A reassessment of the use of herbicide binding to measure

Photosynthesis Research 24: 109-113, 1990. © 1990KluwerAcademic Publishers. Printedin the Netherlands. Short communication

A reassessment of the use of herbicide binding to measure photosystem II reaction centres in plant thylakoids W.S. Chow ~, A.B. Hope 2 & Jan M. Anderson 1 t CSIRO, Division of Plant Industry GPO Box 1600, Canberra, A C T 2601, Australia. :School of Biological Sciences, Flinders University, Bedford Park, S.A. 5042, Australia. Received 8 June 1989;accepted28 September 1989

Key words." atrazine, diuron, photosystem II, photosynthesis, thylakoids Abstract

The binding of the herbicide atrazine to thylakoid membranes is often used to quantify Photosystem II reaction centres. Two atrazine binding sites, with high and low affinities, have been observed on the D1 and D2 polypeptides of Photosystem II, respectively (McCarthy S., Jursinic P. and Stemler A. (1988) Plant Physiol. 86S:46). We have observed that the accessibility of the low-affinity binding sites is variable, being limited in freshly isolated thylakoids or in fresh frozen-thawed thylakoids, but increasing during storage of the membranes on ice. In contrast, the accessibility of the high-affinity binding sites, which are titratable at low concentrations (< 500 nM) of herbicide, is much less variable, although the dissociation constant is greatly influenced by ethanol. We conclude that to quantify Photosystem II reaction centres by atrazine binding, it is sufficient and more reliable to assay only the high-affinity binding sites.

Introduction

The ratio ofPhotosystem II to Photosystem I reaction centres (PSII/PSI) has recently been a topic of considerable interest and debate (see Chow et al. 1988; Jursinic and Dennenberg 1989). While PSI is satisfactorily assayed by the chemical or photochemical oxidation of the reaction centre chlorophyll a (P700), PSII has been determined by a variety of methods. Estimates of PSII have varied widely, ranging from ~ 1 to ~ 3 mmol (mol Chl) - 1. This variation arises partly from the variability in the plant materials and partly from the different methods used. One of the methods used to determine [PSII] is based on the specific binding of certain herbicides to thylakoid membranes, assuming there is a fixed number of herbicide binding sites per PSI1 reaction centre. Using radioactively-labelled herbicides, it has been shown (Tischer and Strotmann 1977; Oettmeier et al. 1982) that herbicide binding was directly related to the inhibition of linear electron

transport. Herbicide-binding is usually analysed by a plot of the reciprocal of bound herbicide, Cb (mol Chl/mol) vs. the reciprocal of free herbicide concentration, Cf (M-~), which gives for a singlespecies of binding site a straight line of the form: 1/Cb =

1/Cb.... -I- (Kd/C b. . . . )'(l/Of)

where Cb.... is the maximum number of binding sites, obtained from the intercept, and Kd is the dissociation constant of the herbicide-PSII complex, and is inversely related to the affinity of the binding sites. Occasionally, a marked deviation from a straight line is evident at high concentrations of atrazine (Tischer and Strotmann 1977; Jursinic and Stemler 1983), diuron (Oettmeier et al. 1982) or phenmedipham (Tischer and Strotmann 1977). Initially, the downward deviation from a straight line obtained with high concentrations of herbicide was attributed to non-specific binding to thylakoid membranes (Tischer and Strotmann 1977; Laasch et al. 1981). Subsequently, McCarthy et al. (1988) showed that there were in fact two

110 classes of atrazine-binding sites in pea thylakoids, one with a high affinity (low Kd) corresponding to the linear region of the double-reciprocal plot, and the other with a low affinity (high Kd) contributing to the non-linearity at high concentrations. The high- and low-affinity sites are believed to be located in the D1 and D2 polypeptides of the PSII reaction centres, respectively (McCarthy et al. 1988). Our previous studies showed an approximate 1: 1 correspondence between herbicide-binding sites and functional PSII complexes (capable of evolving oxygen in repetitive flashing) when a miss factor in the latter measurement was taken into account (Chow and Anderson 1987; Chow and Hope 1987; Chow et al. 1989). Because we wished to avoid non-specific binding of herbicide to thylakoid membranes, we used relativley low concentrations (< 500 nM) of atrazine or diuron (DCMU) in our previous studies. Hence only the high-affinity sites were measured, as indicated by the linearity in the double-reciprocal plots. Recently, however, Jursinic and Dennenberg (1989) have raised the possibility that falsely-low concentrations of the binding sites could be found when insufficient concentrations of ~4C-atrazine are used, because of incomplete binding to both classes of sites. To see whether more accurate estimates of [PSII] are obtained using low or high concentrations of herbicide, and in response to the comments of Jursinic and Dennenbeg (1989), we have investigated herbicide binding over an extended range of concentrations (up to 1.5 or 2.0 #M) under a variety of conditions.

Materials and methods

Chloroplasts were isolated from freshly-harvested leaves of Pisum sativum (cv. Greenfeast) grown under fluorescent light (~100#mol photons m -2 s -1, 16h light/8h dark) or Spinacia oIeracea (Henderson's hybrid 102) grown in a glasshouse. The method of rapidly isolating chloroplasts was described previously (Chow and Hope 1987). Strips of leaf tissue were homogenised at 0°C in a Waring blendor for about 5 s, using an isolation medium containing 400 mM sorbitol, 50 mM MES-NaOH (pH 6.5), 5mM MgC12, 10mM NaC1 and 0.2% (w/v) bovine serum albumin. The homogenate was

filtered through three layers of Miracloth and centrifuged for 30 s at 1200 x g using a swing-out rotor. The pellet was resuspended in a small amount of isolation medium, and was either kept on ice or promptly frozen at 77 K. Herbicide binding was done by the method of Tischer and Strotmann (1977). Binding was initiated by vortexing 1 ml of thylakoid suspension (50pM Chl) containing 400mM sucrose, 50mM TES-NaOH (pH 7.5), 10mM NaC1 and 5mM MgC12, to which was added [ethyl- l -14C] atrazine stock (50 #M in 20% ethanol) or 14C-diuron stock (50/~M in 80% ethanol). Both radiochemicals were obtained form Amersham, England. The final concentration of ethanol was made equal in a series of herbicide concentrations. After equilibrating in darkness for 3 min, the suspensions were centrifuged in an Eppendorf 5414 centrifuge for 3 min. The clear supernatant (0.7 ml) was mixed with 5 ml of Beckman Ready-Solv EP scintillant and counted. The herbicide concentration in (he supernatant was determined from a straight calibration line obtained by processing the standards in the same way without chloroplasts. The difference between the herbicide added to a thylakoid suspension and that remaining in the supernatant was taken as the amount of bound herbicide.

Results and discussion

When thylakoids were rapidly isolated from freshly-harvested pea leaves (see Methods), and used for atrazine-binding assays within 2 h, a straight line in the double reciprocal plot was obtained, even when the added concentration of herbicide was extended up to 2#M (o, Fig. 1). Similarly, if these freshlyisolated thylakoids were immediately frozen in liquid nitrogen, then subsequently thawed and assayed promptly, a straight line double reciprocal plot was also obtained, without any downward deviation at high concentrations of atrazine (A, Fig. 1). Our results indicate that only one class of atrazine-binding sites was titrated in fresh thylakoids or fresh frozen-thawed thylakoids. Further, the immediate freezing of freshly-isolated thylakoids for subsequent assay is an acceptable alternative to the immediate assay of fresh thylakoids. In contrast, upon storage of the thylakoid stock on ice for an appreciable period, a downward de-

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viation from a straight line was observed at high concentrations of atrazine (o, Fig. 1). This result suggests that (a) low-affinity binding sites became accessible to atrazine during storage on ice, indicating conformational changes in the PSII complex, and (b) the deviation from linearity was due to

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specific binding to low-affinity sites rather than "non-specific" binding, which would also have led to non-linearity in freshly-isolated thylakoids. Another critical point for accurate measurement of the herbicide binding concerns the ethanol concentration carried over with the addition of herbicide from an ethanol stock solution. As shown in Fig. 2 for spinach thylakoids with atrazine, when ethanol was increased from 0.6 to 10.0%, the slope of the straight lines increased, whilst the y-intercept remained essentially constant (i.e. constant number of binding sites). This result indicates that the affinity of the single species of binding sites for atrazine decreased with increasing ethanol concentration, e.g., Kd was 53, 69, 107 and 349nM for ethanol concentrations of 0.6, 1.5, 3.0 and 10.0%, respectively. McCarthy et al. (1988) reported that Kd was 73 nM for the high-affinity atrazine-binding sites in pea thylakoids. Presumably it is the same species of sites whose affinity was affected by ethanol here in Fig. 2. In Fig. 2, the regression lines could be interpreted to indicate a 'competitive' inhibition of atrazine binding by ethanol, analogous to the competitive displacement of [3H] D C M U by plastoquinone- 1 in plastoquinone-depleted thylakoids (Oettmeier and Soil 1983). Assuming that the experimentallydetermined apparent dissociation constant K~ for atrazine varies with ethanol concentration [I] due to competitive inhibition according to the relationship (Fersht 1985)

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Fig. 2. Double-reciprocal plots of herbicide binding to frozenthawed thylakoids isolated from a single batch of spinach leaves grown in a glasshouse. The numbers indicate % ethanol (v/v) present in each of a series of samples. Linear regression lines are shown. (A) DCMU binding, y-intercept 312, K'd 32nM. (e, ~ , II, o) atrazine binding, K~ values being 53, 69, 107 and 349 nM and y-intercepts being 377, 373, 377 and 366 for the ethanol concentrations 0.6, 1.5, 3.0 and 10.0%, respectively.

where Kd is the extrapolated value (40 nM) for the dissociation constant for atrazine in the absence of ethanol, a dissociation constant for ethanol Ki = 1.8% (v/v) is obtained in the ethanol concentration range 0.6-3.0%. It is pertinent to point out that, whilst ethanol may competitively inhibit the binding of DCMU, it alone does not affect the capacity for uncoupled electron transport from H20 to ferricyanide unless its concentration exceeds 15% (Masamoto and Nishimura 1978). Methanol has a similar though somewhat smaller effect on Kd (W.S. Chow and J.R. Evans, unpublished data). Therefore, it is surprising that Tischer and Strotmann (1977) obtained similar values for Kd with 5% methanol, and K (inhibition of electron transport) with 2.5% methanol. However, the latter assay was done during il-

112 lumination, which decreases the affinity of the binding sites for herbicide (Jursinic and Stemler 1983), as does an increase in methanol. If in general the alcohol concentration was allowed to increase with the increase in herbicide concentration in binding assays, there would be a tendency for an upward deviation from the straight line in the double-reciprocal plot, tending to offset a downward deviation due to any low-affinity binding. In Fig. 2, where [ethanol] was kept constant in a series of herbicide concentrations, the data points lie on a straight line up to at least 2 #M in added herbicide concentration for both DCMU and atrazine. In some other freshly-isolated batches of spinach thylakoids, however, a low-affinity binding site was detected (data not shown), indicating again that the accessibility of low-affinity sites to DCMU and atrazine may be variable. We have also compared the binding of atrazine and DCMU to see if the number of high-affinity binding sites for the two herbicides is the same. As can be seen from Fig. 2, the DCMU line (bottom) intercepts the vertical axis at 312, and the atrazine lines intercept the vertical axis at ~ 374. That is, there are apparently 20% more DCMU-binding sites than atrazine-binding sites in spinach thylakoids. Oettmeier et al. (1982) also reported that DCMU-binding sites exceeded atrazine-binding sites in Arnarantkus retroflexus by 12%. The reason for the discrepancy between the concentrations of the high-affinity DCMU- and atrazine-binding sites is not clear. It could be that a proportion of PSII reaction centres are unable to bind atrazine with high affinity, but are nevertheless able to bind DCMU as tightly as the majority of PSII reaction centres. Such a sub-population of PSII would be titrated by DCMU but not atrazine at the low concentrations of herbicide added. An extreme example of such a case is the atrazine-resisrant Amaranthus retroflexus which binds DCMU to the same extent as the wild type, but has no highaffinity binding sites for atrazine or metribuzin (Oettmeier et al., 1982). Also consistent with the present proposal is our observation that high concentrations of atrazine competed against DCMU less effectively than expected. For example, in a spinach thylakoid suspension (50#M Chl) with 0.2#M 14C-DCMU added, approximately 80% of the high-affinity sites of PSII bound DCMU; even on adding 5 #M non-radioactive atrazine, a sub-

stantial proportion (about 18 %) of the high-affinity sites still bound DCMU. A further difference between the two herbicides is that their binding to spinach thylakoids responds differently to ferricyanide, with a drastic effect of the oxidant on DCMU binding (Wraight 1985) but little effect on atrazine binding (Renger et al. 1988). Because of such different characteristics of the two herbicides, perhaps an identical number of high-affinity binding sites should not necessarily be expecte& Given that the DCMU-binding sites exceed those of atrazine in isolated thylakoids, one would like to known which herbicide is preferable for use in binding studies with thylakoids. When the functional PSI! centres in leaf discs were assayed directly be repetitive flashing (Chow et al. 1989), the number of functional PSII centres in leaf discs was about 12% lower than the number of DCMUbinding sites. The slightly lower number of functional PSII centres was attributed to a 'miss' factor (Chow and Anderson 1987, Chow and Hope 1987; Chow et al. 1989), but may be more accurately accounted for by back reactions following charge separations (Jursinic and Pearcy 1988). In any case, DCMU used at low concentrations seems to give a satisfactory method to assay PSII in isolated thylakoids. On the other hand, allowing for 'misses' or back reactions in the assay, the number of functional PSII centres in leaf discs would exceed the number of high-affinity atrazine binding sites. Previously, we reported (Chow and Anderson 1987; Chow and Hope 1987) that the functional PSII complexes in isolated thylakoids was 12-16% less abundant than atrazine binding sites. However, it is now realized that in assaying functional PSII complexes in isolated thylakoids, an underestimation can occur from the use of 0.5 mM phenyl-pbenzoquinone; this concentration of the electron acceptor inhibits flash-induced 02 evolution by up to 24% as compared with ferricyanide which yields concentrations of functional PSII in gently-isolated spinach thylakoids very similar to those directly measured in leaf discs (unpublished data). In conclusion, to quantify PSII reaction centres by herbicide binding, it is sufficient to assay the high-affinity binding sites, using low concentrations of herbicides and fixed concentrations of carriedover alcohol. We feel that this approach is preferable to using high concentrations of herbicide to titrate both high- and low-affinity sites and dividing

113 the t o t a l n u m b e r by t w o . I n o u r e x p e r i e n c e , the accessibility o f the l o w - a f f i n i t y b i n d i n g sites m a y v a r y f r o m o n e p r e p a r a t i o n to a n o t h e r a n d w i t h ' a g e i n g ' o f t h y l a k o i d s o n ice, t h u s m a k i n g a r e l i a b l e e s t i m a t e o f t h e s e sites difficult.

Acknowledgements T h i s c o l l a b o r a t i v e p r o j e c t was p r o m o t e d b y a j o i n t C S I R O / F l i n d e r s U n i v e r s i t y g r a n t , f o r w h i c h we are grateful. We thank Ms Stephanie Hossack-Smith f o r e x c e l l e n t t e c h n i c a l assistance, a n d D r T o n y A s h t o n f o r h e l p f u l discussions.

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