Chlorophyll Fluorometer Research Articles

Observation of the scale of patchy stomatal behavior in leaves of Quercus crispula using an Imaging-PAM chlorophyll fluorometer

Patchy stomatal closure occurs in plants with heterobaric leaves, in which vertical extensions of bundle sheath cells delimit the mesophyll and restrict the diffusion of CO(2). The scale of patchy stomatal behavior was investigated in this study. The distribution of PSII quantum yield (Φ(II)) obtained from chlorophyll fluorescence images was used to evaluate the scale of stomatal patchiness and its relationship with leaf photosynthesis in the sun leaves of 2-year-old saplings of Quercus crispula Blume. Fluorescent patches were observed only during the day with low stomatal conductance. Comparison of numerical simulation of leaf gas exchange and chlorophyll fluorescence images showed that heterogeneous distribution of electron transport rate through PSII (J) was observed following stomatal closure with a bimodal manner under both natural and saturated photosynthetic photon flux densities. Thus, fluorescence patterns can be interpreted in terms of patchy stomatal closure. The mapping of J from chlorophyll fluorescence images showed that the scale of stomatal patchiness was approximately 2.5-fold larger than that of anatomical patches (lamina areas bounded by bundle sheath extensions within lamina). Our results suggest the spatial scale of stomatal patches in Q. crispula leaves.

Preliminary investigations on Arctic microalgae by joint application of fluorescent instruments

In vivo fluorescence has a wide application in analyzing microalgae, including assessing phytoplankton biomass, ratesof primary production and physiological status. This study describes a preliminary investigation on the joint application of the threekinds of fluorescence analysis in the physiological study of microalgae. Flow cytometry and fluorescence spectrometry were usedto obtain the in vivo static fluorescence information of pigments, and a Pulsed-Amplitude-Modulation chlorophyll fluorometer wasused to detect the dynamic fluorescence of chlorophyll. The validity of the joint application was proved by analyzing two laboratorycultured Arctic microalgae, Pseudo-nitzschia delicatissima ( Bacillariophyceae ) and Thalassiosira sp. The higher value of minimum fluorescence yield in dark-adapted state ( Fo ), actual photochemical efficiency of PSII (ΦPSII), and electron transportrate (ETR) exhibited positive results in a higher cell abundance and chlorophyll a content of P. delicatissima ; whereas higher β-carotene content of Thalassiosira sp. played an important role in the protection of photosynthesis.

Early Detection of Soybean Plant Injury from Glyphosate by Measuring Chlorophyll Reflectance and Fluorescence

Early detection of crop injury from off-target drift of herbicide is critical in crop production. Subtle changes in canopy reflectance could present useful information to detect the onset of crop stress. This study was conducted in a greenhouse to evaluate a portable spectroradiometer and a portable chlorophyll fluorometer for the detection of crop injury caused by glyphosate spray. Non-glyphosate resistant soybean (Glycine max L. Merr.) plants were sprayed with glyphosate using a pneumatic track sprayer in a spray chamber. Four plants received a rate of 0.86 kg ae/ha glyphosate and four plants received 0.086 kg ae/ha. Additional four non-sprayed plants were used as controls. After the glyphosate spray, the chlorophyll reflectance of the plants was measured with the spectroradiometer at 4, 24, 48, and 72 hours to determine the plant response to herbicide. Simultaneously, fluorescence induction kinetics of the crop under stress was measured with the portable chlorophyll fluorometer. Results of the statistical mean separation indicated that the plant chlorophyll reflectance measurement could be used to differentiate crop stress from glyphosate at 24 hours after spray among treatments and to identify the effect of herbicide at 24 hours after spray in each treatment. Moreover, linear discriminant analysis with the reflectance data showed that the crop stress of the soybean plants from glyphosate could be identified at 24 hours or more post application. Results of the statistical mean separation also indicated that use of plant chlorophyll fluorescence measurement could not differentiate crop stress until 48 hours after spray among treatments while it could identify the effect of herbicide 24 hours after spray in each treatment. These findings demonstrate that chlorophyll reflectance and fluorescence measurements both could be used for early detection of crop stress.

Vertical distribution of phytoplankton and physico-chemical characteristics in the lacustrine zone of Xin'anjiang Reservoir(Lake Qiandao) in subtropic China during summer stratification

The investigation on vertical distribution of phytoplankton and physical-chemical characteristics was conducted in the lacustrine zone during summer stratification in Xin'anjiang Reservoir(Lake Qiandao).The Water-PAM Chlorophyll Fluorometer was used to obtain vertical profiles of phytoplankton photosynthesis.The results showed that depth of thermocline appeared between 10-20m.Besides,the vertical profiles of pH,dissolved oxygen and turbidity were in agreement with chlorophyll-a.In summer,the phytoplankton was dominated by Bacillariophyta,C.meneghiniana was dominant in surface layer,while F.crotonensis was absolutely dominant below the surface layer.High-density of phytoplankton was found around 5-10m below the water surface.However,a sharp decline was observed for phytoplankton density below thermocline layer.Therefore,the thermal stratification might affect the vertical distribution of phytoplankton population.Phytoplankton maximum photosynthetic efficiency had a negative relationship with water depth and the actual maximum value of photosynthetic efficiency was determined at 10m depth.

小型クロロフィル蛍光測定器を用いた水ストレスによる樹勢衰退の客観的診断の可能性

小型クロロフィル蛍光測定器を用いた水ストレスによる樹勢衰退の客観的診断の可能性

Combined effects of lanthanumion and acid rain on growth, photosynthesis and chloroplast ultrastructure in soybean seedlings

Rare earth elements (REEs) have been accumulated in the agricultural environment. Acid rain is a serious environmental issue. In the present work, the effects of lanthanum ion (La 3+) and acid rain on the growth, photosynthesis and chloroplast ultrastructure in soybean seedlings were investigated using the gas exchange measurements system, chlorophyll fluorometer, transmission electron microscopy and some biochemical techniques. It was found that although the growth and photosynthesis of soybean seedlings treated with the low concentration of La 3+ was improved, the growth and photosynthesis of soybean seedlings were obviously inhibited in the combined treatment with the low concentration of La 3+ and acid rain. At the same time, the chloroplast ultrastructure in the cell of soybean seedlings was destroyed. Under the combined treatment with the high concentration of La 3+ and acid rain, the chloroplast ultrastructure in the cell of soybean seedlings was seriously destroyed, and the growth and of photosynthesis were greatly decreased compared with those of the control, the single treatment with the high concentration of La 3+ and the single treatment with acid rain, respectively. The degree of decrease and destruction on chloroplast ultrastructure depended on the increases in the concentration of La 3+ and acid rain (H +). In conclusion, the combined pollution of La 3+ and acid rain obviously destroyed the chloroplast ultrastructure of cell and aggravated the harmful effect of the single La 3+ and acid rain on soybean seedlings. As a new combined pollutant, the harmful effect of REEs ions and acid rain on plant should be paid attention to.

Intra‐leaf gradients of photoinhibition induced by different color lights: implications for the dual mechanisms of photoinhibition and for the application of conventional chlorophyll fluorometers

• We studied how different color lights cause gradients of photoinhibition within a leaf, to attempt to resolve the controversy of whether photon absorption by chlorophyll or by manganese (Mn) is the primary cause of photoinhibition, as suggested by the excess-energy hypothesis or the two-step hypothesis, respectively. • Lincomycin-treated leaf discs were photoinhibited by white, blue, green or red light. Combining a microfiber fluorometer, a fiber-thinning technique and a micro-manipulator enabled us to measure the chlorophyll fluorescence signals within a leaf. Photoinhibition gradients were also compared with results from various conventional fluorometers to estimate their depth of signal detection. • The severity of photoinhibition was in the descending order of blue, red and green light near the adaxial surface, and in the descending order of blue, green and red light in the deeper tissue, which correlated with the chlorophyll and the Mn absorption spectrums, respectively. These results cannot be explained by either hypothesis alone. • These data strongly suggest that both the excess-energy and the two-step mechanisms occur in photoinhibition, and fluorometers with red or blue measuring light give overestimated or underestimated F(v)/F(m) values of photoinhibited leaves compared with the whole tissue average, respectively; that is, they measured deeper or shallower leaf tissue, respectively.

Utilizing the Effective Xanthophyll Cycle for Blooming of Ochromonas smithii and O. itoi (Chrysophyceae) on the Snow Surface

Snow algae inhabit unique environments such as alpine and high latitudes, and can grow and bloom with visualizing on snow or glacier during spring-summer. The chrysophytes Ochromonas smithii and Ochromonas itoi are dominant in yellow-colored snow patches in mountainous heavy snow areas from late May to early June. It is considered to be effective utilizing the xanthophyll cycle and holding sunscreen pigments as protective system for snow algae blooming in the vulnerable environment such as low temperature and nutrients, and strong light, however the study on the photoprotection of chrysophytes snow algae has not been shown. To dissolve how the chrysophytes snow algae can grow and bloom under such an extreme environment, we studied with the object of light which is one point of significance to this problem. We collected the yellow snows and measured photosynthetically active radiation at Mt. Gassan in May 2008 when the bloom occurred, then tried to establish unialgal cultures of O. smithii and O. itoi, and examined their photosynthetic properties by a PAM chlorophyll fluorometer and analyzed the pigment compositions before and after illumination with high-light intensities to investigate the working xanthophyll cycle. This experimental study using unialgal cultures revealed that both O. smithii and O. itoi utilize only the efficient violaxanthin cycle for photoprotection as a dissipation system of surplus energy under prolonged high-light stress, although they possess chlorophyll c with diadinoxanthin.

New insights on obtaining phytoplankton concentration and composition from in situ multispectral Chlorophyll fluorescence

A three‐channel excitation (435 nm, 470 nm, and 532 nm) Chlorophyll fluorometer (695 nm emission) was calibrated and characterized to improve uncertainty in estimated in situ Chlorophyll concentrations. Protocols for reducing sensor‐related uncertainties as well as environmental‐related uncertainties were developed. Sensor calibrations were performed with thirteen monospecific cultures in the laboratory, grown under limiting and saturating irradiance, and sampled at different growth phases. Resulting uncertainties in the calibration slope induced by natural variations in the in vivo fluorescence per extracted Chlorophyll yield were quantified. Signal variations associated with the sensors (i.e., dark current configurations, drift, and stability) and the environment (i.e., temperature dependent dark currents and contamination by colored dissolved organic matter [CDOM] fluorescence) yielded errors in estimating in situ Chlorophyll concentration exceeding 100%. Calibration protocols and concurrent observations of in situ temperature and CDOM fluorescence eliminate these uncertainties. Depending upon excitation channel, biomass calibration slopes varied between 6‐ and 10‐fold between species and as a function of growth irradiance or growth phase. The largest source of slope variability was due to variations in accessory pigmentation, and thus the variance could be reduced among pigment‐based taxonomic lines. Fluorescence ratios were statistically distinct among the pigment‐based taxonomic groups, providing not only a means for approximating bulk taxonomic composition, but also for selecting the appropriate calibration slope to statistically improve the accuracy of in situ Chlorophyll concentration estimates. Application to 5 months of deployment in China Lake, Maine, USA reduced the error in estimating extracted Chlorophyll concentration from > 30% to < 6%.

Expression of yeast Hem1 controlled by Arabidopsis HemA1 promoter enhances leaf photosynthesis in transgenic tobacco

A gene encoding aminolevulinate synthase (ALA-S) in yeast (Saccharomyces cerevisiae YHem1) was introduced into the genome of tobacco (Nicoliana tabacum) under the control of Arabidopsis thaliana HemA1 gene promoter (AtHemA1 P). All transgenic lines transcribed the YHem1 gene, especially under light condition. The capacity to synthesize ALA and therefore chlorophyll was increased in transformed plants. Determination of gas exchange suggested that transgenic plants had significantly higher level of net photosynthetic rate (P ( n )), stomatal conductance (G ( s )) and transpiration rate (T ( r )), compared to the wild type (WT). Analysis with a modulated chlorophyll fluorometer demonstrated that the genetic transformation also caused a significant increase in photochemical efficiency of PSII ([Formula: see text]), actual photochemical efficiency (Ф ( PSII )), photochemical quenching (qP), electron transfer rate (ETR) and the energy proportion in photochemistry (Pc), but decrease in proportion in heat dissipation (Hd). Chlorophyll-a fast fluorescence measurement and JIP-test indicated that photosynthetic performance index on cross section basis (PI ( CS )) and electron transport flux (ET ( o ) /CS) of transgenic tobacco were increased remarkably. And the probability that a trapped exciton can move a electron into the electron transport chain beyond Q ( A ) (-) (Ψ ( o )) and the density of active reaction centers (RC/CS) were also increased obviously in transgenic tobacco. These results imply that transgenic tobacco plants expressing YHem1 gene had higher photosynthetic capacity and energy conversion efficiency than the WT plants.

Photochemical Efficiency and SPAD Value as Indirect Selection Criteria for Combined Selection of Spot Blotch and Terminal Heat Stress in Wheat

AbstractTerminal heat and spot blotch caused by Cochliobolus sativus are important stresses causing significant wheat (Triticum aestivum L.) yield losses in the south Asian plains. Recent studies have shown that chlorophyll‐related traits are correlated with heat stress and spot blotch resistance in wheat. This study was conducted to evaluate leaf photochemical efficiency and leaf greenness (measured as SPAD value) for combined selection of spot blotch and terminal heat stress. The efficiency of photosystem II was measured as ratio of variable to maximal chlorophyll fluorescence, Fv/Fm, using chlorophyll fluorometer build on pulse modulation principle. The study was conducted in three spring wheat populations derived by crossing spot blotch–resistant wheat genotypes ‘Milan/Shanghai#7’, ‘Chirya.3’ and ‘NL971’ with a susceptible cultivar ‘BL 1473’. The F3 and F4 generations were grown under natural epiphytotics of spot blotch either in optimal or in terminal heat stress conditions at Rampur, Nepal. The heritability (h2) of Fv/Fm, SPAD measurements and their genetic correlation with 1000‐kernel weight (TKW) and area under disease progress curve (AUDPC) were estimated. The h2 estimates for Fv/Fm and SPAD measurements were moderate to high. In addition, AUDPC and TKW showed low to high genetic correlation with these traits. These findings suggest that Fv/Fm and SPAD measurements could be used as complementary traits in selecting for spot blotch resistance and heat tolerance in wheat.

LPA19, a Psb27 Homolog in Arabidopsis thaliana, Facilitates D1 Protein Precursor Processing during PSII Biogenesis

The biogenesis and assembly of photosystem II (PSII) are mainly regulated by the nuclear-encoded factors. To further identify the novel components involved in PSII biogenesis, we isolated and characterized a high chlorophyll fluorescence low psii accumulation19 (lpa19) mutant, which is defective in PSII biogenesis. LPA19 encodes a Psb27 homolog (At1g05385). Interestingly, another Psb27 homolog (At1g03600) in Arabidopsis was revealed to be required for the efficient repair of photodamaged PSII. These results suggest that the Psb27 homologs play distinct functions in PSII biogenesis and repair in Arabidopsis. Chloroplast protein labeling assays showed that the C-terminal processing of D1 in the lpa19 mutant was impaired. Protein overlay assays provided evidence that LPA19 interacts with D1, and coimmunoprecipitation analysis demonstrated that LPA19 interacts with mature D1 (mD1) and precursor D1 (pD1). Moreover, LPA19 protein was shown to specifically interact with the soluble C terminus present in the precursor and mature D1 through yeast two-hybrid analyses. Thus, these studies suggest that LPA19 is involved in facilitating the D1 precursor protein processing in Arabidopsis.

Water quality patterns in a river-lake system from multiple drivers (Three Rivers, New York State)

Robust spatial patterns of common water quality metrics are resolved for the downstream 90 km of the Three Rivers system of New York State (USA), extending to its entry into Lake Ontario, to assess water quality status and document signatures from an array of stressors and drivers. The strong longitudinal and vertical signatures documented are based on eight high spatial resolution (vertical profiles at 50 sites) summer surveys of temperature, specific conductance, turbidity, fluorometric chlorophyll a, dissolved oxygen, and nitrate. The water quality of three upstream boundary waters is also documented based on continuous concurrent measurements from robotic monitoring platforms. The spatial signatures along the river system are demonstrated to be driven by: (1) the major differences in the water quality of boundary water inputs, (2) physical modification of the river system that reduces turbulence and promotes vertical stratification, (3) the configuration of three lakes within this system, (4) inputs from waste water treatment plants, and (5) the zebra mussel invasion. The oxygen resources of this river system are shown to be degraded; e.g., water quality standards for dissolved oxygen were not met for 31 km of river length in one survey. The character and causes of the dissolved oxygen problems represent severe challenges for rehabilitation, which may require interventions to recover turbulence. A robotic monitoring program is recommended, with unit deployment locations guided by the surveys of this study, to stay apprised of water quality status and support water quality modeling initiatives. Development of a water quality model is recommended to guide management deliberations for recovery of oxygen resources, which will require a robust hydrodynamic transport framework to represent the complexities of this system.

Recovery from Drought Stress at the Shooting Stage in Oilseed Rape (Brassica napus)

AbstractPeriods of drought frequently affect the development of winter rape at the shooting stage in spring. A growth chamber study was conducted to determine the effects of 13 days of strong water deficit at the shooting stage followed by rewatering on oilseed rape (Brassica napus). The osmolality and the efficiency of photosystem II in leaves were measured by means of a vapour pressure osmometer and a chlorophyll fluorometer, respectively. The activities of sucrose cleaving enzymes in leaves were determined through invertase as well as sucrose synthase tests. A higher osmotic pressure was observed 2 days after water deprivation. Extracellular and vacuolar invertases as well as chlorophyll fluorescence were decreased with delay. Rewatering led to a gradual decrease in the osmotic pressure and a delayed increase in the enzyme activities as well as the photosynthetic efficiency. A linear regression model could predict osmolality in well‐watered plants based on the extracellular enzyme activity and growth stage. The ontogenetic and stress‐related pattern of extracellular invertase activity could indicate a role of this enzyme in balancing source‐sink relations. The onset of flowering was not influenced by drought, but the period of this stage was prolonged. Though, the ontogenetic stages of stressed and unstressed plants were the same at the time of harvest, the development of seed biomass was significantly depressed under stress.

Low-cost photochemical reflectance index measurements of micropropagated plantlets using image analysis

A system for imaging the photochemical reflectance index (PRI) of micropropagated plantlet leaves was developed using a low-cost charge-coupled device (CCD) camera. The reflected light intensities of leaves at 530 and 570nm were imaged using a monochrome CCD camera with respective band-pass filters. The reflection images were used to estimate the PRI values of the leaves. The relationships between the PRI estimated from images and a chlorophyll fluorescence parameter @DF/Fm', determined by pulse-amplitude modulation (PAM) chlorophyll fluorometer and representing PSII quantum yield, were investigated for several plants under various conditions to test the performance of the system. The PRI estimated from images decreased with a decline in the @DF/Fm' for strawberry leaves treated with high intensity light under artificial light condition and lettuce leaves treated with abscisic acid. These results suggest that the system can be used for non-destructive estimation of the PRI of leaves. The system was used to estimate the PRI of micropropagated potato leaves from outside the culture vessels. The time course of PSII quantum yield for the individual leaf could be demonstrated by the PRI estimated from images of potato plantlet leaves treated with high light. The findings suggest that the system has the potential for inexpensive, simple and efficient estimation of PRI for micropropagated plantlets.

Influence of crust-covered paving on photosynthesis and chlorophyll fluorescence parameters of a city landscape plant, Firmiana simplex

In order to explore the influence of crust-covered paving on city landscape plants, the photosynthesis and chlorophyll fluorescence parameters of Firmiana simplex were studied in this paper by the LCI portable plant photosynthesis meter and the OS5-FL-mode pulse modulated chlorophyll fluorometer. The results showed that during the day, the net photosynthetic rates of Firmiana simplex leaves under the crust-covering stress were slightly lower than those of the control, and the differences were significant at 18:00; the transpiration rate of the treatment group showed a single-peak curve, with the peak appearing between 12:00–14:00, while the transpiration rate of the control group showed a bimodal curve with the peak appearing at 10:00 and 16:00, respectively. When the transpiration rate of the control showed a low peak at 14:00, the transpiration rate of treatments was very significantly higher, and the difference was as much as 45.08%. In addition to 16:00 of all day, the water use efficiency of Firmiana simplex leaves under crust-covered condition was slightly higher than that of the control; the differences were not significant in the morning but very significant in the afternoon. The stomatal conductance of treatments was significantly higher than that of the control at 14:00 and slightly lower than the control at any other time. The intercellular CO2 concentration of treatments was higher than that of the control during the day except at 16:00, and the difference was significant at 14:00 and 18:00. The maximal photochemical efficiency of PS II (Fv/Fm) and the potential activity of PS II (Fv/Fo) of treatments were both lower than those of the control during the day except at 18:00, with very significant differences at 14:00. By this token, we concluded that the crust-covered paving had somehow brought a certain stress to the city landscape plant, Firmiana simplex.

Exploring Photosynthesis and Plant Stress Using Inexpensive Chlorophyll Fluorometers

Mastering the concept of photosynthesis is of critical importance to learning plant physiology and its applications, but seems to be one of the more challenging concepts in biology. This teaching challenge is no doubt compounded by the complexity by which plants alter photosynthesis in different environments. Here we suggest the use of chlorophyll fluorometers in the undergraduate plant physiology classroom as one means to enhance conceptual learning of photosynthesis and its acclimation to changing environments. We also provide an overview of current research in photosynthetic acclimation to changing conditions, review the methodological considerations of making good Fv/Fm measurements with small inexpensive fluorometers, and suggest an open‐inquiry activity for teaching concepts of photosynthesis and photo‐acclimation with fluorometers in an undergraduate plant physiology course.

PGRL1 Participates in Iron-induced Remodeling of the Photosynthetic Apparatus and in Energy Metabolism in Chlamydomonas reinhardtii

PGRL1 RNA and protein levels are increased in iron-deficient Chlamydomonas reinhardtii cells. In an RNAi strain, which accumulates lower PGRL1 levels in both iron-replete and -starved conditions, the photosynthetic electron transfer rate is decreased, respiratory capacity in iron-sufficient conditions is increased, and the efficiency of cyclic electron transfer under iron-deprivation is diminished. Pgrl1-kd cells exhibit iron deficiency symptoms at higher iron concentrations than wild-type cells, although the cells are not more depleted in cellular iron relative to wild-type cells as measured by mass spectrometry. Thiol-trapping experiments indicate iron-dependent and redox-induced conformational changes in PGRL1 that may provide a link between iron metabolism and the partitioning of photosynthetic electron transfer between linear and cyclic flow. We propose, therefore, that PGRL1 in C. reinhardtii may possess a dual function in the chloroplast; that is, iron sensing and modulation of electron transfer.

In situ photochemical activity of the phytobenthic communities in two Antarctic lakes

Photochemical activity of phytobenthic communities in two freshwater lakes in East Antarctica was estimated using a submersible pulse-amplitude modulation (PAM) chlorophyll fluorometer, to answer the following questions: (1) Are the communities under bright summer photosynthetically active radiation (PAR) photosynthetically active? (2) If active, which community shows the most active signals? (3) Where is the most productive part (or depth) in the lake? Our limnological measurements indicated the two lakes were ultra-oligotrophic. Diving observations revealed that the phytobenthos of the lakes was moss-dominated which had different life-forms (moss shoots in shallow depths of both lakes, moss-pillars in the shallow lake, pinnacle moss-microbial complex community in the deeper lake). In addition, various mat-forming microbial communities inhabited the lake beds. In situ measurements of photochemical parameters indicated that shoots of mosses living just below the littoral slope, and the apical part of the moss pillars, had the highest photosynthetic activity in open water summer conditions, but mat-forming microbial communities and the other moss-microbial complex communities, showed rather lower activity. Most of the mat-forming phytobenthos surface also showed positive photosynthetic activity, but there were some cases of negligible signals in the shallow depth. This suggests that the photosynthetic activities of mat-forming communities in the shallow water were suppressed by strong ambient light in summer.

Freezing cytorrhysis and critical temperature thresholds for photosystem II in the peat moss Sphagnum capillifolium

Leaflets of Sphagnum capillifolium were exposed to temperatures from -5 degrees C to +60 degrees C under controlled conditions while mounted on a microscope stage. The resultant cytological response to these temperature treatments was successfully monitored using a light and fluorescence microscope. In addition to the observable cytological changes during freezing cytorrhysis and heat exposure on the leaflets, the concomitant critical temperature thresholds for inactivation of photosystem II (PS II) were studied using a micro fibre optic and a chlorophyll fluorometer mounted to the microscope stage. Chlorophyllous cells of S. capillifolium showed extended freezing cytorrhysis immediately after ice nucleation at -1.1 degrees C in the water in which the leaflets were submersed during the measurement. The occurrence of freezing cytorrhysis, which was visually manifested by cell shrinkage, was highly dynamic and was completed within 2 s. A total reduction of the mean projected diameter of the chloroplast containing area during freezing cytorrhysis from 8.9 to 3.8 microm indicates a cell volume reduction of approximately -82%. Simultaneous measurement of chlorophyll fluorescence of PS II was possible even through the frozen water in which the leaf samples were submersed. Freezing cytorrhysis was accompanied by a sudden rise of basic chlorophyll fluorescence. The critical freezing temperature threshold of PS II was identical to the ice nucleation temperature (-1.1 degrees C). This is significantly above the temperature threshold at which frost damage to S. capillifolium leaflets occurs (-16.1 degrees C; LT(50)) which is higher than observed in most higher plants from the European Alps during summer. High temperature thresholds of PS II were 44.5 degrees C which is significantly below the heat tolerance of chlorophyllous cells (49.9 degrees C; LT(50)). It is demonstrated that light and fluorescence microscopic techniques combined with simultaneous chlorophyll fluorescence measurements may act as a useful tool to study heat, low temperature, and ice-encasement effects on the cellular structure and primary photosynthetic processes of intact leaf tissues.