Light-induced chloroplast rearrangements and their action spectra as measured by absorption spectrophotometry

Summary A new combination technique of using both dual-wavelength and opalglas methods for scanning translucent biological samples was applied to leaves of terrestrial plants in order to observe their absorption changes by irradiation and the action spectra for the absorption changes. The measurements of true absorption, free from various effects of scattering, by this technique showed an increase of absorption by weak blue light and a decrease of absorption by strong blue light for almost all of the leaves of 20 plant species examined. These weak- and strong-light responses in absorption were reversible. The fractional increase and decrease of absorbance at 678 nm by weak and strong light were highest, +20% and -31%, for leaves of Begonia semperflorens Link et Otto, and +12% and -13% for leaves of foxtail, Setaria viridis (L.) Beauv., the species examined in further experiments. The response to strong light proceeded to completion earlier than did that to weak light. The strong-light response could be observed separately from the weak-light response by using a leaf pre-irradiated with weak blue light. The responses were measured as a function of light intensity by scanning a single leaf irradiated locally at different intensities, and the action spectra for these responses were measured by scanning a leaf irradiated locally at different wavelengths but at identical intensities. The action spectra for these opposite responses were similar, and showed a band at 450 nm with shoulders but no band in the red region. Microscopic observations of chloroplasts in leaves during irradiation indicated that these changes in absorption are mostly due to rearrangements of chloroplasts in cells caused by irradiation.     

Selective Formation of Photosystem 1 under Illumination at Low Intensity

Analyses of chlorophylls a and b and P700 in the wheat leaves grown for 8 days under illumination with white light at different intensities suggested selective formation of photosystem 1 of the photosynthesis at low light intensities. This was confirmed for the two types of chloroplasts isolated from leaves grown at light intensities of 1.1 and 240 μ W/cm², respectively, by measuring their pigment compositions, activities of photosystems 1 and 2, and absorption and fluorescence spectra. The chloroplasts developed at the low intensity showed properties only of photosystem 1 while those developed at the high intensity showed properties of both photosystems 1 and 2. Only photosystem 1 particles were obtained by fractionation of low intensity chloroplasts by treatment with digitonin followed by centrifugation, while high intensity chloroplasts could be fractionated into photosystem-1 and photosystem-2 particles. When the leaves grown at low light intensity were illuminated with strong light, photosystem 2 was developed. The fluorescence emission spectrum of low intensity chloroplasts at 77°K showed two peaks at 685 and 734 nm, and the spectrum of high intensity chloroplasts showed three peaks at 685, 697 and 740 nm.     

Chloroplasts can move in any direction to avoid strong light

Chloroplasts migrate in response to different light intensities. Under weak light, chloroplasts gather at an illuminated area to maximize light absorption and photosynthesis rates (the accumulation response). In contrast, chloroplasts escape from strong light to avoid photodamage (the avoidance response). Photoreceptors involved in these phenomena have been identified in Arabidopsis thaliana and Adiantum capillus-veneris. Chloroplast behavior has been studied in detail during the accumulation response, but not for the avoidance response. Hence, we analyzed the chloroplast avoidance response in detail using dark-adapted Adiantum capillus-veneris gametophyte cells and partial cell irradiation with a microbeam of blue light. Chloroplasts escaped from an irradiated spot. Both duration of this response and the distance of the migrated chloroplasts were proportional to the total fluence irradiated. The speed of movement during the avoidance response was dependent on the fluence rate, but the speed of the accumulation response towards the microbeam from cell periphery was constant irrespective of fluence rate. When a chloroplast was only partially irradiated with a strong microbeam, it moved away towards the non-irradiated region within a few minutes. During this avoidance response two additional microbeam irradiations were applied to different locus of the same chloroplast. Under these conditions the chloroplast changed the moving direction after a lag time of a few minutes without rolling. Taken together, these findings indicate that chloroplasts can move in any direction and never have an intrinsic polarity. Similar phenomenon was observed in chloroplasts of Arabidopsis thaliana palisade cells.     

Light absorption by isolated chloroplasts and leaves: effects of scattering and ‘packing’

Light absorption was quantified in the following systems: isolated chloroplasts and leaves of spinach (Spinacea oleracea L.), a mutant of geranium (Pelargonium zonale L.) widely differing in pigment content, and coleus (Coleus blumei Benth.) at different stages of leaf ontogenesis. For these species and pea (Pisum sativum L.), scattering-compensated absorption spectra of chloroplast suspensions are presented. Comparison of leaf and chloroplast spectra showed considerable changes in the extent of the ‘package’ effect and the lengthening of the effective optical path in a leaf. The difference between leaf and isolated chloroplast absorption could be quantitatively described by adapting Duysens’s treatment of flattening. It was found that the accumulation of chlorophyll in leaves is accompanied by a monotonous enhancement of the package effect. The results are discussed with special reference to the role of light scattering in leaf optics, light utilization in photosynthesis and wavelength-dependent light gradients in a leaf.     

Cyclic Changes in Chloroplast Structure in Synchronized Euglena gracilis*

SYNOPSIS. In populations of Euglena gracilis strain Z synchronized by cultivation on a repetitive light-dark cycle, chloroplasts undergo cyclic changes in structure. During most of the light period chloroplasts are relatively compact with closely appressed lamellae; during the dark (division) period the chloroplasts become quite distended. This change persists for at least one cycle even when the cells are left in continuous light, suggesting that the periodicity may be related more to the age of the cell than to a direct effect of light. In addition, the pyrenoid in synchronized cells has a transient existence, being present only in the first half of the light period.     

Anthocyanin accumulation in the illuminated surface of maize leaves enhances protection from photo-inhibitory risks at low temperature, without further limitation to photosynthesis

At suboptimal temperatures, anthocyanins accumulate in the illuminated leaf surface of some maize genotypes and, if the anthocyanins shade chloroplasts, they can effectively reduce the risk of photo‐inhibition but also photo‐synthesis. To investigate this phenomenon, gas exchange, fluorescence, superoxide dismutase activity and xantho‐phyll composition of anthocyanin‐containing HOPI and anthocyanin‐deficient W22 maize genotypes were measured in either white or red light, where the latter is not absorbed by anthocyanins. Despite differences in light absorption in chloroplasts, photosynthesis did not differ between HOPI and W22 under either light source, suggesting that neither CO₂ supply nor photochemistry were more limiting in red leaves than in green leaves. In fact, no major differences in transpiration were detected. The ΔF/F_m (photosystem II quantum yield) of HOPI in white light was higher than in red light and higher than ΔF/F_m of W22 with either light source. This probably compensated for the lower white light absorption of HOPI chloroplasts compared with W22 because of the presence of anthocyanins and led to similar rates of calculated electron transport for both genotypes. After exposure to high white light at 5 °C, xanthophyll de‐epoxidation and superoxide dismutase activity were lower in HOPI than in W22. Further, HOPI could be exposed to a much higher irradiance than W22 before F_v/F_m was reduced to that of W22.     

Temperature stress and survival ability of Mediterranean sclerophyllous plants

ABSTRACT

The Mediterranean climate with hot and dry summer periods, and low winter temperatures and episodic frosts in northern, altitudinal and continental districts, demands from evergreen broadleaved woody plants an adequate and flexible acclimation to the climatic constraints.

In this brief survey on some responses of Mediterranean sclerophylls to temperature stress, the following is presented and discussed: criteria for cold and heat limits of photosynthetic function; winter depression and summer photoinactivation of photosynthesis; peculiar patterns of tissue freezing of scleromorphous leaves and limits of frost resistance of various plant parts and ontogenetic stages; heat impairment of chloroplasts and thermotolerance of sclerophyllous species; survival capacity and recovery after damage. Risks of damage to plants in relation to stressful temperatures in Mediterranean regions are estimated. Cold stress and drought stress indices, according to Mitrakos (1980), have been applied to characterise different localities in Italy. Additionally, a heat stress index for the Mediterranean region is proposed. Future research topics are suggested.     

Are Sclerophylls and Malacophylls Hydraulically Different?

This work tests the hypothesis that sclerophylls (i.e. hard-leaved species) would be less efficient than malacophylls (i.e. soft-leaved species) in terms of water transport through the stem as well as within the leaf blade. Mean leaf surface area (A_L), leaf specific mass (LSM) as well as shoot (K_WL), stem (K_SL) and leaf (K_LL) hydraulic conductances were measured in eight Mediterranean evergreen sclerophylls and eight temperate deciduous malacophylls. No difference was observed between the two groups in terms of K_LL and of the contribution of leaves to the overall shoot hydraulic resistance. Leaves represented in all cases 48 to 90 % of the shoot hydraulic resistance, suggesting that the sclerophyllous habitus does not per se lead to low efficiency in water transport within the leaf blade. A weak negative relationship (r² = 0.252) appeared to exist between K_SL and LSM. This might provide an explanation for the lower growth rates of sclerophylls with respect to malacophylls.     

Expression of the CMV‐CP Gene in Synechocystis 6803 Affects Cyanobacterial Photosynthesis

Previous work has shown that the presence of excess coat protein (CP) of cucumber mosaic virus (CMV) in the chloroplasts was related with mosaic symptoms. However, whether these mosaic symptoms are directly induced by the interaction between CP and chloroplasts is unknown. To directly demonstrate the interaction between CP and the chloroplast, Synechocystis sp. PCC 6803 was used as the chloroplast model. The cDNA encoding the CMV‐CP was cloned in a cyanobacterial shuttle vector (pKT‐CP) and transferred to Synechocystis sp. PCC 6803. The CP was expressed in the cyanobacterium with the psbA promoter. The expression of CMV‐CP hindered the growth of transgenic cyanobacterium cells and decreased its photosynthetic rate and the PS II activity. The transgenic cells showed increased fluorescence (F) from the phycobilisome terminal emitters and increased fluorescence (F) from PS II. The absorption spectra at room temperature showed the Chl and the phycocyanin absorption peak of the mutant strain significantly decreased. These results showed that CP may directly affect the cyanobacterium cells and decreased its photosynthesis, especially the PS II activity. These data might provide new evidence for mosaic symptoms being directly induced by the interaction between CP and chloroplasts.     

Trapping at low temperature of oriented chloroplasts: Application to the study of antenna pigments and of the trap of photosystem-1

A technique is described for the preparation of oriented samples from spinach chloroplasts whose linear dichroism is then studied by (flash) absorption spectroscopy. The chloroplasts are suspended in a glycerol-containing medium, oriented in a magnetic field, and slowly cooled in the magnet until the medium is rigid enough to avoid disorientation effects. The absorption spectra in polarized light have been measured at ?50° and ?170°C. They allow the orientation of chlorophyll b to be resolved, and the red transition moment is found to be tilted out of the membrane plane. A study of the flash-induced absorption changes linked to Photosystem-1 activity reveals a progressive evolution of the difference spectra and of the linear dichroism with decreasing temperatures. At ?170°C, the difference spectrum of P700 in the red is well resolved. All transition moments are found to be largely parallel to the membrane plane. The potential use of the technique for other experiments by differential absorption spectroscopy and by EPR techniques is discussed.     

Slow photosynthetic induction and low photosynthesis in Paphiopedilum armeniacum are related to its lack of guard cell chloroplast and peculiar stomatal anatomy

Paphiopedilum and Cypripedium are close relatives in the subfamily Cypripedioideae. Cypripedium leaves contain guard cell chloroplasts, whereas Paphiopedilum do not. It is unclear whether the lack of guard cell chloroplasts affects photosynthetic induction, which is important for understory plants to utilize sunflecks. To understand the role of guard cell chloroplasts in photosynthetic induction of Paphiopedilum and Cypripedium, the stomatal anatomy and photosynthetic induction of Paphiopedilum armeniacum and Cypripedium flavum were investigated at different ratios of red to blue light. The highest stomatal opening and photosynthesis of intact leaves in P. armeniacum were induced by irradiance enriched with blue light. Its stomatal opening could be induced by red light 250 µmol m^?2 s^?1, but the magnitude of stomatal opening was lower than those at the other light qualities. However, the stomatal opening and photosynthesis of C. flavum were highly induced by mixed blue and red light rather than pure blue or red light. The two orchid species did not differ in stomatal density, but P. armeniacum had smaller stomatal size than C. flavum. The stomata of P. armeniacum were slightly sunken into the leaf epidermis, while C. flavum protruded above the leaf surface. The slower photosynthetic induction and lower photosynthetic rate of P. armeniacum than C. flavum were linked to the lack of guard cell chloroplasts and specific stomatal structure, which reflected an adaptation of Paphiopedilum to periodic water deficiency in limestone habitats. These results provide evidence for the morphological and physiological evolution of stomata relation for water conservation under natural selection.     

Studies on Halimeda IV. An endogenous rhythm of chloroplast migration in the siphonous green alga,H. distorta 1

Abstract

Halimeda has been found particularly suitable for studies of long‐distance chloroplast migration by virtue of its coenocytic structure and calcium carbonate skeleton. A circadian rhythm of chloroplast migration in Halimeda distorta was monitored by videography of segment surface pigmentation. In normal 12 h light/12 h dark treatments synchronised with dawn and dusk, the segments were green all day, began to become pale immediately the light was turned off, and then remained almost white for most of the night until beginning to re‐green a few hours before dawn. As a result of that, they were already quite green by the time the light was turned on. In continuous darkness a similar cycle, albeit with reducing amplitude and a period of about 23 hours, was maintained for at least 7 days. However, this cycle differed significantly from the normal one in that the segments did not remain green after the light was not switched on at dawn, but rather began to pale immediately thereafter. Conversely, in continuous light the segments did not become pale at any time. Thus, the rhythmical re‐emergence of the chloroplasts before dawn and their subsequent withdrawal appears to be controlled by an endogenous rhythm which is independent of light. However, light does completely, but reversibly, inhibit the chloroplast withdrawal component of the cycle. This behaviour of the chloroplasts in Halimeda is very similar to that in the related alga, Caulerpa, but it is quite different from that in another extensively Studied but unrelated siphonous green alga, Acetabularia, in which the circadian rhythm of chloroplast migration is maintained in continuous light.     

SEAWEED ANATOMY AND PHOTOSYNTHETIC PERFORMANCE: THE ECOLOGICAL SIGNIFICANCE OF LIGHT GUIGES,HETEROGENEOUS ABSORPTION AND MULTIPLE SCATTER12

Light absorption by two green seaweeds with similar photophysiology but different anatomies are compared: i) Ulva lactuca var. rigida (C. Ag.) Le Jolis, an optically translucent species of two cell layers both bearing chloroplasts; and, ii) Codium fragile subsp. tomentosoides (van Goor) Silva, an optically opaque species with a colorlelss medulla surrounded by a cortex of choloroplast-bearing utriclels. Thallus absorptance (fraction of incident light absorbed) was measured for various pigment contents. Absorptance by U. lactuca was dependent on pigment concentration in an exponential manner and never exceeded 0.6, whereas absorptance by C. fragile was independent of pigment concentration and always approached a balue of 1.0. Water in the medullary tissue of C. fragile is often of the utricles. The utricles appear to be “integrating spheres” enhancing the capture of incident light, aided by the wave-guide function of the thin peripheral layer of cytoplasm and a reflector function at their base. Photosynthitic performance for U. lactuca saturates at high light intensities and attenuates rapidly with decreasing intensities. In contrast, photosynthetic performance for C. fragile saturates at low light intensities and attenuates slowly with diminishing radiation. Extrapolated diel variation in photosynthesis shows that U. lactuca's anatomy is adaptive for high light intensity environments, whereas C. fragile's anatomy is adaptive for low light intensity environments. Both seaweeds fit into the ecological category of “fugitive” species, and compete in the Long Island Sound (Atlantic Ocean) rocky intertidal for free-space. Predictions are presented for relative species abundances along a monotonic gradient of light intensity.     

Light,genotype, and abscisic acid affect chloroplast positioning in guard cells of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> leaves in distinct ways

The goal of this study was to investigate the effects of light intensity, genotype, and various chemical treatments on chloroplast movement in guard cells of Arabidopsis thaliana leaves. After treatment at various light intensities (dark, low, and high light), leaf discs were fixed with glutaraldehyde, and imaged using confocal laser microscopy. Each chloroplast was assigned a horizontal (close to pore, center, or epidermal side) and vertical (outer, middle, inner) position. White light had a distinct effect on chloroplast positioning, most notably under high light (HL) when chloroplasts on the upper leaf surface of wild-type (WT) moved from epidermal and center positions toward the pore. This was not the case for phot1-5/phot2-1 or phot2-1 plants, thus phototropins are essential for chloroplast positioning in guard cells. In npq1-2 mutants, fewer chloroplasts moved to the pore position under HL than in WT plants, indicating that white light can affect chloroplast positioning also in a zeaxanthin-dependent way. Cytochalasin B inhibited the movement of chloroplasts to the pore under HL, while oryzalin did not, supporting the idea that actin plays a role in the movement. The movement along actin cables is dependent on CHUP1 since chloroplast positioning in chup1 was significantly altered. Abscisic acid (ABA) caused most chloroplasts in WT and phot1-5/phot2-1 to be localized in the center, middle part of the guard cells irrespective of light treatment. This indicates that not only light but also water stress influences chloroplast positioning.     

Moderate heat stress of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> leaves causes chloroplast swelling and plastoglobule formation

Photosynthesis is inhibited by heat stress. This inhibition is rapidly reversible when heat stress is moderate but irreversible at higher temperature. Absorbance changes can be used to detect a variety of biophysical parameters in intact leaves. We found that moderate heat stress caused a large reduction of the apparent absorbance of green light in light-adapted, intact Arabidopsis thaliana leaves. Three mechanisms that can affect green light absorbance of leaves, namely, zeaxanthin accumulation (absorbance peak at 505 nm), the electrochromic shift (ECS) of carotenoid absorption spectra (peak at 518 nm), and light scattering (peak at 535 nm) were investigated. The change of green light absorbance caused by heat treatment was not caused by changes of zeaxanthin content nor by the ECS. The formation of non-photochemical quenching (NPQ), chloroplast movements, and chloroplast swelling and shrinkage can all affect light scattering inside leaves. The formation of NPQ under high temperature was not well correlated with the heat-induced absorbance change, and light microscopy revealed no appreciable changes of chloroplast location because of heat treatment. Transmission electron microscopy results showed swollen chloroplasts and increased number of plastoglobules in heat-treated leaves, indicating that the structural changes of chloroplasts and thylakoids are significant results of moderate heat stress and may explain the reduced apparent absorbance of green light under moderately high temperature.     

Comparison of chlorophyll a spectra in wild-type and mutant barley chloroplasts grown under day or intermittent light

The absorption (640–710 nm) and fluorescence emission (670–710 nm) spectra (77 K) of wild-type and Chl b-less, mutant, barley chloroplasts grown under either day or intermittent light were analysed by a RESOL curve-fitting program. The usual four major forms of Chl a at 662, 670, 678 and 684 nm were evident in all of the absorption spectra and three major components at 686, 693 and 704 nm in the emission spectra. A broad Chl a component band at 651 nm most likely exists in all chlorophyll spectra in vivo. The results show that the mutant lacks not only Chl b, but also the Chl a molecules which are bound to the light-harvesting, Chl a/b, protein complex of normal plants. It also appears that the absorption spectrum of this antenna complex is not modified appreciably by its isolation from thylakoid membranes.Abbreviations Chl chlorophyll - DL daylight - ImL intermittent light - WT wildtype - LHC light-harvesting Chl a/b protein complex - S.E. standard error of the mean DBP-CIW No. 763.     

The kinetics of P515 in relation to the lipid composition of the thylakoid membrane

Flash-induced P515 absorbance changes have been studied in dark-adapted chloroplasts isolated from spinach plants grown under two different light intensities. The slow component (reaction 2), normally present in the P515 response of chloroplasts isolated from plants grown at an intensity of 60 W · m^–2, was largely reduced in chloroplasts isolated from plants grown at an intensity of 6 W · m^–2. This reduction of the slow component in the P515 response appeared to be coincident with an alteration in the lipid composition of the thylakoid membrane. Mainly the ratio monogalactosyldiacylglycerol to digalactosyldiacylglycerol appeared to be altered. In thylakoids from plants grown at 6 W · m^–2, the ratio was approximately 35% lower than that of plants grown at 60 W · m^–2. The amount of both cytochromeb ₅₆₃ and cytochromef was largely reduced in chloroplasts isolated from plants grown at low light intensity. These results may indicate a possible correlation between structural organization of the thylakoid membrane and the kinetics of the flash-induced P515 response.     

Action Spectra of Photosynthetic Activities of Chlorella ellipsoidea

• 1) Suspensions of Chlorella show an even stronger light scattering than suspensions of chloroplasts of spinach. The bands of absorption are thus broadened and, at higher concentrations, moved to lower wave-lengths. The intensity of the photosynthesis closely follows the curves of light scattering, a fact partly explaining the high efficiency of green light. Calculated per unit thermoelectrically measured incident energy the action spectrum shows bands at 660–670 nm and c. 500 nm and a comparatively high level of the whole region 500–560 nm.
• 2) Flash experiments show the existence of a steady state carotene/xanthophyll that is moved to reduction (c/x > 1) in blue and green light and to oxidation (c/x < 1) in red light. All experiments point to the existence of two light reactions, the first one involving excitation of carotenoids, with ferredoxin-TPN as acceptor, the second one involving excitation of chlorophyll, with the cytochrome system of the chloroplasts acting as donors of electrons and thus completing an energy converting circulation between pigments and enzyme systems.
• 3) The operation of combined light reactions appears also from experiments with simultaneous or succedaneous illumination with monochromatic light of different wave-lengths. Some effects may be explained from separate excitations of carotenoids and chlorophylls, others may depend on still unknown photic reactions.
• 4) The action spectrum in ultrared shows a positive band at c. 900 nm but no or only very small effects in the region 950–1400 nm. Ultrared radiation has on the other hand an enhancing effect on the light excitation in the visible spectrum. A combination of infrared and visible radiation shows a roughly linear relation between incident energy and photosynthetic effect.
• 5) All experiments were performed in the region of linear relation between intensity of incident light and O₂-production. Induced effects of combined monochromatic regions show a very rapid initial change in the steady states that in one or two minutes simmers down to a balanced state of continued photosynthesis. No change was observed in the total quantity of the pigments.

     

The response of ultrastructure and function of chloroplasts from cycads to doubled CO2 concentration

This study examines the effects of doubled CO₂ concentration on the ultrastructure and function of chloroplasts from cycads and, for control from two other herbaceous angiosperms. Under a doubled CO₂ concentration condition, the chloroplast ultrastructure of the two cycads (Cycas multipinnata with a shade-type chloroplast andC. panzhihuaensis with a sun-type chloroplast) changed little: The conformation of the thylakoid membrane system kept well, and almost no starch grains accumulated. In contrast, under the same conditions the chloroplast ultrastructure of soybean and foxtail millet changed considerably, with starch grains accumulating in their chloroplasts and some of thylakoids (especially stroma thylakoid) membranes being destroyed to some degree by the more numerous and larger starch grains that accumulated in the chloroplasts. Interestingly, the changes in the ultrastructure of the chloroplasts from the two cycads was correlated with the 77K fluorescence emission spectra of their chlorophyll; i.e., the F685/F734 (PS II / PS I) ratio within the chloroplasts, which were minimal. The absorption spectrum showed decreases in the red and blue peaks. These changes in the absorption spectrum may be related to changes in the structural arrangement of the thylakoid membranes. Preliminarily, this experimental result shows that the cycads may adapt themselves to environmental changes under doubled CO₂ concentration in the coming centuries. However, more studies on this aspect are necessary.