Plant Physiol. (1983)72, 925-930 0032-0889/83/72/0925/06/$00.5/0

Comparison of Photosynthetic Performance in Triazine-Resistant and Susceptible Biotypes of Amaranthus hybridus' Received for publication March 2, 1983 and in revised form April 11, 1983

DONALD R. ORT, WILLIAM H. AHRENS2, BJORN MARTIN, AND EDWARD W. STOLLER Department ofPlant Biology (D. R. O., B. M.), Department ofAgronomy (W H. A., E. W S.), and United States Department ofAgriculture/Agricultural Research Service (D. R O., E. W S.), University ofIllinois, Urbana, Illinois 61801

ABSTRACT weed species growing on agricultural lands (for review, see 16). Investigations into the biochemical basis for the lower sensitivity The rate of CO2 reduction in the S-triazine-resistant biotype of smooth of these weeds to triazine herbicides have clearly established that pigweed (Amaranthus hybridus L.) was lower at all levels ofirradiance than the mode of resistance is at the level of the interaction of the the rate of CO2 reduction in the susceptible biotype. The intent of this herbicide with the photosynthetic electron transport chain (16). study was to determine whether or not the lower rates of CO2 reduction Maternal inheritance (9, 12) of triazine resistance indicates control are a direct consequence of the same factors which confer triazine resist- by a chloroplast rather than a nuclear gene(s). The implication to ance. The quantum yield of CO2 reduction was 23 ± 2% lower in the agriculture of herbicide selectivity based on a seemingly minor resistant biotype of pigweed and the resistant biotype of pigweed had about alteration in an intrinsic chloroplast membrane protein is truly 25% fewer active photosystem II centers on both a chlorophylH and leaf exciting. The emerging technology of molecular genetics makes area basis. This quantum inefficiency of the resistant biotype can be plausible the prospect of designed alterations in the chloroplast accounted for by a decrease in the equilibrium constant between the genome of crop plants to establish desired herbicide resistance. primary and secondary quinone acceptors of the photosystem II reaction Unfortunately, the weed biotypes resistant to triazine herbicides centers which in turn would lead to a higher average level of reduced often display other characteristics absent from their triazine-sus- primary quinone acceptor in the resistant biotype. Thus, the photosystem ceptible counterparts, characteristics which are decidedly not ad- II quantum inefficiency of the resistant biotype appears to be a direct vantageous. These resistant biotypes are reported to be competi- consequence of those factors responsible for triazine resistance but a tively less successful (1, 7, 24) with a rate of light- and C02- caveat to this conclusion is discussed. The effects of the quantum ineffi- saturated photosynthesis which is significantly depressed (1, 20). ciency of photosystem II on CO2 reduction should be overcome at high Arntzen and colleagues (16, 17) demonstrated that herbicide re- light and therefore cannot account for the lower light-saturated rate of sistance was manifested by a markedly decreased binding of the CO2 reduction in the resistant biotype. Chloroplast lameliar membranes inhibitor molecule to a 32 to 34 kD intrinsic membrane protein of isolated from both triazine-resistant and triazine-susceptible pigweed sup- the PSII reaction center complex. Subsequently, Bowes et al. (5) port equivalent rates of whole chain electron transfer and these rates are demonstrated that electron transfer from the primary quinone sufficient to account for the rate of light-saturated CO2 reduction. This acceptor (QA3) ofthe PSII reaction center to the secondary quinone observation shows that the slower transfer of electrons from the primary acceptor (QB) was as much as 10-fold slower in chloroplasts from to the secondary quinone acceptor of photosystem II, a trait which is triazine-resistant pigweed. These data are consistent with the characteristic of the resistant biotype, is nevertheless still more rapid than notion that the 32 to 34 kD protein is the apoprotein of QB (3). It subsequent reactions of photosynthetic CO2 reduction. Thus, it appears might be thought, based on these and related observations, that that the lower rate of light-saturated CO2 reduction ofthe resistant biotype the lower rate of CO2 fixation observed in resistant biotypes is a is not limited by electron transfer capacity and therefore is not a direct consequence of the increase in QA -* QB electron transfer time consequence of those factors which confer triazine resistance. and consequently a trait inseparable from the trait of herbicide resistance. If so, the impact on agriculture of engineering a tria- zine-resistant crop employing this mode of resistance would be significantly diminished. In this paper, we report on an investigation of photosynthesis in triazine-susceptible and triazine-resistant biotypes of Amaranthus hybridus. Based on the measurements ofthe quantum yield ofCO2 As many as one-half of all commercially available herbicides reduction by attached leaves and the flash-induced turnover of used in agriculture act by interfering with photosynthetic electron PSII in isolated chloroplasts, we conclude that (a) the resistant transfer reactions. The margin of selectivity of many of these biotype has about 25% ofits PSII reaction centers in a photochem- herbicides between the crop and unwanted plant species is disap- ically inactive state and (b) that this quantum inefficiency can be pointingly low, although the metabolic detoxification of triazines by corn is an exception of immense economic importance. In 3 Abbreviations used: QA, primary quinone acceptor of PSII: QB, sec- recent years, dramatically lower sensitivities of photosynthesis to ondary quinone acceptor of PSII; PQ, plastoquinone/plastoquinol; DAD, S-triazine herbicides have appeared in populations of numerous diaminodurene; DADox, diiminodurene; DMMDBQ, dimethyl-methyle- nedioxy-p-benzoquinone; DHQ, duroquinol, tetramethyl-p-hydroquinone; ' Supported in part by United States Department of Agriculture/Com- MV, methylviologen; DMQ, 2,5-dimethyl-p-benzoquinone; TMPD, petitive Research Grants Office Grant AG 82-CRCR-1-1075 to D. R. 0. N,N,N',N'-tetramethyl-p-phenylenediamine. 2HCI; DBMIB, 2,5-dibromo- 2 Present address: Plant Science Department, University of Delaware, 3-methyl-6-isopropyl-p-benzoquinone; DCPIP, 2,6-dichlorophenolindo- Newark, DE 19711. phenol. 925 926 ORT ET AL. Plant Physiol. Vol. 72, 1983 accounted for by a modest decrease in the equilibrium constant Electron Transport Studies. Light-saturated rates of 02 evolu- between the quinone acceptors of the PSII reaction center in the tion or uptake in isolated chloroplast membranes were measured resistant biotype. In addition, measurement of the light-saturated polarographically with a Clark-type 02 electrode in a continuously rates ofelectron transfer in isolated chloroplasts demonstrates that stirred 2-ml reaction at 20°C. Illumination was provided by a 500 the slower electron transfer between the two quinone acceptors of w tungsten incandescent lamp focused by a 500-ml round bottom the PSII reaction center of the resistant plant probably cannot flask containing 150 mm Fe(NH4)2(SO4)2 in 1 N H2SO4 (18), and account for the lower rate of light-saturated CO2 fixation. then filtered through two Coming filters (CS 1-75, CS 2-62). The detailed reaction conditions for individual reactions are given in MATERIALS AND METHODS the legend of Table I. Determination of the Chl to Photosystem Ratio. The number of Plant Material. Seed was obtained from a triazine-resistant and 02-evolving PSII reaction centers in a sample was measured by a triazine-susceptible population of smooth pigweed (Amaranthus exposing isolated chloroplast membranes to a series of 50 saturat- hybridus L.) which originated in Washington state. The plants ing flashes (6 ps half width, 11 J total discharge energy) from a were grown from seed in a soil-peat-vermiculite mixture in a xenon lamp (FX-193 Flashtube, EG&G, Salem, MA). Protons controlled environment chamber. The temperature during the 14- released due to the flash-induced oxidation of water were meas- h light period (500,uE*m-2.s-I PAR) was 30°C, and the chamber ured with a pH electrode (Orion 9103 combination electrode and was cooled to 20°C during the night. The RH was maintained a Keithley 6lOC electrometer) in uncoupled membranes incapable between 50 and 60%o. Plants were watered twice a day and nutrients of accumulating hydrogen ions. The 4-ml reaction solution con- were added three times a week. Some of the plants used for tained 100 mm sorbitol, 0.5 mm Tricine-NaOH (pH 7.8), 5 mm isolation of chloroplasts were grown in a glasshouse with a 15-h MgCl2, 25 mi KCI, 0.4 mm K3Fe(CN)6, 2 Atm gramicidin D, 0.2 light period supplemented by metal halide vapor lamps. The mM DMQ, and 155 nmol Chl. The pH changes were calibrated by glasshouse temperature was 24 ± 3C, RH was 40 to 60%, and the the injection of 20 nmol HCI. maximum light intensities were 1200 to 1400 ,uE* The technique used for determining the number of active PSI Isolation of Stroma-Free Chloroplast Lamellar Membranes. centers was similar to that described above for PSII centers. About 15 g of leaves from plants 15 to 30 cm tall were deveined However, in this case PSII turnover was inhibited by DCMU and and washed twice with chilled deionized H20. The leaves were TMPD was used to introduce electrons into PSI. Flash-induced then homogenized for 5 s at low speed in a Waring Blendor in 100 proton uptake associated with H202 formation during the aerobic ml of a solution containing 0.3 M NaCl, 50 mm Tricine-NaOH oxidation of photosynthetically reduced MV was monitored. The (pH 7.8), 3 mi MgCl2, 10 mm ascorbic acid, and 2 mg/ml BSA. 4-ml reaction mixture contained 100 mm sorbitol, 0.5 mM Tricine- The homogenate was filtered through 16 layers of cheesecloth and NaOH (pH 7.8), 5 mM MgCl2, 25 mM KCI, 1 mM EDTA, 0.1 mM then centrifuged at 2200g for 3 min. After discarding the super- MV, 5 ym DCMU, 2 IM gramicidin D, 1 yimol NaOH, 0.25 mM natant, the pellet was resuspended using a soft paint brush in TMPD.2 HCI, and 155 nmol Chl. approximately 2 ml of resuspending solution containing 100 mm Chi Content Per Unit of Leaf Area. Leaves from plants grown MgCl2, 10 mM sorbitol, 10 mm Tricine-NaOH (pH 7.8), 3 mM as described above were harvested and a 4 cm2 disc was taken KCI, and 1 mg/ml BSA. This was filtered through a Kimwipe, from the central portion of the leaf avoiding the midrib. The disc diluted with resuspending solution to about 30 ml, and centrifuged was homogenized in a tissue grinder in 80%o acetone (v/v). The at 3000g for 5 min. The supematant was again discarded and the extract was filtered through Whatman No. 4 filter paper. The Chl chloroplasts were stored in a few ml of resuspending solution concentration was determined using the extinction coefficients of giving a final Chl concentration of 1 to 2,umol/ml. This prepa- MacKinney (13). Five extractions representing five different plants ration procedure gave chloroplast lamellar membranes predomi- each biotype. nantly from mesophyll cells. were done for Measurement of CO2 Fixation. The uppermost leaf on the plant RESULTS that was almost fully expanded was enclosed in an assimilation chamber. The rate of light-saturated CO2 fixation was measured Comparison of the Light Intensity Dependence of the Rate of at 20°C in a closed compensating system at 300 ,lI/I or 1500 ,il/l CO2 Reduction in Triazine-Resistant versus Triazine-Susceptible ambient CO2 as described earlier (14). Biotypes of A. hybridus. Figure 1 shows that the rate of CO2 The quantum yield for photosynthetic CO2 reduction of an reduction in the resistant biotype of pigweed was lower at all attached leaf was measured at 300 p1/i ambient CO2 with the same levels of irradiance than the rate of CO2 reduction in the suscep- apparatus used for measurement of the light-saturated rates. All tible biotype. Inasmuch as these measurements were made at a light intensities used for quantum yield determinations were near CO2 concentration that is saturating for photosynthesis (i.e. 1500 or below the light compensation point. CO2 exchange was calcu- .d1-1) the difference in rate between the two biotypes cannot be lated from the rates at which the CO2 concentration changed in accounted for by a difference in stomatal aperture. The maximum the closed assimilation chamber. Total or gross photosynthesis rate of CO2 fixation was extraordinarily high in both biotypes of was calculated by subtracting the rate of CO2 exchange in the light pigweed being nearly equal to the highest rate for any species from the rate ofdark respiration. The amount of incident radiation previously reported (15). Light saturation was only barely attained that was absorbed was calculated by correcting for the portion of in the experiment depicted in Figure 1, at which point there was light which was either reflected or transmitted as described pre- a 15% difference in the rate of net photosynthesis between the two viously (14). The measurements were performed in an atmosphere biotypes. In a series of additional measurements made at 25% containing 21% 02 at 25°C and 90Yo RH. The light intensity was higher incident light intensity (i.e. 2300 ,IE-m-2-s-') and con- varied by inserting metal screens and diffusive glass plates between ducted on four plants of each biotype, the average difference the assimiliation chamber and a quartz iodide lamp (1000w, between the maximum rates of photosynthesis was 17% with a SE 3200K filament; General Electric)4 positioned above the chamber. of ±3%. The light saturation profile for the susceptible biotype had the 4 Mention of a trademark, proprietary product, or vendor does not normal hyperbolic appearance with half-saturation attained at an constitute a guarantee or warranty of the product by the United States incident light intensity of about 375 uE m.2s . The incident Department of Agriculture or the University of Illinois and does not imply light intensity required for half-saturation of the CO2 reduction its approval to the exclusion of other products or vendors that may also be rate in the resistant biotype was somewhat higher, about 500 uE. suitable. m . s', and the shape ofthe light saturation profile was distinctly PHOTOSYNTHESIS IN TRIAZINE-RESISTANT AMARANTHUS 927

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0 500 1000 1500 2000 0 Incident Light Intensity 0 5 10 15 20 (pE m-2 s-1) Absorbed Light (E * m-2 * s' ) FIG. 1. Comparison of the dependence of C02-saturated photosyn- FIG. 2. Comparison of the quantum yield for photosynthetic CO2 thesis on the incident light intensity in triazine-resistant and triazine- fixation in attached leaves of triazine-resistant and triazine-susceptible susceptible biotypes of A. hybridus. The ambient CO2 concentration was biotypes of A. hybridus. The ambient CO2 concentration was 300 tLll ', 1,500 L -.I', the ambient temperature was 20°C, and the RH 90%o. the ambient temperature was 25°C, and the RH 90%o. The absolute quantum yield was calculated from the slopes of the lines by linear least different. There is a remarkable similarity in the shape of the light squares analysis of the data points. intensity dependence curve of CO2 reduction of the resistant biotype of pigweed shown in Figure 1 and that of the resistant varied from 100 ms to 1 s. The same result was obtained when the biotype of Senecio vulgaris reported by Sims Holt et al. (20). flash energy was increased or decreased by 25% showing that the Comparison of the Quantum Yield of CO2 Reduction in Tria- flashes were saturating (data not shown). The data in Figure 3 zine-Resistant versus Triazine-Susceptible Biotypes ofA. hybridus. show that the Chl to PSII ratio was 25% higher in the resistant The absolute quantum yield ofCO2 reduction was calculated from biotype. the slope of the dependence of the CO2 reduction rate on the The number of active PSI centers was determined in a similar amount of light absorbed by the leaf (Fig. 2). All measurements fashion but in this case PSII turnover was prevented by the were made at irradiances near or below the light compensation presence of DCMU and electrons were supplied to PSI by the low point to avoid any involvement of differing stomatal conductance potential reductant TMPD. Since the oxidation of this compound to CO2 (i.e. the quantum flux density was the only factor limiting at pH values above 6 involves no change in protonation (19), the the rate of CO2 reduction in these determinations). The data in number of active PSI centers is equal to the number of protons Figure 2 show that the quantum yield of CO2 reduction was 25% taken up in H202 formation from the aerobic oxidation of pho- lower in the resistant biotype of pigweed. In three separate exper- tosynthetically reduced MV. The data (Fig. 3) show that the Chl iments, the mean of the difference in the quantum yield between to PSI ratio was very close to 600 in both biotypes. Virtually the the two biotypes was 23% with a SE of ±2%. This finding agrees same value was obtained for the ratio of Chl to Cytf in the two well with the 20%/o lower quantum yield in the resistant biotype of biotypes (630:1). This determination (data not shown) was made S. vulgaris reported by Sims Holt et aL (20). from measurements of the flash-induced turnover of Cyt f as Comparison of the Chl to Photosystem Ratio in Chloroplast described by Whitmarsh et al. (25) and calculated employing the Lameilar Membranes of Triazine-Resistant versus Triazine-Sus- extinction coefficient of Cytfestablished for spinach. ceptible Biotypes of A. hybridus. The ratio of Chl to active PSII The Chl concentration on a leaf area basis for the growth centers in chloroplast lamellar membranes isolated from both chamber grown pigweed used in this study was 8.6 ± 0.8 ,umol Chl triazine-resistant and triazine-susceptible biotypes ofpigweed was dm 2 in susceptible plants and 8.4 ± 0.7 ptmol Chl dm-2 in determined from measurements of proton release associated with resistant plants. Because there is no difference in the Chl content flash-induced oxidation of water with ferricyanide as the terminal of the leaves of the two biotypes, it is clear from the data in Figure electron acceptor. Each flash in the series was sufficiently intense 3 that there were about 25% more active PSII centers on a leaf to excite every reaction center in the sample yet short enough to area basis in the susceptible biotype. ensure that each reaction center turned over only a single time Comparison of Electron Transport Activities in Chloroplast (less than l1o double hits expected). Consequently, the number Lamellar Membranes Isolated from Triazine-Resistant versus of protons released from water oxidation on each flash was equal Triazine-Susceptible Biotypes ofA. hybridus. The rate of electron to the number of active PSII centers present in the sample. In transport required to support the maximum observed rate of CO2 Figure 3, H+ production due to water oxidation was measured fixation in attached leaves oftriazine-susceptible pigweed was 250 from a series of 50 flashes in which the intervening dark time was mmol e- mol Chl-P's-'. This was calculated on the basis of a 928 ORT ET AL. Plant Physiol. Vol. 72, 1983 Table I. Comparison of Electron Transport Activities in Chloroplasts 15C) Susceptible Isolatedfrom Triazine-Susceptible and Triazine-Resistant Biotypes of Amaranthus hybridus Light-saturated electron transport rates were measured polarographi- cally with a Clark-type 02 electrode at 20°C. Electron Transport Rate 125 PSI 550 System Photosystem - Susceptible Resistant )_* 3 A 650 mmole- *mol Chl -'ss-1 PS 31 H20-+DAD0xa II 270 + 15 140 + 10 H20--DMMDBQh 11 230 + 20 110 + 5 70 DAD *MVC I 630 + 50 660 ± 60 't DHQ-.MVC I 380 ± 30 390 ± 40 T I H20--+MVd I + 11 270 ± 15 265 ± 20 .4. a The 2-ml reaction consisted of 100 mm sorbitol, 20 mM Tricine-NaOH ) Resistant 715C o (pH 7.8), 3 mM MgCl2, 10 mm KCI, I tiM DBMIB, and chloroplast

4.. membranes containing 22 nmol Chl. The reaction rate was maximized in C.Z 15C resistant chloroplasts at 2.5 mM DAD and 5.25 mm K3Fe(CN)6 while the maximum rate in susceptible chloroplasts was attained at 2.0 mm DAD and 4.25 mm K3Fe(CN)6. The measurement was made three times with - o each of four PSI 550 t separate chloroplast preparations. Z1 lOC)- a A A b The 2-ml reaction consisted of 100 mm sorbitol, 50 mm Tricine-NaOH 75 _ 650_ (pH 8.0), 3 iM MgC12, 10 mm KCI, 0.1 mm MnCl2, 5 mm NaN3, 1 /LM DBMIB, 0.25 mm DMMDBQ, and chloroplast membranes containing 22 750 $ nmol Chl. The 5 PS3 measurement was made four times on each of three 850 chloroplast preparations. I_ c Light-induced electron transfer from DHQ (0.5 mm) or DAD (0.5 mm T plus 1 mm ascorbate) to MV was measured in a reaction mixture consisting of 50 mm sorbitol, 50 mm Tricine-NaOH mM mM 0 5 10 (pH 8.0), 3 MgCl2, 10 Flash Frequency (Hz) KCI, 5 mm NaN3, 0.1 mm MV, 5 ,Mt DCMU, 2 ,UM gramicidin D, 300 units/ml of superoxide dismutase, and chloroplasts containing 28 nmol FIG. 3. Comparison of the Chl to PSI and PSII ratios in chloroplast Chl. The measurement was made three times on each of four different membranes isolated from triazine-resistant and triazine-susceptible bio- chloroplast of A. The number of PSII centers preparations. types hybridus. 02-evolving reaction d The reaction mixture differed from that given in footnote c only in a was determined from the flash-induced H+ release by present sample the absence of DCMU and the exogenous associated with water-oxidation. The number of active PSI centers in the electron donor. sample was determined from H+ uptake associated with the oxidation of photosynthetically reduced methylviologen when electrons were supplied to PSI by exogenous TMPD. See "Materials and Methods" for a detailed DISCUSSION description of these measurements. There is good evidence (16, 17) that the resistance to inhibition of electron transfer by triazine herbicides in numerous weed requirement of four electrons per C02 molecule reduced and using species is due to an alteration in the PSII reaction center protein the Chl per leaf area data cited above. The data in Table I which binds the secondary quinone acceptor, QB. Our intent in demonstrate that the photosynthetic reduction of MV with elec- this study was to determine if the lower rates of light-saturated trons originating from water, a reaction which involves both CO2 reduction, which seems to be characteristic of triazine-resist- photosystems, slightly exceeded the requirement of C02 fixation ant biotypes (Fig. 1; Refs. 1, 20), are a direct consequence of an in both biotypes. In the presence of DCMU, the photosynthetic effect on electron transfer brought about by the alteration of the reduction of MV with electrons originating from DAD or DHQ QB binding site. In addition, we wanted to investigate the recent involves only PSI. These are very rapid reactions and no statisti- report (20) that not only is there an alteration in the QB binding cally significant difference in rate was detected in chloroplast protein but also an affect of triazine-resistance on the oxidizing membranes isolated from the two biotypes. side of PSII as well. Comparison of the rate of PSII electron transport in chloroplasts The equilibrium constant for the QA to QB electron transfer of the two was made the oxidants reaction (i.e. QKQB ±1 QAQB) has been estimated to be approxi- biotypes using lipophilic strong mately 20 for spinach chloroplasts (8) and it is expected, judging DADoX (10) and DMMDBQ (21). In these reactions, electron flow from the similarity in the oxidation kinetics ofQA (4), that triazine- beyond plastoquinone was prevented by dibromothymoquinone susceptible pigweed chloroplasts would have a very similar value. and electrons were intercepted from the reducing side of PSII Since an equilibrium constant is the quotient of the forward and prior to plastoquinone by the lipophilic oxidant present in the reverse rate constants, if no change occurred in rate of the reverse membrane (10). The DAD was then rapidly oxidized by ferricy- reaction then the 10-fold smaller rate constant for QA to QB anide present in the suspension media and the reduced DMMDBQ electron transfer reported for triazine-resistant chloroplasts (5) was oxidized by molecular oxygen. In chloroplasts from the would result in a corresponding smaller equilibrium constant for susceptible plants, the rate of these PSII-dependent reactions was the reaction. That is, for an electron shared between QA and QB, closely comparable to the rate of the H20 to MV reaction (Table the probability of the electron residing on QA at any given time I). However, the rate of electron transfer supported by these would be higher in reaction centers of the resistant biotype. exogenous PSII electron acceptors was reduced to 50%o in chloro- Reaction center semiquinone anions are exceptionally stable (for plasts from the resistant biotype. instance, the half-time for the disappearance of QB after PHOTOSYNTHESIS IN TRIAZINE-RESISTANT AMARANTHUS 929 a single flash is greater than 20 s in spinach chloroplasts [8]) and reduction of DADox (or DMMDBQ) by resistant chloroplasts consequently the percentage ofinactive, or closed, reaction centers could be accounted for by a lower binding constant for the in chloroplasts from resistant plants would be greater than in exogenous oxidant to the QB binding site. Clearly, the activity of chloroplasts from susceptible plants by an amount proportional to the natural acceptor, plastoquinone, would have to be greater than the change in the equilibrium constant. Our data for smooth that of these exogenous oxidants to be adequate to support the pigweed (Fig. 2) show that the quantum yield of CO2 reduction is, observed rates of MV reduction (Table I). Burke et al. (6) and on average, 23% lower in resistant plants, indicating that nearly a Sims Holt et al. (20) found that DCPIP reduction by chloroplasts quarter of the reaction centers are inoperative at any given instant. of resistant plants of Brassica campestris and S. vulgaris was A relatively modest change in the equilibrium constant between significantly less than that of susceptible plants; we too found a QA and QB is sufficient to account for the observed decrease in 30%o lower rate of DCPIP reduction in resistant pigweed chloro- quantum yield ofthe resistant biotype. Considering the case where plasts (data not shown). Although these data were taken as one electron is shared between QA and QB and QA + QA = QIB + evidence for an electron transport limitation of photosynthesis in QB, 25% of the reaction centers would be closed (i.e. in the form resistant biotypes (6, 20) it appears much more likely that the QAQB) with an equilibrium constant for the reaction of 9 assuming explanation lies at least in part with a differential reactivity bimolecular kinetics or 3 for a first-order reaction. Thus, it appears of the that the lower quantum yield of CO2 reduction in resistant bio- indophenol with the QB binding site. types can be adequately accounted for by the already known The origin of the lower rates of light- and C02-saturated CO2 alterations on the reducing side of PSII. Sims Holt et al. (20) reduction in triazine-resistant biotypes is unknown. The level of argued for an additional malfunction on the oxidizing side of PSII quantum inefficiency observed in the resistant biotype, should be based on the measurement of a 5-fold lower flash-induced oxygen largely, if not entirely, overcome at high light intensity since other yield in the chloroplasts from the resistant biotype of S. vulgaris. reactions in the photosynthetic reduction of CO2 that are subse- In contrast, our measurements with pigweed showed that the quent to PSII are more strongly rate limiting (see 14 for discus- number of PSII centers of resistant chloroplasts turning over in sion). The fact that chloroplasts isolated from both biotypes saturating flashes (Fig. 3) was exactly in line with the diminished support equivalent rates of whole chain electron transfer and that quantum yield. Since the resistant biotypes of both species showed these rates are sufficient to support the rate of CO2 reduction about a 20 to 25% lower quantum yield of CO2 reduction, we do almost eliminates electron transfer as the direct underlying cause not believe that the discrepancy in flash-induced PSII turnover of the lower CO2 reduction rates in the resistant biotypes. How- represents a fundamental difference in the mechanism of triazine ever, it leaves open the possibility that alterations in the acceptor resistance between these two weed species. Instead, it appears that side of the PSII reaction center may be involved in a more subtle severely impaired flash-induced PSII turnover observed by Sims fashion. For instance, activity in this region of the electron transfer Holt et al. (20) was the result of the chloroplast isolation procedure chain has been implicated in the regulation of membrane protein which in some fashion magnified the in vivo differences in quan- phosphorylation (2). Perhaps the answer to the underlying cause tum efficiency, a possibility which these workers themselves raised. of lower CO2 fixation rates in the resistant biotypes does lie with Pfister and Arntzen (16) and later Bowes et al. (5) inferred from some sort of subtle effect of PSII but it seems at least as likely that measurements of Chl fluorescence that the QA to QB electron the resistant and susceptible biotypes selected from field popula- transfer time was as much as 10-fold greater in chloroplast mem- tions have genetic variations in traits in addition to that controlling branes ofthe resistant biotypes. It should be appreciated, however, the triazine-receptor protein. For instance, in the pigweed biotypes that although the half-time for electron transfer between the PSII used in this study, the resistant plants could be easily identified by quinone acceptors in resistant chloroplasts may be as long as a heavy anthocyanin pigmentation of vascular tissue, epinastic leaf few ms, it is nevertheless likely to be more rapid than the normally posture, and a more elongated leaf shape. Other, less obvious, rate-limiting oxidation of plastoquinol. This is in fact the case genetic differences between the resistant and susceptible biotypes since the rate of reduction of MV with electrons originating from which have no direct relationship to the components of the water is indistinguishable in the two biotypes even when the rate photosynthetic electron transport chain may also exist and may of plastoquinol oxidation is maximally enhanced by uncoupling be the underlying cause of the lower CO2 reduction rates in the (Table I). Because these electron transfer rates measured in vitro resistant biotypes. A more precise determination of the contribu- are adequate to support the light-saturated rate of CO2 reduction tion of the modified chloroplast genome in triazine-resistant weeds measured in vivo at the same temperature, it is apparent that the could be achieved if progeny from crosses slower QA to QB electron transfer cannot directly account for the F, reciprocal between lower CO2 reduction rates observed in resistant plants. susceptible and resistant individuals were compared. In this hybrid It seems probable that the mechanism of electron transfer out the nuclear genotypes would be identical so only differences in of the PSII reaction center occurs by the debinding of fully the cytoplasmic genomes would be under analysis. Although this reduced QB with a subsequent rebinding of plastoquinone (8). is an attractive approach it should be appreciated that even this Furthermore, the simplest explanation for the action of PSII analysis could not discriminate between multiple mutations of the herbicides, such as atrazine, is that the inhibitor displaces the chloroplast chromosome in the resistant biotypes versus pleiotropic quinone form of QB from its binding site (22, 23, 26). As has affects of a single mutation of the herbicide-receptor protein. For already been pointed out, chloroplasts of triazine-resistant plants instance, we cannot be absolutely certain that the slower electron appear to have an altered QB binding site that drastically affects transfer between QA and QB of the PSII reaction centers in the binding constant of several classes of herbicides but most resistant plants arises from the same change in the chloroplast notably symmetrical triazines (16). This change in the QB binding genome as reduced binding of triazine herbicides. Presently, there site is the basis of the most likely explanation for the much lower is not enough information to make a definitive evaluation of rate of reduction of lipophilic strong oxidants, such as DADox, by whether or not a mutation in the triazine-binding protein confers PSII of resistant plants (Table I). Benzoquinone is an example of any direct effect upon the overall process of CO2 fixation by the an exogenous plastoquinone analog which can intercept electrons intact plant. from PSII. Lavergne (11) has presented evidence to indicate that benzoquinone displaces plastoquinone from the QB binding site. We suggest a similar Acknowledgments-The authors thank Drs. C. A. Wraight, J. Whitmarsh, and C. mechanism for reduction of phenylenedi- J. Arntzen for valuable discussions. In addition, we thank Dr. J. S. Boyer for the imines which are also plastoquinone analogs. The lower rate of generous use of his gas exchange instrumentation. 930 ORT ET AL. Plant Physiol. Vol. 72, 1983 LITERATURE CITED inheritance ofchloroplast atrazine tolerance in Brassica campestris. Can J Plant Sci 58: 977-981 l. AHRENS WH, EW STOLLER 1983 Competition, growth rate, and CO2 fixation in 13. MACKINNEY G 1941 Absorption of light by chlorophyll solutions. J Biol Chem triazine-susceptible and resistant smooth pigweed (Amaranthus hybridus L.) 140: 315-322 Weed Sci 31: In press 14. MARTIN B, DR ORT 1982 Insensitivity of water-oxidation and photosystem II 2. ALLEN JF, J BENNETT, KE STEINBACK, CJ ARNTZEN 1981 Chloroplast protein activity in tomato to chilling temperatures. Plant Physiol 70: 689-694 phosphorylation couples plastoquinone redox state to distribution ofexcitation 15. MOONEY HA, 0 BJORKMAN, J EHLERINGER, J BERRY 1976 Photosynthetic energy between photosystems. Nature 291: 25-29 capacity of plants in situ Death Valley plants. Annu Rep Carnegie Inst, pp 3. 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