Pulse Amplitude Modulation Fluorescence Research Articles

Circadian and diel regulation of photosynthesis in the bryophyte Marchantia polymorpha.

Circadian rhythms are 24‐h biological cycles that align metabolism, physiology, and development with daily environmental fluctuations. Photosynthetic processes are governed by the circadian clock in both flowering plants and some cyanobacteria, but it is unclear how extensively this is conserved throughout the green lineage. We investigated the contribution of circadian regulation to aspects of photosynthesis in Marchantia polymorpha, a liverwort that diverged from flowering plants early in the evolution of land plants. First, we identified in M. polymorpha the circadian regulation of photosynthetic biochemistry, measured using two approaches (delayed fluorescence, pulse amplitude modulation fluorescence). Second, we identified that light‐dark cycles synchronize the phase of 24 h cycles of photosynthesis in M. polymorpha, whereas the phases of different thalli desynchronize under free‐running conditions. This might also be due to the masking of the underlying circadian rhythms of photosynthesis by light‐dark cycles. Finally, we used a pharmacological approach to identify that chloroplast translation might be necessary for clock control of light‐harvesting in M. polymorpha. We infer that the circadian regulation of photosynthesis is well‐conserved amongst terrestrial plants.

Heatwave breaks down the linearity between sun-induced fluorescence and gross primary production.

Sun-induced fluorescence in the far-red region (SIF) is increasingly used as a remote and proximal-sensing tool capable of tracking vegetation gross primary production (GPP). However, the use of SIF to probe changes in GPP is challenged during extreme climatic events, such as heatwaves. Here, we examined how the 2018 European heatwave (HW) affected the GPP-SIF relationship in evergreen broadleaved trees with a relatively invariant canopy structure. To do so, we combined canopy-scale SIF measurements, GPP estimated from an eddy covariance tower, and active pulse amplitude modulation fluorescence. The HW caused an inversion of the photosynthesis-fluorescence relationship at both the canopy and leaf scales. The highly nonlinear relationship was strongly shaped by nonphotochemical quenching (NPQ), that is, a dissipation mechanism to protect from the adverse effects of high light intensity. During the extreme heat stress, plants experienced a saturation of NPQ, causing a change in the allocation of energy dissipation pathways towards SIF. Our results show the complex modulation of the NPQ-SIF-GPP relationship at an extreme level of heat stress, which is not completely represented in state-of-the-art coupled radiative transfer and photosynthesis models.

Far-Red Chlorophyll Fluorescence Radiance Tracks Photosynthetic Carbon Assimilation Efficiency of Dark Reactions

Solar-induced chlorophyll fluorescence (SIF) observations from space have shown close relationships with terrestrial photosynthesis rates. SIF originates from the light reactions of photosynthesis, whereas carbon fixation takes place during the dark reactions of photosynthesis. Questions remain regarding whether SIF is able to track changes in the efficiency of the dark reactions in photosynthesis. Using concurrent measurements of leaf-scale gas exchange, pulse amplitude-modulated (PAM) fluorescence, and fluorescence spectral radiances, we found that both far-red fluorescence radiances and PAM fluorescence yields responded rapidly to changes in photosynthetic carbon assimilation due to changes in environmental factors or induced stomatal closure under constant light conditions. Uncertainties in outgoing and incoming irradiance mismatch for SIF measurements may very likely obscure the contributions of the dark reactions, thereby causing the inconsistent findings previously reported, which were no change in far-red SIF and PAM fluorescence yields after clear reductions in the photosynthetic carbon assimilation efficiency of dark reactions. Our results confirm that high-quality SIF measurements have the potential to provide insights into the dark reactions of photosynthesis. This study is particularly relevant for better interpreting satellite SIF observations that are obtained under roughly constant overpass times and relatively stable light intensities.

Simultaneously measuring pulse-amplitude-modulated (PAM) chlorophyll fluorescence of leaves at wavelengths shorter and longer than 700nm.

PAM fluorescence of leaves of cherry laurel (Prunus laurocerasus L.) was measured simultaneously in the spectral range below 700nm (sw) and above 700nm (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.

Solar‐Induced Fluorescence Does Not Track Photosynthetic Carbon Assimilation Following Induced Stomatal Closure

AbstractSince 2006, six satellites measuring solar‐induced chlorophyll fluorescence (SIF) have been launched to better constrain terrestrial gross primary productivity (GPP). The promise of the SIF signal as a proxy for photosynthesis with a strong relationship to GPP has been widely cited in carbon cycling studies. However, chlorophyll fluorescence originates from dynamic energy partitioning at the leaf level and does not exhibit a uniformly linear relationship with photosynthesis at finer scales. We induced stomatal closure in deciduous woody tree branches and measured SIF at a proximal scale, alongside leaf‐level gas exchange, pulse amplitude modulated (PAM) fluorescence, and leaf pigment content. We found no change in SIF or steady‐state PAM fluorescence, despite clear reductions in stomatal conductance, carbon assimilation, and light‐use efficiency in treated leaves. These findings suggest that equating SIF and photosynthesis is an oversimplification that may undermine the utility of SIF as a biophysical parameter in GPP models.

Measuring photosynthesis of both oxygenic and anoxygenic photosynthetic organisms using pulse amplitude modulation (PAM) fluorometry in wastewater ponds

Oxygenic photosynthesis can be measured easily using O2 or CO2 gas exchange, oxygen electrodes, Winkler titration, 14CO2-fixation and by PAM (pulse amplitude modulation) fluorometry. PAM estimates the photosynthetic electron transport rate (ETR) by measuring the variable fluorescence of chlorophyll (Chl) a (> 695 nm) induced by absorption of blue or red light. Anoxygenic photosynthetic bacteria (APB) do not use water as an electron source and are typically photoheterotrophic rather than photoautotrophic and so 14CO2 fixation is a misleading estimate of photosynthetic electron transport in APB photosynthesis. In vivo bacteriochlorophyll a (BChl a) absorbs blue light similar to Chl a but its characteristic longer-wavelength absorption is in the infrared and fluorescence is at > 800 nm. Blue light-induced PAM fluorescence can be used to measure the ETR in purple non-sulphur anoxygenic photobacteria and purple sulphur photobacteria because their RC-2 type BChl a complexes fluoresce similarly to PSII but at longer wavelengths than Chl a. Conventional PAM fluorometers using blue light cannot readily distinguish between oxygenic and RC-2 type anoxygenic photosynthesis because they use a simple > 700 nm highpass filter in front of the detector diode. We modified one fluorometer to use a 695–750-nm bandpass filter to measure Chl a fluorescence from PS-II, representing oxygenic photosynthesis. Similarly, we modified another fluorometer to use a highpass filter (> 830 nm) to measure BChl a fluorescence, representing anoxygenic photosynthesis. However, the fluorescence bands of Chl a and BChl a were found to be too wide to unambiguously distinguish between oxygenic and anoxygenic photosynthesis purely by fluorometry. Treatment with the specific PS-II inhibitor DCMU (Diuron) did enable discrimination of the two types of photosynthesis in a mixture of oxygenic and anoxygenic organisms. Ecological niches made up of both oxygenic and anoxygenic organisms such as microbial mats and hypereutrophic environments such as sewage ponds, wastewater ponds and prawn farm ponds are much more common than often realized. Anoxygenic photosynthesis in such systems is significant yet largely unquantified.

Assessing the suitability of Imaging-PAM fluorometry for monitoring growth of benthic diatoms

High-throughput monitoring of growth using Pulse Amplitude Modulated (PAM) chlorophyll fluorescence imaging (Imaging-PAM) is an important tool for experimental ecological and physiological research on benthic diatom cultures, as due to substrate adhesion non-destructive subsampling for biomass determination is difficult and/or not representative. We evaluated the reliability of Imaging-PAM for quantifying benthic diatom biomass by comparing biomass determinations based on PAM fluorescence parameters to those based on cells counts and chlorophyll a measurements for cultures of the diatoms Cylindrotheca closterium and Seminavis robusta in different growth phases (exponential vs stationary) and cell size ranges. We found that PAM fluorescence imaging is particularly suited to monitor growth during the exponential phase. The dark adapted minimal fluorescence (F0) parameter proved to be the most reliable fluorescence-based biomass proxy, especially for establishing complete growth curves, as it was the most robust parameter to growth phase changes. A dark adaptation period prior to the PAM measurements improved the quantification of the stationary but not of the exponential cultures. Under the right conditions, dark adaptation can thus be omitted for exponentially growing cultures to enhance throughput. The use of multiwell plates for growth monitoring with the Imaging-PAM is feasible even though there can be a slight biomass underestimation near of the edges of the plate. Based on these findings, we formulate specific guidelines for using Imaging-PAM fluorometry in experiments with benthic diatom cultures. We especially recommend taking caution when working under conditions which potentially influence the condition of the photosynthetic apparatus of the algae.

Reduced expression of the proteolytically inactive FtsH members has impacts on the Darwinian fitness of Arabidopsis thaliana.

FtsH (filamentation-temperature-sensitive protein H) proteases are a family of membrane-bound enzymes present in eubacteria, animals, and plants. Besides the 12 genes encoding proteolytically active members of the FtsH family in the genome of Arabidopsis, there are five genes coding for members that are assumed to be proteolytically inactive due to mutations in the protease domain; these are termed FtsHi (i for inactive). Despite their lack of proteolytic activity, these FtsHi members seem to be important for chloroplast and plant development as four out of five homozygous knockout-mutants of FtsHis are embryo-lethal. Here, we analysed the Darwinian fitness of weak homozygous (ftshi1,3,4) and heterozygous (ftshi/FTSHi2,4,5) mutants. We compared the growth and development of these mutants to their respective wild-type Arabidopsis plants under controlled laboratory conditions and in the field, and we also evaluated the photosynthetic efficiency by pulse-amplitude modulation fluorescence. Homologous genotypes were subjected to various stress conditions in a greenhouse and gene co-expression as well as phylogenetic analyses were performed. Analysis of the gene-expression network of the five FTSHi genes indicated common clusters with genes encoding FtsH12, OTP51, and methylase. Phylogenetic analyses pointed to a common evolution (and common disappearance in grasses and gymnosperms) of FtsH12 and multiple presumably proteolytically inactive FtsHi enzymes. Our data show that the FtsHi enzymes are highly important during the seedling stage and for Darwinian fitness analyses in semi-natural conditions.

Evaluating sago palm (Metroxylon sagu Rottb.) photosynthetic performance in waterlogged conditions: utilizing pulse-amplitude-modulated (PAM) fluorometry as a waterlogging stress indicator

Abstract Photosynthetic performance of sago palm (Metroxylon sagu Rottb.) was evaluated in normal and waterlogged conditions. Net assimilation rate (AN), light response curve of net assimilation rate (AN/I) and pulse-amplitude modulation fluorescence vs. irradiance (PAM/I), leaf water potential, leaf chlorophyll content and leaflet area were measured. AN was higher in a particular period of waterlogging than in the normal condition. However, prolonged waterlogging (more than two months) negatively affected sago palm photosynthetic capacity. This was indicated by reduction in several photosynthetic parameters such as AN, maximum value of AN obtained at maximum irradiance (AN (Imax)), quantum yield at zero to 200 µmol m−2 s−1 irradiance (ϕ(Io_I200)), asymptotic photosynthetic efficiency (αo), and maximum electron transport rate (ETRmax). The plants experiencing waterlogging produced high qNmax and NPQmax values. Prolonged waterlogging period also caused significant reduction in leaf water potential (ψleaf) and photosynthetic pigments content. Normal soil conditions with a sufficient amount of water are preferable in order for the plant to generate higher photosynthetic capacity and facilitate stable sago production.

Light-Harvesting Strategy during CO2-Dependent Photosynthesis in the Green Alga Chlamydomonas reinhardtii.

To maximize the efficiency of photosynthesis, photosynthetic organisms must properly balance their light-harvesting ability and CO2 utilization. However, the molecular mechanisms of light harvesting under various CO2 conditions remain unclear. To reveal these mechanisms, we performed new analysis on cells of the green alga Chlamydomonas reinhardtii under different CO2 conditions. The analysis combines three kinds of fluorometries: pulse-amplitude modulated fluorescence, steady-state fluorescence with absolute intensity, and time-resolved fluorescence. Under low CO2 conditions, the main regulatory mechanism was migration of a light-harvesting chlorophyll-protein complex (LHC) II from photosystem (PS) II to PSI. However, under CO2-deficient conditions with carbon supplementation, some of the LHCII separated from the PSI and aggregated with quenching. These different light-harvesting abilities of LHCII may play an important role in the regulation of light harvesting in C. reinhardtii under various CO2 conditions.

Dynamic interplay between photodamage and photoprotection in photosystem II.

Photoinhibition is the light-induced reduction in photosynthetic efficiency and is usually associated with damage to the D1 photosystem II (PSII) reaction centre protein. This damage must either be repaired, through the PSII repair cycle, or prevented in the first place by nonphotochemical quenching (NPQ). Both NPQ and D1 repair contribute to light tolerance because they ensure the long-term maintenance of the highest quantum yield of PSII. However, the relative contribution of each of these processes is yet to be elucidated. The application of a pulse amplitude modulation fluorescence methodology, called protective NPQ, enabled us to evaluate of the protective effectiveness of the processes. Within this study, the contribution of NPQ and D1 repair to the photoprotective capacity of Arabidopsis thaliana was elucidated by using inhibitors and mutants known to affect each process. We conclude that NPQ contributes a greater amount to the maintenance of a high PSII yield than D1 repair under short periods of illumination. This research further supports the role of protective components of NPQ during light fluctuations and the value of protective NPQ and qPd as unambiguous fluorescence parameters, as opposed to qI and Fv /Fm , for quantifying photoinactivation of reaction centre II and light tolerance of photosynthetic organisms.

Connecting active to passive fluorescence with photosynthesis: a method for evaluating remote sensing measurements of Chl fluorescence.

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 , 670-850nm) 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.

Investigating the association between photosynthetic efficiency and generation of biophotoelectricity in autotrophic microbial fuel cells.

Microbial fuel cells operating with autotrophic microorganisms are known as biophotovoltaic devices. It represents a great opportunity for environmentally-friendly power generation using the energy of the sunlight. The efficiency of electricity generation in this novel system is however low. This is partially reflected by the poor understanding of the bioelectrochemical mechanisms behind the electron transfer from these microorganisms to the electrode surface. In this work, we propose a combination of electrochemical and fluorescence techniques, giving emphasis to the pulse amplitude modulation fluorescence. The combination of these two techniques allow us to obtain information that can assist in understanding the electrical response obtained from the generation of electricity through the intrinsic properties related to the photosynthetic efficiency that can be obtained from the fluorescence emitted. These were achieved quantitatively by means of observed changes in four photosynthetic parameters with the bioanode generating electricity. These are the maximum quantum yield (Fv/Fm), alpha (α), light saturation coefficient (Ek) and maximum rate of electron transfer (rETRm). The relationship between the increases in the current density collected by the bioanode to the decrease of the rETRm values in the photosynthetic pathway for the two microorganisms was also discussed.

Kleptoplastic sacoglossan species have very different capacities for plastid maintenance despite utilizing the same algal donors

The sea slug Elysia clarki sequesters photosynthetically functional chloroplasts from at least a dozen algal species and maintains these plastids for up to 4 months. Elysia papillosa and Placida kingstoni also feed on some of the same, or congeneric species such as Bryopsis plumosa and Penicillus lamourouxii, but cannot maintain plastids longer than 2 weeks. Photosystem II maximum quantum efficiency (ϕIIe-max) was measured in freshly fed specimens of E. clarki, E. papillosa, and P. kingstoni using pulse amplitude modulated (PAM) fluorescence. The slugs were then immediately starved, and PAM fluorescence measurements continued with starvation until ϕIIe-max reached near 0 or animals expired. Initially, E. clarki fed either P. lamourouxii or B. plumosa had ϕIIe-max values that were statistically equivalent. However, as length of starvation increased, ϕIIe-max decreased differently. After 12 weeks, E. clarki fed B. plumosa had no ϕIIe-max activity, while those fed P. lamourouxii still had low ϕIIe-max levels. Freshly fed E. papillosa had ϕIIe-max > 0.5 initially, which declined over the next 12 days of starvation. Freshly fed P. kingstoni specimens also had ϕIIe-max > 0.5 immediately, but it rapidly dropped to near 0 over the next 4 h of starvation. Thus, the algal source of sequestered chloroplasts in E. clarki has a minor effect on chloroplast longevity and ϕIIe-max values during prolonged starvation. However, among slug species, specific adaptations account for the tremendous variation in the length and functionality of these kleptoplastic associations.

Decreased capacity for sodium export out of Arabidopsis chloroplasts impairs salt tolerance, photosynthesis and plant performance

Salt stress is a widespread phenomenon, limiting plant performance in large areas around the world. Although various types of plant sodium/proton antiporters have been characterized, the physiological function of NHD1 from Arabidopsis thaliana has not been elucidated in detail so far. Here we report that the NHD1-GFP fusion protein localizes to the chloroplast envelope. Heterologous expression of AtNHD1 was sufficient to complement a salt-sensitive Escherichia coli mutant lacking its endogenous sodium/proton exchangers. Transport competence of NHD1 was confirmed using recombinant, highly purified carrier protein reconstituted into proteoliposomes, proving Na(+) /H(+) antiport. In planta NHD1 expression was found to be highest in mature and senescent leaves but was not induced by sodium chloride application. When compared to wild-type controls, nhd1 T-DNA insertion mutants showed decreased biomasses and lower chlorophyll levels after sodium feeding. Interestingly, if grown on sand and supplemented with high sodium chloride, nhd1 mutants exhibited leaf tissue Na(+) levels similar to those of wild-type plants, but the Na(+) content of chloroplasts increased significantly. These high sodium levels in mutant chloroplasts resulted in markedly impaired photosynthetic performance as revealed by a lower quantum yield of photosystem II and increased non-photochemical quenching. Moreover, high Na(+) levels might hamper activity of the plastidic bile acid/sodium symporter family protein 2 (BASS2). The resulting pyruvate deficiency might cause the observed decreased phenylalanine levels in the nhd1 mutants due to lack of precursors.

Nitrogen fixation and respiratory electron transport in the cyanobacteriumCyanotheceunder different light/dark cycles

Incompatibility of nitrogen fixation and oxygen production compels unicellular diazotrophic cyanobacteria to perform photosynthesis during daytime and restrict nitrogen fixation to nighttime. The marine diazotroph Cyanothece BG 043511 was grown in continuous culture under three light/dark regimes (16L:8D, 12L:12D, and 8L:16Dh); we monitored nitrogen fixation and potential photosynthetic efficiency simultaneously online to reveal how their temporal separation is affected by different LD regimes. An increase in nitrogen fixation rate at night coincided with a rise in pulse-amplitude modulated fluorescence, indicating that the enhanced respiratory electron transport to fuel diazotrophy affects the oxidation state of the plastoquinone pool. This may offer an alternative approach to assess instantaneous nitrogen fixation activity. Regardless of photoperiod, the maximum rate of nitrogen fixation was conserved at about 20h after the onset of the light. Consequently, nitrogen fixation rates peaked at different moments in the dark: relatively early in the 16L:8D cycle, at midnight in 12L:12D, and relatively late in 8L:16D. Under 16L:8D, nitrogen fixation extended into the light, demonstrating the functional plasticity of nitrogen fixation in Cyanothece. Highest daily amounts of nitrogen fixed were obtained in 12L:12D, which is consistent with the natural LD cycle of subtropical latitudes in which Cyanothece thrives.

Monitoring of Pulsed Electric Field-Induced Abiotic Stress on Microalgae by Chlorophyll Fluorescence Diagnostic

Application of a pulsed electric field (PEF) on biological cells, in general, results in stress reactions of the affected organisms. Depending on pulse parameters, reactions such as growth stimulation or apoptosis can be observed for short pulses on the nanosecond (ns) time scale. Cell inactivation usually occurs at longer pulse duration and appropriate high treatment energy values. In this paper, the impact of short PEFs on chloroplasts of green microalgae Auxenochlorella protothecoides was investigated, with closer inspection of the photosystem II (PS II), located in the thylakoid membrane. For this purpose, a pulse amplitude modulated (PAM) fluorescence diagnostic was employed, which is a common method for monitoring changes in the photosynthesis apparatus. In particular, alterations of the PS II can be identified by fluorescence quenching analysis, sensitively. For PEF treatment of microalgae suspensions, the high-voltage pulse duration was adjusted to 100 and 1000 ns. The treatment energy was varied between 2 and 78 kJ/kg. The electric field amplitude was constant throughout the experiments (ECuv=40 kV/cm). After PEF treatment, the samples were periodically analyzed by chlorophyll fluorescence analysis for 1 h, using the saturation pulse method. For the evaluation of the physiological status of the microalgae, the maximum photochemical quantum yield of PS II, Fv/Fm, was chosen. The obtained results showed that the influence of PEFs on PS II is significant. Contrary to commonly accepted explanations that intracellular organelles are predominantly affected by short ns-pulses, a large influence of PEF exposure on chloroplasts, particularly on PS II, could be identified for longer pulses. In this paper, the diagnostic method, applied pulse protocols, and the results of the PAM fluorescence measurements will be discussed.

Laminaria hyperborea photosynthesis-irradiance relationship measured by oxygen production and pulse-amplitude-modulated chlorophyll fluorometry

Information about photosynthesis−irradiance (P−I) relationships is crucial in many primary production studies. This is frequently obtained using incubation experiments, which may not simulate in situ conditions where algae are adjusting their photosynthetic systems to perma- nent light variations. There has been increasing interest in pulse-amplitude-modulated (PAM) chlorophyll fluorometry—which measures instantaneous photosynthetic response—particularly in the use of rapid light curves (RLCs) to minimize the confounding effects of light acclimation encountered with traditional 'steady-state' light curves (SSLC). However, there is still a lack of information about how oxygen SSLC curves and estimations from fluorescence P−I curves are related. The present study addresses some of these topics, using Laminaria hyperborea. The objectives of this study were to (1) determine the effect of light sequence (increasing or decreas- ing) in the estimation of P−I parameters, (2) characterize the daily patterns of P−I curve parame- ters, (3) compare oxygen and fluorescence P−I parameters and (4) evaluate the possibility of con- verting fluorescence P−I parameters into oxygen production measurements. Results showed that light sequence had no significant effect on P−I parameters (fluorescence and oxygen) and that P−I curves exhibited considerable differences over the day (F-ratio analysis). Fluorescence SSLCs and RLCs were good oxygen production predictors only at sub-saturating irradiances, when signifi- cant linear relationships were obtained with results from oxygen incubations. Thus care should be taken when interpreting PAM fluorescence gross production estimations from higher light intensities.

Comparison of chlorophyll fluorescence curves and texture analysis for automatic plant identification

With automatic plant identification methods, the amount of herbicides used in agriculture can be reduced when herbicides are sprayed only on weeds. In the present study, leaves of oat (Avena sativa) and dandelion (Taraxacum officinale, TAROF) were arranged so that there was overlap between the species, imaged with a pulse amplitude modulation fluorescence camera and photographed with a digital color camera. The fluorescence induction curves from each pixel were parameterized to obtain a set of features and from color photographs, texture features were calculated. A support vector algorithm that also performed feature selection was used for pattern recognition of both data sets. Fluorescence-based identification worked well with oat leaves, producing 92.2 % of correctly identified pixels, whereas the texture-based method often mis-identified the central vein of a TAROF leaf as oat, identifying correctly only 66.5 % of oat pixels. With TAROF that shows a clear dicot-type texture, the texture method was slightly better (96.4 % correctly identified pixels) than the fluorescence method (94.6 %). In fluorescence-based identification, the accuracy varied between entire TAROF leaves, probably reflecting the genetic variability of TAROF. The results suggest that the accuracy of identification could be improved by combining two identification methods.

Gene expression indicates a zone of heterocyst differentiation within the thallus of the cyanolichen Pseudocyphellaria crocata

Development of the symbiotic association in the bipartite lichen Pseudocyphellaria crocata was investigated by characterizing two regions of the thallus. Thallus organization was examined using microscopy. A HIP1-based differential display technique was modified for use on Nostoc strains, including lichenized strains. Northern hybridization and quantitative real-time polymerase chain reaction were used to confirm differential display results, and determine expression levels of key cyanobacterial genes. Photosystem II yield across the thallus was measured using pulse-amplitude modulated fluorescence. Microscopy revealed structural differences in the thallus margins compared with the centre and identified putative heterocysts in both regions. Differential display identified altered transcript levels in both Nostoc punctiforme and a lichenized Nostoc strain. Transcript abundance of cox2, atpA, and ribA was increased in the thallus margin compared with the centre. Expression of cox2 is heterocyst specific and expression of other heterocyst-specific genes (hetR and nifK) was elevated in the margin, whereas, expression of psbB and PSII yield were not. Structural organization of the thallus margin differed from the centre. Both regions contained putative heterocysts but gene expression data indicated increased heterocyst differentiation in the margins where photosystem II yield was decreased. This is consistent with a zone of heterocyst differentiation within the thallus margin.