Search

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.

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 C

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.

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 (F0 and FM, 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 F0, FM 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 (F0, FM, \( F^{\prime}_{0} \) and \( F^{\prime}_{\text{M}} \)) depend solely on the spectral properties of the fluorometer used. Under the present conditions, the PSI contribution to F0 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/F0 − 1/FM, up to 50 % in A. thaliana and up to 400 % in Z. mays.

The chelation of Fe2+ and Mg2+ ions forms protoheme IX and Mg-protoporphyrin IX, respectively, and the latter is an intermediate in chlorophyll synthesis. Active magnesium protoporphyrin IX chelatase (Mg-chelatase) is an enzyme complex consisting of three different subunits. To investigate the function of the CHL I subunit of Mg-chelatase and the effects of modified Mg-chelatase activity on the tetrapyrrole biosynthetic pathway, we characterized N. tabacum transformants carrying gene constructs with the Chl I cDNA sequence in antisense and sense orientation under the control of the CaMV 35S promoter. Both elevated and diminished levels of Chl I mRNA and Chl I protein led to reduced Mg-chelatase activities, reflecting a perturbation of the assembly of the enzyme complex. The transformed plants did not accumulate the substrate of Mg-chelatase, protoporphyrin IX, but the leaves contained less chlorophyll and possessed increased chlorophyll a/b ratios, as well as a deficiency of light-harvesting chlorophyll binding proteins of photosystems I and II. The expression and activity of several tetrapyrrolic enzymes were reduced in parallel to lower the Mg-chelatase activity. Consistent with the lower chlorophyll contents, the rate-limiting synthesis of 5-aminolevulinate was also decreased in the transgenic lines analyzed. The consequence of reduced Mg-chelatase on early and late steps of chlorophyll synthesis, and on the organization of light harvesting complexes is discussed.

Chlorophyll a fluorescence characteristics were investigated in 12 species and 2 hybrids from the genus Flaveria exhibiting C3, C3–C4 intermediate, or C4 photosynthesis, and in the C4 species Zea mays. At room temperature, the variable fluorescence divided by the maximum fluorescence (FV/FM) of dark-adapted leaves decreased from C3 to C4 plants. This trend was qualitatively paralleled by an increase of the 735 nm peak relative to the 685 nm peak (F735/F685) of fluorescence emission spectra measured at low temperature (77 K). The variations were analysed using a quantitative model that takes into account higher PS I fluorescence in C4 plants than in C3 plants. The model predicts a linear correlation between 1/(FV/FM) and F735/F685, and was experimentally confirmed. From linear regression analysis, the FV/FM of PS II was calculated to be 0.88. By comparing the FV/FM of PS II with the FV/FM from leaves, the PS I contribution to total F0 fluorescence at wavelengths greater than 700 nm was determined to be about 30% and 50% in C3 and C4 plants, respectively. The corresponding values for the FM fluorescence were 6% and 12%. It is concluded that the effects of PS I fluorescence are significant and should be taken into account when analysing fluorescence data.

Stable assembly of the major LHC-II proteins (22, 26 kDa) is inhibited in the α-carotenoid-free mutant C-2A′-34 of the green alga Scenedesmus obliquus, whereas other LHC-II proteins (22, 23, 29 kDa) seem to be not affected. We conclude that lutein is essential for assembly of the major LHC-II proteins of S. obliquus, but not for assembly of the minor LHC-II proteins. It is proposed that in the minor LHC-II proteins lutein is replaced by violaxanthin, zeaxanthin and antheraxanthin.

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.

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.

This paper discusses biochemical and regulatory aspects of the violaxanthin cycle as well as its possible role in photoprotection. The violaxanthin cycle responds to environmental conditions in the short-term and long-term by adjusting rates of pigment conversions and pool sizes of cycle pigments, respectively. Experimental evidence indicating a relationship between zeaxanthin formation and non-photochemical energy dissipation is reviewed. Zeaxanthin-associated energy dissipation appears to be dependent on transthylakoid ΔpH. The involvement of light-harvesting complex II in this quenching process is indicated by several studies. The current hypotheses on the underlying mechanism of zeaxanthin-dependent quenching are alterations of membrane properties, including conformational changes of the light-harvesting complex II, and singlet-singlet energy transfer from chlorophyll to zeaxanthin.

In higher plants and in most of the eukaryotic algal classes light-dependent reversible interconversions of 5,6-epoxycarotenoids (xanthophyll cycles) were found (see [1,2,3] for a review). Two types of cycle are known. In higher plants as well as in Chlorophyceae and in Phaeophyceae the violaxanthin cycle is known to be present. It starts with the enzymatic de-epoxidation of violaxanthin (5,6,5′,6′-diepoxyzeaxanthin) via antheraxanthin (5,6-monoepoxyzeaxanthin) to zeaxanthin induced by strong light; the back reaction occurring on a different pathway is light-independent. According to the transmembrane model of the violaxanthin cycle, the de-epoxidase which has its optimum at pH 5 and requires ascorbate for its activity in vitro, is located at the loculus side of the thylakoid membrane, while epoxidation takes place at the stroma side of the membrane (optimum at pH 7.5, cosubstrates: NADPH, O2). The de-epoxidation system is present in both grana and stroma thylakoids [2]. The ability to de-epoxi-date violaxanthin has been established in etiolated leaves [4].

We present a simple approach for the calculation of in vivo fluorescence excitation spectra from measured absorbance spectra of the isolated pigments involved. Taking into account shading of the pigments by each other, energy transfer from carotene to chlorophyll a, and light scattering by the leaf tissue, we arrive at a model function with 6 free parameters. Fitting them to the measured fluorescence excitation spectrum yields good correspondence between theory and experiment, and parameter estimates which agree with independent measurements. The results are discussed with respect to the origin and the interpretation of in vivo excitation spectra in general.

In etiolated leaves the occurrence of the enzymatic violaxanthin de-epoxidation to zeaxanthin is shown. The carotenoid transformation is provoked by the infiltration of whole leaves with ascorbate at pH 5 and is susceptible to DTT. Identification of the de-epoxidase activity is achieved by in vivo spectroscopy and pigment analysis (TLC).