PMS absent
Chloroplasts present, PMS absent
Gassing during
first 10 min
dark period
N9
PAN
N,
PAN
-
Pi
Atmoles
4.2
4.2
3.5
0.5
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%
Inhibition
None
86
KOUKOL ET AL.-EFFECT OF PAN ON CYCLIC PHOTOPHOSPHORYLATION
addition of NaNO, was tested over a wide range of
concentrations and had no effect on photophosphorylation. Washing the chloroplasts after they had been
gassed with PAN did not result in reversal of the
inhiibition.
The Effect of ADP and Pi ont the Inhibition by
PAN. Although it has been observed that some
enzymes are protected during PAN gassing by the
presence of the substrates (9), we found that the
inhibition to photophosphorylation by PAN was the
same whether or not ADP and Pi were present
during the dark gassing period. Further, varying
the quantity of ADP and Pi present during either
the dark or the light in,hibition over a 4-fold range
had no effect on the inhibition to photophosphorylation caused by PAN.
The Effect of GSH on the Inhibition of Photophosphorylation by PAN. It was possible to protect
the chloroplast preparation from PAN by adding
GSH to the reaction mixture before the gassing,.
This protection was probably due to the destruction
of the oxidizing power of PAN by GSH. However,
GSH was not able to reverse the inhibition to photophosphorylation caused by PAN if added after the
PAN gassing (table II). Thus, in the dark inhibition, 0.25 pxmole of PAN resulted in about 50 %
inhibition whether or not 10 pjumoles of GSH were
added at the end of the dark gassing -period and before
the light period. In the light inhibition, 1.1 ,umoles
of PAN resulted in about the same inhbition to
photophospbhorylation whether or not 20 jumoles of
GSH were added at the end of the firtst light period.
Neither GSH (tajble II) nor GSSG (not shown)
had any effect on photophosphorylation by this particular chloroplast preparation.
Inhibition to photophosphorylation by sulfhydryl
reagents has, of course, been observed (2). We
found that para-chloromercuriphenylsulfonic acid at
a final, concentration of 0.1 mm (total in flask,
0.2 pmole) conmpletely inhibited photophosphorylation
by bean leaf chloroplasts and a concentration of
0.01 mM inhibited 77 %. The inhibition result.:ng
1421
from the addition of 0.2 ,umole of the sulfhydryl recompletely reversed by 0.6 /Lmole of
GSH. Thus, although a 3-ifold excess of GSH would
reverse the inhibition to photophosphorylation caused
by para-chloromercuriphenylsulfonic acid, a 40-fold
excess of GSH in the case of the dark inhibition,
and an 18-fold excess of GSH in the case of the light
in-hibition, failed to relieve the inhibition caused by
PAN (table II).
agent could be
Discussion
This investigation has shown that PAN inhibits
cyclic photophosphoryliation and is not dependent on
light. The fact that washing the chloroplasts did
not relieve the PAN inhibition suggested that a
chemical modification of a functional group occurred
and
our evidence suggested that the chemical modification was the oxidation of sulfhydryl groups necessary for cyclic photophosphorylation. Mudd et al.
showed that PAN oxidized the cysteine residues of
proteins to cysteic acid (7). Such an oxidation would
account for the observation that GSH did not reverse
the PAN inhibition to cyclic photophosphorylation,
i.e., the oxidation proceeds beyon'd the disulifide stage.
Further, such an oxidation would occur in the dark
as well as in the light.
We 'found that PAN also inhibits the Hill reaction
with ferrieyanide bv isolated spinach chloroplasts in
both the dark and the light. However, catalvtic
amounts of NaNO, equal to the amount of NO9formed from the breakdown of PAN, severely inhibit
the Hill reaction. We found no such effect of
NaNO., in cyclic photophosphorylation. Further, concentrations of p-chloromercuriphenylsul fonic acid
which completely inhibit cyclic photophosiphorylation
have little ef'fect on the Hill reaction. It would
appear that PAN is a:ffecting another functional
in the case of the Hill reaction.
Thus, the relationship between the oxidation of sulfhydry] groups or other chemical structures by PAN
in in vitro systems to the severe l:ght dependent
group or groups
Table II. The Effect of GSH on the Dark and the Light PAN Inhibition
Conditions
Dark inhibition*
1. No PAN, 10,umoles GSH added at end of 10 min dark period
2. PAN, no GSH added at end of 10 min dark period
3. PAN, 10 Amoles GSH added at end of 10 min dark period
Effect
None
43 % Inhibition
50 % Inhibition
Light inhibition**
1. No PAN, 20 umoles GSH added at end of first light period
None
36 % Inhibition
2. PAN, no GSH added at end of first light period
48 % Inhibition
3. PAN, 20 ,umoles GSH added at end of first light period
* Dark Inhibition: At the end of the dark gassing period, all the flasks were removed from the bath, GSH added
where indicated, and the flasks returned to the bath for the light assay. 0.25 ,umole was the total PAN present
per reaction flask. Flasks were run in duplicate. The P, uptake for the N2 control flasks was 5.8 ,moles.
** Light Inhibition: All the flasks were removed from the bath at the end of the first 10 minute light period, and
an additional 5 ,umoles each of ADP anid Pi added. Also, GSH was added where indicated. The flasks were
returned to the bath and assayed for another 10 minutes under N,. 1.1 ,umoles was the total PAN present per
reaction flask. Flasks were run in duplicate. The Pi uptake for the No controls undergoing both 10 minute
light periods was 7.0 ,moles.
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1422
PLANT PHYSIOLOGY
damiage of intact plants caused by PAN remains to
be elutcidated.
5.
6.
Acknowledgments
The authors thank MIrs. Nao Belser, AMrs. Jean
Roberts, and 'Mr. John Eshleman for their technical
7.
assistance.
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