The age dependence of the photosynthetic performance, chlorophyll fluorescence and chloroplast ultrastructure of green form and Chl ft-deficient form (aurea) of tobacco Su/su mutant were compared. The most pronounced differences between the aurea and green tobacco found in young leaves diminished with leaf age. Slower accumulation of the photosynthetic pigments during the development of aurea leaves was accompanied by a slower accumulation of LHC antennae of both photosystems, particularly that of PS2, and by retention of an increase in the capacity of PS2 photochemistry, measured as Fy/FM The ratio Fv/Fm, however, increased rapidly during maturation of aurea leaves, and fmally the mature aurea leaves exhibited higher values of this ratio than the green ones. Rates of photosynthesis at saturating irradiance (Epiax) saturating CO2 concentration (/’sat) decreased with leaf age for both aurea and green tobacco, being always higher in aurea leaves than in leaves of green tobacco of comparable age. AU these characteristics indicated retarded development of aurea leaves. Also the chloroplast ultrastructure, particularly grana formation, exhibited slower development. The decrease in /Wx and with leaf age in both tobacco forms and retardation in the development of aurea leaves can explain higher value of usually found in aurea tobacco.
When exposed to Y-radiation (12, 8 and 3.5 kGy), the growth of beán seedlings {Phaseolus vulgaris L.) was stopped and after some hours or days the plants began to wilt in a dose-dependent manner, starting from the leaf rim. The rate of the dark respiration {R) of leaves increased and that of net photosynthesis {P^ was strongly reduced. The regulation of stomata opening and closure was lost and the stomatal conductance (g^) of the y-ray exposed plants was strongly reduced. The reduced was only partly due to either the partial or almost Ml stomata closure. Chlorophyll (Chl) fluorescence measurements with a two-wavelength fluorometer and a PAM fluorometer showed an increasingly reduced variable fluorescence Fy, lower values of Rfj, of ground fluorescence Fq, and of the fluorescence ratios Fy/F,n and Fy/F^. This indicated a damage to the photosynthetic apparatus. The increasing loss of photosynthetic pigments in the 350 krad exposed plants was also detected via an increase in the fluorescence ratio F690/F730. The performance of the light driven xanthophyll cycle (violaxanthin/zeaxanthin transformation) proceeded in the y-ray treated plants only at reduced rates. The y-ray damage of plants can best be detected by measurements of stomatal conductance, and various Chl fluorescence ratios such as Rf(j, Fy/Fj, and Fy/F^,.
Changes in the chlorophyll (Chl) and carotenoid (Car) contents and photosynthetic activity of leaves of three-year-old maple trees were studied under an extremely high irradiance (HI) (5 000 and 7 000 pmol m-2 s‘*) and subsequent low irradiance (LI) (10 pmol m-2 s‘*). Speciál attention was paid to a possible linear correlation between zeaxanthin (z) accumulation and the decrease in variable Chl fluorescence (ratio Fy/FnO during photoinhibitory treatment. Hl-induced violaxanthin (v) transformation into z was a fast one-step response proceeding within 2-5 min. Changes in the Chl a and b contents or in non-xanthophyll cycle Car were either not observed (30 min at 5 000 pmol m*2 s‘i) or a slight decline of the Chl a, lutein, 3-carotene and z contents only occurred at prolonged exposure (60 min at 7 000 pmol m'^ s'i)- Photosynthetic activity measured via the ratios (Fy/F^, Fy/F^, Rfj 690, Rfj 735) sank during this treatment to a much higher extent at 7 000 than at 5 000 pmol m'^ s'*. Ratios Fy/F<„ Rfd 690 and Rfj 735 proved to be better indicators of photoinhibition with a larger amplitudě ťhan the ratio Fy/F,„. Unlike literatuře reports, no linear correlation was found between Hl-induced decrease of fluorescence ratios and the z accumulation. Decrease of the fluorescence ratios appears to be biphasic in nátuře: fast initial fall which parallels the z formation is followed by a phase when ratios Fy/F^,, Fy/F^,, Rj^ 690 and Rf^ 735 sink further without any changes in the z pool. Regeneration of photosynthetic activity under LI also proceeded in two steps; a relatively fast increase in ratios with little or no changes in the z pool followed by a slow many hours' restoring of the former fluorescence ratios, paralleled by the z transformation into v. The back-reaction of z to v under LI was increasingly retarded with higher irradiance. Z formation and decrease in the fluorescence ratios are thus probably two Hl-induced processes which may be mutually independent.
Primary leaves of barley (Hordeum vulgare L.) plants, growing either in complete (H) oř nitrogen deficient nutrítion (H-N), were subjected to complex chlorophyll (Chl) a fluorescence measurement after an 8 or 14 d cultivation period. Ilie fluorescence spectra of H-N plants exhibited a higher ratio of the two maxima F690/F73S, caused predominantly by drastic decrease in Chl content. The ratio of Chl to carotenoids (a+b/x+c) decreased considerably in H-N leaves. In špite of high Rfd values (ratio of fluorescence decrease) in H-N leaves, indicating high efficiency of photochemical energy conversion, quenching analysis of H-N leaves showed a significantly higher coefiicient of non-photochemicď quenching, qjsip (i.e. higher proportion of heat loss). Fast fluorescence kinetics indicated slower reoxidation of primáty quencher (Q^) in H-N leaves.
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., and H. K. Lichtenthaler ... [et al.].
The content of chlorophylls (Chl) (a+b), total carotenoids (x+c), and the pigment ratios of Chl a/b and Chls to carotenoids (a+b)/(x+c) of green leaves of five C4 plants were determined and compared to those of C3 plants. The C4 plants were: Pacific and Chinese silvergrass (Miscanthus floridulus and Miscanthus sinensis), sugar cane (Saccharum officinarum) as well as feed and sugar maize (Zea mays). The three C3 plants were beech, ginkgo, and oak. C4 plants possess higher values for the ratio Chl a/b (3.4-4.5) as compared to the C3 plants (2.6-3.3). Sugar maize had the highest values for Chl a/b (4.04-4.70) and exceptionally high contents of total carotenoids and consequently lower values for the ratio of (a+b)/(x+c) (mean: 3.75 ± 0.6). During autumnal senescence also C4 plants showed a faster decline of Chl b as compared to Chl a yielding high values for Chl a/b of 6 to 8. Chlorophylls declined faster than carotenoids yielding low (a+b)/(x+c) values below 1.0.
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., and H. K. Lichtenthaler, C. Buschmann, M. Knapp.
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. and C. Buschmann, G. Langsdorf, H. K. Lichtenthaler.
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 Fp and steady state fluorescence Fs of the induction kinetics (Kautsky effect) and by a subsequent determination of the images of the fluorescence decrease ratio RFd and the ratio Fp/Fs. 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. and H. K. Lichtenthaler ... [et al.].
a1_Imaging the four fluorescence bands of leaves, the red (F690) and far-red (F740) chlorophyll (Chl) fluorescence as well as the blue (F440) and green (F520) fluorescence of leaves and the corresponding fluorescence ratios is a fast and excellent nondestructive technique to detect the photosynthetic activity and capacity of leaves, of gradients over the leaf area as well as the effect of various strain and stress parameters on plants. This review primarily deals with the first and pioneering multi-colour fluorescence imaging results obtained since the mid-1990s in a cooperation with French colleagues in Strasbourg and in my laboratory in Karlsruhe. Together we introduced not only the joint imaging of the red and far-red Chl fluorescence but also of the blue and green fluorescence of leaves. The two instrumental setups composed for this purpose were (1) the Karlsruhe-Strasbourg UV-Laser Fluorescence Imaging System (Laser-FIS) and (2) the Karlsruhe Flash-Light Fluorescence Imaging System (FL-FIS). Essential results obtained with these instruments are summarized as well as the basic principles and characteristics of multi-colour fluorescence imaging. The great advantage of fluorescence imaging is that the fluorescence yield in the four fluorescence bands is sensed of several thousand up to 200,000 pixels per leaf area in one image. The multi-colour FIS technique allows to sense many physiological parameters and stress effects in plants at an early stage before a damage of leaves is visually detectable. Various examples of plant stress detection by the multi-colour FIS technique are given. Via imaging the Chl fluorescence ratio F690/F740 it is even possible to determine the Chl content of leaves. The FIS technique also allows to follow the successive uptake of diuron and loss of photosynthetic function and to screen the ripening of apples during storage., a2_Particularly meaningful and of high statistical relevance are the fluorescence ratio images red/far-red (F690/F740), blue/red (F440/F690), and blue/green (F440/F520) as well as images of the fluorescence decrease ratio RFd, which is an indicator of the net CO2 assimilation rates of leaves., H. K. Lichtenthaler., and Obsahuje bibliografické odkazy