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Alteration of the stromal side of photosystem II by diphenylcarbazide
ptan ience [ I SI \ I I . R S( II N F I I i( 1'~ RI I'~HI R~; I R I I g ' q D
Plant Science 94 (1993) 55-59
Alteration of the stromal side of photosystem II by diphenylcarbazide Marc Purcell, Robert Carpentier* Centre de recherche en photobiophysique University; du Qukbec ~ Trois-Rivibres C.P. 500, Trois-Rivibres, Qukbec, Canada G9A 5H7
(Received 8 March 1993: revision received 2 July 1993: accepted 2 July 1993)
Abstract
In the thylakoid membranes, the photosystem II electron donor diphenylcarbazide decreases the inhibitory effect of quinone-type herbicides like atrazine (Purcell et al., 1990, Pest. Biochem. Physiol. 37, 83-89). This behavior is studied in more detail and it is shown that the donor strongly inhibits the binding of radiolabelled atrazine to the Q~ protein. The electron acceptor activity of 2,6-dichlorobenzoquinone (DCBQ) is also impaired but not that of potassium ferricyanide or 2,6-dichlorophenolindophenol (DCIP). Electron transport from photosystem II to photosystem I, which involves the reduction of plastoquinone at the QB site, is only slightly affected. This data indicated that at concentrations where it is commonly used as an electron donor (0.5- 1.0 mM), diphenylcarbazide can alter the binding of herbicides and artificial electron acceptors having overlapping-binding domains in the QB pocket. However, the effect of diphenyl-carbazide on the QB site can affect plastoquinone reduction only at relatively high concentrations (>2 mM). Key words: Diphenylcarbazide; Atrazine; Photosystem II; QB Protein; Electron transport; Plastoquinone
1. Introduction
The core complex of chloroplast photosystem (PS) II is composed of two transmembranous polypeptides of apparent molecular weight of 33 kDa named D1 and D2, respectively [1]. The polypeptide D1 was initially known as the QBpolypeptide because it bears the binding site of the secondary quinone acceptor QB [2]. This site can also be occupied by several herbicides and artificial electron acceptors of quinone type which then * Corresponding author.
results in inhibition of plastoquinone (PQ) photoreduction [3-6]. It was reported that the PSII electron donor diphenylcarbazide (DPC) can decrease the extent of atrazine and 3-(3,4-dichlorophenyl)-l,l-dimethylurea (DCMU) inhibition of 2,6-dichlorophenolindophenol (DCIP) photoreduction in thylakoid membranes [7]. Because these inhibitors bind in the QB pocket located on the stromal side of the photosystem, the decreased inhibition was not due to the electron donor properties of DPC. In fact, this donor is known to transfer electrons directly to Z, the secondary donor of PSII [8-9].
0168-9452/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved. SSDI 0168-9452(93)03700-6
56
This intermediate is probably the tyrosine-161 of the polypeptide D1 and is located on the lumenal side of the thylakoid membrane [10]. We have found that besides its electron donor capabilities in PSII, DPC also alters the binding of atrazine to the stromal side of the photosystem [11]. This explains the reduced inhibitory action of the herbicide in the presence of the donor. In this report, we present a further study of the effect of DPC on the stromal side of PSII. Another quinone-type molecule, the artificial electron acceptor 2,6-dichlorobenzoquinone (DCBQ), also shows a decreased ability to accept electrons from the QB pocket in the presence of DPC whereas potassium ferricyanide (FeCN) photoreduction remains unaffected. It is also shown that DPC inhibits the reduction of endogenous PQ only slightly and does not significantly affect linear electron transport to PSI at concentrations where it is used as electron donor. The data are discussed in terms of a possible overlapping between the binding sites of herbicides, electron acceptors and DPC in the herbicide binding niche. 2. Materials and methods
2.1. Thylakoid membrane preparation Thylakoid membranes were isolated from spinach leaves as described previously [12] and resuspended in a solution containing 50 mM N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (Tes)-NaOH, pH 7.5, 2 mM MgCI2, 1 mM NH4CI , and 300 mM sorbitol at a chlorophyll (Chl) concentration of 2 mg/ml. Chlorophyll was determined according to Arnon [13]. The preparations were used the same day or stored in liquid nitrogen. 2.2. DCIP photoreduction Light minus dark rates of DCIP photoreduction were measured at 600 nm using a Perkin-Elmer model 553 UV/VIS-spectrophotometer as described elsewhere [14]. The reaction mixture was composed of thylakoid membranes at a Chl concentration of 5 ~g/ml, 30 #M DCIP, 50 mM TesNaOH pH 7.5, 2 mM MgC12, 1 mM NHaCI, and 330 mM sorbitol.
M. Purcell, R. ('arpentier / Plant Sci, 94 (1993) 55-59
2.3. Oxygen evolution and uptake measurements Oxygen evolution was monitored with a Clarktype electrode as described previously [15]. The assay medium was composed of thylakoid membranes at a Chl concentration of 11/zg/ml suspended in the above buffer with an electron acceptor as specified in the text. Oxygen uptake was monitored in similar samples but with 0.5 mM methylviologen (MV) as electron acceptor. Samples were illuminated for 1.5 rain in the oxygen electrode cell. Then, 2000 units of catalase were added in the dark. The oxygen release upon addition of catalase was measured to determine the Mehler reaction [16-17]. 2.4. Herbicide binding Herbicide binding experiments were performed under dim light using a procedure similar to that described by Carpentier et al., [18]. The reaction mixture (1 ml) contained thylakoid membranes equivalent to 50/~M Chl, 50 mM Tes-NaOH pH 7.5, 2 mM MgCI2, 1 mM NH4CI, and 330 mM sorbitol. [14C]atrazine (19.5 /zCi/mg, Ciba Geigy, Greensboro, NC) was added to obtain between 90-2000 disintegrations/rain in the counting vials in the presence of specified concentrations of DPC. The samples were incubated for 10 min at room temperature and harvested in an Eppendorf centrifuge (4 min). Aliquots of 0.75 ml were taken from the supernatant fractions for the determination of free atrazine by scintillation counting. 3. Results and discussion
The inhibition of DCIP photoreduction by atrazine is described in Fig. 1. Thylakoid membranes were used with or without 1.6 mM DPC. It is clear from Fig. 1 that the photoactivity strongly decreases as the herbicide concentration is raised. However, there is always less inhibitory action in the presence of DPC. The reduced inhibitory effect is quantified in Fig. 1 (inset). The release of inhibition varies from 100 down to 10% and decreases with increasing atrazine concentrations. As mentioned, this effect on atrazine inhibition cannot be accounted for by the electron donating properties of DPC. Instead, it was found that atra-
M, Purcell, R. Carpentier / Plant Sci. 94 (1993) 55-59
57
o~ v
100 z 0
F--
80
¢0
12C ~) 108 60
a LU
.~ 8o
n-
~ 6o ~ 4o
O
~0
40
_
2o'- Ol
'I-
2O
20
_1
¢0 Q
^
008'1
'
1 0 .7
10 .6
ATRAZINE
10 -s
0 .4
(M)
Fig. 1. DCIP photoreduction with (Q), and without (O) 1.6 mM DPC. The rates corresponding to 100% were 300 and 270 ~mol DCIP/mg Chl" h with and without DPC, respectively. Inset: relief of inhibition in the presence of 1.6 mM DPC calculated from the data of Fig. 1.
zine binding was affected by the donor [11]. It is shown in Fig. 2 that the amount of [14C]atrazine bound to the thylakoid membranes decreases in proportion to the concentration of DPC at the two atrazine concentrations used. The DPC concentration required to decrease atrazine binding by 50% is 1.4 ± 0.1 mM. However, the effect is already seen within the range of concentrations used for
E
electron transport studies when DPC serves as a donor. DPC also alters the binding of the PSII electron acceptor DCBQ which receives its electrons from the QB site. In fact, DPC strongly inhibits oxygen evolution when DCBQ is used as acceptor (Table 1). However, if FeCN is used as acceptor instead of DCBQ, there is no detectable effect of DPC on the initial rates of oxygen evolution. Surprisingly, DPC does not reduce the extent of DCIP photoreduction (Fig. 1). DCIP accepts electrons directly from the pool of reduced plastoquinone and does not interact directly with the QB site. This implies that the reduction of plastoquinone must proceed freely in the presence of DPC. To verify this point more precisely, electron transport from water to MV was measured with and without DPC. Oxygen uptake was measured without interference from oxygen evolution using the catalase assay (see Materials and methods). The rates of MV reduction were not affected by 1 mM DPC but they were slightly decreased (by 15%) in the presence of 2.5 mM DPC (Table 2). In order to reduce MV, PSI must receive its electrons from PSII with plastoquinone as a mandatory intermediate. It was verified for this experiment that DPC could not act as an electron donor to PSI. In fact, in samples inhibited by 1.2 mM D C M U (in order to obtain full inhibition of PSII, high DCMU concentrations were required to overcome
Table 1 Effect of DPC on oxygen evolution
e- 2 0
Additions
Oxygen evolution a (p,g mol 02/ mg Chl" h)
DCBQ b (600 ttM) DCBQ (600 t~M) + DPC c (0.5 mM) DCBQ (600 #M) + DPC (1.0 mM) FeCN d (10 mM) FeCN (10 raM) + DPC (0.5 raM) FeCN (10 mM) + DPC (1.0 mM)
319 55 16 170 168 171
Z
O "'10 Z N
nI--
6 0
1
2
DPC
(mM)
3
Fig. 2. Inhibition of atrazine binding by DPC in the presence of 0,05 (O), or 0.25 (Q) t~M atrazine.
aThe values represent the average of three separate experiments and the variations were within 4%. b2,6-Dichlorobenzoquinone. CDiphenylcarbazide. dpotassium ferricyanide.
58
M. Purcell. R. Carpentier / Plant Sci. 94 (1993) 55-59
Table 2 Effect of DPC on oxygen uptake by PS! through the Mehler reaction (H20-- MV) using the catalase assay~ Addition
Oxygen uptakeb (#mol O~/ mg Chl" h)
None DPC (1.0 mM) DPC (2.5 mM) DCBQ (600 #M) DCBQ (600 t~M)+ DPC (I.0 mM)
110 108 93 27 60
aConditions are given in Materials and methods. bThe values represent the average of three separate experiments and the variations were within 5'7,,.
the adverse effect of DPC) there was no oxygen uptake activity even in the presence of 2 mM DPC but the addition of the donor system DCIP/ascorbate resulted in an 02 uptake activity of 550 t~mol/mg Chl" h. As a comparison, in Table 2, DCBQ is shown to strongly inhibit oxygen uptake; this is because it prevents plastoquinone reduction when it is serving as electron acceptor. If DPC is added together with DCBQ, the inhibitory effect of the acceptor is greatly released as predicted from the data of Table 1. The experiments presented in Table 2 indicate that DPC affects plastoquinone binding but only at high concentrations. Inhibition of atrazine, DCBQ, and PQ binding could proceed via two different mechanisms. The first possibility would be that DPC may modify the QB site through a transmembrane conformational change. Eventhough DPC is known to interact at the lumenal side of PSII to reduce the secondary donor Z, both the Q8 site and the DPC oxidation site are presumably located on the same transbilayer polypeptide complex which contains the reaction center of PSII [1]. Furthermore, the acceptor side of PSII was already shown to be affected by modifications of the lumenal side in several types of experiments [19-21]. However, maximal inhibition of atrazine binding could be observed only at relatively high DPC concentrations (>2.5 mM) (Fig. 2) whereas electron donation is usually optimal between 0.5-1 raM. Thus,
the action of DPC on atrazine binding seems to originate from the binding DPC at a site having a weaker affinity in comparison with the usual electron donation site. A second hypothesis would be that DPC directly competes with atrazine at the QB site. Diphenylcarbazide would not be the first member of the family of electron donors and acceptors to affect herbicide binding. Silicomolybdate, which was initially though to accept electrons from PSII at a DCMU insensitive site [22,23] was later reported to compete with atrazine and D C M U binding [24,25]. In line with this second hypothesis the selective action of DPC on the binding of various compounds acting in the QB pocket could be understood in terms of various degrees of overlapping between their binding sites, a remaining more specific binding domain being reserved to each one, similar to the binding of all the herbicides acting at QB [26,27]. In that case, DPC would affect only part of the QB pocket at concentrations where it is used as an electron donor, leaving almost unaffected the binding sites of plastoquinone and FeCN.
4. Acknowledgments This work was supported in part by a grant to R.C. from the Natural Sciences and Engineering Research Council of Canada (NSERC). M.P. was supported by a postgraduate fellowship from NSERC.
5. References l O. Nanba and K. Satoh, Isolation of a photosystem II reaction center consisting of D1 and D2 polypeptides and cytochrome b-559. Proc. Natl. Acad. Sci USA, 84 (1987) 109-112. 2 D.J. Kyle, The 32 000 dalton QB protein of photosystem II. Photochem. Photobiol., 41 (1985) 107-116. 3 W.F.J. Vermaas, G. Renger and C.J. Arntzen, Herbicide/quinone binding interactions in photosystem 11. Z. Naturforsch., C39 (1984) 368-373. 4 J.E. Mullet and C.J. Arntzen, Identification of a 32-34 kilodalton polypeptide as a herbicide receptor protein in photosystem II. Biochim. Biophys. Acta, 635 (1981) 236-248. 5 K. Pfister and C.J. Arntzen, The mode of action of
M. Purcell, R. Carpentier /Plant Sci. 94 (1993) 55-59
6
7
8
9
10
11
12
13
14
15
photosystem II-specific inhibitors in herbicide-resistant weed biotypes. Z. Naturforsch Teil, C34 (1979) 996-1009. A.K. Mattoo, U. Pick, H. Hoffmann-Falk and M. Edelman, The rapidly metabolized 32 000 dalton polypeptide of chloroplast is the 'proteinaceous shield' regulating photosystem 11 electron transport and mediating diuron herbicide sensitivity. Proc. Natl. Acad. Sci. USA, 78 (1981) 1572-1576. M. Purcell, G.D. Leroux and R. Carpentier, Atrazine action on the donor side of photosystem II in triazineresistant and -susceptible weed biotypes. Pest. Biochem. Physiol., 37 (1990) 83-89. G.T. Babcock, The photosynthetic oxygen-evolving process, in: J. Amesz (Ed.), Photosynthesis, Elsevier, Amsterdam, 1987, pp. 125-158. J.G. Metz, P.J. Nixon, M. Rogner, G.M. Brudvig and B.A. Diner, Directed alteration of the DI polypeptide of photosystem II evidence that tyrosine-161 is the redox component, Z, connecting the oxygen-evolving complex to the primary electron-donor, P680. Biochemistry, 28 (1989) 6960-6969. R.J. Debus, B.A. Barry, 1. Sithole, G.T. Babcock and L. Mclntosh, Directed mutagenesis indicates that the donor to P+680 in photosystem II is tyrosine-161 of the D1 polypeptide. Biochemistry, 27 (1988) 9071-9074. M. Purcell, G.D. Leroux and R. Carpentier, Interaction of the electron donor diphenylcarbazide with the herbicide-binding niche of photosystem II. Biochim. Biophys. Acta, 1058 (1991) 374-378. R. Carpentier, R.M. Leblanc and M. Mimeault, Photoinhibition and chlorophyll photobleaching in immobilized thylakoid membranes. Enzyme Microb. Technol., 9 ( 1987~ 489-493. D.I. Arnon, Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol., 24 (1949) 1-15. A. Rashid and R. Carpentier, The 16 and 23 kDa extrinsic polypeptides and the associated Ca 2+ and CI modify atrazine interaction with the photosystem II core complex. Photosynth. Res., 24 (1990)221-227. R. Carpentier, B. LaRue and R.M. Leblanc, Photoacoustic spectroscopy of Ana~Tstis nidulans III. Detection of photosynthetic activities. Arch. Biochem. Biophys., 228 (1984) 534-543.
59 16
17
18
19
20
21
22
23
24
25
26
27
D.A. Walker, L.J. Ludwig and D.G. Whitehouse, Oxygen evolution in the dark following illumination of chloroplasts in the presence of added manganese. FEBS Lett., 6 (1970) 281-284. R.D. Ort and S. Izawa, Studies on the energy-coupling sites of photophosphorylation. Plant Physiol., 53 (1974) 370-376. R. Carpentier, P. Euerst, H.Y. Nakatani and C.l. Arntzen, A second site for herbicide action in photosystem II. Biochim. Biophys. Acta, 808 (1985) 293-299. A. Rashid and R. Carpentier, CaCI 2 inhibition of H202 electron donation to photosystem II in submembrane preparations depleted in extrinsic polypeptides. FEBS Lett., 258 (1989) 331-334. J.G. Metz, H.B. Pakrasi, M. Siebert and C.J. Arntzen, Evidence for a dual function of the herbicide-binding DI protein in photosystem If. FEBS Lett., 205 (1985) 269-274. G. Renger, R. Hagemann and R. Fromme, The susceptibility of the p-benzoquinone-mediated electron transport and atrazine binding to trypsin and its modification by CaCI 2 in thylakoids and PSII membrane fragments. EEBS Lett., 203 (1986) 210-214. G. Girault and J.M. Galmiche, Restoration by silicotungstic acid of DCMU-inhibited photoreactions in spinach chloroplasts. Biochim. Biophys. Acta, 333 (1974) 314-319. S.P. Berg and S. Izawa, Pathways of silicomolybdate photoreduction and the associated photophosphorylation in tobacco chloroplasts. Biochim. Biophys. Acta, 460 (1977) 206-219. P. B6ger, Replacement of photosynthetic electron transport inhibitors by silicomolybdate. Physiol. Plant, 54 (1982) 221-224. Y. Graan, The interaction of silicomolybdate with the photosystem II herbicide binding site. FEBS Lett., 20 (1986) 9-12. K. Pfister, K.E. Steinkack, G. Gardner and C.J. Arntzen, Photoaffinity labeling of an herbicide receptor protein in chloroplast membranes. Proc. Natl. Acad. Sci. USA, 78 (1981) 981-985. A. Trebst, The three-dimentional structure of the herbicide binding niche on the reaction center polypeptides of photosystem II. Z. Naturforsch, Teil C42 (1987) 724-750.
Plant Science 94 (1993) 55-59
Alteration of the stromal side of photosystem II by diphenylcarbazide Marc Purcell, Robert Carpentier* Centre de recherche en photobiophysique University; du Qukbec ~ Trois-Rivibres C.P. 500, Trois-Rivibres, Qukbec, Canada G9A 5H7
(Received 8 March 1993: revision received 2 July 1993: accepted 2 July 1993)
Abstract
In the thylakoid membranes, the photosystem II electron donor diphenylcarbazide decreases the inhibitory effect of quinone-type herbicides like atrazine (Purcell et al., 1990, Pest. Biochem. Physiol. 37, 83-89). This behavior is studied in more detail and it is shown that the donor strongly inhibits the binding of radiolabelled atrazine to the Q~ protein. The electron acceptor activity of 2,6-dichlorobenzoquinone (DCBQ) is also impaired but not that of potassium ferricyanide or 2,6-dichlorophenolindophenol (DCIP). Electron transport from photosystem II to photosystem I, which involves the reduction of plastoquinone at the QB site, is only slightly affected. This data indicated that at concentrations where it is commonly used as an electron donor (0.5- 1.0 mM), diphenylcarbazide can alter the binding of herbicides and artificial electron acceptors having overlapping-binding domains in the QB pocket. However, the effect of diphenyl-carbazide on the QB site can affect plastoquinone reduction only at relatively high concentrations (>2 mM). Key words: Diphenylcarbazide; Atrazine; Photosystem II; QB Protein; Electron transport; Plastoquinone
1. Introduction
The core complex of chloroplast photosystem (PS) II is composed of two transmembranous polypeptides of apparent molecular weight of 33 kDa named D1 and D2, respectively [1]. The polypeptide D1 was initially known as the QBpolypeptide because it bears the binding site of the secondary quinone acceptor QB [2]. This site can also be occupied by several herbicides and artificial electron acceptors of quinone type which then * Corresponding author.
results in inhibition of plastoquinone (PQ) photoreduction [3-6]. It was reported that the PSII electron donor diphenylcarbazide (DPC) can decrease the extent of atrazine and 3-(3,4-dichlorophenyl)-l,l-dimethylurea (DCMU) inhibition of 2,6-dichlorophenolindophenol (DCIP) photoreduction in thylakoid membranes [7]. Because these inhibitors bind in the QB pocket located on the stromal side of the photosystem, the decreased inhibition was not due to the electron donor properties of DPC. In fact, this donor is known to transfer electrons directly to Z, the secondary donor of PSII [8-9].
0168-9452/93/$06.00 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved. SSDI 0168-9452(93)03700-6
56
This intermediate is probably the tyrosine-161 of the polypeptide D1 and is located on the lumenal side of the thylakoid membrane [10]. We have found that besides its electron donor capabilities in PSII, DPC also alters the binding of atrazine to the stromal side of the photosystem [11]. This explains the reduced inhibitory action of the herbicide in the presence of the donor. In this report, we present a further study of the effect of DPC on the stromal side of PSII. Another quinone-type molecule, the artificial electron acceptor 2,6-dichlorobenzoquinone (DCBQ), also shows a decreased ability to accept electrons from the QB pocket in the presence of DPC whereas potassium ferricyanide (FeCN) photoreduction remains unaffected. It is also shown that DPC inhibits the reduction of endogenous PQ only slightly and does not significantly affect linear electron transport to PSI at concentrations where it is used as electron donor. The data are discussed in terms of a possible overlapping between the binding sites of herbicides, electron acceptors and DPC in the herbicide binding niche. 2. Materials and methods
2.1. Thylakoid membrane preparation Thylakoid membranes were isolated from spinach leaves as described previously [12] and resuspended in a solution containing 50 mM N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (Tes)-NaOH, pH 7.5, 2 mM MgCI2, 1 mM NH4CI , and 300 mM sorbitol at a chlorophyll (Chl) concentration of 2 mg/ml. Chlorophyll was determined according to Arnon [13]. The preparations were used the same day or stored in liquid nitrogen. 2.2. DCIP photoreduction Light minus dark rates of DCIP photoreduction were measured at 600 nm using a Perkin-Elmer model 553 UV/VIS-spectrophotometer as described elsewhere [14]. The reaction mixture was composed of thylakoid membranes at a Chl concentration of 5 ~g/ml, 30 #M DCIP, 50 mM TesNaOH pH 7.5, 2 mM MgC12, 1 mM NHaCI, and 330 mM sorbitol.
M. Purcell, R. ('arpentier / Plant Sci, 94 (1993) 55-59
2.3. Oxygen evolution and uptake measurements Oxygen evolution was monitored with a Clarktype electrode as described previously [15]. The assay medium was composed of thylakoid membranes at a Chl concentration of 11/zg/ml suspended in the above buffer with an electron acceptor as specified in the text. Oxygen uptake was monitored in similar samples but with 0.5 mM methylviologen (MV) as electron acceptor. Samples were illuminated for 1.5 rain in the oxygen electrode cell. Then, 2000 units of catalase were added in the dark. The oxygen release upon addition of catalase was measured to determine the Mehler reaction [16-17]. 2.4. Herbicide binding Herbicide binding experiments were performed under dim light using a procedure similar to that described by Carpentier et al., [18]. The reaction mixture (1 ml) contained thylakoid membranes equivalent to 50/~M Chl, 50 mM Tes-NaOH pH 7.5, 2 mM MgCI2, 1 mM NH4CI, and 330 mM sorbitol. [14C]atrazine (19.5 /zCi/mg, Ciba Geigy, Greensboro, NC) was added to obtain between 90-2000 disintegrations/rain in the counting vials in the presence of specified concentrations of DPC. The samples were incubated for 10 min at room temperature and harvested in an Eppendorf centrifuge (4 min). Aliquots of 0.75 ml were taken from the supernatant fractions for the determination of free atrazine by scintillation counting. 3. Results and discussion
The inhibition of DCIP photoreduction by atrazine is described in Fig. 1. Thylakoid membranes were used with or without 1.6 mM DPC. It is clear from Fig. 1 that the photoactivity strongly decreases as the herbicide concentration is raised. However, there is always less inhibitory action in the presence of DPC. The reduced inhibitory effect is quantified in Fig. 1 (inset). The release of inhibition varies from 100 down to 10% and decreases with increasing atrazine concentrations. As mentioned, this effect on atrazine inhibition cannot be accounted for by the electron donating properties of DPC. Instead, it was found that atra-
M, Purcell, R. Carpentier / Plant Sci. 94 (1993) 55-59
57
o~ v
100 z 0
F--
80
¢0
12C ~) 108 60
a LU
.~ 8o
n-
~ 6o ~ 4o
O
~0
40
_
2o'- Ol
'I-
2O
20
_1
¢0 Q
^
008'1
'
1 0 .7
10 .6
ATRAZINE
10 -s
0 .4
(M)
Fig. 1. DCIP photoreduction with (Q), and without (O) 1.6 mM DPC. The rates corresponding to 100% were 300 and 270 ~mol DCIP/mg Chl" h with and without DPC, respectively. Inset: relief of inhibition in the presence of 1.6 mM DPC calculated from the data of Fig. 1.
zine binding was affected by the donor [11]. It is shown in Fig. 2 that the amount of [14C]atrazine bound to the thylakoid membranes decreases in proportion to the concentration of DPC at the two atrazine concentrations used. The DPC concentration required to decrease atrazine binding by 50% is 1.4 ± 0.1 mM. However, the effect is already seen within the range of concentrations used for
E
electron transport studies when DPC serves as a donor. DPC also alters the binding of the PSII electron acceptor DCBQ which receives its electrons from the QB site. In fact, DPC strongly inhibits oxygen evolution when DCBQ is used as acceptor (Table 1). However, if FeCN is used as acceptor instead of DCBQ, there is no detectable effect of DPC on the initial rates of oxygen evolution. Surprisingly, DPC does not reduce the extent of DCIP photoreduction (Fig. 1). DCIP accepts electrons directly from the pool of reduced plastoquinone and does not interact directly with the QB site. This implies that the reduction of plastoquinone must proceed freely in the presence of DPC. To verify this point more precisely, electron transport from water to MV was measured with and without DPC. Oxygen uptake was measured without interference from oxygen evolution using the catalase assay (see Materials and methods). The rates of MV reduction were not affected by 1 mM DPC but they were slightly decreased (by 15%) in the presence of 2.5 mM DPC (Table 2). In order to reduce MV, PSI must receive its electrons from PSII with plastoquinone as a mandatory intermediate. It was verified for this experiment that DPC could not act as an electron donor to PSI. In fact, in samples inhibited by 1.2 mM D C M U (in order to obtain full inhibition of PSII, high DCMU concentrations were required to overcome
Table 1 Effect of DPC on oxygen evolution
e- 2 0
Additions
Oxygen evolution a (p,g mol 02/ mg Chl" h)
DCBQ b (600 ttM) DCBQ (600 t~M) + DPC c (0.5 mM) DCBQ (600 #M) + DPC (1.0 mM) FeCN d (10 mM) FeCN (10 raM) + DPC (0.5 raM) FeCN (10 mM) + DPC (1.0 mM)
319 55 16 170 168 171
Z
O "'10 Z N
nI--
6 0
1
2
DPC
(mM)
3
Fig. 2. Inhibition of atrazine binding by DPC in the presence of 0,05 (O), or 0.25 (Q) t~M atrazine.
aThe values represent the average of three separate experiments and the variations were within 4%. b2,6-Dichlorobenzoquinone. CDiphenylcarbazide. dpotassium ferricyanide.
58
M. Purcell. R. Carpentier / Plant Sci. 94 (1993) 55-59
Table 2 Effect of DPC on oxygen uptake by PS! through the Mehler reaction (H20-- MV) using the catalase assay~ Addition
Oxygen uptakeb (#mol O~/ mg Chl" h)
None DPC (1.0 mM) DPC (2.5 mM) DCBQ (600 #M) DCBQ (600 t~M)+ DPC (I.0 mM)
110 108 93 27 60
aConditions are given in Materials and methods. bThe values represent the average of three separate experiments and the variations were within 5'7,,.
the adverse effect of DPC) there was no oxygen uptake activity even in the presence of 2 mM DPC but the addition of the donor system DCIP/ascorbate resulted in an 02 uptake activity of 550 t~mol/mg Chl" h. As a comparison, in Table 2, DCBQ is shown to strongly inhibit oxygen uptake; this is because it prevents plastoquinone reduction when it is serving as electron acceptor. If DPC is added together with DCBQ, the inhibitory effect of the acceptor is greatly released as predicted from the data of Table 1. The experiments presented in Table 2 indicate that DPC affects plastoquinone binding but only at high concentrations. Inhibition of atrazine, DCBQ, and PQ binding could proceed via two different mechanisms. The first possibility would be that DPC may modify the QB site through a transmembrane conformational change. Eventhough DPC is known to interact at the lumenal side of PSII to reduce the secondary donor Z, both the Q8 site and the DPC oxidation site are presumably located on the same transbilayer polypeptide complex which contains the reaction center of PSII [1]. Furthermore, the acceptor side of PSII was already shown to be affected by modifications of the lumenal side in several types of experiments [19-21]. However, maximal inhibition of atrazine binding could be observed only at relatively high DPC concentrations (>2.5 mM) (Fig. 2) whereas electron donation is usually optimal between 0.5-1 raM. Thus,
the action of DPC on atrazine binding seems to originate from the binding DPC at a site having a weaker affinity in comparison with the usual electron donation site. A second hypothesis would be that DPC directly competes with atrazine at the QB site. Diphenylcarbazide would not be the first member of the family of electron donors and acceptors to affect herbicide binding. Silicomolybdate, which was initially though to accept electrons from PSII at a DCMU insensitive site [22,23] was later reported to compete with atrazine and D C M U binding [24,25]. In line with this second hypothesis the selective action of DPC on the binding of various compounds acting in the QB pocket could be understood in terms of various degrees of overlapping between their binding sites, a remaining more specific binding domain being reserved to each one, similar to the binding of all the herbicides acting at QB [26,27]. In that case, DPC would affect only part of the QB pocket at concentrations where it is used as an electron donor, leaving almost unaffected the binding sites of plastoquinone and FeCN.
4. Acknowledgments This work was supported in part by a grant to R.C. from the Natural Sciences and Engineering Research Council of Canada (NSERC). M.P. was supported by a postgraduate fellowship from NSERC.
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