Chromatic photoacclimation, photosynthetic electron transport and oxygen evolution in the chlorophyll d-containing oxyphotobacterium Acaryochloris marina

Changes in photosynthetic pigment ratios showed that the Chlorophyll d-dominated oxyphotobacterium Acaryochloris marina was able to photoacclimate to different light regimes. Chl d per cell were higher in cultures grown under low irradiance and red or green light compared to those found when grown under high white light, but phycocyanin/Chl d and carotenoid/Chl d indices under the corresponding conditions were lower. Chl a, considered an accessory pigment in this organism, decreased respective to Chl d in low irradiance and low intensity non-white light sources. Blue diode PAM (Pulse Amplitude Modulation) fluorometry was able to be used to measure photosynthesis in Acaryochloris. Light response curves for Acaryochloris were created using both PAM and O(2) electrode. A linear relationship was found between electron transport rate (ETR), measured using a PAM fluorometer, and oxygen evolution (net and gross photosynthesis). Gross photosynthesis and ETR were directly proportional to one another. The optimum light for white light (quartz halogen) was about 206+/-51 micromol m(-2) s(-1) (PAR) (Photosynthetically Active Radiation), whereas for red light (red diodes) the optimum light was lower (109+/-27 micromol m(-2) s(-1) (PAR)). The maximum mean gross photosynthetic rate of Acaryochloris was 73+/-7 micromol mg Chl d(-1) h(-1). The gross photosynthesis/respiration ratio (P(g)/R) of Acaryochloris under optimum conditions was about 4.02+/-1.69. The implications of our findings will be discussed in relation to how photosynthesis is regulated in Acaryochloris.     

How to correctly determine the different chlorophyll fluorescence parameters and the chlorophyll fluorescence decrease ratio R<Subscript>Fd</Subscript> of leaves with the PAM fluorometer

This contribution is a practical guide to the measurement of the different chlorophyll (Chl) fluorescence parameters and gives examples of their development under high-irradiance stress. From the Chl fluorescence induction kinetics upon irradiation of dark-adapted leaves, measured with the PAM fluorometer, various Chl fluorescence parameters, ratios, and quenching coefficients can be determined, which provide information on the functionality of the photosystem 2 (PS2) and the photosynthetic apparatus. These are the parameters F_v, F_m, F₀, F_m′, F_v′, NF, and ΔF, the Chl fluorescence ratios F_v/F_m, F_v/F₀, ΔF/F_m′, as well as the photochemical (q_P) and non-photochemical quenching coefficients (q_N, q_CN, and NPQ). q_N consists of three components (q_N = q_E + q_T + q_I), the contribution of which can be determined via Chl fluorescence relaxation kinetics measured in the dark period after the induction kinetics. The above Chl fluorescence parameters and ratios, many of which are measured in the dark-adapted state of leaves, primarily provide information on the functionality of PS2. In fully developed green and dark-green leaves these Chl fluorescence parameters, measured at the upper adaxial leaf side, only reflect the Chl fluorescence of a small portion of the leaf chloroplasts of the green palisade parenchyma cells at the upper outer leaf half. Thus, PAM fluorometer measurements have to be performed at both leaf sides to obtain information on all chloroplasts of the whole leaf. Combined high irradiance (HI) and heat stress, applied at the upper leaf side, strongly reduced the quantum yield of the photochemical energy conversion at the upper leaf half to nearly zero, whereas the Chl fluorescence signals measured at the lower leaf side were not or only little affected. During this HL-stress treatment, q_N, q_CN, and NPQ increased in both leaf sides, but to a much higher extent at the lower compared to the upper leaf side. q_N was the best indicator for non-photochemical quenching even during a stronger HL-stress, whereas q_CN and NPQ decreased with progressive stress even though non-photochemical quenching still continued. It is strongly recommended to determine, in addition to the classical fluorescence parameters, via the PAM fluorometer also the Chl fluorescence decrease ratio R_Fd (F_d/F_s), which, when measured at saturation irradiance is directly correlated to the net CO₂ assimilation rate (_{P N}) of leaves. This R_Fd-ratio can be determined from the Chl fluorescence induction kinetics measured with the PAM fluorometer using continuous saturating light (cSL) during 4–5 min. As the R_Fd-values are fast measurable indicators correlating with the photosynthetic activity of whole leaves, they should always be determined via the PAM fluorometer parallel to the other Chl fluorescence coefficients and ratios.     

两种生境条件下6种牧草叶绿素含量及荧光参数的比较

昆仑山前山牧场海拔较高, 策勒绿洲海拔相对较低, 两者生境差异较大。以昆仑山前山牧场和策勒绿洲边缘两种不同生境条件下生长的6种牧草: 冰草(Agropyron cristatum)、无芒雀麦(Bromus inermis)、矮生高羊茅(Festuca elata)、披碱草(Elymus dahuricus )、红豆草(Onobrychis pulchella)及和田大叶(Medicago sativa var. luxurians)为试验材料, 研究了不同生境条件下牧草叶片叶绿素含量及叶绿素荧光动力学参数的变化情况。结果显示: (1)在两种生境条件下, 昆仑山前山牧场生境生长的牧草叶绿素a、叶绿素b、总叶绿素的含量明显较高, 生长在策勒绿洲生境的牧草品种叶绿素a/b值较高; (2)昆仑山前山牧场生境牧草最大荧光、光系统II (PSII)最大光化学效率、PSII潜在活性和单位面积反应中心的数量的值明显高于策勒绿洲生境品种, 而初始荧光、单位反应中心吸收的光能、单位反应中心捕获的能量、单位反应中心耗散的能量、荧光诱导曲线初始斜率值则低于策勒绿洲生境品种。因此, 两种生境下环境因子发生了改变, 对牧草产生综合的胁迫作用; 策勒绿洲生境明显对牧草生长产生了抑制, 策勒绿洲生境牧草的色素含量降低以及PSII的机构遭到损坏, 导致反应中心一部分失活或裂解, 剩余有活性的反应中心的效率增加, 昆仑山生境则相对比较适宜牧草生长; 两种生境不同牧草叶绿素含量和叶绿素荧光参数的变化幅度不同。     

Carbon:chlorophyll <Emphasis Type="Italic">a</Emphasis> ratio,assimilation numbers and turnover times of Lake Kinneret phytoplankton

Carbon to chlorophyll a (C:Chl) ratios, assimilation numbers (A.N.) and turnover times of natural populations of individual species and taxonomic groups were extracted from a long-term database of phytoplankton wet-weight biomass, chlorophyll a concentrations, and primary production in Lake Kinneret, Israel. From a database spanning more than a decade, we selected data for samples dominated by a single species or taxonomic group. The overall average of C:Chl was highest for cyanophytes and lowest for diatoms, while chlorophytes and dinoflagellates showed intermediate values. When converting chlorophyll a to algal cellular carbon this variability should be taken into account. The variability in C:Chl within each phylum and species (when data were available) was high and the variability at any particular sampling date tended to be greater than the temporal variability. The average chlorophyll a-normalized rate of photosynthetic activity of cyanophytes was higher and that of the dinoflagellates lower than that of other phyla. Turnover time of phytoplankton, calculated using primary productivity data at the depth of maximal photosynthetic rate, was longest in dinoflagellates and shortest in cyanophytes, with diatoms and chlorophytes showing intermediate values. The more extreme C:Chl and turnover times of dinoflagellates and cyanobacteria in comparison with chlorophytes and diatoms should be taken into consideration when employed in ecological modeling.     

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

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

Phototrophic microendoliths bloom during coral “white syndrome”

Following rapid lesion progression of white syndrome in tabular Acropora spp., the white bare skeleton gradually changes to green, a result of endolithic algae blooms (primarily Ostreobium spp.). Endolithic algal biomass and chlorophyll concentration were found to be an order of magnitude higher in the green zone compared with healthy appearing parts of each colony. Chl b to Chl a ratio increased from 1:1.6 in the healthy area to 1:2 and 1:3.5 in the white exposed skeleton and green zones, respectively. These observations together with pulse amplitude modulated (PAM) fluorometry suggest photoacclimation of the endoliths in the green zone. Histopathological microscopy revealed that the endolithic algal filaments penetrate the coral tissue. This study highlights the interaction of endolithic algae with both the skeleton and host tissue. This may have a critical role in the processes that accompany the post-disease state in reef-building corals.     

Formyl group modification of chlorophyll a: a major evolutionary mechanism in oxygenic photosynthesis

We discuss recent advances in chlorophyll research in the context of chlorophyll evolution and conclude that some derivations of the formyl side chain arrangement of the porphyrin ring from that of the Chl a macrocycle can extend the photosynthetic active radiation (PAR) of these molecules, for example, Chl d and Chl f absorb light in the near‐infrared region, up to ～750 nm. Derivations such as this confer a selective advantage in particular niches and may, therefore, be beneficial for photosynthetic organisms thriving in light environments with particular light signatures, such as red‐ and near‐far‐red light‐enriched niches. Modelling of formyl side chain substitutions of Chl a revealed yet unidentified but theoretically possible Chls with a distinct shift of light absorption properties when compared to Chl a.     

植物叶绿素合成、分解代谢及信号调控

叶绿素(Chlorophyll, Chl)合成是决定植物光合效率的重要性状, 是决定作物产量的重要因素。参与植物Chl合成、分解代谢及信号调控的基因数目众多, 其中任何一个基因发生突变都有可能引起Chl含量的变化, 从而表现为各种叶色异常甚至导致植株死亡。自然或人工创造突变体, 对于Chl相关基因的功能分析非常必要。目前, Chl突变体己广泛应用于基础研究和生产实践。文章就该研究领域内的最新研究进展进行了概述。     

Pigment Content of Selected Planktonic Algae in Response to Simulated Natural Light Fluctuations and a Short Photoperiod

The lipophilic photosynthetic pigments in Limnothrix redekei, Planktothrix agardhii (cyanobacteria), Stephanodiscus minutulus, Synedra acus (diatoms), Scenedesmus acuminatus, and Scenedesmus armatus (chlorophycean) all isolated from an eutrophic lake were quantitatively determined by HPLC. The algae were grown semi-continuously under nutrient sufficient conditions at 20°C at a 12/12 h light/dark cycle with constant irradiance or with simulated natural light fluctuations as well as at a 6/18 h light/dark cycle with constant irradiance, all at the same daily light exposure. The zeaxanthin and the myxoxanthophyll contents of cyanobacteria were not influenced by fluctuating light, a short photoperiod or a different sampling time. The chlorophyll b/a ratio, the lutein/chlorophyll a ratio, and the neoxanthin content of chlorophycean as well as the chlorophyll c/a and the fucoxanthin/chlorophyll a ratio of diatoms were only slightly influenced by these factors. Therefore in some cases marker pigment contents and in other cases marker pigment/chlorophyll a ratios may be more useful for quantifying the relative importance of different taxonomic groups in natural phytoplankton. Simulated natural light fluctuations or the length of the photoperiod only slightly influenced the pigment content or the marker pigment/chlorophyll a ratio.     

Quantification of algal biofilms colonising building materials: chlorophyll a measured by PAM-fluorometry as a biomass parameter

Abstract

The aim of this study was to quantify algal colonisation on anthropogenic surfaces (viz. building facades and roof tiles) using chlorophyll a (chl a) as a specific biomarker. Chl a was estimated as the initial fluorescence F₀ of ‘dark adapted’ algae using a pulse-modulated fluorometer (PAM-2000). Four isolates of aeroterrestrial green algae and one aquatic isolate were included in this study. The chl a concentration and F₀ showed an exponential relationship in the tested range between 0 and 400 mg chl a m^?2. The relationship was linear at chl a concentrations <20 mg m^?2. Exponential and linear models are presented for the single isolates with large coefficients of determination (exponential: r² > 0.94, linear: r² > 0.92). The specific power of this fluorometric method is the detection of initial algal colonisation on surfaces in thin or young biofilms down to 3.5 mg chl a m^?2, which corresponds to an abundances of the investigated isolates between 0.2 and 1.5 million cells cm^?2.     

Regulation by light of chlorophyll synthesis in the cotyledons of Scots pine (Pinus sylvestris) seedlings

Seedlings of gymnosperms, unlike angiosperms, synthesize chlorophyll(ide) (Chl) in darkness (D). In Scots pine cotyledons ( Pinus sylvestris L.) Chl accumulation ceases in D at a low level but Chl accumulation is strongly increased by light, red light (R) being more effective than blue light (B), whereas in Pinus maritima Chi synthesis is almost light-independent. In Scots pine the capacity to form Chl can be increased by R pulses, fully reversible by far-red light, demonstrating the involvement of phytochrome. However, when B- or R–grown seedlings were transferred to D, Chl accumulation stopped immediately irrespective of the level of P_fr (far-red light absorbing form of phytochrome), indicating that the conversion of protochlorophyllide (PChl) is light-dependent. Dose response curves in R and B and simultaneous irradiation with R and B show that R and B are perceived by separate photoreceptors. The immunodetected NADPH-dependent protochlorophyllide oxidoreductase (POR, EC 1.6.99.1), assumed to regulate light-dependent Chl synthesis in angiosperms, is not correlated with the capacity of gymnosperm Chi accumulation in darkness. While two FOR bands could be separated in extracts from dark grown material (38 and 36 kDa) of Pinus sylvestris and P. maritima , only the 38 kDa band disappeared consistently in the light. However. the significance of the more light resistant 36 kDa band for chlorophyll synthesis remains unclear as well.     

On the limits of applicability of spectrophotometric and spectrofluorimetric methods for the determination of chlorophyll a/b ratio

The concentration limits for spectrophotometric and spectrofluorimetric determinations of the chlorophyll (Chl) a/b ratio in barley leaves were studied using 80% acetone extracts at room temperature. The optimum sample absorbances (at 663.2 nm – maximum of the Q_Y) band of Chl a) for the Chl a/b determination were determined. For given spectrometers and sample positions, these absorbances ranged between 0.2 and 1.0 and 0.008–0.1 for the absorption and fluorescence methods, respectively. Precision of the measurements and the distorting effects are discussed. The lower limits of both absorption and fluorescence methods depend on sensitivity of the spectrometers for the Chl b detection. The spectrophotometric determination of Chl a/b ratio at higher Chl concentrations can be distorted by the chlorophyll fluorescence signal. The extent of this distortion depends on sample-detector geometry in any given type of the spectrometer. The effect of inner filter of Chl molecules and the detection instrumental function affect the value of the upper limit for the spectrofluorimetric method. Both methods were applied to estimate the Chl a/b ratio in pigment extracts from greening barley leaves, which are characterized by a low Chl concentration and a high Chl a/b ratio at the beginning of greening process.     

An automatic device for in vivo absorption spectra acquisition and chlorophyll estimation in phytoplankton cultures

In order to aid the study of photoacclimation, a new programmable deviceis described which provides automatic on-line acquisition of in vivo cellabsorption in phytoplankton cultures. The system was used for a long-termstudy of Rhodomonas salina grown at constant photon flux density ina nitrate-limited continuous culture with different dilution rates. Particulate absorption measured at the red chlorophyll a (Chl a)maximum was not a good proxy of biomass, because of the large variabilityof cellular chlorophyll induced by nitrogen limitation. However, thedevice is well suited to automatic assessment of Chl a andphycoerythrin (PE) concentrations in phytoplankton cultures, if algal cellsize and concentration are measured in parallel to correct the packagingeffect. The effects of nitrogen limitation on Chl a and PE contentsand particle absorbance are discussed.     

Adaptation of a PAM-fluorometer for remote sensing of chlorophyll fluorescence

The Laser-PAM described in this paper is an adaptation of the PAM 101 fluorometer (Heinz Walz, Effeltrich, Germany) designed for remote sensing and non-invasive laboratory measurements of chlorophyll fluorescence. It is based on a 5 mW laser diode, emitting at 638 nm, and a Fresnel lens coupled to the ED-101 detection unit. Due to these modifications, measurements can be performed at a distance ranging from 0.3 to 2 m. The ED-101 detection unit has been modified to perform simultaneous measurements of both modulated fluorescence and light reflected by the leaf. Reflected light showed a good estimation of the photosynthetically active radiation measured exactly at the same area as the fluorescence. A particular advantage of the Laser-PAM fluorometer is its suitability for remote measurements under field conditions. Simultaneous fluorescence and gas-exchange measurements, performed on grapevine leaves, are reported as an example of an application for the Laser-PAM. This revised version was published online in June 2006 with corrections to the Cover Date.     

Acclimation of barley to changes in light intensity: chlorophyll organization

Barley seedlings (Hordeum vulgare L. cv. Boone) were grown at 20°C with a 16h/8h light/dark cycle of either high (H) intensity (550 mole m^-2 s^-1) or low (L) intensity (55 mole m^-2 s^-1) white light. Plants were transferred from high to low (H L) or low to high (L H) light intensity at various times from 4 to 8 d after leaf emergence from the soil. Primary leaves were harvested at the beginning of the photoperiod and a 3 cm apical segment removed for analysis. H control plants had greater chlorophyll (Chl) per leaf area and higher Chl a/b ratios than L controls. Analysis of Chl-protein complexes revealed that H and L plants had the same percentage of total Chl (62–65%) associated with Photosystem II (PS II), but that the organization of Chl within PS II was different. H plants contained lower levels of light-harvesting complex (LHC-II) and higher levels of the PS II complex CPa compared with L plants. Leaf Chl content and Chl organization within PS II were sensitive to changes in light intensity. In H L plants, leaf Chl content decreased, Chl a/b ratio decreased, and a redistribution of Chl from CPa to LHC-II occurred during acclimation to low light. Acclimation of L H plants to high light involved an increase in leaf Chl content, an increase in Chl a/b ratio, and a decrease in LHC-II. In contrast, the level of photosystem I related Chl-protein complexes (CP1 + CP1a) was similar in all light treatments. The light acclimation process occurred slowly over a period of 6 to 8 d in H L and L H plants.Abbreviations DMF dimethylformamide - H control plants grown under high light intensity - H L plants transferred from high to low light intensity - L control plants grown under low light intensity - L H plants transferred from low to high light intensity Cooperative investigations of the United States Department of Agriculture, Agricultural Research Service, and the North Carolina Agricultural Research Service, Raleigh, NC. Paper No. 11989 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695-7643, USA.     

Determining algal assemblages in oligotrophic lakes and streams: comparing information from newly developed pigment/chlorophyll a ratios with direct microscopy

1. Pigment analyses by high performance liquid chromatography (HPLC) are commonly used for determining algal groups in marine and estuarine areas but are underdeveloped in freshwaters. In this study, 15 characteristic pelagic algal species (representing five algal groups) of oligo‐ / mesotrophic lakes were cultured and pigment / Chl a ratios determined at three light intensities. 2. With the exception of cyanophytes, light treatment had little effect on pigment / Chl a ratios. This justifies the use of the same pigment / Chl a ratios during seasonal studies where light conditions may change. 3. The determined pigment / Chl a ratios were tested on seasonal samples from five oligo‐ / mesotrophic lakes and three streams using CHEMTAX software. Pigment ratios of both pelagic and benthic algal communities from the lakes and streams were analysed to determine whether the pelagic algae‐based ratios can be used for benthic algal communities. 4. HPLC combined with CHEMTAX was useful for identifying freshwater phytoplankton classes and for quantifying the abundance of phytoplankton groups. However, although correlations were significant for six of seven phytoplankton classes studied, they were weak and varied with season. 5. HPLC was valid for quantifying benthic diatom groups in stream samples, whereas for lakes more benthic algal groups were recorded with HPLC than with microscopy and correlations between the two methods were not significant. 6. The use of both HPLC and microscopy is recommended as a cost‐efficient method for analysing many samples. It is crucial, however, that the CHEMTAX software is calibrated with the correct information, and the user is aware of the limitations.     

Effects of different excitation and detection spectral regions on room temperature chlorophyll <Emphasis Type="Italic">a</Emphasis> fluorescence parameters

The effects of different spectral region of excitation and detection of chlorophyll (Chl) a fluorescence at room temperature on the estimation of excitation energy utilization within photosystem (PS) 2 were studied in wild-type barley (Hordeum vulgare L. cv. Bonus) and its Chl b-less mutant chlorina f2 grown under low and high irradiances [100 and 1 000 μmol(photon) m⁻² s⁻¹]. Three measuring spectral regimes were applied using a PAM 101 fluorometer: (1) excitation in the red region (maximum at the wavelength of 649 nm) and detection in the far-red region beyond 710 nm, (2) excitation in the blue region (maximum at the wavelength of 461 nm) and detection beyond 710 nm, and (3) excitation in the blue region and detection in the red region (660– 710 nm). Non-photochemical quenching of maximal (NPQ) and minimal fluorescence (SV₀), determined by detecting Chl a fluorescence beyond 710 nm, were significantly higher for blue excitation as compared to red excitation. We suggest that this results from higher non-radiative dissipation of absorbed excitation energy within light-harvesting complexes of PS2 (LHC2) due to preferential excitation of LHC2 by blue radiation and from the lower contribution of PS1 emission to the detected fluorescence in the case of blue excitation. Detection of Chl a fluorescence originating preferentially from PS2 (i.e. in the range of 660–710 nm) led to pronounced increase of NPQ, SV₀, and the PS2 photochemical efficiencies (F_V/F_M and F_V′/F_M′), indicating considerable underestimation of these parameters using the standard set-up of PAM 101. Hence PS1 contribution to the minimal fluorescence level in the irradiance-adapted state may reach up to about 80 %.     

Imaging of chlorophyll a fluorescence: theoretical and practical aspects of an emerging technique for the monitoring of photosynthetic performance

The development of chlorophyll (Chl) a fluorescence imaging systems has greatly increased the versatility of Chl a fluorometry as a non-invasive technique for the investigation of photosynthesis in plants and algae. For example, systems that image at the microscopic level have made it possible to measure PSII photochemical efficiencies from chloroplasts within intact leaves and from individual algal cells within mixed populations, while systems that image over much larger areas have been used to investigate heterogeneous patterns of photosynthetic performance across leaves and in screening programmes that image tens or even hundreds of plants simultaneously. In addition, it is now practical to use fluorescence imaging systems as real-time, multi-channel fluorometers, which can be used to record continuous fluorescence traces from multiple leaves, plants, or algal cells. This paper discusses some of the theoretical and practical issues associated with the imaging of Chl a fluorescence and with Chl a fluorometry in general. This discussion includes a review of the most commonly used Chl a fluorescence parameters.     

Evidence for a light-harvesting chlorophyll a-protein complex in a chlorophyll b-less barley mutant

Chloroplasts of a chlorophyll (Chl) b-less barley mutant were solubilized with digitonin and fractionated by polyacrylamide gel electrophoresis with sodium deoxycholate in the running buffer. By this procedure, in contrast to using sodium dodecylsulfate (SDS) for solubilization, a Chl a-protein analogous to the major light-harvesting Chl a-b protein complex from wildtype chloroplasts was recovered. This mutant Chl a-protein comprises about fifty percent of the total Chl a, and is very similar in carotenoid, amino acid, protein and polypeptide composition to the major wildtype antenna Chl a-b protein. The only major differences we have found is its instability in the presence of SDS and sensitivity to protease action. Even with deoxycholate, the mutant Chl a complex often dissociates during electrophoresis into two green bands. The lack of Chl b appears to affect the normal organization of Chl a and protein in such a way as to render the complex more unstable.CIW-DPB No. 917.     

Practical aspects of the measurements of non-photochemical chlorophyll fluorescence quenching in green microalgae Chlamydomonas reinhardtii using Open FluorCam

Methods of chlorophyll fluorescence measurements are widely used in the research on photosynthesis and ecophysiology of plants and algae. Among them, a very popular technique is pulse-amplitude-modulation (PAM) flourometry, which is simple to carry out, fast and non-invasive. However, this method is also prone to generate artifacts if the experiments were not planned and executed properly. Application of this technique to algae brings additional complications, which need to be taken into consideration. Some of them are connected with sample preparation and setting of the protocols used, while another origin from the differences in the photosynthetic apparatus and regulation of photosynthesis in various algal groups when compared to vascular plants. In the present paper, some important practical aspects concerning PAM fluorometry measurements in the green microalga Chlamydomonas reinhardtii have been described, including the equipment settings and sample preparation. The impact of growth conditions, such as light, temperature and medium type on the induction of non-photochemical quenching of chlorophyll fluorescence have been also tackled, as well as the question of state transitions occurring in darkness.