Differential Light Responses of Photosynthesis by Triazine-resistant and Triazine-susceptible Senecio vulgaris Biotypes

Studies were conducted to determine a physiological basis for competitive differences between Senecio vulgaris L. biotypes which are either resistant or susceptible to triazine herbicides. Net carbon fixation of intact leaves of mature plants was higher at all light intensities in the susceptible biotype than in the resistant biotype. Quantum yields measured under identical conditions for each biotype were 20% lower in the resistant than in the susceptible biotype. Oxygen evolution in continuous light measured in stroma-free chloroplasts was also higher at all light intensities in the susceptible biotype than in the resistant biotype. Oxygen evolution in response to flashing light was measured in stroma-free chloroplasts of both biotypes. The steady-state yield per flash of resistant chloroplasts was less than 20% that of susceptible chloroplasts. Susceptible chloroplasts displayed oscillations in oxygen yield per flash typically observed in normal chloroplasts, whereas the pattern of oscillations in resistant chloroplasts was noticeably damped. It is suggested that modification of the herbicide binding site which confers s-triazine resistance may also affect the oxidizing side of photosystem II, making photochemical electron transport much less efficient. This alteration has resulted in a lowered capacity for net carbon fixation and lower quantum yields in whole plants of the resistant type.     

Inhibition of Photosystem II by formate. Possible evidence for a direct role of bicarbonate in photosynthetic oxygen evolution

In broken chloroplasts the presence of 100 mM sodium formate at pH 8.2 will specifically lengthen the Photosystem II relaxation times of the reactions S′₂ → S₃ and S′₃ → S₀. Rates of reactions S′₀ → S₁ and S′₁ → S₂ remain unaffected. Evidence is presented which indicates the discrimination among S-states by formate cannot be attributed to a block imposed on the reducing side of Photosystem II. The results are interpreted in context of the known interaction of formate and CO₂ which is bound to the Photosystem II reaction center complex. It is proposed that those S-state transitions which show extended relaxation times in the presence of formate must result in the momentary release and rebinding of CO₂. Furthermore since formate is acting on the oxygen-evolving side of Photosystem II, it would seem that CO₂ is released in reactions that occur there. A chemical model of oxygen evolution is presented. It is based on the hypothesis that hydrated CO₂ is the immediate source of photosynthetically evolved oxygen and explains why, under certain conditions formate slows only the S-state transitions S′₂ → S₃ and S′₃ → S₀.     

On the Mechanism of Resistance to Paraquat in Hordeum glaucum and H. leporinum: Delayed Inhibition of Photosynthetic O(2) Evolution after Paraquat Application

The mechanism of resistance to paraquat was investigated in biotypes of Hordeum glaucum Steud. and H. leporinum Link. with high levels of resistance. Inhibition of photosynthetic O₂ evolution after herbicide application was used to monitor the presence of paraquat at the active site. Inhibition of photosynthetic O₂ evolution after paraquat application was delayed in both resistant biotypes compared with the susceptible biotypes; however, this differential was more pronounced in the case of H. glaucum than in H. leporinum. Similar results could be obtained with the related herbicide diquat. Examination of the concentration dependence of paraquat-induced inhibition of O₂ evolution showed that the resistant H. glaucum biotype was less affected by herbicide compared with the susceptible biotype 3 h after treatment at most rates. The resistant H. leporinum biotype, in contrast, was as inhibited as the susceptible biotype except at the higher rates. In all cases photosynthetic O₂ evolution was dramatically inhibited 24 h after treatment. Measurement of the amount of paraquat transported to the young tissue of these plants 24 h after treatment showed 57% and 53% reductions in the amount of herbicide transported in the case of the resistant H. glaucum and H. leporinum biotypes, respectively, compared with the susceptible biotypes. This was associated with 62% and 66% decreases in photosynthetic O₂ evolution of young leaves in the susceptible H. glaucum and H. leporinum biotypes, respectively, a 39% decrease in activity for the resistant H. leporinum biotype, but no change in the resistant H. glaucum biotype. Photosynthetic O₂ evolution of leaf slices from resistant H. glaucum was not as inhibited by paraquat compared with the susceptible biotype; however, those of resistant and susceptible biotypes of H. leporinum were equally inhibited by paraquat. Paraquat resistance in these two biotypes appears to be a consequence of reduced movement of the herbicide in the resistant plants; however, the mechanism involved is not the same in H. glaucum as in H. leporinum.     

Modification of Herbicide Binding to Photosystem II in Two Biotypes of Senecio vulgaris L

The present study compares the binding and inhibitory activity of two photosystem II inhibitors: 3-(3,4-dichlorophenyl)-1,1-dimethylurea (diuron [DCMU]) and 2-chloro-4-(ethylamine)-6-(isopropyl amine)-S-triazene (atrazine). Chloroplasts isolated from naturally occurring triazine-susceptible and triazine-resistant biotypes of common groundsel (Senecio vulgaris L.) showed the following characteristics. (a) Diuron strongly inhibited photosynthetic electron transport from H₂O to 2,6-dichlorophenolindophenol in both biotypes. Strong inhibition by atrazine was observed only with the susceptible chloroplasts. (b) Hill plots of electron transport inhibition data indicate a noncooperative binding of one inhibitor molecule at the site of action for both diuron and atrazine. (c) Susceptible chloroplasts show a strong diuron and atrazine binding (¹⁴C-radiolabel assays) with binding constants (K) of 1.4 × 10⁻⁸ molar and 4 × 10⁻⁸ molar, respectively. In the resistant chloroplasts the diuron binding was slightly decreased (K = 5 × 10⁻⁸ molar), whereas no specific atrazine binding was detected. (d) In susceptible chloroplasts, competitive binding between radioactively labeled diuron and non-labeled atrazine was observed. This competition was absent in the resistant chloroplasts.     

Studies on the water-oxidizing system by the effects of different treatments in chloroplasts

Treatments such as trypsinization (50 μg/ml per mg Chl for 1 h), osmotic shock of the chloroplasts or mild heating altered the oxygen evolution in such a way that the properties of the Photosystem II were simplified. After these treatments, the damping of the oscillation pattern of O₂ yields induced by a flash series remained the same, irrespective of the level of inhibition induced by the treatment. This damping did not decrease with increasing flash energy, as observed in untreated chloroplasts. The light saturation curve of the S₂ → S₃ transition of the O₂ evolving system no more exhibited the slow-increasing phase at high flash energy observed under normal conditions. The kinetic properties of the O₂-evolving system were also simplified. After the treatments cited above, deactivation of S₂ and S₃ were identical and accelerated with respect to untreated chloroplasts. Turnover kinetics of the transitions S₁ → S₂ and S₂ → S₃ were also similar and simpler without a lag for S₂ → S₃. These results indicate that the treatments mentioned above disconnect one donor from the O₂-evolving complex. This donor, under normal conditions, contributes to the increase of the quantum yield of the transition S₂ → S₃ at high flash energy. This donor is here denoted by D. Our results are in agreement with the following working hypothesis: the large miss, observed on the S₂ → S₃ transition without any contribution of the donor D, may be due to the fact that the system needs a conformation change of the O₂-evolving complex in the S₂ state, so that the main donor Y can oxidize the second H₂O molecule in the water-splitting complex. In the inactive state corresponding to the absence of a conformation change, the donor D, being different in configuration, is likely to oxidize the S₂ state into an S₃ state at high light intensity.     

Triazine Resistance in Senecio vulgaris Parental and Nearly Isonuclear Backcrossed Biotypes Is Correlated with Reduced Productivity

Isonuclear triazine-susceptible and triazine-resistant Senecio vulgaris L. biotypes were developed by making reciprocal crosses between susceptible and resistant biotypes to obtain F₁ hybrids and backcrossing the hybrids to the appropriate pollen parent. The electrophoretic isozyme patterns of the enzyme aconitase obtained from leaf extracts of triazine-susceptible parental (S) and backcrossed (S×R_BC6) biotypes, and triazine-resistant parental (R) and backcrossed (R×S_BC6) biotypes verified that the biotypes had the expected nuclear genomes. Atrazine inhibition of chloroplast whole chain electron transport from water to methyl viologen was measured to verify susceptibility or resistance to triazine herbicides. The photosynthetic rate and biomass accumulation of greenhouse grown susceptible and resistant S. vulgaris biotypes were measured 28, 35, 42, 50, 57, and 64 days after planting to determine the effect of altered chloroplast function. S and S×R_BC6 biotypes had CO₂ assimilation rates of 16.2 and 16.6 micromoles CO₂ per square meter per second, respectively, and I₅₀ values (herbicide concentration producing 50% inhibition) of about 0.49 micromolar atrazine. The corresponding values for the R and R×S_BC6 biotypes were 14.7 and 14.6 micromoles CO₂ per square meter per second with I₅₀ values of 65.0 micromolar atrazine. The S biotype was larger and more productive than the R biotype at all harvests. At the harvest 57 days after planting, mean shoot dry weight was 33.2 and 8.7 grams for the S and R biotypes, respectively. The growth effect associated with chloroplast differences was shown in comparisons of the S biotype with the R×S_BC6 biotype and of the S×R_BC6 biotype with the R biotype. The R×S_BC6 biotype had 72% of the shoot dry weight of the S biotype while the R biotype had 55% of the shoot dry weight of the S×R_BC6 biotype. The R×S_BC6 and R biotypes produced about 73 and 62% of the leaf area of the S and S×R_BC6 biotypes, respectively. Relative growth rate was similar in biotypes with the same nuclear genome; however, instantaneous unit leaf rate was higher in the S compared to the R×S_BC6 biotype and in the S×R_BC6 compared to the R biotype. At 57 days after planting, the cumulative leaf area duration (i.e. photosynthetic opportunity) of the R×S_BC6 and R biotypes was 86 and 66% of that of the S and S×R_BC6 biotypes, respectively. Our data indicate that impaired chloroplast function in triazine resistant S. vulgaris biotypes limits growth and productivity at the whole plant level.     

Determination of the Rate Limiting Step for Photosynthesis in a Nearly Isonuclear Rapeseed (Brassica napus L.) Biotype Resistant to Atrazine

Plant biotypes that are resistant to S-triazines under most conditions often grow less vigorously and have lower quantum yields and lower maximum rates of photosynthesis. The photosynthetic reactions responsible for these effects were identified in whole leaves and thylakoids of nearly isonuclear lines of oilseed rape (Brassica napus L.). The lower quantum yield was a result of poor efficiency in the use of separated charge at the photosystem II reaction center. Charge separation occurred normally, but over 30% of the charges recombined instead of being used for oxygen evolution and for reduction capacity in photosystem I. The lower maximum rate of photosynthesis in the resistant biotype was set by the transfer of electrons between the primary, Q_A, and secondary, Q_B, acceptors of photosystem II. This charge transfer reaction became rate limiting in resistant biotypes. The decreased quantum yield and decreased maximum rate of photosynthesis are both believed to be consequences of changes in the 32 kilodalton herbicide binding protein. As such, it is likely that these traits will not be genetically separable.     

Atrazine Resistance in a Velvetleaf (Abutilon theophrasti) Biotype Due to Enhanced Glutathione S-Transferase Activity

We previously reported that a velvetleaf (Abutilon theophrasti Medic) biotype found in Maryland was resistant to atrazine because of an enhanced capacity to detoxify the herbicide via glutathione conjugation (JW Gronwald, Andersen RN, Yee C [1989] Pestic Biochem Physiol 34: 149-163). The biochemical basis for the enhanced atrazine conjugation capacity in this biotype was examined. Glutathione levels and glutathione S-transferase activity were determined in extracts from the atrazine-resistant biotype and an atrazine-susceptible or “wild-type” velvetleaf biotype. In both biotypes, the highest concentration of glutathione (approximately 500 nanomoles per gram fresh weight) was found in leaf tissue. However, no significant differences were found in glutathione levels in roots, stems, or leaves of either biotype. In both biotypes, the highest concentration of glutathione S-transferase activity measured with 1-chloro-2,4-dinitrobenzene or atrazine as substrate was in leaf tissue. Glutathione S-transferase measured with 1-chloro-2,4-dinitrobenzene as substrate was 40 and 25% greater in leaf and stem tissue, respectively, of the susceptible biotype compared to the resistant biotype. In contrast, glutathione S-transferase activity measured with atrazine as substrate was 4.4- and 3.6-fold greater in leaf and stem tissue, respectively, of the resistant biotype. Kinetic analyses of glutathione S-transferase activity in leaf extracts from the resistant and susceptible biotypes were performed with the substrates glutathione, 1-chloro-2,4-dinitrobenzene, and atrazine. There was little or no change in apparent K_m values for glutathione, atrazine, or 1-chloro-2,4-dinitrobenzene. However, the V_max for glutathione and atrazine were approximately 3-fold higher in the resistant biotype than in the susceptible biotype. In contrast, the V_max for 1-chloro-2,4-dinitrobenzene was 30% lower in the resistant biotype. Leaf glutathione S-transferase isozymes that exhibit activity with atrazine and 1-chloro-2,4-dinitrobenzene were separated by fast protein liquid (anion-exchange) chromatography. The susceptible biotype had three peaks exhibiting activity with atrazine and the resistant biotype had two. The two peaks of glutathione S-transferase activity with atrazine from the resistant biotype coeluted with two of the peaks from the susceptible biotype, but peak height was three- to fourfold greater in the resistant biotype. In both biotypes, two of the peaks that exhibit glutathione S-transferase activity with atrazine also exhibited activity with 1-chloro-2,4-dinitrobenzene, with the peak height being greater in the susceptible biotype. The results indicate that atrazine resistance in the velvetleaf biotype from Maryland is due to enhanced glutathione S-transferase activity for atrazine in leaf and stem tissue which results in an enhanced capacity to detoxify the herbicide via glutathione conjugation.     

Similar Photosynthetic Performance in Low Light-Grown Isonuclear Triazine-Resistant and -Susceptible Brassica napus L

Triazine-resistant plants grown under moderate to high photon flux density (PFD) conditions exhibit decreased photon yield, decreased light-saturated O₂ evolution and slower growth than triazine-susceptible plants. In this study we tested the hypothesis that the comparable growth previously observed in resistant and susceptible Brassica napus L. lines grown under low PFD was accompanied by comparable photon yield and light-saturated O₂ evolution. We measured photon yield, O₂ flash yield, fluorescence decay kinetics, fluorescence transient kinetics, and quenching components, F_v/F_m and light saturated O₂ evolution in leaf disks of low PFD-grown triazine-resistant and susceptible B. napus isogenic lines. Results indicated that slow electron transfer from the primary to secondary quinone electron acceptors of photosystem II was still present in the resistant line but photon yield and light-saturated O₂ evolution were similar in the two B. napus lines. We conclude that the alteration in the D1 protein that confers resistance does not necessarily cause decreased photosynthetic performance. Decreased photon yield in resistant plants grown at high PFD is not a direct consequence of the alteration in D1, but represents secondary damage.     

Increased Tolerance to Photoinhibitory Light in Paraquat-Resistant Conyza bonariensis Measured by Photoacoustic Spectroscopy and CO(2)-Fixation

Tolerance to photoinhibition was compared between a paraquat-resistant and a sensitive biotype of Conyza bonariensis (L.). Cronq. Photoinhibitory damage was measured as a decrease in oxygen evolution or energy storage using photoacoustic spectroscopy, or as a decrease of ¹⁴CO₂-fixation. Prior to exposure to high fluence rates, both biotypes had similar quantum yields of oxygen evolution and energy storage. After exposure to high intensity light, the resistant biotype continued to evolve oxygen and to store energy with a high quantum yield while both energy storage and oxygen evolution were severely reduced in the sensitive biotype. CO₂-fixation was less rapidly inhibited in the resistant biotype compared to the sensitive one. The data show that the paraquat resistant biotype with its high constitutive levels of the chloroplast localized enzymes of the oxygen detoxification pathway, is also partially protected from photoinhibition. This supports the theory that an enhanced radical scavenging system can give temporary protection against photooxidative damage from a variety of sources.     

Altered Leaf Structure and Function in Triazine-Resistant Common Groundsel (Senecio vulgaris)

Anatomical and physiological characteristics of leaves of triazinesusceptible and -resistant biotypes of common groundsel (Senecio vulgaris L.) were studied in order to explain the differences in light-saturated photosynthetic rates previously reported. Leaves were of uniform leaf plastochron index from greenhouse-grown plants. Susceptible plants had greater leaf fresh and dry weights and leaf areas, while resistant plants had greater specific leaf mass (mg fresh weight/cm²). Susceptible plants had greater amounts of total chlorophyll per unit leaf weight and a higher chlorophyll a/b ratio. Soluble protein in leaves was higher in susceptible chloroplasts on a weight and area basis, but similar to resistant chloroplasts on a unit chlorophyll basis. Activity of ribulose 1,5-bisphosphate carboxylase was higher in resistant plants on a fresh weight, leaf area, and milligram chlorophyll basis. Stomatal frequency, length, and arrangement were similar between biotypes, as were transpiration and conductance. Resistant leaves had less air space (v/v), more cells in palisade and spongy mesophyll, and a greater volume of palisade tissue than spongy, when compared to susceptible leaves. Differences in leaf structure and function between biotypes are probably due to a complex of developmental adaptations which may be only indirectly related to modified photosystem II in resistant plants. These results indicate that the consistently lower rates of net photosynthesis and yield in resistant plants cannot be explained solely on the basis of these leaf characteristics. Several possible mechanisms to account for reduced productivity are suggested.     

Oxygen-evolution patterns from spinach Photosystem II preparations

Patterns of O₂ evolution resulting from sequences of short flashes are reported for Photosystem (PS) II preparations isolated from spinach and containing an active, O₂-evolving system. The results can be interpreted in terms of the S-state model developed to explain the process of photosynthetic water splitting in chloroplasts and algae. The PS II samples display damped, oscillating patterns of O₂ evolution with a period of four flashes. Unlike chloroplasts, the flash yields of the preparations decay with increasing flash number due to the limited plastoquinone acceptor pool on the reducing side of PS II. The optimal pH for O₂ evolution in this system (pH 5.5–6.5) is more acidic than in chloroplasts (pH 6.5–8.0). The O₂-evolution, inactivation half-time of dark-adapted preparations was 91 min (on the rate electrode) at room temperature. Dark-inactivation half-times of 14 h were observed if the samples were aged off the electrode at room temperature. Under our conditions (experimental conditions can influence flash-sequence results), deactivation of S₃ was first order with a half-time of 105 s while that of S₂ was biphasic. The half-times for the first-order rapid phase were 17 s (one preflash) and 23 s (two preflashes). The longer S₂ phase deactivated very slowly (the minimum half-time observed was 265 s). These results indicate that deactivation from S₃ → S₂ → S₁, thought to be the dominant pathway in chloroplasts, is not the case for PS II preparations. Finally, it was demonstrated that the ratio of S₁ to S₀ can be set by previously developed techniques, that S₀ is formed mostly from activated S₃ (S₄), and that both S₀ and S₁ are stable in the dark.     

Electron transfer and proton pumping under excitation of dark-adapted chloroplasts with flashes of light

Proton release inside thylakoids, which is linked to the action of the water-oxidizing enzyme system, was investigated spectrophotometrically with the dye neutral red under conditions when the external phase was buffered. Under excitation of dark-adapted chloroplasts with four short laser flashes in series, the pattern of proton release as a function of the flash number was recorded and interpreted in the light of the generally accepted scheme for consecutive transitions of the water-oxidizing enzyme system: S₀ → S₁, S₁ → S₂, S₂ → S₃, S₃ → S₄, S₀. It was found that the proton yield after the first flash varied in a reproducible manner, being dependent upon the dark pretreatment given. In terms of the proton-electron reaction during these transitions, the pattern was as follows. In strictly dark-adapted chloroplasts (frozen chloroplasts thawed in darkness and kept for at least 7 min in the dark after dilution), it was fitted well by a stoichiometry of 1:0:1:2. In a less stringently dark-adapted preparation (as above but thawed under light), it was fitted by 0:1:1:2. Mechanistically this is not yet understood. However, it is a first step towards resolving controversy over this pattern among different laboratories. Under conditions where the 1:0:1:2 stoichiometry was observed, proton release was time resolved. Components with half-rise times of 500 and 1000 μs could be correlated with the S₂ → S₃ and S₃ → S₄ transitions, respectively. Proton release during the S₀ → S₁ transition is more rapid, but is less well attributable to the transitions due to error proliferation. A distinct component with a half-rise time of only 100 μs was observed after the second flash. Since it did not fit into the expected kinetics (based on literature data) for the S_i → S_i+1 transitions, we propose that it reflects proton release from a site which is closer to the reaction center of Photosystem (PS) II than the water-splitting enzyme system. This is supported by the observation of rapid proton release under conditions where water oxidation is blocked. Related experiments on the pattern of proton uptake at the reducing side of PS II indicated that protons act as specific counterions for semiquinone anions without binding to them.     

Correlation between the activity of membrane-bound ATPase and the decay rate of flash-induced 515-nm absorbance change in chloroplasts in intact leaves,assayed by means of rapid isolation of chloroplasts

(1) The relationship between activation of the membrane-bound ATPase and the stimulation of dissipation of the flash-induced membrane potential by preillumination was studied in intact spinach leaves by measuring the ATPase activity of rapidly isolated chloroplasts and the decay of the flash-induced 515-nm absorbance change (ΔA₅₁₅) in intact leaves. (2) The decay of ΔA₅₁₅ was accelerated by preillumination. The ΔA₅₁₅ decay in leaves treated with N,N′-dicyclohexylcarbodiimide (DCCD) became slower and was not accelerated by preillumination. However, treatment with DCCD did not lower the intensity of delayed fluorescence. (3) Membrane-bound ATPase of chloroplasts which were rapidly isolated from the preilluminated leaves (90 s preparation time) showed a higher activity (over 200 μmol P_i/mg chlorophyll per h in the case of 2-min preillumination) than that of chloroplasts isolated from dark-adapted leaves. (4) The acceleration of ΔA₅₁₅ decay and the activation of ATPase showed similar dependences on illumination time in intact leaves. 3-(3′,4′-Dichlorophenyl)-1,1-dimethylurea, carbonyl cyanide p-chlorophenylhydrazone and DCCD inhibited the activation of ATPase and the acceleration of the ΔA₅₁₅ decay by preillumination. (5) The ATPase activity of chloroplasts isolated from illuminated leaves showed a single exponential decay (‘dark inactivation in vitro’). The ATPase activity induced by illuminating the leaves became lower as the dark interval between illumination and the isolation of chloroplasts was increased (‘dark inactivation in vivo’). The time course of the decay of activity had a lag and showed a sigmoidal curve when plotted semilogarithmically. The decay had an apparent half-time of 25 min. (6) The recovery of the accelerated ΔA₅₁₅ decay in preilluminated leaves to the original slow rate showed a sigmoidal decay similar to that of the activity of ATPase in intact leaves with a half-time of about 23 min in the dark. (7) It was concluded that the decay rate of ΔA₅₁₅ reflected the chloroplast ATPase activity in intact leaves and that the ion conductance of thylakoid membrane was mainly determined by the H⁺ flux through the ATPase, the activity of which was increased after the formation of the high-energy state.     

Characterization of intermediate biotypes in atrazine-susceptible populations of Chenopodium polyspermum L. and Amaranthus bouchonii thell. in Hungary

Intermediate biotypes for atrazine herbicide resistance in Chenopodium polyspermum and Amaranthus bouchonii were characterized by a peculiar chlorophyll fluorescence induction curve. The intermediate biotypes were isolated from progenies of susceptible plants in maize grown in alternate years without atrazine. The lethal dose in seedling treatments was lower than that of the resistant plants but higher than for susceptible plants. Atrazine at 10 μM was near the I₅₀-value for in vivo nitrite reductase activity in both intermediate biotypes. The activity of nitrite reducttase in the intermediate biotypes was about 75% of that of susceptible biotypes. These characteristics of intermediate biotypes were maternally inherited in crosses.     

Investigations of mechanisms of resistance to bipyridyl herbicides in Arctotheca calendula (L.) Levyns

The mechanism of resistance to diquat and paraquat was investigated in a bipyridyl-herbicide-resistant biotype of Arctotheca calendula (L.) Levyns. No differences were observed in the interactions of these herbicides with Photo-system I, the active site, in thylakoids isolated from resistant and susceptible biotypes. Likewise, absorption of herbicide through the cuticle and gross translocation were identical in plants of the two biotypes. Foliar application of either 25 g ha⁻¹ diquat or 200 g ha⁻¹ paraquat rapidly inhibited CO₂-dependent O₂ evolution of leaf segments of the susceptible biotype. O₂ evolution of leaf segments of the resistant biotype was less affected by these treatments. Fluorescence imaging was used to observe visually, as fluorescence quenching, the penetration of herbicide to the active site. These experiments demonstrated that diquat appears at the active site more slowly in the resistant biotype compared to the susceptible biotype. HCO₃-dependent O₂ evolution of thin leaf slices was less inhibited by diquat in the resistant biotype than in the susceptible biotype. The mechanism of resistance to the bipyridyl herbicides in this biotype of A. calendula is not a result of changes at the active site, decreased herbicide absorption or decreased translocation, but appears to be due to reduced herbicide penetration to the active site.     

Oxidation versus Reductive Detoxification of SO(2) by Chloroplasts

Intact chloroplasts isolated from spinach (Spinacia oleracea L. cv Yates) both oxidized and reduced added sulfite in the light. Oxidation was fast only when endogenous superoxide dismutase was inhibited by cyanide. It was largely suppressed by scavengers of oxygen radicals. After addition of O-acetylserine, chloroplasts reduced sulfite to cysteine and exhibited sulfite-dependent oxygen evolution. Cysteine synthesis from sulfite was faster than from sulfate. The results are discussed in relation to species-specific differences in the phytotoxicity of SO₂.     

Comparison of Triazine-Resistant and -Susceptible Biotypes of Senecio vulgaris and Their F(1) Hybrids

The relationship of triazine resistance to decreased plant productivity was investigated in Senecio vulgaris L. F₁ reciprocal hybrids were developed from pure-breeding susceptible (S) and resistant (R) lines. The four biotypes (S, S × R, R, R × S) were compared in terms of atrazine response, electron transport, carbon fixation, and biomass production. Atrazine response, carbon fixation rate, and PSII and whole-chain electron transport rates of hybrids were nearly identical to those of their respective maternal parents. Significant differences occurred between the two susceptible (S, S × R) and two resistant (R, R × S) biotypes in atrazine response (I₅₀), carbon fixation rate, and PSII and whole-chain electron transport rates; PSI rates were identical in all four biotypes. Coupled and uncoupled, whole-chain electron transport rates of thylakoids of the two susceptible biotypes were approximately 50% greater than those of the two resistant biotypes at photon flux densities greater than 215 micromoles per square meter per second. Carbon exchange rates of the two susceptible biotypes were 23% greater than those of the two resistant biotypes. Hybrid biotypes (S × R, R × S) were not identical to their maternal parents in biomass production. The S, S × R, and R × S plants all achieved greater biomass than R plants. These results suggest that while the resistance mutation influences thylakoid performance, reduced productivity of triazine-resistant plants cannot be ascribed solely to decreases in electron transport or carbon assimilation rates brought about by the altered binding protein. Since the F₁ hybrids differed from their maternal parents only in nuclear genes, it appears that the detrimental effects of the triazine resistance mutation on plant growth may be attenuated by interactions of the plastid and nuclear genomes.     

Regulation of Photosynthesis in Triazine-Resistant and -Susceptible Brassica napus

The response of photosynthetic carbon assimilation and chlorophyll fluorescence quenching to changes in intercellular CO₂ partial pressure (C_i), O₂ partial pressure, and leaf temperature (15-35°C) in triazine-resistant and -susceptible biotypes of Brassica napus were examined to determine the effects of the changes in the resistant biotype on the overall process of photosynthesis in intact leaves. Three categories of photosynthetic regulation were observed. The first category of photosynthetic response, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)-limited photosynthesis, was observed at 15, 25, and 35°C leaf temperatures with low C_i. When the carbon assimilation rate was Rubisco-limited, there was little difference between the resistant and susceptible biotypes, and Rubisco activity parameters were similar between the two biotypes. A second category, called feedback-limited photosynthesis, was evident at 15 and 25°C above 300 microbars C_i. The third category, photosynthetic electron transport-limited photosynthesis, was evident at 25 and 35°C at moderate to high CO₂. At low temperature, when the response curves of carbon assimilation to C_i indicated little or no electron transport limitation, the carbon assimilation rate was similar in the resistant and susceptible biotypes. With increasing temperature, more electron transport-limited carbon assimilation was observed, and a greater difference between resistant and susceptible biotypes was observed. These observations reveal the increasing importance of photosynthetic electron transport in controlling the overall rate of photosynthesis in the resistant biotype as temperature increases. Photochemical quenching of chlorophyll fluorescence (q_P) in the resistant biotype never exceeded 60%, and triazine resistance effects were more evident when the susceptible biotype had greater than 60% q_P, but not when it had less than 60% q_P.     

Primary and secondary electron donors in Photosystem II of chloroplasts. Rates of electron transfer and location in the membrane

Absorption changes at 820 or 515 nm after a short laser flash were studied comparatively in untreated chloroplasts and in chloroplasts in which oxygen evolution is inhibited.In chloroplasts pre-treated with Tris, the primary donor of Photosystem II (P-680) is oxidized by the flash, as observed by an absorption increase at 820 nm. After the first flash it is re-reduced in a biphasic manner with half-times of 6 μs (major phase) and 22 μs. After the second flash, the 6 μs phase is nearly absent and P-680⁺ decays with half-times of 130 μs (major phase) and 22 μs. Exogenous electron donors (MnCl₂ or reduced phenylenediamine) have no direct influence on the kinetics of P-680⁺.In untreated chloroplasts the 6 and 22 μs phases are of very small amplitude, either at the 1st, 2nd or 3rd flash given after dark-adaptation. They are observed, however, after incubation with 10 mM hydroxylamine.These results are interpreted in terms of multiple pathways for the reduction of P-680⁺: a rapid reduction (<1 μs) by the physiological donor D₁; a slower reduction (6 and 22 μs) by donor D′₁, operative when O₂ evolution is inhibited; a back-reaction (130 μs) when D′₁ is oxidized by the pre-illumination in inhibited chloroplasts. In Tris-treated chloroplasts the donor system to P-680⁺ has the capacity to deliver only one electron.The absorption change at 515 nm (electrochromic absorption shift) has been measured in parallel. It is shown that the change linked to Photosystem II activity has nearly the same magnitude in untreated chloroplasts or in chloroplasts treated with hydroxylamine or with Tris (first and subsequent flashes). Thus we conclude that all the donors (P-680, D₁, D′₁) are located at the internal side of the thylakoid membrane.