Heinz Walz GmbH
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Recent publications
Photosynthetic Control is defined as the control imposed on photosynthetic electron transport by the lumen-pH-sensitive re-oxidation of plastoquinol (PQH2) by cytochrome b6f. Photosynthetic Control leads at higher actinic light intensities to an electron transport chain with a (relatively) reduced photosystem (PS) II and PQ pool and a (relatively) oxidized PS I. Making Light Curves of more than 33 plant species with the recently introduced DUAL-KLAS-NIR (Chl a fluorescence + the redox states of plastocyanin (PC), P700, and ferredoxin (Fd)) the light intensity-dependent induction of Photosynthetic Control was probed and characterized. It was observed that PC became completely oxidized at light intensities ≤ 400 µmol photons m⁻² s⁻¹ (at lower light intensities in shade than in sun leaves). The relationship between qP and P700(red) was used to determine the extent of Photosynthetic Control. Instead of measuring the whole Light Curve, it was shown that a single moderate light intensity can be used to characterize the status of a leaf relative to that of other leaves. It was further found that in some shade-acclimated leaves Fd becomes again more oxidized at high light intensities indicating that electron transfer from the PQ pool to P700 cannot keep up with the outflow of electrons on the acceptor side of PS I. It was observed as well that for NPQ-induction a lower light intensity (less acidified lumen) was needed than for the induction of Photosynthetic Control. The measurements were also used to make a comparison between the parameters qP and qL, a comparison suggesting that qP was the more relevant parameter.
The photosynthetic electron transport chain is mineral rich. Specific mineral deficiencies can modify the electron transport chain specifically. Here, it is shown that on the basis of 2 short Chl fluorescence and P700+ measurements (approx. 1 s each), it is possible to discriminate between 10 out of 12 different mineral deficiencies: B, Ca, Cu, Fe, K, Mg, Mn, Mo, N, P, S, and Zn. B- and Mo-deficient plants require somewhat longer measurements to detect the feedback inhibition they induce. Eight out of twelve deficiencies mainly affect PS I and NIR measurements are, therefore, very important for this analysis. In Cu- and P-deficient plants, electron flow from the plastoquinone pool to PS I, is affected. In the case of Cu-deficiency due to the loss of plastocyanin and in the case of P-deficiency probably due to a fast and strong generation of Photosynthetic Control. For several Ca-, K-, and Zn-deficient plant species, higher levels of reactive oxygen species have been measured in the literature. Here, it is shown that this not only leads to a loss of Pm (maximum P700 redox change) reflecting a lower PS I content, but also to much faster P700+ re-reduction kinetics during the I2-P (~30–200 ms) fluorescence rise phase. The different mineral deficiencies affect the relation between the I2-P and P700+ kinetics in different ways and this is used to discuss the nature of the relationship between these two parameters.
Most non-destructive methods for plant stress detection do not measure the primary stress response but reactions of processes downstream of primary events. For instance, the chlorophyll fluorescence ratio Fv/Fm, which indicates the maximum quantum yield of photosystem II, can be employed to monitor stress originating elsewhere in the plant cell. This article describes the properties of a sensor to quantify herbicide and pathogen stress in agricultural plants for field applications by the Fv/Fm parameter. This dedicated sensor is highly mobile and measures images of pulse amplitude modulated (PAM) chlorophyll fluorescence. Special physical properties of the sensor are reported, and the range of its field applications is defined. In addition, detection of herbicide resistant weeds by employing an Fv/Fm-based classifier is described. The PAM-imaging sensor introduced here can provide in-field estimation of herbicide sensitivity in crops and weeds after herbicide treatment before any damage becomes visible. Limitations of the system and the use of a classifier to differentiate between stressed and non-stressed plants based on sensor data are presented. It is concluded that stress detection by the Fv/Fm parameter is suitable as an expert tool for decision making in crop management.
PAM fluorescence of leaves of cherry laurel (Prunus laurocerasus L.) was measured simultaneously in the spectral range below 700 nm (sw) and above 700 nm (lw). A high-sensitivity photodiode was employed to measure the low intensities of sw fluorescence. Photosystem II (PSII) performance was analyzed by the saturation pulse method during a light response curve with subsequent dark phase. The sw fluorescence was more variable, resulting in higher PSII photochemical yields compared to lw fluorescence. The variations between sw and lw data were explained by different levels of photosystem I (PSI) fluorescence: the contribution of PSI fluorescence to minimum fluorescence (F0) was calculated to be 14% at sw wavelengths and 45% at lw wavelengths. With the results obtained, the validity of an earlier method for the quantification of PSI fluorescence (Genty et al. in Photosynth Res 26:133–139, 1990, https://doi.org/10.1007/BF00047085) was reconsidered. After subtracting PSI fluorescence from all fluorescence levels, the maximum PSII photochemical yield (FV/FM) in the sw range was 0.862 and it was 0.883 in the lw range. The lower FV/FM at sw wavelengths was suggested to arise from inactive PSII reaction centers in the outermost leaf layers. Polyphasic fluorescence transients (OJIP or OI1I2P kinetics) were recorded simultaneously at sw and lw wavelengths: the slowest phase of the kinetics (IP or I2P) corresponded to 11% and 13% of total variable sw and lw fluorescence, respectively. The idea that this difference is due to variable PSI fluorescence is critically discussed. Potential future applications of simultaneously recording fluorescence in two spectral windows include studies of PSI non-photochemical quenching and state I–state II transitions, as well as measuring the fluorescence from pH-sensitive dyes simultaneously with chlorophyll fluorescence.
Robert John Porra (7.8.1931–16.5.2019) is probably best known for his substantial practical contributions to plant physiology and photosynthesis by addressing the problems of both the accurate spectroscopic estimation and the extractability of chlorophylls in many organisms. Physiological data and global productivity estimates, in particular of marine primary productivity, are often quoted on a chlorophyll basis. He also made his impact by work on all stages of tetrapyrrole biosynthesis: he proved the C5 pathway to chlorophylls, detected an alternative route to protoporphyrin in anaerobes and the different origin of the oxygen atoms in anaerobes and aerobes. A brief review of his work is supplemented by personal memories of the authors.
Short-term effects of pCO 2 (700-380 ppm; HC-LC) and nitrate content (50-5 M; HN-LC) on photosynthesis, estimated by different pulse amplitude modulated (PAMs) fluorometers and by oxygen evolution, were investigated in Ulva rigida (Chlorophyta) under solar radiation (ex-situ) and in the laboratory under artificial light (in-situ). After 6-days of incubation at ambient temperature (AT), algae were subjected to a 4 o C-temperature increase (AT+4 o C) for 3 d. Both in-situ and ex-situ, maximal electron transport rate (ETR max) and in situ gross photosynthesis (GP) measured by O 2 evolution presented the highest values under HCHN, and the lowest under HCLN, across all measuring systems. Maximal quantum yield (F v /F m), and ETR max of PSII (ETR(II) max) and of PSI (ETR(I) max), decreased under HCLN under AT+4°C. Ex situ ETR was higher than in situ ETR. At noon, F v /F m decreased (indicating photoinhibition), whereas ETR(II) max and maximal non-photochemical quenching (NPQ max) increased. ETR(II) max decreased under AT+4 o C in contrast to F v /F m , photosynthetic efficiency ( ETR) and saturated irradiance (E K). Thus, U. rigida exhibited a decrease in photosynthetic production under acidification, LN levels and AT+4 o C. These results emphasize the importance of studying the interactive effects between environmental parameters using in-situ vs. ex-situ conditions when aiming to evaluate the impact of global change on marine macroalgae. A c c e p t e d M a n u s c r i p t Abbreviations A Absorptance AT Ambient temperature ETR Electron transport rate Fv/Fm Maximal quantum yield HC High pCO 2 (700 ppm) HN High nitrate levels (50 M) LC Low pCO 2 (380 ppm) LN Low nitrate levels (5 M) NPQ Non photochemical quenching PAM Pulse amplitude modulated PAR Photosynthetic active radiation PSI Photosystem I PSII Photosystem II RLC Rapid light curves Y(II) Effective quantum yield
The saturation pulse method provides a means to distinguish between photochemical and non-photochemical quenching, based on the assumption that the former is suppressed by a saturating pulse of light (SP) and that the latter is not affected by the SP. Various types of non-photochemical quenching have been distinguished by their rates of dark relaxation in the time ranges of seconds, minutes, and hours. Here we report on a special type of non-photochemical quenching, which is rapidly induced by a pulse of high-intensity light, when PS II reaction centers are closed, and rapidly relaxes again after the pulse. This high-intensity quenching, HIQ, can be quantified by pulse-amplitude-modulation (PAM) fluorimetry (MULTI-COLOR-PAM, high sensitivity combined with high time resolution) via the quasi-instantaneous post-pulse fluorescence increase that precedes recovery of photochemical quenching in the 100–400-µs range. The HIQ amplitude increases linearly with the effective rate of quantum absorption by photosystem II, reaching about 8% of maximal fluorescence yield. It is not affected by DCMU, is stimulated by anoxic conditions, and is suppressed by energy-dependent non-photochemical quenching (NPQ). The HIQ amplitude is close to proportional to the square of maximal fluorescence yield, Fm′, induced by an SP and varied by NPQ. These properties are in line with the working hypothesis of HIQ being caused by the annihilation of singlet excited chlorophyll a by triplet excited carotenoid. Significant underestimation of maximal fluorescence yield and photosystem II quantum yield in dark-acclimated samples can be avoided by use of moderate SP intensities. In physiologically healthy illuminated samples, NPQ prevents significant lowering of effective photosystem II quantum yield by HIQ, if excessive SP intensities are avoided.
Low light (LL) and high light (HL)-acclimated plants of A. thaliana were exposed to blue (BB) or red (RR) light or to a mixture of blue and red light (BR) of incrementally increasing intensities. The light response of photosystem II was measured by pulse amplitude-modulated chlorophyll fluorescence and that of photosystem I by near infrared difference spectroscopy. The LL but not HL leaves exhibited blue light-specific responses which were assigned to relocation of chloroplasts from the dark to the light-avoidance arrangement. Blue light (BB and BR) decreased the minimum fluorescence ([Formula: see text]) more than RR light. This extra reduction of the [Formula: see text] was stronger than theoretically predicted for [Formula: see text] quenching by energy dissipation but actual measurement and theory agreed in RR treatments. The extra [Formula: see text] reduction was assigned to decreased light absorption of chloroplasts in the avoidance position. A maximum reduction of 30% was calculated. Increasing intensities of blue light affected the fluorescence parameters NPQ and qP to a lesser degree than red light. After correcting for the optical effects of chloroplast relocation, the NPQ responded similarly to blue and red light. The same correction method diminished the color-specific variations in qP but did not abolish it; thus strongly indicating the presence of another blue light effect which also moderates excitation pressure in PSII but cannot be ascribed to absorption variations. Only after RR exposure, a post-illumination overshoot of [Formula: see text] and fast oxidation of PSI electron acceptors occurred, thus, suggesting an electron flow from stromal reductants to the plastoquinone pool.
Recent advances in the retrieval of Chl fluorescence from space using passive methods (solar-induced Chl fluorescence, SIF) promise improved mapping of plant photosynthesis globally. However, unresolved issues related to the spatial, spectral, and temporal dynamics of vegetation fluorescence complicate our ability to interpret SIF measurements. We developed an instrument to measure leaf-level gas exchange simultaneously with pulse-amplitude modulation (PAM) and spectrally resolved fluorescence over the same field of view – allowing us to investigate the relationships between active and passive fluorescence with photosynthesis. Strongly correlated, slope-dependent relationships were observed between measured spectra across all wavelengths (Fλ, 670–850 nm) and PAM fluorescence parameters under a range of actinic light intensities (steady-state fluorescence yields, Ft) and saturation pulses (maximal fluorescence yields, Fm). Our results suggest that this method can accurately reproduce the full Chl emission spectra – capturing the spectral dynamics associated with changes in the yields of fluorescence, photochemical (ΦPSII), and nonphotochemical quenching (NPQ). We discuss how this method may establish a link between photosynthetic capacity and the mechanistic drivers of wavelength-specific fluorescence emission during changes in environmental conditions (light, temperature, humidity). Our emphasis is on future research directions linking spectral fluorescence to photosynthesis, ΦPSII, and NPQ.
Arundo donax has attracted interest as a potential bioenergy crop due to a high apparent productivity. It uses C3 photosynthesis yet appears competitive with C4 grass biomass feedstock’s and grows in warm conditions where C4 species might be expected to be that productive. Despite this there has been no systematic study of leaf photosynthetic properties. This study determines photosynthetic and photorespiratory parameters for leaves in a natural stand of A. donax growing in southern Portugal. We hypothesise that A. donax has a high photosynthetic potential in high and low light, stomatal limitation to be small and intrinsic water use efficiency unusually low. High photosynthetic rates in A. donax resulted from a high capacity for both maximum Rubisco (Vc,max 117 μmol CO2 m−2 s−1) and ribulose-1:5-bisphosphate limited carboxylation rate (Jmax 213 μmol CO2 m−2 s−1) under light-saturated conditions. Maximum quantum yield for light-limited CO2 assimilation was also high relative to other C3 species. Photorespiratory losses were similar to other C3 species under the conditions of measurement (25%), while stomatal limitation was high (0.25) resulting in a high intrinsic water use efficiency. Overall the photosynthetic capacity of A. donax is high compared to other C3 species, and comparable to C4 bioenergy grasses.
A Monitoring-PAM fluorometer with high temporal resolution (every 5 min) was used to assess the effects on photosynthesis in Ulva rigida (Chlorophyta) during exposure to 2 different CO2 conditions: current (‘LC’, 390 ppm), and the predicted level for the year 2100 (‘HC’, 700 ppm) in a crossed combination with 2 different daily pulsed nitrate concentrations (‘LN’, 5 µM and ‘HN’, 50 µM) and 2 temperature regimes (ambient and ambient +4°C). Effective quantum yield (ΔF/Fm’) in the afternoon was lower under HCLN conditions than under the other treatments. The decrease in ΔF/Fm’ from noon to the afternoon was significantly lower under +4°C compared to ambient temperature. Maximal quantum yield (Fv/Fm) decreased during the night with a transient increase 1 to 3 h after sunset, whereas a transient increase in ΔF/Fm’ was observed after sunrise. These transient increases have been related to activation/deactivation of the electron transport rate and the relaxation of non-photochemical quenching. Relative electron transport rate was higher under the LC and +4°C treatment, but the differences were not significant due to high variability in daily irradiances. Redundancy analysis on the data matrix for the light periods indicates that photosynthetically active radiation through the day is the main variable determining the physiological responses. The effects of nutrient levels (mainly carbon) and experimental increase of temperature were low but significant. During the night, the effect of nutrient availability is of special importance with an opposite effect of nitrogen compared to carbon increase. The application of the Monitoring-PAM to evaluate the effects of environmental conditions by simulating climate change variations under outdoor-controlled, semi-controlled conditions is discussed.
Chlorophyll a fluorescence (ChlF) has been used for decades to study the organization, functioning, and physiology of photosynthesis at the leaf and subcellular levels. ChlF is now measurable from remote sensing platforms. This provides a new optical means to track photosynthesis and gross primary productivity of terrestrial ecosystems. Importantly, the spatiotemporal and methodological context of the new applications is dramatically different compared with most of the available ChlF literature, which raises a number of important considerations. Although we have a good mechanistic understanding of the processes that control the ChlF signal over the short term, the seasonal link between ChlF and photosynthesis remains obscure. Additionally, while the current understanding of in vivo ChlF is based on pulse amplitude-modulated (PAM) measurements, remote sensing applications are based on the measurement of the passive solar-induced chlorophyll fluorescence (SIF), which entails important differences and new challenges that remain to be solved. In this review we introduce and revisit the physical, physiological, and methodological factors that control the leaf-level ChlF signal in the context of the new remote sensing applications. Specifically, we present the basis of photosynthetic acclimation and its optical signals, we introduce the physical and physiological basis of ChlF from the molecular to the leaf level and beyond, and we introduce and compare PAM and SIF methodology. Finally, we evaluate and identify the challenges that still remain to be answered in order to consolidate our mechanistic understanding of the remotely sensed SIF signal.
Technical features and examples of application of a special emitter-detector module for highly sensitive measurements of the electrochromic pigment absorbance shift (ECS) via dual-wavelength (550-520 nm) transmittance changes (P515) are described. This device, which has been introduced as an accessory of the standard, commercially available Dual-PAM-100 measuring system, not only allows steady-state assessment of the proton motive force (pmf) and its partitioning into ΔpH and ΔΨ components, but also continuous recording of the overall charge flux driven by photosynthetic light reactions. The new approach employs a double-modulation technique to derive a continuous signal from the light/dark modulation amplitude of the P515 signal. This new, continuously measured signal primarily reflects the rate of proton efflux via the ATP synthase, which under quasi-stationary conditions corresponds to the overall rate of proton influx driven by coupled electron transport. Simultaneous measurements of charge flux and CO2 uptake as a function of light intensity indicated a close to linear relationship in the light-limited range. A linear relationship between these two signals was also found for different internal CO2 concentrations, except for very low CO2, where the rate of charge flux distinctly exceeded the rate of CO2 uptake. Parallel oscillations in CO2 uptake and charge flux were induced by high CO2 and O2. The new device may contribute to the elucidation of complex regulatory mechanisms in intact leaves.
The effect of stepwise increments of red light intensities on pulse-amplitude modulated (PAM) chlorophyll (Chl) fluorescence from leaves of A. thaliana and Z. mays was investigated. Minimum and maximum fluorescence were measured before illumination (F 0 and F M, respectively) and at the end of each light step (\( F^{\prime}_{0} \) and \( F^{\prime}_{\text{M}} \), respectively). Calculated \( F^{\prime}_{0} \) values derived from F 0, F M and \( F^{\prime}_{\text{M}} \) fluorescence according to Oxborough and Baker (1997) were lower than the corresponding measured \( F^{\prime}_{0} \) values. Based on the concept that calculated \( F^{\prime}_{0} \) values are under-estimated because the underlying theory ignores PSI fluorescence, a method was devised to gain relative PSI fluorescence intensities from differences between calculated and measured \( F^{\prime}_{0} \). This method yields fluorometer-specific PSI data as its input data (F 0, F M, \( F^{\prime}_{0} \) and \( F^{\prime}_{\text{M}} \)) depend solely on the spectral properties of the fluorometer used. Under the present conditions, the PSI contribution to F 0 fluorescence was 0.24 in A. thaliana and it was independent on the light acclimation status; the corresponding value was 0.50 in Z. mays. Correction for PSI fluorescence affected Z. mays most: the linear relationship between PSI and PSII photochemical yields was clearly shifted toward the one-to-one proportionality line and maximum electron transport was increased by 50 %. Further, correction for PSI fluorescence increased the PSII reaction center-specific parameter, 1/F 0 − 1/F M, up to 50 % in A. thaliana and up to 400 % in Z. mays.
The MULTI-COLOR-PAM Multiple Excitation Wavelength Chlorophyll Fluorescence Analyzer is unique in provid-ing 6 different wavelengths of pulse-modulated measuring light, ML (400, 440, 480, 540, 590 and 625 nm), as well as 6 different wavelengths of actinic light, namely 440, 480, 540, 590, 625 and 420-640 nm (white). The variously col-ored actinic light can be used for continuous illumination (AL), maximal intensity single turnover pulses (ST), high intensity multiple turnover pulses (MT) and Saturation Pulses (SP). In addition, far-red light (FR, peaking at 725 nm) is provided for preferential excitation of PS I. This article outlines the properties and applications of the various colors of measuring and actinic light provided by the MULTI-COLOR-PAM. The pulse-modulated ML can be applied with vastly different pulse frequencies ranging between 10 to 200000 Hz, thus enabling continuous monitoring of quasi-dark fluorescence yield (Fo) as well as measurement of fast induction kinetics in the sub-ms time range using the same ML of various colors. Analysis of the O-I 1 fluorescence rise kinetics in saturating light allows determination of the wavelength and sample specific functional absorption cross-section of PS II, Sigma(II) λ , with which the PS II turnover rate at a given incident PAR can be calculated. Vastly different light response curves can be obtained with AL of different colors. Based on Sigma(II) λ the usual PAR, in units of µmol quanta/(m² · s), can be converted into PAR(II), in units of PS II effective quanta/s, in order to compare the responses obtained with differently colored AL. A fluorescence-based electron transport rate ETR(II) = PAR(II) · Y(II)/Y(II) max is defined, which in contrast to the usual relative ETR can describe the rate of electron transport even in dilute suspensions of unicellular algae and cyanobacteria. When chlorophyll content is known, an absolute rate of O 2 evolution can be estimated from ETR(II). The MULTI-COLOR-PAM is also well suited for measurements with leaves, for which a special optical unit with clip-holder is provided. For this application the use of 440 nm ML/AL/ST/MT/SP and detection of F < 710 nm are recommended.
The F(0) and F(M) level fluorescence from a wild-type barley, a Chl b-less mutant barley, and a maize leaf was determined from 430 to 685 nm at 10 nm intervals using pulse amplitude-modulated (PAM) fluorimetry. Variable wavelengths of the pulsed excitation light were achieved by passing the broadband emission of a Xe flash lamp through a birefringent tunable optical filter. For the three leaf types, spectra of F(V)/F(M) (=(F(M) - F(0))/F (M)) have been derived: within each of the three spectra of F(V)/F(M), statistically meaningful variations were detected. Also, at distinct wavelength regions, the (V)/F(M) differed significantly between leaf types. From spectra of F(V)/F (M), excitation spectra of PS I and PS II fluorescence were calculated using a model that considers PS I fluorescence to be constant but variable PS II fluorescence. The photosystem spectra suggest that LHC II absorption results in high values of F(V)/F(M) between 470 and 490 nm in the two wild-type leaves but the absence of LHC II in the Chl b-less mutant barley leaf decreases the F(V)/F(M) at these wavelengths. All three leaves exhibited low values of F(V)/F(M) around 520 nm which was tentatively ascribed to light absorption by PS I-associated carotenoids. In the 550-650 nm region, the F(V)/F(M) in the maize leaf was lower than in the barley wild-type leaf which is explained with higher light absorption by PS I in maize, which is a NADP-ME C(4) species, than in barley, a C(3) species. Finally, low values of F(V)/F(M) at 685 in maize leaf and in the Chl b-less mutant barley leaf are in agreement with preferential PS I absorption at this wavelength. The potential use of spectra of the F(V)/F(M) ratio to derive information on spectral absorption properties of PS I and PS II is discussed.
Greenhouse studies were initiated to determine the duration of time after herbicide treatment required to render johnsongrass physiologically noncompetitive. Nicosulfuron, imazapic, clethodim, and glyphosate were applied to rhizomatous johnsongrass at 35, 70, 140, and 840 g ai ha−1, respectively. Net carbon assimilation, stomatal conductance, chlorophyll meter readings, and maximum (dark adapted) efficiency of photosystem II were measured. Net carbon assimilation (AN) was assumed to be the best indicator of johnsongrass competitiveness. Johnsongrass was considered to be physiologically noncompetitive when AN declined below 50% of that of nontreated check. From these data, it was concluded that glyphosate rendered johnsongrass noncompetitive most readily, 4.3 d after treatment, whereas no differences were detected between nicosulfuron, imazapic, or clethodim throughout the experiment. Stomatal conductance (gs) was highly correlated to AN and was determined to be an adequate substitute for AN when determining johnsongrass competitiveness. It was concluded that chlorophyll meter readings and photosystem II efficiency were poor indicators of johnsongrass competitiveness. Nomenclature: Clethodim; glyphosate; imazapic; nicosulfuron; johnsongrass, Sorghum halepense L., SORHA.
We present and evaluate the performance of a new field monitoring PAM fluorometer (MONI-PAM) which is intended for short- and long-term monitoring of the acclimation of photosystem II (PSII). The instrument measures chlorophyll fluorescence, photosynthetic photon flux density (PPFD), and temperature in the field, and monitors exactly the same leaf area over prolonged periods of time, facilitating the estimation of both rapidly reversible and sustained non-photochemical quenching (NPQ). The MONI-PAM performance is evaluated in the lab and under natural conditions in a Scots pine canopy during spring recovery of photosynthesis. The instrument provides a new tool to study in detail the acclimation of PSII to the environment under natural field conditions.
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