G. Heinrich Krause

Chlorophyll fluorescence as a tool in plant physiology. II. Interpretation of fluorescence signals.

Revue et interpretation des analyses de la fluorescence chlorophyllienne et interet pour les processus photosynthetiques dans les feuilles intactes ou chez les Algues

Protective systems against active oxygen species in spinach: response to cold acclimation in excess light.

Spinach (Spinacia oleracea L.) plants were acclimated to 1° C or maintained at 18° C under the same light regime (260–300 μmol photons·m−2·s−1). The cold acclimation led to several metabolic and biochemical changes that apparently include improved protection of the photosynthetic apparatus against active oxygen species. In particular, cold-acclimated leaves exhibited a considerably higher ascorbate content and significantly increased activities of superoxide dismutase, ascorbate peroxidase, and monodehydroascorbate reductase in the chloroplasts. The level of dehydroascorbate reductase did not alter. Catalase activity decreased. The photosynthetic pigment composition of cold-acclimated spinach was characterized by increased levels of the xanthophylls lutein + zeaxanthin and violaxanthin. The observed changes are discussed in terms of their possible relevance for plant resistance to photoinhibition at chilling temperatures.

Two mechanisms of recovery from photoinhibition in vivo : reactivation of photosystem II related and unrelated to D1-protein turnover

Recovery (at 20° C) of spinach (Spinacia oleracea L.) leaf sections from photoinhibition of photosynthesis was monitored by means of the fluorescence parameter FV/FM of intact leaf tissue and of PSII-driven electron-transport activity of isolated thylakoids. Different degrees of photoinactivation of PSII were obtained by preillumination in ambient air (at 4 or 20° C), CO2-free air or at low and high O2 levels (2 or 41 %) in N2. The kinetics of recovery exhibited two distinct phases. The first phase usually was completed within about 20-60 min and was most pronounced after preillumination in low O2. The slow phase proceeded for several hours leading to almost complete reactivation of PSII. Preincubation of the leaves with streptomycin (SM), which inhibits chloroplast-encoded protein synthesis, inhibited the slow recovery phase only, indicating the dependence of this phase on resynthesis of the reaction-centre protein, D1. The fast recovery phase remained largely unaffected by SM. Both phases were strongly but not totally dependent on irradiation of the leaf with low light. When SM was absent, net degradation of the D1 protein could neither be detected upon photoinhibitory irradiation nor during following incubation of the leaf sections in low light or darkness. In the presence of SM, net D1 degradation was seen and tended to increase with O2 concentration during photoinhibition treatment. Based on these data, we suggest that photoinactivation of PSII in vivo occurs in at least two steps. From the first step, reactivation appears possible in low light without D1 turnover (fast recovery phase). Action of oxygen then may lead to a second step, in which the D1 protein is affected and reactivation requires its removal and replacement (slow phase).

Increased xanthophyll cycle activity and reduced D1 protein inactivation related to photoinhibition in two plant systems acclimated to excess light

Young and mature canopy leaves of tropical forest trees (Anacardium excelsum, Castilla elastica) and cold- and non-acclimated leaves of spinach (Spinacia oleracea) grown in temperate climate were used as systems of differing susceptibility to high light stress to assess the role of xanthophyll cycle activity and D1 protein turnover in photoinhibition of photosynthesis. Previous investigations with spinach revealed two distinct stages of photoinhibition (indicated by a decline in the ratio of variable to maximum chlorophyll fluorescence) which were reflected in different kinetics of recovery in low light. An initial fast recovery phase of unknown biochemical mechanism (about 1 h) was followed by a slow phase (several hours), which may be based upon resynthesis of the D1 protein in the photosystem II reaction center. All leaves studied exhibited these two phases. However, the kinetics and relative amplitude of the two phases varied strongly and were dependent on leaf age and light acclimation. Recovery was rapid in young canopy sun leaves and cold-acclimated leaves of spinach due to a pronounced initial phase. On the other hand, the slow, probably D1 related phase dominated in mature canopy leaves and non-acclimated leaves of spinach. The fast phase of recovery and epoxidation of zeaxanthin via the xanthophyll cycle were closely correlated in all leaves studied. In addition, recovery following photoinhibition in the presence of dithiothreitol, which prevented formation of zeaxanthin, occurred only in a slow phase. On the other hand, leaf incubation prior to photoinhibition with streptomycin, an inhibitor of chloroplast-encoded protein synthesis, eliminated slow recovery by preventing resynthesis of the D1 protein. The young tropical sun leaves and the cold-acclimated spinach leaves exhibited a higher pool of xanthophyll cycle pigments per chlorophyll and in response to strong light converted a higher percentage of violaxanthin to zeaxanthin than the mature and the non-acclimated leaves, respectively. Quantification of D1 protein in photoinhibited spinach leaves by means of electrophoresis and Western blotting revealed a strongly diminished D1 protein degradation in the cold-acclimated compared to the non-acclimated state. High xanthophyll cycle activity and pool sizes observed in response to high light with young canopy and cold-acclimated spinach leaves may serve to protect these leaves against inactivation of the D1 protein.

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 (FV) 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.

On the mechanism of photoinhibition in chloroplasts : relationship between changes in fluorescence and activity of photosystem II

Summary Spinach ( Spinacia oleracea L.) leaves were subjected to photoinhibitory treatments. Thylakoid membranes isolated from such leaves showed inhibition of the optimal quantum yield and of the capacity of photosystem (PS)II-dependent electron transport to the same extent. The decline in PS II capacity was linearly related to the decrease in the ratio of variable to maximum fluorescence (F v /F M ) of the leaves. Chlorophyll fluorescence properties (in the presence of 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea, DCMU) and PS II activity were studied in thylakoid membranes released from photoinhibited spinach chloroplasts (or mesophyll protoplasts of Valerianella locusta L.). PS II electron transport capacity was linearly related to F v /F M and to the DCMU binding capacity. The maximum complementary area above the fluorescence induction curve (A max ) declined with progressing photoinhibition to the same extent as F v . Area growth analysis indicated a predominant photoinhibition of PS IIα, but no transformation of a to β units. The results support the view that photoinhibition is based on transformation of active reaction centers to photochemically inactive fluorescence quenchers, which convert excitation energy to heat. Indications for increased thermal deactivation in the antenna system of PS II were not observed.

Xanthophyll cycle and energy-dependent fluorescence quenching in leaves from pea plants grown under intermittent light

The possible role of zeaxanthin formation and antenna proteins in energy-dependent chlorophyll fluorescence quenching (qE) has been investigated. Intermittent-light-grown pea (Pisum sativum L.) plants that lack most of the chlorophyll a/b antenna proteins exhibited a significantly reduced qE upon illumination with respect to control plants. On the other hand, the violaxanthin content related to the number of reaction centers and to xanthophyll cycle activity, i.e. the conversion of violaxanthin into zeaxanthin, was found to be increased in the antenna-protein-depleted plants. Western blot analyses indicated that, with the exception of CP 26, the content of all chlorophyll a/b-binding proteins in these plants is reduced to less than 10% of control values. The results indicate that chlorophyll a/b-binding antenna proteins are involved in the energy-dependent fluorescence quenching but that only a part of qE can be attributed to quenching by chlorophyll a/b-binding proteins. It seems very unlikely that xanthophylls are exclusively responsible for the qE mechanism.

Sun-shade patterns of leaf carotenoid composition in 86 species of neotropical forest plants

A survey of photosynthetic pigments, including 86 species from 64 families, was conducted for leaves of neotropical vascular plants to study sun-shade patterns in carotenoid biosynthesis and occurrence of α-carotene (α-Car) and lutein epoxide (Lx). Under low light, leaves invested less in structural components and more in light harvesting, as manifested by low leaf dry mass per area (LMA) and enhanced mass-based accumulation of chlorophyll (Chl) and carotenoids, especially lutein and neoxanthin. Under high irradiance, LMA was greater and β-carotene (β-Car) and violaxanthin-cycle pool increased on a leaf area or Chl basis. The majority of plants contained α-Car in leaves, but the α- to β-Car ratio was always low in the sun, suggesting preference for β-Car in strong light. Shade and sun leaves had similar β,ε-carotenoid contents per unit Chl, whereas sun leaves had more β,β-carotenoids than shade leaves. Accumulation of Lx in leaves was found to be widely distributed among taxa: >5 mmol mol Chl-1 in 20% of all species examined and >10 mmol mol Chl-1 in 10% of woody species. In Virola elongata (Benth.) Warb, having substantial Lx in both leaf types, the Lx cycle was operating on a daily basis although Lx restoration in the dark was delayed compared with violaxanthin restoration.

Capacity of protection against ultraviolet radiation in sun and shade leaves of tropical forest plants

Protection of leaves of tropical forest plants against UV-A and -B radiation was studied in three lowland forests, a montane cloud forest and a mangrove stand in Panama. Leaves were classified as sun or shade leaves according to their chlorophyll a / b ratio, pool size of xanthophyll cycle pigments and α- and β-carotene contents. The capacity of the leaves for protection against UV radiation was assessed by estimating epidermal UV-A shielding, by a non-invasive fluorometric method, and by the absorbance of ethanolic / aqueous leaf extracts in the UV spectral region. In all sun leaves tested, UV-A shielding by the adaxial epidermis was high, usually above 90%, whereas in shade leaves the epidermal UV-A shielding was markedly lower and varied widely between species. In most cases UV-A shielding by the abaxial epidermis was lower than by the adaxial epidermis. UV absorbance of the leaf extracts was generally higher in sun than in shade leaves, and the absorbance was much higher in the UV-B spectral region at 305 nm than in the UV-A region at 375 nm. The data demonstrate that sun leaves of tropical plants are well protected against solar UV-A and UV-B radiation. However, UV-induced damage may occur when shade leaves become exposed to full solar radiation.

Xanthophyll Cycle And Thermal Energy Dissipation In Photosystem II: Relationship between Zeaxanthin Formation, Energy-Dependent Fluorescence Quenching and Photoinhibition

Summary The influence of zeaxanthin formation on energy-dependent (qE) and photoinhibitory fluorescence quenching (decrease in Fv/Fm ratio) was studied with isolated thylakoids of spinach ( Spinacia oleracea L.) suspended in a medium of pH 7.6. It was found that in the absence of any qE (due to addition of uncoupler) the degree of photoinhibition and maximum fluorescence yield were unaffected by the presence of zeaxanthin. Thylakoid samples with and without added methylviologen both exhibited these effects. In the absence of any artificial electron acceptor, substantial qE occurred only in the presence of zeaxanthin. When methylviologen was added, which increased electron flow and pH gradient, substantial zeaxanthin-independent qE was exhibited. This qE was significantly stimulated (i.e. nearly doubled) by formation of zeaxanthin. The minimum luminal pH required for occurrence of zeaxanthin-independent qE was estimated as about 4.6, which was attained in the presence of methylviologen only. Antheraxanthin formation did not correlate with zeaxanthin-independent qE. Dithiothreitol, an inhibitor of violaxanthin de-epoxidase, stimulated photoinhibition even in the absence of zeaxanthin, indicating a secondary effect on photoinhibition that is not related to its influence on zeaxanthin formation. Photoinhibition was decreased when qE had been formed, showing the protective function of the qE process. Under all applied conditions photoinhibition was increased by the presence of methylviologen compared with its absence. The hypothesis is advanced that zeaxanthin acts as photoprotector only in the energized (coupled) thylakoid system via stimulation of the qE process and is not a quencher of excessive energy per se .