Claus Buschmann

Photosynthetic activity, chloroplast ultrastructure, and leaf characteristics of high-light and low-light plants and of sun and shade leaves

The photosynthetic CO2-fixation rates, chlorophyll content, chloroplast ultrastructure and other leaf characteristics (e.g. variable fluorescence, stomata density, soluble carbohydrate content) were studied in a comparative way in sun and shade leaves of beech (Fagus sylvatica) and in high-light and low-light seedlings.1.Sun leaves of the beech possess a smaller leaf area, higher dry weight, lower water content, higher stomata density, higher chlorophyll a/b ratios and are thicker than the shade leaves. Sun leaves on the average contain more chlorophyll in a leaf area unit; the shade leaf exhibits more chlorophyll on a dry weight basis. Sun leaves show higher rates for dark respiration and a higher light saturation of photosynthetic CO2-fixation. Above 2000 lux they are more efficient in photosynthetic quantum conversion than the shade leaves.2.The development of HL-radish plants proceeds much faster than that of LL-plants. The cotyledons of HL-plants show a higher dry weight, lower water content, a higher ratio of chlorophyll a/b and a higher gross photosynthesis rate than the cotyledons of the LL-plants, which possess a higher chlorophyll content per dry weight basis. The large area of the HL-cotyledon on the one hand, as well as the higher stomata density and the higher respiration rate in the LL-cotyledon on the other hand, are not in agreement with the characteristics of sun and shade leaves respectively.3.The development, growth and wilting of wheat leaves and the appearance of the following leaves (leaf succession) is much faster at high quanta fluence rates than in weak light. The chlorophyll content is higher in the HL-leaf per unit leaf area and in the LL-leaf per g dry weight. There are no differences in the stomata density and leaf area between the HL- and LL-leaf. There are fewer differences between HL- and LL-leaves than in beech or radish leaves.4.The chloroplast ultrastructure of shade-type chloroplasts (shade leaves, LL-leaves) is not only characterized by a much higher number of thylakoids per granum and a higher stacking degree of thylakoids, but also by broader grana than in sun-type chloroplasts (sun leaves, HL-leaves). The chloroplasts of sun leaves and of HL-leaves exhibit large starch grains.5.Shade leaves and LL-leaves exhibit a higher maximum chlorophyll fluorescence and it takes more time for the fluorescence to decline to the steady state than in sun and HL-leaves. The variable fluorescence VF (ratio of fluorescence decrease to steady state fluorescence) is always higher in the sun and HL-leaf of the same physiological stage (maximum chlorophyll content of the leaf) than in the shade and LL-leaf. The fluorescence emission spectra of sun and HL-leaves show a higher proportion of chlorophyli fluorescence in the second emission maximum F2 than shade and LL-leaves.6.The level of soluble carbohydrates (reducing sugars) is significantly higher in sun and HL-leaves than in shade and LL-leaves and even reflects changes in the amounts of the daily incident light.7.Some but not all characteristics of mature sun and shade leaves are found in HL- and LL-leaves of seedlings. Leaf thickness, dry weight, chlorophyll content, soluble carbohydrate level, photosynthetic CO2-fixation, height and width of grana stacks and starch content, are good parameters to describe the differences between LL- and HL-leaves; with some reservations concerning age and physiological stage of leaf, a/b ratios, chlorophyll content per leaf area unit and the variable fluorescence are also suitable.

How to correctly determine the different chlorophyll fluorescence parameters and the chlorophyll fluorescence decrease ratio RFd 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 Fv, Fm, F0, Fm′, Fv′, NF, and ΔF, the Chl fluorescence ratios Fv/Fm, Fv/F0, ΔF/Fm′, as well as the photochemical (qP) and non-photochemical quenching coefficients (qN, qCN, and NPQ). qN consists of three components (qN = qE + qT + qI), 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, qN, qCN, and NPQ increased in both leaf sides, but to a much higher extent at the lower compared to the upper leaf side. qN was the best indicator for non-photochemical quenching even during a stronger HL-stress, whereas qCN 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 RFd (Fd/Fs), which, when measured at saturation irradiance is directly correlated to the net CO2 assimilation rate (PN) of leaves. This RFd-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 RFd-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.

Application of chlorophyll fluorescence in ecophysiology

SummaryIn 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.

The chlorophyll fluorescence ratio F735/F700 as an accurate measure of the chlorophyll content in plants

Abstract A remote sensing technique is presented to estimate the chlorophyll content in higher plants. The ratio between chlorophyll fluorescence at 735 nm and in the range 700–710 nm, F735/F700 was found to be linearly proportional to the chlorophyll content (with determination coefficient, r2, more than 0.95), and, thus, this ratio can be used as a precise indicator of chlorophyll content in plant leaves. This new chlorophyll fluorescence ratio indicates chlorophyll levels with high precision- the error in chlorophyll prediction over a wide range of chlorophyll content (from 41 to 675 mg m−2) was less than 40 mg m−2. The technique was tested and validated in three plant species: beech (Fagus sylvatica L.), elm (Ulmus minor Miller), and wild vine (Parthenocissus tricuspidata L.).

Leaf chlorophyll fluorescence corrected for re-absorption by means of absorption and reflectance measurements

Summary The in vivo emission spectra of chlorophyll (Chi) fluorescence of green leaves, taken at room temperature, show two maxima near 685 nm and 735 nm. The shape of the spectra is modified by re-absorption processes and depends on the Chl content of leaves. By performing reflectance, transmission and fluorescence measurements, we investigated the reason for the repeatedly reported increase of the fluorescence ratio F685/F735 during stress or damage-induced breakdown of chlorophyll in plants. The in vivo spectra of Chi fluorescence (F), transmittance (T) and reflectance (R) were taken at room temperature at the same leaf spot. Leaves with a wide range of green colour (Chl content varied from 70 to 670 mg m 2 ) were chosen from a beech ( Fagus sylvatica L.) and an elm tree ( Ulmus minor Miller) as well as from a wild vine shrub ( Parthenocissus tricuspidata L.). Strict linear correlations (with a determination coefficient of r 2 >0.92) were found between the ratio F685/F735 on the one hand, and (i) the ratio of the non-absorbed radiation (R685+T685)/(R735+T735), (ii) the reflectance ratio R685/R735, and (iii) the reflectance at 685 nm, on the other hand. The results demonstrate that 92% or more of the ratio F685/F735 variation in leaves during development or at damage and stress events is determined by the variation in Chl content and corresponding changes of the optical properties of leaves. The Chi fluorescence emission spectrum has been corrected at each wavelength for re-absorption by means of non-absorbed radiation (R + T) and reflectance yielding the actually emitted retrieved Chl fluorescence. The retrieved Chl fluorescence at 685 and 735 nm is found to linearily increase with the Chl content. The shape of the retrieved Chl fluorescence spectrum was very similar to that of strongly diluted Chl a in solution, only the position of the emission maxima is different (673 nm of Chl a in ethanol solution, and 685 nm in leaves). Studies on the actually emitted «true» Chl fluorescence, as retrieved from room temperature and outdoor fluorescence measurements, may show new ways of plant stress detection.

Imaging of the blue, green, and red fluorescence emission of plants: an overview.

An overview is given on the fluorescence imaging of plants. Emphasis is laid upon multispectral fluorescence imaging in the maxima of the fluorescence emission bands of leaves, i.e., in the blue (440 nm), green (520 nm), red (690 nm), and far-red (740 nm) spectral regions. Details on the origin of these four fluorescence bands are presented including emitting substances and emitting sites within a leaf tissue. Blue-green fluorescence derives from ferulic acids covalently bound to cell walls, and the red and far-red fluorescence comes from chlorophyll (Chl) a in the chloroplasts of green mesophyll cells. The fluorescence intensities are influenced (1) by changes in the concentration of the emitting substances, (2) by the internal optics of leaves determining the penetration of excitation radiation and partial re-absorption of the emitted fluorescence, and (3) by the energy distribution between photosynthesis, heat production, and emission of Chl fluorescence. The set-up of the Karlsruhe multispectral fluorescence imaging system (FIS) is described from excitation with UV-pulses to the detection with an intensified CCD-camera. The possibilities of image processing (e.g., formation of fluorescence ratio images) are presented, and the ways of extraction of physiological and stress information from the ratio images are outlined. Examples for the interpretation of fluorescence images are given by demonstrating the information available for the detection of different developmental stages of plant material, of strain and stress of plants, and of herbicide treatment. This novel technique can be applied for near-distance screening or remote sensing.

Adaptation of Chloroplast-Ultrastructure and of Chlorophyll- Protein Levels to High-Light and Low-Light Growth Conditions

Abstract Adaptation In saturating light radish seedlings grown in high-light growth conditions (90 W · m-2) possess a much higher photosynthetic capacity on a chlorophyll and leaf area basis than the low-light grown plants (10 W · m-2). The higher CO2-fixation rate of HL-plants is due to the presence of HL-chloroplasts which possess a different ultrastructure and also different levels of the individual chlorophyll-carotenoid-proteins than the LL-chloroplasts of LL-seedlings. 1. Ultrastructure: The high-light adapted chloroplasts are characterized by fewer photo synthetic membranes per chloroplast section, by low grana stacks (only few thylakoids per granum), a lower stacking degree of thylakoids, a higher proportion of non-appressed membranes (stroma thylakoids + end grana membranes) and a high starch content. The LL-chloroplasts possess no starch, their grana stacks are higher (up to 17 thylakoids per granum) and also significantly broader than that of HL-chloroplasts. 2. Chlorophyll-proteins: The photosynthetic apparatus of HL-chloroplasts contains a larger proportion of chlorophyll a-proteins of photosystem I (CPIa + CPI) and of photosystem II (CPa, the presumable reaction center of PS II) than the LL-chloroplasts which possess a higher proportion of light-harvesting chlorophyll a/b-proteins (LHCP1, LHCP2, LHCP3, LHCPy). The higher levels of LHCPs in LL-plants are associated with a higher ground fluorescence fo and maximum fluorescence fp of the in vivo chlorophyll. 3. Chlorophyll and carotenoid ratios: The chloroplasts of HL-plants possess a higher proportion of chlorophyll a and β-carotene (higher values for the ratios chlorophyll a /b and lower values for a/c and x/c) which reflect the increased level of the chlorophyll a/β-carotene-proteins CPIa, CPI and CPa. The higher level of light-harvesting chlorophyll a/b-xanthophyll-proteins (LHCPs) in LL-plants is also indicated by an increased content of xanthophylls and chlorophyll b as seen from lower a/b and higher x/c and a/c ratios. 4. The results indicate that plants possess the capacity for an ontogenetic adaptation of their photosynthetic apparatus to the incident light intensity. The HL-chloroplasts of HL-plants which contain less antenna chlorophyll, are adapted for a more efficient photosynthetic quantum conversion at light saturation than the LL-chloroplasts with high grana stacks. The correlation between higher levels of light-harvesting chlorophyll a/b-proteins (LHCPs) and a higher stacking degree of thylakoids, and the involvement of LHCPs in stacking is discussed.

REGULATION OF CHLOROPLAST DEVELOPMENT BY RED AND BLUE LIGHT

There are specific differences between red and blue light greening of etiolated seedlings of Hordevm vulgare L. Blue light results in a different prenyl lipid composition of chloroplast as compared to red light of equal quanta density. This is documented by a much higher prenylquinone content, higher chlorophyll a/b ratios, and lower values for the ratio xanthophylls to carotenes (x/c). The photosynthetic activity of “blue light” chloroplasts (Hill reaction) is higher than that of “red light” chloroplasts. These differences in prenylquinone composition and Hill‐activity are associated with a different ultrastructure of chloroplasts. “Red light” chloroplasts exhibit a much higher grana content than “blue light” chloroplasts. The difference in thylakoid composition, photosynthetic activity and chloroplast structure found between blue and red light greening are similar to those found between sun and shade leaves and those between plants grown under high and low light intensities.

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, RFd (defined as Chl fluorescence decrease Fd from maximum to steady state fluorescence Fs:Fd/Fs), that is determined by the Chl fluorescence induction kinetics (Kautsky effect). The height of the values of the Chl fluorescence decrease ratio RFd is linearly correlated to the net photosynthetic CO2 fixation rate PN as is indicated here for sun and shade leaves of various trees that considerably differ in their PN. Imaging the RFd-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.

The Importance of Blue Light for the Development of Sun-Type Chloroplasts

Depending on environmental factors, the development of chloroplasts from either pro-plastids or etioplasts will lead to two distinctive types of chloroplast, which are different in composition, ultrastructure, and photosynthetic activity [1, 8]. At low light intensities and in shade leaves the shade-type chloroplast with high grana stacks, lower Hill activity rates and a higher level of chlorophyll b is formed, indicating more light-harvesting complex CPII [6, 10]. Sun leaves and plants grown at high light intensities, in turn, develop sun-type chloroplasts with less lamellar material and only few and low grana stacks. Their higher Hill activity is correlated with a higher level of prenylquinones, which function as potential photosynthetic electron carriers, and can also be seen in a changed chlorophyll (higher a/b ratios) and carotenoid composition (lower x/c ratios) [6].