Zeaxanthin and the Induction and Relaxation Kinetics of the Dissipation of Excess Excitation Energy in Leaves in 2% O(2), 0% CO(2)

The relationship between the carotenoid zeaxanthin, formed by violaxanthin de-epoxidation, and nonphotochemical fluorescence quenching (q_NP) in the light was investigated in leaves of Glycine max during a transient from dark to light in 2% O₂, 0% CO₂ at 100 to 200 micromoles of photons per square meter per second. (a) Up to a q_NP (which can vary between 0 and 1) of about 0.7, the zeaxanthin content of leaves was linearly correlated with q_NP as well as with the rate constant for radiationless energy dissipation in the antenna chlorophyll (k_D). Beyond this point, at very high degrees of fluorescence quenching, only k_D was directly proportional to the zeaxanthin content. (b) The relationship between zeaxanthin and k_D was quantitatively similar for the rapidly relaxing quenching induced in 2% O₂, 0% CO₂ at 200 micromoles of photons per square meter per second and for the sustained quenching induced by long-term exposure of Nerium oleander to drought in high light (B Demmig, K Winter, A Krüger, F-C Czygan [1988] Plant Physiol 87: 17-24). These findings suggest that the same dissipation process may be induced by very different treatments and that this particular dissipation process can have widely different relaxation kinetics. (c) A rapid induction of strong nonphotochemical fluorescence quenching within about 1 minute was observed exclusively in leaves which already contained a background level of zeaxanthin.     

Light Response of CO(2) Assimilation, Dissipation of Excess Excitation Energy, and Zeaxanthin Content of Sun and Shade Leaves

Intact attached sun leaves of Helianthus annuus and shade leaves of Monstera deliciosa and Hedera helix were used to obtain light response curves of CO₂ uptake, the content of the carotenoid zeaxanthin (formed by violaxanthin de-epoxidation), as well as nonphotochemical quenching (q_NP), and the rate constant of radiationless energy dissipation (k_D). The latter two parameters were calculated from the decrease of chlorophyll a fluorescence at closed photosystem II traps in saturating pulses in the light. Among the three species, the light-saturated capacity of CO₂ uptake differed widely and light saturation of CO₂ uptake occurred at very different photon flux densities. Fluorescence quenching and zeaxanthin content exhibited features which were common to all three species: below light-saturation of CO₂ uptake nonphotochemical quenching occurred in the absence of zeaxanthin and was not accompanied by a decrease in the yield of instantaneous fluorescence. Nonphotochemical quenching, q_NP, increased up to values which ranged between 0.35 and 0.5 when based on a control value of the yield of variable fluorescence determined after 12 hours of darkness. As light saturation of CO₂ uptake was approached, q_NP showed a secondary increase and the zeaxanthin content of the leaves began to rise. This was also the point from which the yield of instantaneous fluorescence began to decrease. The increase in zeaxanthin was paralleled by an increase in the rate constant for radiationless energy dissipation k_D, which opens the possibility that zeaxanthin is related to the rapidly relaxing “high-energy-state quenching” in leaves.     

Inhibition of zeaxanthin formation and of rapid changes in radiationless energy dissipation by dithiothreitol in spinach leaves and chloroplasts

Dithiothreitol, which completely inhibits the de-epoxidation of violaxanthin to zeaxanthin, was used to obtain evidence for a causal relationship between zeaxanthin and the dissipation of excess excitation energy in the photochemical apparatus in Spinicia oleracea L. In both leaves and chloroplasts, inhibition of zeaxanthin formation by dithiothreitol was accompanied by inhibition of a component of nonphotochemical fluorescence quenching. This component was characterized by a quenching of instantaneous fluorescence (F_o) and a linear relationship between the calculated rate constant for radiationless energy dissipation in the antenna chlorophyll and the zeaxanthin content. In leaves, this zeaxanthin-associated quenching, which relaxed within a few minutes upon darkening, was the major component of nonphotochemical fluorescence quenching determined in the light, i.e. it represented the `high-energy-state' quenching. In isolated chloroplasts, the zeaxanthin-associated quenching was a smaller component of total nonphotochemical quenching and there was a second, rapidly reversible high-energy-state component of fluorescence quenching which occurred in the absence of zeaxanthin and was not accompanied by F_o quenching. Leaves, but not chloroplasts, were capable of maintaining the electron acceptor, Q, of photosystem II in a low reduction state up to high degrees of excessive light and thus high degrees of nonphotochemical fluorescence quenching. When ascorbate, which serves as the reductant for violaxanthin de-epoxidation, was added to chloroplast suspensions, zeaxanthin formation at low photon flux densities was stimulated and the relationship between nonphotochemical fluorescence quenching and the reduction state in chloroplasts then became more similar to that found in leaves. We conclude that the inhibition of zeaxanthin-associated fluorescence quenching by dithiothreitol provides further evidence that there exists a close relationship between zeaxanthin and potentially photoprotective dissipation of excess excitation energy in the antenna chlorophyll.     

A Comparison of the Effects of Chilling on Leaf Gas Exchange in Pea (Pisum sativum L.) and Cucumber (Cucumis sativus L.)

The effects of chilling on the photosynthesis of a chilling-resistant species, pea (Pisum sativum L. cv Alaska) and a chilling-sensitive species, cucumber (Cucumis sativus L. cv Ashley) were compared in order to determine the differences in the photosynthetic chilling sensitivity of these two species. For these experiments, plants were chilled (5°C) for different lengths of time in the dark or light. Following a 1 hour recovery period at 25°C, photosynthetic activity was measured by gas exchange (CO₂ uptake and H₂O release), quantum yield, and induced chlorophyll fluorescence. The results show that pea photosynthesis was largely unaffected by two consecutive nights of chilling in the dark, or by chilling during a complete light and dark cycle (15 hours/9 hours). Cucumber gas exchange was reduced by one night of chilling, but its quantum yield and variable fluorescence were unaffected by dark chilling. However, chilling cucumber in the light led to reduced CO₂ fixation, increased internal leaf CO₂ concentration, decreased quantum yield, and loss of variable fluorescence. These results indicate that chilling temperatures in conjunction with light damaged the light reactions of photosynthesis, while chilling in the dark did not.     

The Influence of Dark Adaptation Temperature on the Reappearance of Variable Fluorescence following Illumination

The effect of chilling temperatures (5°C) on chlorophyll fluorescence transients was used to study chilling-induced inhibition of photosynthesis in plant species with differing chilling sensitivities. A Brancker SF-20 fluorometer was used to measure induced fluorescence transients from both attached and detached leaves of chilling-sensitive cucumber (Cucumis sativus L. cv Ashley) and chilling-resistant pea (Pisum sativum L. cv Alaska). The rate of reappearance of the variable component of fluorescence (F_v), following a period of illumination at 25°C, was dependent on the temperature at which the leaf was allowed to dark adapt in chilling-sensitive cucumber, but not in chilling-resistant pea. In cucumber, dark adaptation at 25°C following illumination resulted in a much faster return of F_v than dark adaptation at 5°C following illumination. However, F_v reappearance during the dark adaptation period in chilling-resistant pea was temperature independent. The difference in the temperature response of F_v following illumination correlated with temperature sensitivity of these two species. The process responsible for the difference in F_v may represent a site of chilling sensitivity in the photosynthetic apparatus.     

Stress tolerance and stress-induced injury in crop plants measured by chlorophyll fluorescence in vivo: chilling, freezing, ice cover, heat, and high light

The proposition is examined that measurements of chlorophyll fluorescence in vivo can be used to monitor cellular injury caused by environmental stresses rapidly and nondestructively and to determine the relative stress tolerances of different species. Stress responses of leaf tissue were measured by F_R, the maximal rate of the induced rise in chlorophyll fluorescence. The time taken for F_R to decrease by 50% in leaves at 0°C was used as a measure of chilling tolerance. This value was 4.3 hours for chilling-sensitive cucumber. In contrast, F_R decreased very slowly in cucumber leaves at 10°C or in chilling-tolerant cabbage leaves at 0°C. Long-term changes in F_R of barley, wheat, and rye leaves kept at 0°C were different in frost-hardened and unhardened material and in the latter appeared to be correlated to plant frost tolerance. To simulate damage caused by a thick ice cover, wheat leaves were placed at 0°C under N₂. Kharkov wheat, a variety tolerant of ice encapsulation, showed a slower decrease in F_R than Gatcher, a spring wheat. Relative heat tolerance was also indicated by the decrease in F_R in heated leaves while changes in vivo resulting from photoinhibition, ultraviolet radiation, and photobleaching can also be measured.     

Low measuring temperature induced artifactual increase in chlorophyll a fluorescence

Measurement of in vivo chlorophyll a fluorescence at temperatures lower than 20°C can cause an artifactual, nonphotochemically related overestimation of variable fluorescence leading to the calculation of negative values for the nonphotochemical quenching parameter and an underestimation of the photochemical quenching parameter. This artifact is observed only upon exposure of the leaf sample to actinic light. We suggest that a temperature differential between the fiber-optic probe and the leaf sample results in the deposition of water vapor on the probe that distorts the light path such that an increased modulated fluorescence signal is observed. This artifact is eradicated by ensuring that the end of the fiber-optic probe is kept free of condensation.     

Effect of growth temperature and temperature shifts on spinach leaf morphology and photosynthesis

The growth kinetics of spinach plants (Spinacia oleracea L. cv Savoy) grown at 5°C or 16°C were determined to allow us to compare leaf tissues of the same developmental stage rather than chronological age. The second leaf pairs reached full expansion at a plant age of 32 and 92 days for the 16°C and 5°C plants, respectively. Growth at 5°C resulted in an increased leaf area, dry weight, dry weight per area, and leaf thickness. Despite these changes, pigment content and composition, room temperature in vivo fluorescence, and apparent quantum yield and light-saturated rates of CO₂ exchange or O₂ evolution were not affected by the growth temperature. Furthermore, 5°C expanded leaves were found to be more resistant to photoinhibition at 5°C than were 16°C expanded leaves. Thus, it is concluded that spinach grown at low temperature is not stressed. However, shifting spinach leaves from 5°C to 16°C or from 16°C to 5°C for 12 days after full leaf expansion had occurred resulted in a 20 to 25% reduction in apparent quantum yields and 50 to 60% reduction in light saturated rates of both CO₂ exchange and O₂ evolution. This was not accompanied by a change in the pigment content or composition or in the room temperature in vivo fluorescence. It appears that leaf aging during the temperature shift period can account for the reduction in photosynthesis. Comparison of cold-hardened and non-hardened winter rye (Secale cereale L. cv Muskateer) with spinach by in vivo fluorescence indicated that rye is more sensitive to both short term and longer duration temperature shifts than is spinach. Thus, susceptibility to an abrupt temperature shift appears to be species dependent.     

Photoinhibition and zeaxanthin formation in intact leaves : a possible role of the xanthophyll cycle in the dissipation of excess light energy

Comparative studies of chlorophyll a fluorescence, measured with a pulse amplitude modulated fluorometer, and of the pigment composition of leaves, suggest a specific role of zeaxanthin, a carotenoid formed in the xanthophyll cycle, in protecting the photosynthetic apparatus against the adverse effects of excessive light. This conclusion is based on the following findings: (a) exposure of leaves of Populus balsamifera, Hedera helix, and Monstera deliciosa to excess excitation energy (high light, air; weak light, 2% O₂, 0% CO₂) led to massive formation of zeaxanthin and a decrease in violaxanthin. Over a wide range of conditions, there was a linear relationship between either variable, F_v, or maximum fluorescence, F_m, and the zeaxanthin content of leaves. (b) When exposed to photoinhibitory light levels in air, shade leaves of H. helix had a higher capacity for zeaxanthin formation, at the expense of β-carotene, than shade leaves of M. deliciosa. Changes in fluorescence characteristics suggested that, in H. helix, the predominant response to high light was an increase in the rate of nonradiative energy dissipation, whereas, in M. deliciosa, photoinhibitory damage to photosystem II reaction centers was the prevailing effect. (c) Exposure of a sun leaf of P. balsamifera to increasing photon flux densities in 2% O₂ and 0% CO₂ resulted initially in increasing levels of zeaxanthin (matched by decreases in violaxanthin) and was accompanied by fluorescence changes indicative of increased nonradiative energy dissipation. Above the light level at which no further increase in zeaxanthin content was observed, fluorescence characteristics indicated photoinhibitory damage. (d) A linear relationship was obtained between the ratio of variable to maximum fluorescence, F_v/F_m, determined with the modulated fluorescence technique at room temperature, and the photon yield of O₂ evolution, similar to previous findings (O Björkman, B Demmig 1987 Planta 170: 489-504) on chlorophyll fluorescence characteristics at 77 K and the photon yield of photosynthesis.     

Mehler-peroxidase reaction mediates zeaxanthin formation and zeaxanthin-related fluorescence quenching in intact chloroplasts

Induction of zeaxanthin formation and the associated nonphotochemical quenching in iodoacetamide-treated, non-CO₂-fixing intact chloroplasts of Lactuca sativa L. cv Romaine is reported. The electron transport needed to generate the required ΔpH for zeaxanthin formation and nonphotochemical quenching are ascribed to the Mehler-ascorbate peroxidase reaction. KCN, an inhibitor of ascorbate peroxidase, significantly affected these activities without affecting linear electron transport to methyl viologen or violaxanthin deepoxidase activity. At 1 millimolar KCN, zeaxanthin formation and ΔpH were inhibited 60 and 55%, respectively, whereas ascorbate peroxidase activity was inhibited almost totally. The KCN-resistant activity, which apparently was due to electron transport mediated by the Mehler reaction alone, however, was insufficient to support a high level of nonphotochemical quenching. We suggest that in vivo, as CO₂ fixation becomes limiting, the Mehler-peroxidase reaction protects photosystem II against the excess light by supporting the electron transport needed for zeaxanthin-dependent nonphotochemical quenching and concomitantly scavenging H₂O₂. Ascorbate is essential for this process to occur.     

Adaptation of the Photosynthetic Apparatus in Maize Leaves as a Result of Nitrogen Limitation : Relationships between Electron Transport and Carbon Assimilation

In maize (Zea mays L., cv Contessa), nitrogen (NO₃⁻) limitation resulted in a reduction in shoot growth and photosynthetic capacity and in an increase in the leaf zeaxanthin contents. Nitrogen deficiency had only a small effect on the quantum yield of CO₂ assimilation but a large effect on the light-saturated rate of photosynthesis. Linear relationships persisted between the quantum yield of CO₂ assimilation and that of photosystem II photochemistry in all circumstances. At high irradiances, large differences in photochemical quenching and nonphotochemical quenching of Chl a fluorescence as well as the ratio of variable to maximal fluorescence (Fv/Fm) were apparent between nitrogen-deficient plants and nitrogen-replete controls, whereas at low irradiances these parameters were comparable in all plants. Light intensity-dependent increases in nonphotochemical quenching were greatest in nitrogen-deficient plants as were the decreases in Fv/Fm ratio. In nitrogen-deficient plants, photochemical quenching decreased with increasing irradiance but remained higher than in controls at high irradiances. Thermal dissipative processes were enhanced as a result of nitrogen deficiency (nonphotochemical quenching was elevated and Fv/Fm was lowered) allowing PSII to remain relatively oxidised even when carbon metabolism was limited via nitrogen limitation.     

Topography of photosynthetic activity of leaves obtained from video images of chlorophyll fluorescence

The distribution of photosynthetic activity over the area of a leaf and its change with time was determined (at low partial pressure of O₂) by recording images of chlorophyll fluorescence during saturating light flashes. Simultaneously, the gas exchange was being measured. Reductions of local fluorescence intensity quantitatively displayed the extent of nonphotochemical quenching; quench coefficients, q_N, were computed pixel by pixel. Because rates of photosynthetic electron transport are positively correlated with (1 − q_N), computed images of (1 − q_N) represented topographies of photosynthetic activity. Following application of abscisic acid to the heterobaric leaves of Xanthium strumarium L., clearly delineated regions varying in nonphotochemical quenching appeared that coincided with areoles formed by minor veins and indicated stomatal closure in groups.     

Analysis of the Relative Increase in Photosynthetic O2 Uptake When Photosynthesis in Grapevine Leaves Is Inhibited following Low Night Temperatures and/or Water Stress

We found similarities between the effects of low night temperatures (5°C–10°C) and slowly imposed water stress on photosynthesis in grapevine (Vitis vinifera L.) leaves. Exposure of plants growing outdoors to successive chilling nights caused light- and CO₂-saturated photosynthetic O₂ evolution to decline to zero within 5 d. Plants recovered after four warm nights. These photosynthetic responses were confirmed in potted plants, even when roots were heated. The inhibitory effects of chilling were greater after a period of illumination, probably because transpiration induced higher water deficit. Stomatal closure only accounted for part of the inhibition of photosynthesis. Fluorescence measurements showed no evidence of photoinhibition, but nonphotochemical quenching increased in stressed plants. The most characteristic response to both stresses was an increase in the ratio of electron transport to net O₂ evolution, even at high external CO₂ concentrations. Oxygen isotope exchange revealed that this imbalance was due to increased O₂ uptake, which probably has two components: photorespiration and the Mehler reaction. Chilling- and drought-induced water stress enhanced both O₂ uptake processes, and both processes maintained relatively high rates of electron flow as CO₂ exchange approached zero in stressed leaves. Presumably, high electron transport associated with O₂ uptake processes also maintained a high ΔpH, thus affording photoprotection.     

Photophosphorylation after chilling in the light : effects on membrane energization and coupling factor activity

The response of in situ photophosphorylation in attached cucumber (Cucumis sativus L. cv Ashley) leaves to chilling under strong illumination was investigated. A single-beam kinetic spectrophotometer fitted with a clamp-on, whole leaf cuvette was used to measure the flash-induced electrochromic absorbance change at 518 minus 540 nanometers (ΔA_518−540) in attached leaves. The relaxation kinetics of the electric field-indicating ΔA_518−540 measures the rate of depolarization of the thylakoid membrane. Since this depolarization process is normally dominated by proton efflux through the coupling factor during ATP synthesis, this technique can be used, in conjuction with careful controls, as a monitor of in situ ATP formation competence. Whole, attached leaves were chilled at 5°C and 1000 microeinsteins per square meter per second for up to 6 hours then rewarmed in the dark at room temperature for 30 minutes and 100% relative humidity. Leaf water potential, chlorophyll content, and the effective optical pathlength for the absorption measurements were not affected by the treatment. Light- and CO₂-saturated leaf disc oxygen evolution and the quantum efficiency of photosynthesis were inhibited by approximately 50% after 3 hours of light chilling and by approximately 75% after 6 hours. Despite the large inhibition to net photosynthesis, the measurements of ΔA_518−540 relaxation kinetics showed photophosphorylation to be largely unaffected by the chilling and light exposure. The amplitude of the ΔA_518-540 measures the degree of energization of the photosynthetic membranes and was reduced significantly by chilling in the light. The cause of the decreased energization was traced to impaired turnover of photosystem II. Our measurements showed that the chilling of whole leaves in the light caused neither an uncoupling of photophosphorylation from photosynthetic electron transport nor any irreversible inhibition of the chloroplast coupling factor in situ. The sizeable inhibition in net photosynthesis observed after chilling in the light cannot, therefore, be attributed to any direct effect on photophosphorylation competence.     

Thermal Acclimation of Photosynthetic Electron Transport Activity by Thylakoids of Saxifraga cernua

Thermal acclimation by Saxifraga cernua to low temperatures results in a change in the optimum temperature for gross photosynthetic activity and may directly involve the photosynthetic apparatus. In order to test this hypothesis photosynthetic electron transport activity of S. cernua thylakoids acclimated to growth temperatures of 20°C and 10°C was measured in vitro. Both populations exhibited optimum temperatures for whole chain and PSII electron transport activity at temperatures close to those at which the plants were grown. Chlorophyll a fluorescence transients from 10°C-acclimated leaves showed higher rates in the rise and subsequent quenching of variable fluorescence at low measuring temperatures; 20°C-acclimated leaves showed higher rates of fluorescence rise at higher measuring temperatures. At these higher temperatures, fluorescence quenching rates were similar in both populations. The kinetics of State 1-State 2 transitions in 20°C- and 10°C-acclimated leaf discs were measured as changes in the magnitude of the fluorescence emission maxima measured at 77K. Leaves acclimated at 10°C showed a larger F730/F695 ratio at low temperatures, while at higher temperatures, 20°C-acclimated leaves showed a higher F730/F695 ratio after the establishment of State 2. High incubation temperatures also resulted in a decrease in the F695/F685 ratio for 10°C-acclimated leaves, suggesting a reduction in the excitation transfer from the light-harvesting complex of photosystem II to photosystem II reaction centers. The relative amounts of chlorophyll-protein complexes and thylakoid polypeptides separated electro-phoretically were similar for both 20°C- and 10°C-acclimated leaves. Thus, photosynthetic acclimation to low temperatures by S. cernua is correlated with an increase in photosynthetic electron transport activity but does not appear to be accompanied by major structural changes or different relative amounts in thylakoid protein composition.     

Relative contributions of zeaxanthin-related and zeaxanthin-unrelated types of ;high-energy-state' quenching of chlorophyll fluorescence in spinach leaves exposed to various environmental conditions

We have identified two rapidly relaxing components of non-photochemical fluorescence quenching which suggests that dissipative processes occur in two different sites in the photochemical system of leaves. Under a variety of treatment conditions involving different leaf temperatures, photon flux densities (PFD), exposure times, and in the presence of 5% CO₂ or 2% O₂, no CO₂, the components of nonphotochemical fluorescence quenching were characterized with respect to their sensitivity to dithiothreitol (DTT, which completely inhibits zeaxanthin formation), the effect on instantaneous fluorescence, and the rapidity of relaxation upon darkening. Under most circumstances the DTT-sensitive component (associated with a quenching of instantaneous fluorescence and correlated with zeaxanthin) represented the majority of the rapidly relaxing portion of fluorescence quenching. A DTT-insensitive (zeaxanthin-independent) component, which also relaxed rapidly upon darkening but was not associated with a quenching of instantaneous fluorescence, became proportionally greater in an atmosphere of 2% O₂ and no CO₂, at elevated leaf temperatures, and to some degree during the induction of photosynthesis (1 minute after the onset of illumination). A third component which was also DTT-insensitive and was sustained upon darkening, was largely suppressed in 2% O₂, O% CO₂. We conclude that, under conditions favorable for photosynthesis, energy dissipation occurred mainly in the chlorophyll antennae whereas, under conditions less favorable for photosynthesis, a second dissipation process, probably in or around the reaction center of photosystem II, also developed. Furthermore, evidence is presented that the zeaxanthin-associated dissipation process prevents sustained inactivation of photochemistry by excessive light.     

Carbon Dioxide-Induced Oscillations in Fluorescence and Photosynthesis: Role of Thylakoid Membrane Energization in Regulation of Photosystem II Activity

The response of CO₂ fixation to a sudden increase in ambient CO₂ concentration has been investigated in intact leaf tissue from spinach (Spinacia oleracea) using a dual channel infrared gas analyzer. Simultaneous with these measurements, changes in fluorescence emission associated with a weak, modulated measuring beam were recorded. Application of brief (2-3 seconds) dark intervals enabled estimation of the dark fluorescence level (F_o) under both steady state and transient conditions. The degree of suppression of F_o level fluorescence in the light was strongly correlated with nonphotochemical quenching under all conditions. During CO₂-induced oscillations in photosynthesis under 2% O₂ the changes in nonphotochemical quenching anticipate changes in the rate of uptake of CO₂. At such low levels of O₂ and constant illumination, changes in the relative quantum efficiency of open photosystem II units were estimated as the ratio of the rate of CO₂ uptake and the photochemical quenching coefficient. Under the same conditions the relative quantum efficiency of photosystem II was found to vary inversely with the degree of nonphotochemical quenching. The relationship between changes in the rate of CO₂ uptake: photochemical quenching coefficient and nonphotochemical quenching was altered somewhat when the same experiment was conducted under 20% O₂. The results suggest that electron transport coupled to reduction of O₂ occurs to varying degrees with time during oscillations, especially when ambient O₂ concentrations are high.     

Photoinactivation of Catalase Occurs under Both High- and Low-Temperature Stress Conditions and Accompanies Photoinhibition of Photosystem II

Severe photoinactivation of catalase (EC 1.11.1.6) and a decline of variable fluorescence (F_v), indicating photoinhibition of photosynthesis, were observed as rapid and specific symptoms in leaves exposed to a high heat-shock temperature of 40°C as well as in leaves exposed to low chilling temperatures in white light of only moderately high photosynthetic photon flux density of 520 μE m⁻² s⁻¹. Other parameters, such as peroxidase (EC 1.11.1.7), glycolate oxidase (EC 1.1.3.1), glutathione reductase (EC 1.6.4.2), or the chlorophyll content, were hardly affected under these conditions. At a compatible temperature of 22°C, the applied light intensity did not induce severe photoinactivations. In darkness, exposures to high or low temperatures did not affect catalase levels. Also, decline of F_v in light was not related to temperature sensitivity in darkness. The effective low-temperature ranges inducing photoinactivation of catalase differed significantly for chilling-tolerant and chilling-sensitive plants. In leaves of rye (Secale cereale L.) and pea (Pisum sativum L.), photoinactivation occurred only below 15°C, whereas inactivation occurred at 15°C in cucumber (Cucumis sativus L.) and maize (Zea mays L.). The behavior of F_v was similar, but the difference between chilling-sensitive and chilling-tolerant plants was less striking. Whereas the catalase polypeptide, although photoinactivated, was not cleaved at 0 to 4°C, the D1 protein of photosystem II was greatly degraded during the low-temperature treatment of rye leaves in light. Rye leaves did not exhibit symptoms of any major general photodamage, even when they were totally depleted of catalase after photoinactivation at 0 to 4°C, and catalase recovered rapidly at normal temperature. In cucumber leaves, the decline of catalase after exposures to bright light at 0 to 4°C was accompanied by bleaching of chlorophyll, and the recovery observed at 25°C was slow and required several days. Similar to the D1 protein of photosystem II, catalase differs greatly from other proteins by its inactivation and high turnover in light. Inasmuch as catalase and D1 protein levels depend on continuous repair synthesis, preferential and rapid declines are generally to be expected in light whenever translation is suppressed by stress actions, such as heat or chilling, and recovery will reflect the repair capacity of the plants.     

The Xanthophyll Cycle Modulates the Kinetics of Nonphotochemical Energy Dissipation in Isolated Light-Harvesting Complexes, Intact Chloroplasts, and Leaves of Spinach

We analyzed the kinetics of nonphotochemical quenching of chlorophyll fluorescence (qN) in spinach (Spinacia oleracea) leaves, chloroplasts, and purified light-harvesting complexes. The characteristic biphasic pattern of fluorescence quenching in dark-adapted leaves, which was removed by preillumination, was evidence of light activation of qN, a process correlated with the de-epoxidation state of the xanthophyll cycle carotenoids. Chloroplasts isolated from dark-adapted and light-activated leaves confirmed the nature of light activation: faster and greater quenching at a subsaturating transthylakoid pH gradient. The light-harvesting chlorophyll a/b-binding complexes of photosystem II were isolated from dark-adapted and light-activated leaves. When isolated from light-activated leaves, these complexes showed an increase in the rate of quenching in vitro compared with samples prepared from dark-adapted leaves. In all cases, the quenching kinetics were fitted to a single component hyperbolic function. For leaves, chloroplasts, and light-harvesting complexes, the presence of zeaxanthin was associated with an increased rate constant for the induction of quenching. We discuss the significance of these observations in terms of the mechanism and control of qN.     

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.