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.].
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.
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
The thermo-sensitivity of two new pea (Pisum sativum L.) cultivars-Afila (mutant in the gene transforming leaves into mustaches) and Ranen (mutant for early ripening)-as compared to the control cultivar Pleven-4 to either low (4 °C, T4) or high temperature (38 °C, T38) was investigated by means of chlorophyll (Chl) fluorescence kinetics. The low temperature treatment decreased the photosynthetic activity, measured via a decline of the Chl fluorescence decrease ratios RFd690 and RFd735, and this was mainly due to a decline of the Chl fluorescence decrease parameter Fd and maximum Chl fluorescence Fm. In the new cv. Ranen the RFd ratios at first decreased and increased again after 24-h exposure to 4 °C, indicating its good acclimation ability to low temperature. The cold-induced changes in the photosynthetic performance of all cultivars were reversed after transferring plants back to 23 °C for 48 h. In the Chl and carotenoid (Car) contents no or little changes occurred during the T4 treatment, except for a slight but clear increase of the ratio Chl a/b and a decrease in the ratio Chl/Car. In contrast to this, the T38 treatment for 72 h decreased the RFd ratios more strongly than the T4 exposure did. In fact, an irreversible injury of the photosynthetic apparatus was caused in the control pea cv. Pleven-4 by a 48-h T38 exposure and for the new cv. Afila after a 72-h T38 exposure. In contrast, the cv. Ranen was less and little sensitive to the T38 exposure. In the heat-sensitive cvs. Pleven-4 and Afila, the decrease in RFd values at T38 was associated with a strong decline of the Chl a+b and total Car contents. The Chl a+b decline could also be followed via an increase of the Chl fluorescence ratio F690/F735. Parallel to this, a strong decline of Chl a/b from ca. 3.0 (range 2.85-3.15) to ca. 1.9 (range 1.85-1.95) occurred indicating a preferential decline of the Chl a-pigment proteins but not of the Chl a/b-pigment protein LHC2. In the relatively heat-tolerant cv. Ranen, however, the ratio Chl a/b declined only partially. After the T4 treatment the stress adaptation index Ap was higher in cv. Ranen than in controls and reached in heat-treated Ranen plants almost the starting value indicating a cold and heat stress hardening of the treated plants. The Chl fluorescence parameters and pigment contents were influenced by T38 and T4 treatments in various ways indicating that the mechanisms of low and high temperature injury of the photosynthetic apparatus are different. The new cv. Ranen exhibited a cross tolerance showing a fairly good acclimation ability to both T4 and T38, hence it is a very suitable plant for outdoor growth and for clarification of the accl, imation mechanisms to unfavourable temperatures., and K. Georgieva, H. K. Lichtenthaler.