Fluorescence kinetic parameters and cyclic electron transport in guard cell chloroplasts of chlorophyll-deficient leaf tissues from variegated weeping fig (<Emphasis Type="Italic">Ficus benjamina</Emphasis> L.)

Residual chlorophyll in chlorophyll-deficient (albino) areas of variegated leaves of Ficus benjamina originates from guard cell chloroplasts. Photosynthetic features of green and albino sectors of F. benjamina were studied by imaging the distribution of the fluorescence decrease ratio Rfd within a leaf calculated from maximum (Fm) and steady-state leaf chlorophyll fluorescence (Fs) at 690 and 740 nm. Local areas of albino sectors demonstrated an abnormally high Rfd₇₄₀/Rfd₆₉₀ ratio. Fluorescence transients excited in albino sectors at red (640 and 690 nm) wavelengths showed an abrupt decrease of the Rfd values (0.4 and 0.1, correspondingly) as compared with those excited at blue wavelengths (1.7–2.4). This “Red Drop” was not observed for green sectors. Normal and chlorophyll-deficient leaf sectors of F. benjamina were also tested for linear and cyclic electron transport in thylakoids. The tests have been performed studying fluorescence at a steady-state phase with CO₂-excess impulse feeding, photoacoustic signal generated by pulse light source at wavelengths selectively exciting PSI, fluorescence kinetics under anaerobiosis and fluorescence changes observed by dual-wavelength excitation method. The data obtained for albino sectors strongly suggest the possibility of a cyclic electron transport simultaneously occurring in guard cell thylakoids around photosystems I and II under blue light, whereas linear electron transport is absent or insufficient.     

Age-dependent response of maize leaf segments to cadmium treatment: effect on chlorophyll fluorescence and phytochelatin accumulation

The relationship between the age of leaf tissue and response of the photosynthetic apparatus and phytochelatin accumulation to Cd treatment was studied. Studies were carried out with seedlings of Zea mays L. cv. Hidosil grown in the presence of 100-200 mumol/L Cd for 14 days under low light conditions. The third leaf was divided into 3 segments of equal length differing in the stage of tissue maturity and used for measurements of chlorophyll content, chlorophyll fluorescence, glutathione and phytochelatin content and Cd accumulation. A close relationship between the age of leaf tissue and response of the photosynthetic apparatus to Cd was shown. Cadmium (200 mumol/L) reduced photochemical processes more in older than younger leaf segments as seen in the Chl fluorescence parameters Fv/F0, and t1/2, while the chlorophyll fluorescence decrease ratio (Rfd) was inhibited more strongly in younger ones. Fv/Fm was slightly affected. Cd-induced enhancement of GSH content was correlated with higher phytochelatin accumulation to a greater extent in younger than in older leaf segments. Phytochelatin level corresponded to changes of photochemical processes in older leaves. The peptide thiol:Cd molar ratio for the phytochelatins varied depending on Cd concentration and age of leaf segments. The protective role of phytochelatins for the photosynthetic apparatus is discussed.     

Chlorophyll fluorescence induction and estimation of plant resistance to stress factors

The usage of chlorophyll fluorescence induction (CFI) for estimating various types of plant resistance (primary, general, initial, adaptive) to stress factors is reviewed. The necessity of ontogenetic approach (considering the age-specific properties of the photosynthetic apparatus) in determining general and adaptive resistance of plants to prolonged action of stress factors by the CFI method is argued. In the plant Cucumbis sativus L., the possibility is shown of using age-specific qualitative and quantitative traits of leaf CFI (changes in the shape of chlorophyll fluorescence induction curves and in the dynamics of CFI parameters in the course of leaf ontogeny) for comparative study of differences between fully active and stressed plants. Possible criteria are suggested for estimating the effect of outer stress factors by the presence or absence of a steady-state phase in the dynamics of CFI parameters during leaf ontogeny. It is also suggested to use the duration of the steady-state phase following the termination of leaf growth (estimated by the dynamics of the slow phase of CFI as the ratio of fluorescence intensity at the peak P and the steady-state fluorescence intensity, Fp/Fs, or as the viability index Rfd) and the variability of CFI parameters during this period as qualitative estimates of plant resistance to prolonged action of stress factors.     

Developmental changes in spatial distribution of in vivo fluorescence and epidermal UV absorbance over Quercus petraea leaves

Background and Aims

Epidermal phenolic compounds (mainly flavonoids) constitute a vital screen that protects the leaf from damage by natural ultraviolet (UV) radiation. The effectiveness of epidermal UV-screening depends on leaf anatomy, the content of UV-screening compounds and their spatial uniformity over the leaf area. To investigate in vivo the spatial pattern of the epidermal UV-screen during leaf development, a fluorescence imaging method was developed to map the epidermal UV-absorbance at a microscopic scale. This study was done on oak (Quercus petraea) leaves that were used as a model of woody dicotyledonous leaves.

Methods

The leaf development of 2-year-old trees, grown outdoors, was monitored, at a macroscopic scale, by in vivo measurements of chlorophyll content per unit area and epidermal UV-absorbance using two optical leaf-clip meters. The distribution of pigments within leaves was assessed in vivo spectroscopically. The microscopic images of UV-induced fluorescence and UV-absorbance acquired in vivo during leaf development were interpreted from spectral characteristics of leaves.

Key Results

At a macroscopic scale, epidermal UV-absorbance was high on the upper leaf side during leaf development, while it increased on the lower leaf side during leaf expansion and reached the adaxial value at maturity. At a microscopic scale, in immature leaves, for both leaf sides, the spatial distribution of epidermal UV-absorbance was heterogeneous, with a pattern depending on the flavonoid content of vacuoles in developing epidermal cells. At maturity, epidermal UV-absorbance was uniform.

Conclusions

The spatial pattern of epidermal UV-screen over the area of oak leaves is related to leaf anatomy during development. In vivo spectroscopy and fluorescence imaging of the leaf surface showed the distribution of pigments within the leaf and hence can provide a tool to monitor optically the leaf development in nature.Key words: Blue-green fluorescence, chlorophyll fluorescence, epidermis, flavonoids, leaf development, microscopic imaging, polyphenols, Quercus petraea     

Image analysis of chlorophyll fluorescence transients for diagnosing the photosynthetic system of attached leaves

A new image instrumentation system for quantitative analysis of the rapid change in intensity of chlorophyll fluorescence during dark-light transition (CFI, chlorophyll fluorescence induction), which is a sensitive indicator of the various reactions of photosynthesis, was developed and its performance was evaluated. This system made it possible to resolve CFI at any small leaf area (about 1 square millimeter) of a whole leaf when the plant was illuminated by blue-green light at more than 50 micromoles photons per square meter per second. In order to test the usefulness of this system, we applied it to analyze the effect of SO₂ on photosynthetic apparatus in attached sunflower leaves. Dynamic CFI imaging over the whole single leaf, where there was no visible injury, indicated not only the local changes in photosynthetic activity but also the site of inhibition in photosynthetic electron transport system in chloroplasts. The new instrumentation system will be useful for the analytical diagnosis of various stress-actions on plants in situ.     

Blue,Green and Red Fluorescence Signatures and Images of Tobacco Leaves*

Laser-induced fluorescence images of the leaf of an aurea mutant of Nicotiana tabacum were recorded for the blue and green fluorescence at 440 and 520 nm and the red chlorophyll fluorescence at 690 and 735 nm. The results obtained were compared with direct measurements of the fluorescence emission spectra of leaves using a conventional spectrofluorometer. The highest emission of blue (F440) and green fluorescence (F520) within the leaf was found in the leaf veins, particularly the main leaf vein. In contrast, the intercostal fields of leaves, which exhibited the highest chlorophyll content, showed only a very low blue and green fluorescence emission, which was much lower than the red and far-red chlorophyll fluorescence emission bands (F690 and F735). Correspondingly, the ratio of blue to red leaf fluorescence F440/F690 of upper and lower leaf side was much higher in the leaf veins (values 1.2 to 1.5) than in intercostal fields (values of 0.6 to 0.7). The results also demonstrated that in the intercostal fields the major part of the blue-green fluorescence was reabsorbed by chlorophylls and carotenoids. A partial reabsorption of the red fluorescence band near 690 nm by leaf chlorophyll took place, but did not affect the far-red fluorescence band near F735. As a consequence the chlorophyll fluorescence ratio F690/F735 exhibited significantly higher values in the chlorophyll-poor leaf vein regions (1.7 to 1.8) than in the chlorophyll-rich intercostal fields (0.8 to 1.3). Imaging spectroscopy of leaves was shown to be much more precise than the screening of fluorescence signatures by conventional fluorometers. It clearly demonstrated that the blue-green fluorescence and the red chlorophyll fluorescence of leaves exhibit an inverse contrast to each other. The advantage of the fluorescence imaging spectroscopy, which allows the simultaneous screening of the whole leaf surface and distinct parts of it, and its possible application in the detection of stress effects or local damage by insects and pathogens, is discussed.     

A system for imaging transverse distribution of scattered light and chlorophyll fluorescence in intact rice leaves

In order to examine the transverse distribution of scattered light and chlorophyll fluorescence in intact rice leaves, a micro-fluorescence imaging system was devised using a microscope, a CCD camera with an image intensifier, an Ar and a He-Ne laser light source, an image processor, and a microcomputer. A laser light was projected vertically on to the surface of a rice leaf segment at a cut-edge, and scattered light and induced fluorescence were observed at the cut-section from a 90° angle to the axis of the laser beam. The intensity of scattered light showed a maximum at several micrometres depth from the leaf surface and a steep gradient afterwards. Fluorescence reached a maximum crossing with the decline curve of the scattered light. The maximum of fluorescence measured at 741 nm was observed at a greater depth from the leaf surface than that at 687 nm, suggesting that part of the fluorescence of the longer wavelength was emitted due to absorption of fluorescence of the shorter wavelength. Profiles of the scattered light and the chlorophyll fluorescence depended on leaf anatomy.     

Leaf anatomy of the mosaic <Emphasis Type="Italic">Ficus benjamina</Emphasis> cv. Starlight and interaction of source and sink chimera components

Leaf anatomy was studied in the mosaic Ficus benjamina cv. Starlight and non-chimeric Ficus benjamina cv. Daniel. The number of chloroplasts in a white, chlorophyll-deficient tissue declines as compared to the green tissue. However, their functional activity is retained. The leaf of the mosaic F. benjamina contains two or, sometimes, three subepidermal layers. Mesophyll forms one layer in the green and white parts of leaf palisade and one white and one green layer in the transitional zone (edge). In the transitional zone, green spongy mesophyll is located between two white spongy layers and the proportion of photosynthesizing cells varies. In cv. Daniel, there are two subepidermal layers and one layer of columnar mesophyll cells. According to the morphometry data, the proportion of white zone in the leaf correlates with the leaf position in the whole shoot: the higher the branch order, the larger the proportion of white zone. The total leaf area depends also on its position in the shoot. No such correlation was found in non-chimeric F. benjamina cv. Daniel. In the mosaic chimera, the source-sink status appears to depend on the leaf position in the shoot. Experiments with individual shoots of the same order and elimination of all lateral shoots have shown that the proportion of white zone in new leaves on the shoot increases with the total area of green zone. Thus, the area of assimilating shoot surface affects the formation of leaves in the meristem. A hypothesis was put forward that the source-sink state affects the ratio of green and white parts in the leaf primordium. Products of photosynthesis (carbohydrates) are a possible metabolic signal affecting the meristem. It cannot be excluded as well that the hormonal state undergoes changes in the chimeric plant.     

用体内叶绿素a荧光诱导动力学鉴定番茄的抗冷性

研究了冷害温度对具有不同抗冷性品种的番茄叶片的体内叶绿素a荧光诱导动力曲线的影响。实验结果指出,在低温处理(8℃,5℃,2℃下,暗中24小时)后,番茄叶片的体内叶绿素a荧光诱导动力学曲线有了明显的改变,Fv／Fo值、Rfd值降低了,光系统II原初光能转换效率和潜在的光合活力均受到抑制。我们在苗期和开花期得到的实验结果均表明,在番茄叶片的叶绿素a荧光诱导动力学曲线和这些荧光参数改变的程度与该品种的已知抗冷性之间呈现较好的相关性。我们认为,体内叶绿素a荧光诱导动力学方法是鉴定番茄抗冷性的一个快速、灵敏和可靠的方法,并可用于其他绿色植物的抗冷性鉴定中。     

Measurement of Differences in Red Chlorophyll Fluorescence and Photosynthetic Activity between Sun and Shade Leaves by Fluorescence Imaging

With a flash-lamp chlorophyll (Chl) fluorescence imaging system (FL-FIS) the photosynthetic activity of several thousand image points of intact shade and sun leaves of beech were screened in a non-destructive way within a few seconds. The photosynthetic activity was determined via imaging the Chl fluorescence at maximum F_p and steady state fluorescence F_s of the induction kinetics (Kautsky effect) and by a subsequent determination of the images of the fluorescence decrease ratio R_Fd and the ratio F_p/F_s. Both fluorescence ratios are linearly correlated to the photosynthetic CO₂ fixation rates. This imaging method permitted to detect the gradients in photosynthetic capacity and the patchiness of photosynthetic quantum conversion across the leaf. Sun leaves of beech showed a higher photosynthetic capacity and differential pigment ratios (Chl a/b and Chls/carotenoids) than shade leaves. Profile analysis and histogram of the Chl fluorescence yield and the Chl fluorescence ratios allow to quantify the differences in photosynthetic activity between different leaf parts and between sun and shade leaves with a high statistical significance.     

Uptake of the Herbicide Diuron as Visualised by the Fluorescence Imaging Technique

A new fluorescence imaging system for monitoring the uptake of the PSII-herbicide diuron (OCMU) was tested in tobacco leaves. UV-laser-induced (Λ_exc = 355 nm) fluorescence images were collected for blue fluorescence F440 (Λ_em = 440 nm), green fluorescence F520 (Λ_em = 520 nm), red chlorophyll fluorescence F690 (Λ_em = 690 nm) and for far-red chlorophyll fluorescence F740 (Λ_em = 740 nm). Diuron-treated leaf parts exhibited a higher red and far-red chlorophyll fluorescence emission (F690 and F740) than untreated leaf halves, whereas the blue and green fluorescence, F440 and F520, remained unaffected. As a consequence, the fluorescence ratios blue/red (F440/F690) and blue/far-red (F440/F740) significantly decreased in diuron-treated leaf parts. The time course of diuron uptake into the leaf could be followed by fluorescence images taken 10 and 30 min after diuron application. The novel high resolution fluorescence imaging method supplies information on the herbicide uptake of each point of the leaf area. Its great advantage as compared to the point data fluorescence measurements applied so far is discussed.     

Chlorophyll fluorescence imaging for disease-resistance screening of sugar beet

Both biotic and abiotic stresses cause considerable crop yield losses worldwide (Chrispeels, Sadava Plants, genes, and crop biotechnology 2003; Oerke, Dehne Crop Prot 23:275–285 2004). To speed up screening assays in stress resistance breeding, non-contact techniques such as chlorophyll fluorescence imaging can be advantageously used in the quantification of stress-inflicted damage. In comparison with visual spectrum images, chlorophyll fluorescence imaging reveals cell death with higher contrast and at earlier time-points. This technique has the potential to automatically quantify stress-inflicted damage during screening applications. From a physiological viewpoint, screening stress-responses using attached plant leaves is the ideal approach. However, leaf growth and circadian movements interfere with time-lapse monitoring of leaves, making it necessary to fix the leaves to be studied. From this viewpoint, a method to visualise the evolution of chlorophyll fluorescence from excised leaf pieces kept in closed petri dishes offers clear advantages. In this study, the plant–fungus interaction sugar beet–Cercospora beticola was assessed both in attached leaf and excised leaf strip assays. The attached leaf assay proved to be superior in revealing early, pre-visual symptoms and to better discriminate between the lines with different susceptibility to Cercospora.     

Application of chlorophyll fluorescence in ecophysiology

Summary In vivo chlorophyll fluorescence measurements have become a valuable tool in ecophysiology. Fluorescence emission spectra are influenced by the reabsorption of the tissue and indicate the composition of the antenna system and are influenced by the chlorophyll content per leaf area. The fluorescence induction kinetics (Kautsky effect) can be used to study photosynthetic activity. These rapid, non-destructive methods can be applied for ecophysiological field research to check the vitality of plants and to document stress effects on the photosynthetic apparatus. The Rfd-values (Rfd=fd/fs), the ratio of the fluorescence decrease (fd) to the steady state fluorescence (fs), can be taken as a rapid vitality index of the leaves and trees. We here describe fundamental chlorophyll fluorescence results of leaves which are needed for the interpretation of in vivo fluorescence signatures in stress physiology and in the forest dieback research.     

Development of a digital image analysis method for real-time estimation of chlorophyll content in micropropagated potato plants

The present work describes a digital image analysis method based on leaf color analysis to estimate chlorophyll content of leaves of micropropagated potato plantlets. For estimation of chlorophyll content, a simple leaf digital analysis procedure using a simple digital still camera was applied in parallel to a SPAD chlorophyll content meter. RGB features were extracted from the image and correlated with the SPAD values. None of the mean brightness parameters (RGB) were correlated with the actual chlorophyll content following simple correlation studies. However, a correlation between the chromaticity co-ordinates ‘r’, ‘b’ and chlorophyll content was observed, while co-ordinate ‘g’ was not significantly correlated with chlorophyll content. Linear regression and artificial neural networks (ANN) were applied for correlating the mean brightness (RGB) and mean brightness ratio (rgb) features to chlorophyll content of plantlet leaves determined through a SPAD meter. The chlorophyll content as determined by the SPAD meter was significantly correlated (RMSE = 3.97 and 3.59, respectively, for linear and ANN models) to the rgb values of leaf image analysis. Both the models indicate successful prediction of chlorophyll content of leaves of micropropagated plants with high correlation. The developed RGB-based digital image analysis has the advantage over conventional subjective methods for being objective, fast, non-invasive, and inexpensive. The system could be utilized for real-time estimation of chlorophyll content and subsequent analysis of photosynthetic and hyperhydric status of the micropropagated plants for better ex vitro survival.     

Profiles of light absorption and chlorophyll within spinach leaves from chlorophyll fluorescence

Chlorophyll fluorescence was used to estimate profiles of absorbed light within chlorophyll solutions and leaves. For chlorophyll solutions, the intensity of the emitted fluorescence declined in a log–linear manner with the distance from the irradiated surface as predicted by Beer's law. The amount of fluorescence was proportional to chlorophyll concentration for chlorophyll solutions given epi‐illumination on a microscope slide. These relationships appeared to hold for more optically complex spinach leaves. The profile of chlorophyll fluorescence emitted by leaf cross sections given epi‐illumination corresponded to chlorophyll content measured in extracts of leaf paradermal sections. Thus epifluorescence was used to estimate relative chlorophyll content through leaf tissues. Fluorescence profiles across leaves depended on wavelength and orientation, reaching a peak at 50–70 µm depth. By infiltrating leaves with water, the pathlengthening due to scattering at the airspace : cell wall interfaces was calculated. Surprisingly, the palisade and spongy mesophyll had similar values for pathlengthening with the value being greatest for green light (550 > 650 > 450 nm). By combining fluorescence profiles with chlorophyll distribution across the leaf, the profile of the apparent extinction coefficient was calculated. The light profiles within spinach leaves could be well approximated by an apparent extinction coefficient and the Beer–Lambert/Bouguer laws. Light was absorbed at greater depths than predicted from fibre optic measurements, with 50% of blue and green light reaching 125 and 240 µm deep, respectively.     

Extended depth-of-focus imaging of chlorophyll fluorescence from intact leaves

Imaging dynamic changes in chlorophyll a fluorescence provides a valuable means with which to examine localised changes in photosynthetic function. Microscope-based systems provide excellent spatial resolution which allows the response of individual cells to be measured. However, such systems have a restricted depth of focus and, as leaves are inherently uneven, only a small proportion of each image at any given focal plane is in focus. In this report we describe the development of algorithms, specifically adapted for imaging chlorophyll fluorescence and photosynthetic function in living plant cells, which allow extended-focus images to be reconstructed from images taken in different focal planes. We describe how these procedures can be used to reconstruct images of chlorophyll fluorescence and calculated photosynthetic parameters, as well as producing a map of leaf topology. The robustness of this procedure is demonstrated using leaves from a number of different plant species. This revised version was published online in June 2006 with corrections to the Cover Date.     

The ontogenetic approach to chlorophyll fluorescence studies of plant photosynthetic apparatus under stressful conditions

Based on the analysis of reasons limiting the application of the method of chlorophyll fluorescence induction for estimating the state of the leaf photosynthetic apparatus under prolonged stress, the necessity of the ontogenetic approach consisting in a more exact determination of leaf age was substantiated. A comparison of the calendar and ontogenetic ways of determination of age of cucumber leaves under controlled conditions revealed essential distinctions in the estimation of plant leaf photosynthetic apparatus by the method of chlorophyll fluorescence induction for two variants distinguishing by the cultivation light regime ("white", 400-700 nm, and "red", 600-700 nm). It was shown that, in the case of prolonged effect of the stress factor on the plant, the unambiguity of the interpretation of chlorophyll fluorescence induction parameters in the estimation of the state of their photosynthetic apparatus depends essentially on the choice of the ontogenetic period of leaves of plants being compared and the accuracy of determination of leaf age.     

Chlorophyll fluorescence imaging of photosynthetic activity with the flash-lamp fluorescence imaging system

A flash-lamp chlorophyll (Chl) fluorescence imaging system (FL-FIS) is described that allows to screen and image the photosynthetic activity of several thousand leaf points (pixels) of intact leaves in a non-destructive way within a few seconds. This includes also the registration of several thousand leaf point images of the four natural fluorescence bands of plants in the blue (440 nm) and green (520 nm) regions as well as the red (near 690 nm) and far-red (near 740 nm) Chl fluorescence. The latest components of this Karlsruhe FL-FIS are presented as well as its advantage as compared to the classical single leaf point measurements where only the fluorescence information of one leaf point is sensed per each measurement. Moreover, using the conventional He-Ne-laser induced two-wavelengths Chl fluorometer LITWaF, we demonstrated that the photosynthetic activity of leaves can be determined measuring the Chl fluorescence decrease ratio, R_Fd (defined as Chl fluorescence decrease F_d from maximum to steady state fluorescence F_s:F_d/F_s), that is determined by the Chl fluorescence induction kinetics (Kautsky effect). The height of the values of the Chl fluorescence decrease ratio R_Fd is linearly correlated to the net photosynthetic CO₂ fixation rate P _N as is indicated here for sun and shade leaves of various trees that considerably differ in their P _N. Imaging the R_Fd-ratio of intact leaves permitted the detection of considerable gradients in photosynthetic capacity across the leaf area as well as the spatial heterogeneity and patchiness of photosynthetic quantum conversion within the control leaf and the stressed plants. The higher photosynthetic capacity of sun versus shade leaves was screened by Chl fluorescence imaging. Profile analysis of fluoresence signals (along a line across the leaf area) and histograms (the signal frequency distribution of the fluorescence information of all measured leaf pixels) of Chl fluorescence yield and Chl fluorescence ratios allow, with a high statistical significance, the quantification of the differences in photosynthetic activity between various areas of the leaf as well as between control leaves and water stressed leaves. The progressive uptake and transfer of the herbicide diuron via the petiole into the leaf of an intact plant and the concomitant loss of photosynthetic quantum conversion was followed with high precision by imaging the increase of the red Chl fluorescence F₆₉₀. Differences in the availability and absorption of soil nitrogen of crop plants can be documented via this flash-lamp fluorescence imaging technique by imaging the blue/red ratio image F₄₄₀/F₆₉₀, whereas differences in Chl content are detected by collecting images of the fluorescence ratio red/far-red, F₆₉₀/F₇₄₀.     

Fluorescence emission spectra of Photosystem I,Photosystem II and the light-harvesting chlorophyll a/b complex of higher plants

Fluorescence emission spectra excited at 514 and 633 nm were measured at ?196 °C on dark-grown bean leaves which had been partially greened by a repetitive series of brief xenon flashes. Excitation at 514 nm resulted in a greater relative enrichment of the 730 nm emission band of Photosystem I than was obtained with 633 nm excitation. The difference spectrum between the 514 nm excited fluorescence and the 633 nm excited fluorescence was taken to be representative of a pure Photosystem I emission spectrum at ?196 °C. It was estimated from an extrapolation of low temperature emission spectra taken from a series of flashed leaves of different chlorophyll content that the emission from Photosystem II at 730 nm was 12% of the peak emission at 694 nm. Using this estimate, the pure Photosystem I emission spectrum was subtracted from the measured emission spectrum of a flashed leaf to give an emission spectrum representative of pure Photosystem II fluorescence at ?196 °C. Emission spectra were also measured on flashed leaves which had been illuminated for several hours in continuous light. Appreciable amounts of the light-harvesting chlorophyll a/b protein, which has a low temperature fluorescence emission maximum at 682 nm, accumulate during greening in continuous light. The emission spectra of Photosystem I and Photosystem II were subtracted from the measured emission spectrum of such a leaf to obtain the emission spectrum of the light-harvesting chlorophyll a/b protein at ?196 °C.     

Chlorophyll a fluorescence and carbon assimilation in developing leaves of light-grown cucumber

The development of photochemical activity and carbon assimilation in light-grown cucumber (Cucumis sativus L. cv Natsusairaku) leaves was studied to determine the pattern of acquisition and its relationship to leaf growth and expansion. Measurements of chlorophyll a fluorescence showed that leaves acquire photochemical function over a period of 6 or more days, and gas exchange studies showed increases in carbon assimilation over a parallel time period. As leaves expand and mature, they undergo a sequential, three-step series of changes in fluorescence response. The initial kinetics show the absence of wholly functional quenching mechanisms. Dynamic imaging of fluorescence kinetics showed that a temporal series of changes occurred within defined areas of individual developing leaves. The spatial acquisition of photochemical activity in leaves was basipetal as is their directional expansion, development of air spaces and stomata, and the cessation of imported carbon.