Chlorophyll fluorescence imaging of photosynthetic activity with the flash-lamp fluorescence imaging system

A flash-lamp chlorophyll (Chl) fluorescence imaging system (FL-FIS) is described that allows to screen and image the photosynthetic activity of several thousand leaf points (pixels) of intact leaves in a non-destructive way within a few seconds. This includes also the registration of several thousand leaf point images of the four natural fluorescence bands of plants in the blue (440 nm) and green (520 nm) regions as well as the red (near 690 nm) and far-red (near 740 nm) Chl fluorescence. The latest components of this Karlsruhe FL-FIS are presented as well as its advantage as compared to the classical single leaf point measurements where only the fluorescence information of one leaf point is sensed per each measurement. Moreover, using the conventional He-Ne-laser induced two-wavelengths Chl fluorometer LITWaF, we demonstrated that the photosynthetic activity of leaves can be determined measuring the Chl fluorescence decrease ratio, R_Fd (defined as Chl fluorescence decrease F_d from maximum to steady state fluorescence F_s:F_d/F_s), that is determined by the Chl fluorescence induction kinetics (Kautsky effect). The height of the values of the Chl fluorescence decrease ratio R_Fd is linearly correlated to the net photosynthetic CO₂ fixation rate P _N as is indicated here for sun and shade leaves of various trees that considerably differ in their P _N. Imaging the R_Fd-ratio of intact leaves permitted the detection of considerable gradients in photosynthetic capacity across the leaf area as well as the spatial heterogeneity and patchiness of photosynthetic quantum conversion within the control leaf and the stressed plants. The higher photosynthetic capacity of sun versus shade leaves was screened by Chl fluorescence imaging. Profile analysis of fluoresence signals (along a line across the leaf area) and histograms (the signal frequency distribution of the fluorescence information of all measured leaf pixels) of Chl fluorescence yield and Chl fluorescence ratios allow, with a high statistical significance, the quantification of the differences in photosynthetic activity between various areas of the leaf as well as between control leaves and water stressed leaves. The progressive uptake and transfer of the herbicide diuron via the petiole into the leaf of an intact plant and the concomitant loss of photosynthetic quantum conversion was followed with high precision by imaging the increase of the red Chl fluorescence F₆₉₀. Differences in the availability and absorption of soil nitrogen of crop plants can be documented via this flash-lamp fluorescence imaging technique by imaging the blue/red ratio image F₄₄₀/F₆₉₀, whereas differences in Chl content are detected by collecting images of the fluorescence ratio red/far-red, F₆₉₀/F₇₄₀.     

Chlorophyll fluorescence imaging reveals genetic variation and loci for a photosynthetic trait in diploid potato

Physiology and genetics are tightly interrelated. Understanding the genetic basis of a physiological trait such as the quantum yield of the photosystem II, or photosynthetic responses to environmental changes will benefit the understanding of these processes. By means of chlorophyll fluorescence (CF) imaging, the quantum yield of photosystem II can be determined rapidly, precisely and non‐invasively. In this article, the genetic control and variation in the steady‐state quantum yield of PSII (Φ_PSII) is analyzed for diploid potato plants. Current progress in potato research and breeding is slow due to high levels of heterozygosity and complexity of tetraploid genetics. Diploid potatoes offer the possibility of overcoming this problem and advance research for one of the globally most important staple foods. With the help of a diploid genetic mapping population two genetic loci that were strongly associated with differences in Φ_PSII were identified. This is a proof of principle that genetic analysis for Φ_PSII can be done on potato. The effects of three different stress conditions that are important in potato cultivation were also tested: salt stress, low temperature and deficiency in the macronutrient phosphate. For the last two stresses, significant decreases in photosynthetic activity could be shown, revealing potential for stress detection with CF based tools. In general, our findings show the potential of high‐throughput phenotyping for physiological research and breeding in potato.     

Chlorophyll fluorescence imaging of photosynthetic activity in sun and shade leaves of trees

The differences in pigment levels, photosynthetic activity and the chlorophyll fluorescence decrease ratio R _Fd (as indicator of photosynthetic rates) of green sun and shade leaves of three broadleaf trees (Platanus acerifolia Willd., Populus alba L., Tilia cordata Mill.) were compared. Sun leaves were characterized by higher levels of total chlorophylls a + b and total carotenoids x + c as well as higher values for the weight ratio chlorophyll (Chl) a/b (sun leaves 3.23–3.45; shade leaves: 2.74–2.81), and lower values for the ratio chlorophylls to carotenoids (a + b)/(x + c) (with 4.44–4.70 in sun leaves and 5.04–5.72 in shade leaves). Sun leaves exhibited higher photosynthetic rates P _N on a leaf area basis (mean of 9.1–10.1 μmol CO₂ m⁻² s⁻¹) and Chl basis, which correlated well with the higher values of stomatal conductance G _s (range 105–180 mmol m⁻² s⁻¹), as compared to shade leaves (G _s range 25–77 mmol m⁻² s⁻¹; P _N: 3.2–3.7 μmol CO₂ m⁻² s⁻¹). The higher photosynthetic rates could also be detected via imaging the Chl fluorescence decrease ratio R _Fd, which possessed higher values in sun leaves (2.8–3.0) as compared to shade leaves (1.4–1.8). In addition, via R _Fd images it was shown that the photosynthetic activity of the leaves of all trees exhibits a large heterogeneity across the leaf area, and in general to a higher extent in sun leaves than in shade leaves.     

Ribonucleotide reductase activity in green algae

A ribonucleoside diphosphate reductase is demonstrated in the algae, $\text{Scenedesmus}\text{obliquus}$ and $\text{Chlorella}\text{pyrenoidosa}$. In synchronized cultures an activity maximum at the 12th hour of the cell cycle coincides with maximum DNA production. Induction of reductase activity is prevented by cycloheximide. The enzyme requires dithiols for reduction of CDP $\text{in}\text{vitro}$; it is not significantly stimulated by iron or magnesium ions nor dependent upon deoxyadenosylcobalamin. ATP stimulates the reaction but dATP or dTTP act as inhibitors. The ribonucleotide reductase of green algae differs from the B₁₂-requiring enzyme characterized in $\text{Euglena}\text{gracilis}$.     

Chlorophyll and peptide compositions in the two photosystems of marine green algae

The molar ratios of chlorophyll a to b in the thalli of marine green algae were between 1.5 and 2.2, being appreciably lower than the ratio between 2.8 and 3.4 found for the leaves of higher plants and the cells of fresh-water green algae. The ratio of chlorophylls to P-700 in these marine algae was also lower than that in higher plants. The $\text{a}\text{b}$ ratios in the pigment proteins of Photosystems 1 and 2 separated by polyacrylamide-gel electrophoresis from sodium dodecyl sulfate-solubilized chloroplasts of four species of marine green algae, Bryopsis maxima, Cheatomorpha spiralis, Enteromorpha compress and Ulva conglobata, were approximately 5 and 1, which are considerably smaller than the ratios, 7 and 2, respectively, found for the pigment proteins of the two photosystems of higher plants separated by the same technique. The chloroplasts of Bryopsis maxima and Cheatomorpha spiralis lacked two of the peptides associated with Photosystem II, which are present in the chloroplasts of Spinacia oleracea and Taraxacum officinale.     

Chlorophyll and peptide compositions in the two photosystems of marine green algae.

The molar ratios of chlorophyll a to b in the thalli of marine green algae were between 1.5 and 2.2, being appreciably lower than the ratio between 2.8 and 3.4 found for the leaves of higher plants and the cells of fresh-water green algae. The ratio of chlorophylls to P-700 in these marine algae was also lower than that in higher plants. The a/b ratios in the pigment proteins of Photosystems 1 and 2 separated by polyacrylamide-gel electrophoresis from sodium dodecyl sulfate-solubilized chloroplasts of four species of marine green algae, Bryopsis maxima, Cheatomorpha spiralis, Enteromorpha compressa and Ulva conglobata, were approximately 5 and 1, which are considerably smaller than the ratios, 7 and 2, respectively, found for the pigment proteins of the two photosystems of higher plants separated by the same technique. The chloroplasts of Bryopsis maxima and Cheatomorpha spiralis lacked two of the peptides associated with Photosystem II, which are present in the chloroplasts of Spinacia oleracea and Taraxacum officinale.     

Prompt and delayed fluorescence in pigment-protein complexes of a green photosynthetic bacterium

Excitation spectra were measured at 4 K of bacteriochlorophyll a fluorescence in reaction center containing pigment-protein complexes obtained from the green photosynthetic bacterium Prosthecochloris aestuarii. Excitation spectra for the longest-wave emission (838 nm) showed bands of bacteriochlorophyll a, carotenoid, and of a pigment with absorption bands at 670, 438 and possibly near 420 nm, which is probably identical to an unidentified porphyrin described in the preceding paper (Swarthoff, T., Kramer, H.J.M. and Amesz, J. (1982) Biochim. Biophys. Acta 681, 354–358). At room temperature the longest-wave emission is stimulated by a magnetic field, which indicates that at least part of the emission is delayed fluorescence brought about by a reversal of the primary charge separation. Below about 150 K no stimulation was observed. The excitation spectra for short-wave emission (828 nm) were very similar to the absorption spectrum of the isolated antenna bacteriochlorophyll a-protein complex, and showed bands of bacteriochlorophyll a only. This indicates that two forms of the antenna protein exist that are spectroscopically similar: a soluble form that is released by treatment with guanidine hydrochloride and a bound form that remains attached to the reaction center complex. The bands of the antenna complexes were weak in the excitation spectra of the 838 nm fluorescence, which indicates that the efficiency of energy transfer to the reaction center complex is low.     

Motility and photosynthetic responses of the green microalga Tetraselmis subcordiformis to visible and UV light levels

To test the effects of photosynthetic active radiation (PAR, 400–700 nm) and ultraviolet radiation (UVR, 280–400 nm) on phototaxis and photosynthesis of free swimming microalgae, experiments were performed with Tetraselmis subcordiformis (Wille) Butcher under a solar simulator. In particular, we evaluated the effects of different PAR levels and radiation regimes (i.e., PAR only and PAR+UVR) on those two processes. We found that the cells preferred to move to a particular area (e.g., receiving 100 W m^?2 PAR) with little photochemical suppression or inhibition of carbon fixation. Adding UV-A to high PAR decreased its swimming capacity and photosynthetic capability, and further adding UV-B led to more inhibition. The suppression of the moving capability of T. subcordiformis was reversible but the cells exposed to PAR combined with UVR needed longer time intervals to recover their motility as compared with those irradiated only with PAR. Based on the above results, we postulate that in nature, the motile capability and photosynthesis of free swimming the green microalga might be impaired by enhanced solar UVR. On the other hand, the cells can reduce the damage caused by high irradiances (and even get the optimum light level for photosynthesis) by a behavioral swimming response.     

Iron supply and hydrogenase activity in green algae

Summary An addition of EDTA to the culture medium considerably increases H₂ metabolism of green algae. However, even under these conditions, several hours of anaerobic incubation (adaptation) are necessary for the optimum of hydrogenase activity to be reached. In addition, the stability of H₂ metabolism during longer periods of anaerobiosis is much better.     

Quantitative fluorescence correlation spectroscopy reveals a 1000-fold increase in lifetime of protein functionality

We have investigated dilute protein solutions with fluorescence correlation spectroscopy (FCS) and have observed that a rapid loss of proteins occurs from solution. It is commonly assumed that such a loss is the result of protein adsorption to interfaces. A protocol was developed in which this mode of protein loss can be prevented. However, FCS on fluorescent protein (enhanced green fluorescent protein, mCherry, and mStrawberry) solutions enclosed by adsorption-protected interfaces still reveals a decrease of the fluorescent protein concentration, while the diffusion time is stable over long periods of time. We interpret this decay as a loss of protein functionality, probably caused by denaturation of the fluorescent proteins. We show that the typical lifetime of protein functionality in highly dilute, approximately single molecule per femtoliter solutions can be extended more than 1000-fold (typically from a few hours to >40 days) by adding compounds with surfactant behavior. No direct interactions between the surfactant and the fluorescent proteins were observed from the diffusion time measured by FCS. A critical surfactant concentration of more than 23 μM was required to achieve the desired protein stabilization for Triton X-100. The surfactant does not interfere with DNA-protein binding, because similar observations were made using DNA-cutting restriction enzymes. We associate the occurrence of denaturation of proteins with the activity of water at the water-protein interface, which was recently proposed in terms of the “water attack model”. Our observations suggest that soluble biomolecules can extend an influence over much larger distances than suggested by their actual volume.     

Relationship between action spectra for chlorophyll a fluorescence and photosynthetic O2 evolution in algae

Relative excitation spectra of chlorophyll a fluorescence areshown to be very close to relative action spectra of photosystemII O₂ evolution in seven micro- and macro-algae of five phyla.The conditions under which this correspondence should hold,based on theoretical considerations, and the applications ofthis correspondence to interpretation of fluorescence excitationspectra of chlorophyll a from natural populations are discussed.The spectra presented are interpreted in relation to the light-harvestingpigments and their association with the two photosystems.     

Effect of nitrite and nitrate on chlorophyll fluorescence in green algae

Summary The influence of nitrite and nitrate on chlorophyll fluorescence, a very sensitive indicator for the redox state of the primary acceptor of photosystem II of photosynthesis, was studied in green algae (several species of Chlorella, and Ankistrodesmus braunii). In phosphate solution under an atmosphere of nitrogen (i.e., in the absence of O₂ and CO₂, and without nitrite or nitrate), fluorescence shows a pronounced induction and then rises to a high steady-state level. In the presence of nitrite, however, fluorescence decreases after a rather short induction peak to a much lower steady-state. Nitrate, on the other hand, does not have any influence on either induction or steady-state of fluorescence. These results clearly demonstrate that nitrite reduction in the light is very closely coupled to the photosynthetic electron transport system, whereas nitrate is not reduced photosynthetically in vivo.     

Chlorophyll fluorescence induction and relaxation system for the continuous monitoring of photosynthetic capacity in photobioreactors

The development of high‐performance photobioreactors equipped with automatic systems for non‐invasive real‐time monitoring of cultivation conditions and photosynthetic parameters is a challenge in algae biotechnology. Therefore, we developed a chlorophyll (Chl) fluorescence measuring system for the online recording of the light‐induced fluorescence rise and the dark relaxation of the flash‐induced fluorescence yield (Qa^? ? re‐oxidation kinetics) in photobioreactors. This system provides automatic measurements in a broad range of Chl concentrations at high frequency of gas‐tight sampling, and advanced data analysis. The performance of this new technique was tested on the green microalgae Chlamydomonas reinhardtii subjected to a sulfur deficiency stress and to long‐term dark anaerobic conditions. More than thousand fluorescence kinetic curves were recorded and analyzed during aerobic and anaerobic stages of incubation. Lifetime and amplitude values of kinetic components were determined, and their dynamics plotted on heatmaps. Out of these data, stress‐sensitive kinetic parameters were specified. This implemented apparatus can therefore be useful for the continuous real‐time monitoring of algal photosynthesis in photobioreactors.     

A staining method for the detection of impaired photosynthetic electron transport in green algae strains

The utilization of strain CW 15 ofChlamydomonas reinhardtii enables the biochemical staining test for characterizing a d efective photosynthetic electron-transport chain on colonies grown on agar medium. It may be used in order to screen for mutants specifically blocked in different parts of the transport chain.     

Chlorophyll fluorescence measured using the Fraunhofer line-depth principle and relationship to photosynthetic rate in the field

Abstract. A field study was conducted to determine the relationship of solar-excited chlorophyll a fluorescence to net CO₂ assimilation rate in attached leaves. The Fraunhofer line-depth principle was used to measure fluorescence at 656.3 nm wavelength while leaves remained exposed to full sunlight and normal atmospheric pressures of CO₂ and O₂. Fluorescence induction kinetics were observed when leaves were exposed to sunlight after 10 min in darkness. Subsequently, fluorescence varied inversely with assimilation rate. In the C₄ Zea mays , fluorescence decreased from 2.5 to 0.8 mW m^-2 nm^-1 as CO₂ assimilation rate increased from 1 to 8 μmol m^-2 s^-1 (r²= 0.520). In the C₃ Liquidambar styraciflua and Pinus taeda , fluorescence decreased from 6 to 2 mW m^-2 nm^-1 as assimilation rate increased from 2 to 5 or 0 to 2 μmol m^-2 s^-1 (r²= 0.44 and 0.45. respectively). The Fraunhofer line-depth principle enables the simultaneous measurement of solar-excited fluorescence and CO₂ assimilation rate in individual leaves, but also at larger scales. Thus, it may contribute significantly to field studies of the relationship of fluorescence to photosynthesis.     

The xanthophyll cycle in green algae (chlorophyta): its role in the photosynthetic apparatus

Light-dependent conversion of violaxanthin to zeaxanthin, the so-called xanthophyll cycle, was shown to serve as a major, short-term light acclimation mechanism in higher plants. The role of xanthophylls in thermal dissipation of surplus excitation energy was deduced from the linear relationship between zeaxanthin formation and the magnitude of non-photochemical quenching. Unlike in higher plants, the role of the xanthophyll cycle in green algae (Chlorophyta) is ambiguous, since its contribution to energy dissipation can significantly vary among species. Here, we have studied the role of the xanthophyll cycle in the adaptation of several species of green algae (Chlorella, Scenedesmus, Haematococcus, Chlorococcum, Spongiochloris) to high irradiance. The xanthophyll cycle has been found functional in all tested organisms; however its contribution to non-photochemical quenching is not as significant as in higher plants. This conclusion is supported by three facts: (i) in green algae the content of zeaxanthin normalized per chlorophyll was significantly lower than that reported from higher plants, (ii) antheraxanthin + zeaxanthin content displayed different diel kinetics from NPQ and (iii) in green algae there was no such linear relationship between NPQ and Ax + Zx, as found in higher plants. We assume that microalgae rely on other dissipation mechanism(s), which operate along with xanthophyll cycle-dependent quenching.     

Comparison of the long-wave chlorophyll fluorescence in various green and blue-green algae and diatoms

Two types of long-wave fluorescence bands with similar band shape occur at room temperature in various algae: F_II700 and F_I715. F_II700 occurs in a limited number of algae, follows PS II transients, increases with culture age and is moderately increased by cooling to 83 K. F_I715 occurs in most algae, especially Anabaena, but much less in most diatoms and Tribonema. It does not follow PS II transients, does not increase with culture age and is much increased by cooling to 83 K.An interpretation for the characteristics of F_II700 and F_I715 is given.