Fluorescence imaging as a diagnostic tool for plant stress Under normal physiological conditions, the major part of light absorbed by the photo- synthetic pigments, chlorophylls and carotenoids is used for photosynthetic quantum conversion, and only a small pro- portion is de-excited via emission as heat or as red and far-red chlorophyll fluorescence. In contrast, under many stress conditions, the photosynthetic quantum conversion declines, with a concomitant increase in red and far-red chlorophyll fluorescence. Blue and green fluorescence emissions also change under stress. Recently, a high res- olution, ultraviolet (UV) laser-induced fluor- escence (LIF) imaging system was devel- oped, which images all four fluorescence bands: blue, green, red and far-red. Even small fluorescence gradients are detected, so permitting the early detection of stress. The fluorescence images and the correspond- ing fluorescence ratio images blue : red
and blue : far-red are particularly sensitive to environmental change and stress. This noninvasive imaging technique can be used to assess the photosynthetic activity of leaves, to monitor the uptake of herbicides by plants, to screen for mineral deficiencies o r as a general indicator of plant stress.
Chlorophyll fluorescence analysis The inverse relationship between photo- synthetic performance and chlorophyll fluorescence emission was first described by Kautsky and Hirsch 1, and chlorophyll fluorescence analysis has since made a large contribution to the understanding of photosynthesis and electron transport reactions 2'3. The great advantage of chloro- phyll fluorescence analysis is that it can be applied to intact leaves in a noninvasive, nondestructive manner 3-6. Upon illumi- nation, chlorophyll fluorescence has an induction kinetic with two phases: a very fast rise (within 100 ms) via the ground fluorescence (F o) to a maximum fluor- escence level (F~) and then, with the onset of photosynthesis, a slow decline (within minutes) to a steady state fluorescence (F~) (Fig. la).
The rise and decline in light-induced 'variable chlorophyll fluorescence' (also known as the fast and slow components of the Kautsky effect, respectively) are only seen under physiological photosynthetic
(a) (b)
. . . . . . . . . . . . . . . . . . . . . . r" E
c
co
~, Fv = Fo/5 o=
69
0 . ,
Dark Illumination time (min)
1400 .~'1200 I F690 F740
1000 ~ ~ / ~ Fo o; 800
600
4°°11 JIl\ 200 Fs 0
400 500 600 700 800 Dark
Emission wavelength (rim)
Fig. 1. The kinetics of fluorescence. (a) Chlorophyll fluorescence induction kinetics (slow component of the Kautsky effect) in a green leaf darkened for 20 rain prior to illumination, as measured with a fluorometer in the 690 nm fluorescence band. The variable fluorescence decrease ratio (RF~) of the fluorescence decrease (F d) to the steady state fluorescence (F~) (RF~= FJFs) is an indicator of the potential photosyn- thetic activity of leaves, and is, wSth open stomata, linearly correlated with the net photosynthetic CO 2 assimilation 85'36. Abbreviations: Fo, ground fluorescence; Fro, maximum fluorescence; Fv, variable fluorescence. (b) Ultraviolet-radiation-induced fluorescence emission spectrum of a green maize leaf with fluorescence maxima in the blue [F440 (i.e. near 440 nm)], green (F520), red (F690) and far-red (F740) spec- tral regions. The four fluorescence bands, applied in the fluorescence imaging of leaves, are indicated by bars.
conditions. Under stress conditions, when the photosynthetic quantum conversion is disturbed or fully inhibited (e.g. by the her- bicide diuron), the chlorophyll fluorescence level remains high and the decrease from F m to F~ is slow or does not occur at all 4'7. Various processes are responsible for the chlorophyll fluorescence induction kinetics (including impaired activity of the two pho- tosystems in darkness, light-induced state 1-state 2 transitions, phosphorylation of light-harvesting proteins, the build-up of a proton gradient, onset of electron transport and CO s fixation) 1'5's'~. Here, the focus is on the influence of stress on chlorophyll and blue-green fluorescence and how the fluor- escence imaging technique can be used as a diagnostic tool to assess plant stress.
Origin and characteristics of chlorophyll fluorescence
The red to far-red chlorophyll fluorescence emission spectrum of intact dark-green leaves is characterized at room tempera- ture by two distinct maxima near 690 and 740 nm, which are termed F690 and F740 (Fig. lb). As isolated pigments, both chloro- phylls a and b possess the ability to emit red fluorescence 1°. However, the chloro- phyll fluorescence emitted by intact leaves originates from chlorophyll a molecules only, because chlorophyll b molecules transfer their excited states to chlorophyll a (Refs 4, 5 and 11). It is generally accepted that at room temperature chlorophyll fluor- escence is derived from photosystem II. The ground fluorescence (F o) emanates pri- marily from the light-harvesting pigment antenna, and the variable chlorophyll fluor- escence (F~) (Fig. la) from the reaction centre of photosystem II (although the situ- ation is different at extremely low subzero temperatures12).
The relative height of the chlorophyll fluorescence emission bands F690 and F740 (as well as their exact wavelength position) depends upon the chlorophyll con- tent of the leaves. At very low concen- trations of chlorophylls a and b (<5 mg cm -2 leaf area), the red F690 band (then near to 684 nm) is much higher than the far-red fluorescence band F740, which only exists as a shoulder between 730 and 740 nm (Refs 4, 13 and 14). With increasing chloro- phyll a and b content, the red F690 band shifts to longer wavelengths (from 684 to 693 nm) and decreases to yield a low peak or a shoulder, whereas the far-red fluor- escence F730-740 increases to a distinct maximum; in dark-green leaves this is at 740 nm. This change in the F690 and F740 emission is caused by overlap between the emitted F690 fluorescence band and the in vivo absorption bands of chlorophyll a,
causing a re-absorption of the F690 band 4'5. As a consequence, the chlorophyll fluor- escence ratio F690 : F740 is a noninvasive indicator of the in vivo chlorophyll content of leaves 4'~3-~5.
Chlorophyll fluorescence and plant stress Throughout their life, plants are exposed to a multitude of biotic and abiotic stress con- ditions 16. Because stress can lead, directly or indirectly, to modification or damage of the photosynthetic apparatus, various types of chlorophyll fluorescence signa- tures have been considered to be indicative of stress 3'4's'9~z. These parameters include: components of the induction kinetics and the variable chlorophyll fluorescence ratios, such as F~ : F=, F~ : Fo, and the variable fluorescence decrease ratio R E. (= FJF~), measured at 690 and 735 nln)~6's'9'lT'ls; the quenching coefficients qP and qN (Refs 5, 8 and 9); and the chlorophyll fluorescence ratio F690 : F740 (Refs 13, 14 and 15).
Under stress conditions, the photosyn- thetic apparatus exhibits characteristic symptoms, including: an increase in total chlorophyll fluorescence; a strong decline in F~, Fd, and in the ratios of variable fluorescence F~ : F m and R~d (at 690 and 735 nm); a 30% increase of the ratio F690 : F740; and an increase of qN. Short-term stress events (minutes to hours) disturb the photosynthetic performance. Long- term stress events (days to weeks) result in a decline in the chlorophyll content,~which can easily be monitored via the increase in the chlorophyll fluorescence ratio F690 : F740.
Characteristics and origin of blue-green fluorescence
The UV-radiation-induced, blue-green fluorescence emission of leaves and plants is characterized by a maximum in the blue region near 430-450 nm, and a shoulder in the green region near 520-530 nm (Fig. lb). The blue-green fluorescence of leaves was noted by Kautsky 1 and by botanists working with the fluorescence microscope, but its spectral composition has only been studied recently ~s-22. In contrast to chloro- phyll fluorescence, blue-green fluorescence does not exhibit any induction kinetics 23. Grasses and crop plants of the Poaceae (e.g. wheat, barley and maize) exhibit a high blue-green fluorescence, which can be greater than the chlorophyll fluorescence (Fig. lb). In contrast, leaves of dicoty- ledons (including spinach, tobacco and beech) ls'2°'22'24 exhibit a very low blue-green fluorescence that is much lower than the chlorophyll fluorescence ls'2°'2~.
Blue and green fluorescence are pri- marily emitted from two cinnamic acids,
White light
]~' S ~ ~ .....
Fig. 2. Set-up of the Karlsruhe/Strasbourg laser-induced fluorescence (LIF) imag- ing system for detection of small stress-induced gradients in blue, green, red and far-red fluorescence emission over the whole leaf area 2s,84. The fluorescence is excited by a pulsed Nd:YAG-laser at 355 nm; this wavelength is a compromise to excite the blue-green and the red fluorescence simultaneously 33. The fluorescence emitted, shown here for one leaf pixel, is simultaneously collected from several hundred pixels of the leaf area by a CCD (charge-coupled device) camera. The fluorescence bands F440 (i.e. near 440 nm), F520, F690 and F740 are selected by appropriate filters, the signals intensified, and then stored by the image processing system. In most cases leaves are illuminated by white light (or sunlight) in order to screen the red and far-red chlorophyll fluorescence in the steady state.
p-coumaric acid and ferulic acid, which are covalently bound to the cellulosic cell wall 2~. In cell walls of maize leaves, a third as yet unidentified cinnamic acid is present in lower amounts along with a blue-green- fluorescent flavone. The cell walls of plants from the Poaceae possess a higher concen- tration of cinnamic acids than the cell walls of dicotyledons (H. Lichtenthaler and J. Schweiger, unpublished); this is in agreement with their higher blue-green fluorescence emission. Although the blue- green fluorescence signal is primarily emit- ted by the cell walls 24'26, it is evident that the soluble and extractable cinnamic acids, flavones, flavonols and their glycosides, which are bound to cell vacuoles (par- ticularly in epidermal cells), can also contribute to the emission of blue-green fluorescence.
Blue-green fluorescence is emitted not only by green leaves, but also by nongreen plant tissue such as roots, white flower petals or white parts of variegated leaves t6'22'27,2s. In green leaves, the blue- green fluorescence emanates predomi- nantly from the chlorophyll-free epidermal cells and the major leaf veins, whereas the cell walls of the green mesophyll cells con- tribute very little 24'29. This is because in green mesophyll cells the blue-green fluor- escence is reabsorbed by the photosynthetic pigments 1~'2~, which possess broad absorp- tion bands in the blue-green region. White leaves exhibit the highest blue-green fluor- escence, and the lowest signal is found in
fully green leaves with a high chlorophyll content 2~.
Blue-green fluorescence and stress Long-term stress events and mineral deficiencies eventually reduce the chloro- phyll and carotenoid content of leaves. The blue-green fluorescence emission thus increases as the reabsorption of blue-green fluorescence is reduced. In addition, the composition of soluble plant phenolics (including flavones, flavonols, cinnamic acids, coumarins and stilbens) in the epi- dermal cells can change under stress condi- tions, so that the blue and/or green fluor- escence emission of leaves increases. This increase may precede the loss of chloro- phyll or carotenoid content in the meso- phy]l cells. In Rhododendron leaves exposed to severe high-light stress, the level of total flavonols was more than twice as high as in the half-shade leaves, and the green fluorescence signal increased to a much higher degree than the blue fluor- escence 3°. Other green-fluorescing sub- stances include the flavonoid quercetin, the alcaloid berberine and riboflavin ~1. These observations indicate that the ratio of blue to green fluorescence F440 : F520 can change under certain stress conditions.
Following herbicide treatment (diuron), photosynthetic electron transport becomes blocked, and red and far-red chlorophyll fluorescence strongly increase 13'1s, but blu6- green fluorescence either remains the same or only varies slightly 18'32. In such Cases,
August 1997, VoL 2, No, 8 3 1 7
update
Fig. 3. Laser-induced fluorescence images of a tobacco aurea mutant lower leaf surface. The upper four panels show blue [F440 (i.e. near 440 nm] and green (F520) fluor- escence, as well as red (F690) and far-red (F740) chlorophyll fluor- escence. The fluorescence intensity is shown by false colour from blue (zero fluorescence yield) via green and yellow to red (highest fluores- cence yield) 2s. The lower four panels show images of the three fluorescence ratios blue : red (F440/F690), blue : far-red (F440/F740) and blue : green (F440/F520), and of the chlorophyll fluorescence ratio red : far-red (F690/F740). The colour scales shown are numbered using arbitrary units. Fluorescence images taken by M. Sowinska, F. Heisel and M. Lang.
blue-green fluorescence emission can be used as an internal fluorescence standard. In fact, the corresponding fluorescence ratios blue : red (F440 : F690) and blue : far-red (F440 : F740) significantly decline, whereas the chlorophyll fluorescence ratio (F690 : F740), as initiated by the diuron- enhanced chlorophyll fluorescence emis- sion, increases by about 30% (Refs 18, 27 and 32).
Under various stress conditions (high light exposure, enhanced UV radiation, drought and temperature stress) additional UV-absorbing and/or UV-scattering sub- stances are formed in the epidermal cells, which then form a barrier to the pen- etration of UV radiation into the mesophyll cells. The proportion of UV radiation that passes through the epidermis into the leaf mesophyll is strongly reduced and results in a dramatic reduction in chlorophyll
fluorescence emission 25's~. As a conse- quence, the fluorescence ratios blue : red (F440 : F690) and blue : far-red (F440 : F740) strongly increase ~5'27'3z. Indeed, these two fluorescence ratios have proved to be very sensitive to changes in growth condi- tions as well as stress events, and appear to change even before any damage to the photosynthetic apparatus is apparent ls'25.
Fluorescence imaging and point data measurements
In the conventional spectrofluorometers hitherto used to assess chlorophyll fluor- escence and blue-green fluorescence, emis- sion spectra are either recorded or the fluor- escence is screened in one or all of the four bands blue, green, red and far-red (Fig. lb). Stress can then be detected via changes in the fluorescence intensity and fluorescence ratios. However, this analysis is restricted to a point on a single leaf, and it is doubtful whether the fluorescence information at this point is really representative of the whole leaf area. Gradients in the fluor- escence emission between the margins and the central lamella, or between the tip and the petiole (which might be early stress indicators) remain hidden by such point data measurements. In fact, early stress events or attacks by pathogens rarely affect the whole leaf area at once. This disadvan- tage of point data measurements, which prohibit an early stress diagnosis, can be overcome by simultaneously recording the fluorescence information at all points of the leaf area using a new approach, fluor- escence imaging.
Fluorescence imaging of leaves and plants
A LIF imaging system has been developed that screens the blue, green, red and far- red fluorescence of leaves 2s-3°'34 (Fig. 2). The advantage of this system is that it simul- taneously records the LIF signatures for several hundred pixels over the whole leaf area, or even of several leaves or plants. With this technique it has been shown that the blue and green fluorescence emission is not evenly distributed over the leaf area, but primarily emanates from the main and side veins (Fig. 3, upper panelsfi 8,29. In con- trast, the red and far-red chlorophyll fluor- escence is predominantly emitted from the vein-free leaf regions, showing a negative contrast with the blue-green fluorescence. Using the image processing system, the various fluorescence ratios (blue : red, blue : far-red, blue : green) and the chlorophyll fluorescence ratio (red : far-red) are calcu- lated for each of the several hundred pixels of the leaf area 27'2s. In this way, the differ- ences in fluorescence emission in the four
Fig . 4. False-colour fluorescence images of the laser-induced red [F690 (i.e. near 690 nm)] and far-red (F740) chlorophyll fluorescence emission in a green tobacco leaf after drought stress and a 6 h heat stress treatment, showing the gradient in chlorophyll fluorescence from the margins to the centre of the leaf. The fluorescence intensity is shown by false colour, increasing from blue via green and yellow to red (highest intensity) s° . Fluorescence images taken by M. Sowinska, F. Heisel and M. Lang.
fluorescence bands can be quantitated in relative proportions (Fig. 3, lower panels).
Fluorescence imaging of plant stress Fluorescence images and LIF ratio images of the whole leaf area permit an analysis of stress-induced changes in fluorescence emission (e.g. gradients within the leaf and local rises or inconsistencies) at a very early stage of stress. This may be before damage symptoms become visible, and at a time when counter measures can still be taken to protect the plant. An example of an early heat and drought stress, which is seen as a gradient in the LIF image from the leaf margin to the leaf centre, is shown for the red and far-red chlorophyll fluor- escence in Fig. 4. After 6 h heat stress, both fluorescences, F690 and F740, had increased at the leaf margin, because of a heat-induced decline in the photosynthetic quantum conversion; the chlorophyll fluor- escence yield of the centre of the leaf was not yet affected. The heat treatment had almost no effect on the blue-green fluor- escence emission, and thus the fluor- escence ratio F440 : F690 decreased at the leaf rim parts by 60%. In contrast, the chlorophyll fluorescence ratio F690 : F740 only increased slightly (<20%), indicating that chlorophyll breakdown had not yet occurred. When these tobacco plants were watered only one week after the heat stress treatment, visible necrosis of the leaf margin developed within two to three weeks. In contrast, in plants watered directly after the heat treatment, the necrosis did not appear.
Using LIF images and LIF ratio images, various other stresses have also been
3 1 8 August 1997, Vol. 2, No. 8
update
screened, including: mite attack in bean plants2~; combined light, heat and drought stress in sun-exposed leaves of Prunus and Rhododendron29; long-term drought stress in tobacco2~; and herbicide uptake 2s'32 and nutrient deficiencies (Fe, Mg, Zn, N) in maize 34. The type of stress and the response mechanisms of plants determine which fluorescence ratios decline and which increase. However, although the changes in fluorescence ratios allow early stress detection, they do not directly identify the type of imposed stress. Nevertheless, from the way in which the four fluorescence ratios change, it is at least possible to exclude several stresses.
Herbicide uptake and decline in photosynthetic activity
The photosynthetic activity of leaves and the uptake of the herbicide diuron, which blocks photosynthetic electron transport and quantum conversion by binding to the D1 protein of photosystem II, can easily be studied by fluorescence imaging. In this analysis, the measurements centre on the imaging of the red chlorophyll fluorescence (F690) at F m and F~ (reached after 5 min illumination with white light) (see Fig. 1), In the control leaf, the high chlorophyll fluorescence yield at F m decreased evenly over the whole leaf area to F~ (Fig. 5, upper panels). In contrast, in the leaf of the Digitalis plant, which had been treated with 10 -° M dinron (via the roots), F m only decreased to F S in those leaf parts that were still free of the herbicide. Thus the uptake of the herbicide diuron is visualized in the LIF images by a failure of the chloro- phyll fluorescence to decline from F~ to F~.
This decline in chlorophyll fluorescence from F m to F, can be quantified via the vari- able fluorescence decrease ratio RFd, which is a function of the potential photosynthetic capacity of leaves 3~'3~. Such Rra ratio images (Fig. 5, lower panels) show in the leaf of the control plant, high R~d values being evenly distributed over the leaf area, thus indicating that all leaf parts are active in photosynthetic quantum conversion. In contrast, in the diuron-treated plant, the increasing loss of photosynthetic function with increasing exposure time (48-84 h) is seen in the increasing decline of the R~ values to zero in the centre of the leaf an~ near the leaf veins through which diuron is successively transported into the leaf.
Conclusion Fluorescence imaging with LIF images and LIF ratio images represents a superior means of detecting early stress in plants. This high resolution method is more pre- cise and provides stress information at an
Fig. 5. The upper four panels show differences in the decline of the red chlorophyll fluorescence [F690 (i.e. near 690 nm)] from maximum fluorescence, Fro, to steady state fluorescence, F,, in green attached Digitalis leaves, from a control plant and a plant treated with the herbi- cide diuron (10 -5 M) via the roots. Fm was measured upon illumination of a leaf kept in darkness for 20 rain, and F, in the steady state of the fluor- escence kinetics 6 rain after the introduction of the light. The fluor- escence intensity is shown by false colour, with increasing values from blue via green and yellow to red. The four lower panels show fluor- escence images of the variable chloro- phyll fluorescence ratio RE, of a green attached Digitalis leaf;, indi- cating the loss of photosynthetic activity with increasing uptake time of diuron applied via the roots (10 -5 M). The decrease and loss in photosynthetic function are indicated by the decline in the R F values measured at 690 nm, showr~ here by false colour. Fluorescence images taken by M. Sowinska, F. Heisel and M. Lang.
earlier stage than point data fluorescence measurements. The LIF imaging technique is noninvasive, does not require a pretreat- ment of the plant tissue and can be applied to a screen of whole leaves and plants. For this reason, it is also superior to other new techniques such as the imaging of stress- induced changes in cellular calcium levels after transformation with recombinant
aequorins 37'38. The LIF imaging system already works at 20 cm to 10 m, and thus it should be relatively easy to develop for field experiments and remote sensing of ter- restrial vegetation. There are wide possi- bilities for LIF imaging in agriculture, forest decline and environmental research:
Acknowledgements The authors would like to thank Joachim Schweiger and Oliver Wenzel for assis- tance, Inge Jansche and Doris MSller for typing the manuscript, and Gabrielle Johnson for checking the English. Financial support by a grant from the European Union within the lnco-Copernicus program is gratefully acknowledged.
Dedication This article is dedicated to Prof. Dr Benno Parthier, on the occasion of his 65th birthday.
Hartmut K. Lichtenthaler* The Botanical Institute, University of Karlsruhe, Kaiserstra6e 12, D-76128 Kadsruhe, Germany
Joseph A. Mieh~ Groupe d'Optique Appliquee, Centre des Recherches Nucl6aires, 23 rue du Loess, F-67037 Strasbourg Cedex 2, France
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book reviews
Xerophytes re-evaluated Structure-Function Relations of Warm Desert Plants by A.C. Gibson Springer, Adaptations of Desert Organisms, 1996. DM188.00 (xi + 215 pages) ISBN 3 540 59267 9
In this book, the author, who is a well- known researcher on the anatomy of cacti, presents a re-evaluat ion of the s t ruc ture- funct ion relat ions of warm desert plants. The thesis is that the struc- tural designs related to storing or conserv- ing water - which are often referred to as xerophytic or xeromorphic features - have mainly been derived from plants of semi- arid regions, and are not at all typical of plants from warm, lowland deserts. Warm deserts are defined as arid regions con- tained within the 200 mm isohyet (an iso- hyet is a line on a map connecting places with equal rainfall), and having mean monthly temperatures that exceed 20°C in the hottest month and 9°C in the coldest month, without frost except for very short periods. As admitted by the author, this definition is problematic, because it unites deserts of quite different types - ranging from those with only erratic, unreliable precipitation to those with predictable win- ter or summer rainfall, and fog deserts. A quick look through descriptions of the veg- etation of various deserts reveals that dif- ferent climatic regimes almost certainly triggered the evolution of different life forms and survival strategies. For example, in the warm desert of southwestern Africa (broadly, the Namib desert), the winter rainfall portion is dominated by leaf succu- lents; the summer rainfall portion is domi- nated by woody plants, and succulents are only represented by stem succulents
