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.     

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.     

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.     

Paraquat resistance in conyza

A biotype of Conyza bonariensis (L.) Cronq. (identical to Conyza linefolia in other publications) originating in Egypt is resistant to the herbicide 1,1′-dimethyl-4,4′-bipyridinium ion (paraquat). Penetration of the cuticle by [¹⁴C]paraquat was greater in the resistant biotype than the susceptible (wild) biotype; therefore, resistance was not due to differences in uptake. The resistant and susceptible biotypes were indistinguishable by measuring in vitro photosystem I partial reactions using paraquat, 6,7-dihydrodipyrido [1,2-α:2′,1′-c] pyrazinediium ion (diquat), or 7,8-dihydro-6H-dipyrido [1,2-α:2′,1′-c] [1,4] diazepinediium ion (triquat) as electron acceptors. Therefore, alteration at the electron acceptor level of photosystem I is not the basis for resistance. Chlorophyll fluorescence measured in vivo was quenched in the susceptible biotype by leaf treatment with the bipyridinium herbicides. Resistance to quenching of in vivo chlorophyll fluorescence was observed in the resistant biotype, indicating that the herbicide was excluded from the chloroplasts. Movement of [¹⁴C] paraquat was restricted in the resistant biotype when excised leaves were supplied [¹⁴C]paraquat through the petiole. We propose that the mechanism of resistance to paraquat is exclusion of paraquat from its site of action in the chloroplast by a rapid sequestration mechanism. No differential binding of paraquat to cell walls isolated from susceptible and resistant biotypes could be detected. The exact site and mechanism of paraquat binding to sequester the herbicide remains to be determined.     

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.     

Russian wheat aphid biotype RWASA2 causes more vascular disruption than RWASA1 on resistant barley lines

We investigated the comparative effects of the feeding damage caused by two Russian wheat aphid (RWA, Diuraphis noxia Kurdjumov) biotypes, RWASA1 and RWASA2, on leaves of three RWA-resistant barley (Hordeum vulgare L.) lines from the USDA-ARS, and used a South African non-resistant cultivar as control. The relationship between aphid breeding capacity and the structural damage inflicted by the aphids was studied, using wide-field fluorescence and transmission electron microscopy (TEM). Colonies of the two biotypes grew rapidly on all four barley lines during a 10 day feeding exposure but as expected, population size and density were generally lower on the resistant lines than on the non-resistant cultivar. The new South African biotype, RWASA2, bred significantly faster than the original RWASA1 biotype. The feeding and water uptake-related damage sustained by phloem and xylem tissues of the resistant lines suggest that RWASA2 was a more aggressive feeder and caused substantially more cell damage than RWASA1. Examination of wound callose distribution after aphid feeding revealed that high levels of wound callose occurred in non-resistant and in resistant lines. Reduction in aphid population size, as well as ultrastructural damage during feeding by RWA biotypes on resistant lines, signals potential antibiotic and tolerant responses of the barley lines to aphid feeding. We infer from callose distribution and ultrastructural studies, that phloem transport would be substantially reduced in the non-resistant PUMA and to a lesser extent in the resistant STARS lines, which suggests that the STARS lines may be a potential source of RWASA1 and RWASA2-resistance.     

Responses of Two CAM Species to Different Irradiances during Growth and Susceptibility to Photoinhibition by High Light

Two CAM species, Kalanchoë daigremontiana Hamet et Perrier and Hoya carnosa (L.) R. Br., were grown under a range of five photon flux area densitites (PFD) and then characterized. Significant acclimation to shade was indicated by progressive decreases in leaf thickness, rates of respiratory O₂ uptake, light compensation point, maximum rates of photosynthetic O₂ evolution, nocturnal acid accumulation, and δ¹³C values, and increases in chlorophyll concentration and absolute levels of room temperature (25°C) and 77K fluorescence. Quantum yields (as measured by O₂ exchange) and the ratio of variable 77K fluorescence over the maximum yield (F_v/F_m) were relatively constant across the treatments. The only significant deviation from the above characteristics was in H. carnosa grown under full glasshouse PFD, where it apparently experienced photoinhibition. Following a photoinhibitory treatment, K. daigremontiana exhibited increases in the light compensation point and progressively greater reductions in the quantum yield, maximum photosynthetic rate, F_v/F_m, and the variable component of room temperature fluorescence with increasing shade during growth. Thus although Crassulacean acid metabolism plants can adjust to shaded conditions, they are susceptible to photoinhibition when exposed to higher PFD than that experienced during growth.     

Studies on the Mechanism of Photoinhibition in Higher Plants: I. EFFECTS OF HIGH LIGHT INTENSITY ON CHLOROPLAST ACTIVITIES IN CUCUMBER ADAPTED TO LOW LIGHT

Cucumber plants (Cucumis sativus L.), grown at low quantum flux density (120-150 microeinsteins per square meter per second) were photoinhibited by a three-hour exposure in air to ten times the light intensity experienced during growth. Chloroplasts were isolated from photoinhibited and control leaves and the following activities determined: O₂ evolution in the presence of ferricyanide, photosystem I activity, noncyclic and cyclic photophosphorylation, and light-induced proton uptake. Chlorophyll and chloroplast absorbance spectra, and chloroplast fluorescence were also measured. It was found that photosystem II electron transport and non-cyclic photophosphorylation were inhibited by about 50%, while cyclic photophosphorylation was less inhibited and photosystem I electron transport and light-induced proton uptake were unaffected. Electron transport to methylviologen could not be fully restored by electron donation to photosystem II. Chloroplast fluorescence induction at room temperature was strongly reduced following photoinhibition. There was no difference in the absorption spectra of the extracted chlorophylls from control and photoinhibited chloroplasts, but an increase of the absorption in the blue wavelength region was observed in the photoinhibited chloroplasts. It is suggested that high light stress does not result in alteration of the membrane properties, as is the case in low-temperature stress for example, but affects directly the photosynthetic reaction centers, primarily of photosystem II.     

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.     

Reversible photoinhibition of unhardened and cold-acclimated spinach leaves at chilling temperatures

The photoinhibition of photosynthesis at chilling temperatures was investigated in cold-acclimated and unhardened (acclimated to +18° C) spinach (Spinacia oleracea L.) leaves. In unhardened leaves, reversible photoinhibition caused by exposure to moderate light at +4° C was based on reduced activity of photosystem (PS) II. This is shown by determination of quantum yield and capacity of electron transport in thylakoids isolated subsequent to photoinhibition and recovery treatments. The activity of PSII declined to approximately the same extent as the quantum yield of photosynthesis of photoinhibited leaves whereas PSI activity was only marginally affected. Leaves from plants acclimated to cold either in the field or in a growth chamber (+1° C), were considerably less susceptible to the light treatment. Only relatively high light levels led to photoinhibition, characterized by quenching of variable chlorophyll a fluorescence (F_V) and slight inhibition of PSII-driven electron transport. Fluorescence data obtained at 77 K indicated that the photoinhibition of cold-acclimated leaves (like that of the unhardened ones) was related to increased thermal energy dissipation. But in contrast to the unhardened leaves, 77 K fluorescence of cold-acclimated leaves did not reveal a relative increase of PSI excitation. High-light-treated, cold-acclimated leaves showed increased rates of dark respiration and a higher light compensation point. The photoinhibitory fluorescence quenching was fully reversible in low light levels both at +18° C and +4° C; the recovery was much faster than in unhardened leaves. Reversible photoinhibition is discussed as a protective mechanism against excess light based on transformation of PSII reaction centers to fluorescence quenchers.Abbreviations F_O initial fluorescence - F_M maximal fluorescence - F_V devariable fluorescence (f_m-f_o) - PFD photon flux density - PS photosystem - SD standard deviation The authors thank the Deutsche Forschungsgemeinschaft and the Academy of Finland for financial support.     

Photoinhibition of photosynthesis: effect of O2 and selective excitation of the photosystems in intact Lemna gibba plants

Intact Lemna gibba plants were photoinhibited under anaerobic conditions on illumination with monochromatic light which selectively excited the photosystems. Photoinhibition was less when PS 1 was excited and greatest when mainly PS 2 was excited, which suggests that PS 2 was most damaged by photoinhibition induced in complete absence of O₂ and CO₂.The illumination of plants with monochromatic light exciting PS 1, at different O₂ concentrations (in CO₂ deficient conditions), showed that PS 1 photoinhibition was increased at the low O₂ concentrations. The damage to PS 1 was more evident at 2% O₂ than at the higher O₂ concentrations.CO₂ as well as O₂ at atmospheric concentration, (air), was necessary for complete protection of the plant from photoinhibition when both photosystems were excited either separately or together.Abbreviations I irradiance, photon fluence rate - PCO photosynthetic carbon oxidation cycle - PCR photosynthetic carbon reduction cycle - PS 1 photosystem 1 - PS 2 photosystem 2     

The Effect of Light and Temperature on Competition between Atrazine Susceptible and ResistantBrassica rapa

Biotypes ofBrassica rapasusceptible (S) and resistant (R) toatrazine were grown in competitive replacement series in allpossible combinations of two light levels and three temperatureregimes in controlled growth cabinets. Photosystem II functionwas investigated in all conditions by fluorescence-inductiontechniques. There were no significant differences in the dryweight of the two biotypes when grown in pure stands. In purestands both biotypes produced more biomass under the high lightlevel. Under high light both biotypes yielded more biomass athigh temperature; in low light they did so at medium temperature.Under high light conditions at high and medium temperaturesthe susceptible biotype had a greater photon yield and relativecompetitive ability than the resistant due to the greater vulnerabilityof triazine-resistant biotypes to photoinhibition. However,surprisingly, the resistant biotype was the better competitor,and had a higher photon yield, in the high light/low temperatureregime. In low light no photoinhibition was expected and indeedthere were no significant differences in any fluorescence parametersbetween the resistant and susceptible biotypes. Nevertheless,there were differences in the whole plant performance; the susceptiblebiotype was a better competitor at low and medium temperatures,but the resistant biotype was better at high temperature. Relativelysmall variations in both light and temperature, well withinthe range encountered during British summer time, can have largeeffects on the relative competitiveness of triazine R and Sbiotypes in this species with implications for the spread ofresistance genes through semi-natural communities. In lightof predicted climate changes, interactions between climate andresistance should be studied across a wider range of herbicidetypes and weed species.Copyright 1997 Annals of Botany Company Brassica rapa; chlorophyll fluorescence; competition; light; navew; temperature; triazine resistance     

Fluorescence characteristics of photoinhibition and recovery in a sun and a shade species of the red algal genus porphyra

The effects of light treatment (2000 micromole photons per square meter per second) for varying periods (up to 60 minutes) on chlorophyll fluorescence characteristics and light-limited rates of O₂ evolution were examined in two Porphyra species. Brief light exposures (5-60 seconds) produced a large decrease in variable fluorescence which was not accompained by photoinhibition of light-limited O₂ evolution rates. This rapid decrease in variable fluorescence was suppressed by carbonylcyanide m-chlorophenylhydrazone, indicating that it was related to formation of a proton gradient across the thylakiod membranes. A second phase of fluorescence quenching started after 5 minutes of illumination in the case of the shade species, Porphyra nereocystis Anderson, and after 30 minutes of illumination in the case of the sun species, Porphyra perforata J. Agardh. The rate of fluorescence quenching in the second phase was similar to the rate of photoinhibition of light-limited O₂ evolution in both cases. The dark recovery of variable fluorescence in light-treated plants was also biphasic consisting of a rapid first phase and a slower second phase in both the Porphyra species. Recovery of P. perforata was more complete than that of P. nereocystis over the same recovery period. This greater capacity for recovery could represent a mechanism by which P. perforata is more resistant to photoinhibition than P. nereocystis.     

Photoinhibition of photosynthesis on a coral reef

Photoinhibition of macroalgae in the epilithic algal community (KAC) of coral reefs was studied using chlorophyll fluorescence techniques at One Tree Island, Great Barrier Reef, Australia. F_v/F_m (variable to maximum fluorescence, darkened samples) of shallow macroalgae declined by 50% on fine summer and winter days, recovering in late afternoon. Within a species, thalli from low-light habitats were more photoinhibited (2h at 1400μimol m^?2 s^?1) than those from high-light habitats. The sensitivity of Lobophora variegata (Phacophyta) and Chlorodesmis fastigiata (Chlorophyta) increased with depth (1 versus 20 m). However, shallow Halimeda tuna (Chlorophyta) plants growing between corals were more photoinhibited than those from deep, open areas. Photoinhibition and recovery were depth- and species-specific. Shallow Lobophora and Chlorodesmis maintained a greater degree of Q _A oxidation during photoinhibition. In deep thalli, reduced effective quantum yield of open photosystem II centres reflected lower proportions and excitation capture efficiencies of open centres. In Lobophora, zeaxanthin formation accompanied non-photochemical fluorescence quenching (NPQ), but in Chlorodesmis NPQ was limited and no zeaxanthin or antherxanthin formed. Higher photosynthetic efficiency in the lower storey of the EAC may compensate for photoinhibition in the upper storey, thereby reconciling photoinhibition of individual thalli with previous observations of no net inhibition of community productivity.     

Kinetics of prolonged photoinhibition revisited: photoinhibited Photosystem II centres do not protect the active ones against loss of oxygen evolution

Photoinhibition of Photosystem II (PSII) in lincomycin-treated leaves begins as a first-order reaction, but fluorescence measurements have suggested that after prolonged illumination, the number of active PSII centres stabilizes to 15–20% of control. The stabilization has been interpreted to indicate that photoinhibited PSII centres protect the remaining active centres against photoinhibition (Lee, Hong and Chow, Planta 212:332–342, 2001). In an attempt to study the mechanism of this protection, we measured the reaction kinetics of photoinhibition in lincomycin-treated pumpkin (Cucurbita pepo L.) and pepper (Capsicum annuum L.) leaves in vivo. The light-saturated rate of PSII oxygen evolution, assayed from thylakoids and isolated from the treated leaves, was used as a direct measure of the number of remaining active PSII centres, and the fluorescence parameters F _V/F _M and (F _V/F _M)/F ₀ (=1/F ₀ − 1/F _M) were measured for comparison. To our surprise, no stabilization of PSII activity was observed and photoinhibition followed first-order kinetics until PSII activity had virtually declined to zero. A series of in vitro experiments was carried out to see whether stabilization of PSII activity occurs if a particular combination of light intensity and wavelength range is applied, or if a specific PSII preparation is used as experimental material. The results of the in vitro experiments confirmed the in vivo result about persistent first-order kinetics. We conclude that photoinhibited PSII centres offer no measurable protection against photoinhibition.     

Photoinhibition of CO(2)-Dependent O(2) Evolution by Intact Chloroplasts Isolated from Spinach Leaves

Intact spinach (Spinacia oleracea L.) chloroplasts, when pre-illuminated at 4 millimoles quanta per square meter per second for 8 minutes in a CO₂-free buffer at 21% O₂, showed a decrease (30-70%) in CO₂-dependent O₂ evolution and ¹⁴CO₂ uptake. This photoinhibition was observed only when the O₂ concentration and the quantum fluence rate were higher than 4% and 1 millimole per square meter per second, respectively. There was only a small decrease in the extent of photoinhibition when the CO₂ concentration was increased from 0 to 25 micromolar during the treatment, but photoinhibition was abolished when the CO₂ concentration was increased to 30 micromolar. Addition of small quantities of P-glycerate (40-200 micromolar) or glycerate (160 micromolar) was found to prevent photoinhibition. Other intermediates of the Calvin cycle (fructose-6-P, fructose-1,6-P, ribose-5-P, ribulose-5-P) also prevented photoinhibition to various extents. Oxaloacetate was not effective in preventing photoinhibition in these chloroplasts. The amount of O₂ evolved during treatments with 3-P-glycerate or glycerate was no more than 65% of that measured in the presence of low CO₂ concentrations (9-12 micromolar) which did not prevent photoinhibition. In all cases, the extent to which photoinhibition was prevented by these metabolites was not correlated to the amount of O₂ evolved during the photoinhibitory treatment. It is concluded that in these chloroplasts the prevention of the O₂-dependent photoinhibition of light saturated CO₂ fixation capacity is not linked to the dissipation of excitation energy via the photosynthetic electron transport nor to ATP utilization. The requirement of O₂ for photoinhibition of CO₂ fixation capacity in isolated chloroplasts may be explained by an effect of O₂ in allowing metabolic depletion of Calvin cycle intermediates.     

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.     

Photoinhibition at low temperature in chilling-sensitive and -resistant plants

Photoinhibition resulting from exposure at 7°C to a moderate photon flux density (300 micromoles per square meter per second, 400-700 nanometers) for 20 hours was measured in leaves of annual crops differing widely in chilling tolerance. The incidence of photoinhibition, determined as the decrease in the ratio of induced to total chlorophyll fluorescence emission at 693 nanometers (F_v/F_max) measured at 77 Kelvin, was not confined to chilling-sensitive species. The extent of photoinhibition in leaves of all chilling-resistant plants tested (barley [Hordeum vulgare L.], broad bean [Vicia faba L.], pea [Pisum sativum L.], and wheat [Triticum aestivum L.]) was about half of that measured in chilling-sensitive plants (bean [Phaseolus vulgaris L.], cucumber [Cucumis sativus L.], lablab [Lablab purpureus L.], maize [Zea mays L.], pearl millet [Pennisetum typhoides (Burm. f.) Stapf & Hubbard], pigeon pea [Cajanus cajun (L.) Millsp.], sesame [Sesamum indicum L.], sorghum [Sorghum bicolor L. Moench], and tomato [Lycopersicon esculentum Mill.]). Rice (Oryza sativa L.) leaves of the indica type were more susceptible to photoinhibition at 7°C than leaves of the japonica type. Photoinhibition was dependent both on temperature and light, increasing nonlinearly with decreasing temperature and linearly with increasing light intensity. In contrast to photoinhibition during chilling, large differences, up to 166-fold, were found in the relative susceptibility of the different species to chilling injury in the dark. It was concluded that chilling temperatures increased the likelihood of photoinhibition in leaves of both chilling-sensitive and -resistant plants. Further, while the photoinhibition during chilling generally occurred more rapidly in chilling-sensitive plants, this was not related directly to chilling sensitivity.     

Chilling Sensitivity in Oryza sativa: The Role of Protein Phosphorylation in Protection against Photoinhibition

The effects of exposure to low temperature on photosynthesis and protein phosphorylation in chilling-sensitive and cold-tolerant plant species were compared. Chilling temperatures resulted in light-dependent loss of photosynthetic electron transport in chilling-sensitive rice (Oryza sativa L.) but not in cold-tolerant barley (Hordeum vulgare L.). Brief exposure to chilling temperatures (0-15°C, 10 min) did not cause a significant difference in photosynthetic O₂ evolution capacity in vivo between rice and barley. Analysis of in vivo chlorophyll fluorescence in chilling-sensitive rice suggests that low temperatures cause an increased reduction of the plastoquinone pool that could result in photoinhibitory damage to the photosystem II reaction centers. Analysis of ³²P incorporation into thylakoid proteins both in vivo and in vitro demonstrated that chilling temperature inhibited protein phosphorylation in rice, but not in barley. Low temperature (77 K) fluorescence analysis of isolated thylakoid membranes indicated that state I to state II transitions occurred in barley, but not in rice subjected to chilling temperatures. These observations suggest that protein phosphorylation may play an important role in protection against photoinhibition caused by exposure to chilling temperatures.     

Internal CO(2) Supply during Photosynthesis of Sun and Shade Grown CAM Plants in Relation to Photoinhibition

Leaves of Kalanchoë pinnata were exposed in the dark to air (allowing the fixation of CO₂ into malic acid) or 2% O₂, 0% CO₂ (preventing malic acid accumulation). They were then exposed to bright light in the presence or absence of external CO₂ and light dependent inhibition of photosynthetic properties assessed by changes in 77 K fluorescence from photosystem II (PSII), light response curves and quantum yields of O₂ exchange, rates of electron transport from H₂O through Q_B (secondary electron acceptor from the PSII reaction center) in isolated thylakoids, and numbers of functional PSII centers in intact leaf discs. Sun leaves of K. pinnata experienced greater photoinhibition when exposed to high light in the absence of CO₂ if malic acid accumulation had been prevented during the previous dark period. Shade leaves experienced a high degree of photoinhibition when exposed to high light regardless of whether malic acid had been allowed to accumulate in the previous dark period or not. Quantum yields were depressed to a greater degree than was 77 K fluorescence from PSII following photoinhibition.