Journal of Experimental Botany, Vol. 52, No. 363, pp. 2007-2014,
October 1, 2001
© 2001 Oxford University Press
Original Papers |
UV-excited chlorophyll fluorescence as a tool for the assessment of UV-protection by the epidermis of plants
1 Department of Biology and Nature Conservation, Agricultural University of Norway, PO Box 5014, N-1432 Ås, Norway
2 Julius-von-Sachs-Institut für Biowissenschaften, Julius-von-Sachs-Platz 2, D-97082 Würzburg, Germany
Received 12 February 2001; Accepted 31 May 2001
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Abstract |
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 Top Abstract IntroductionThe fluorescence method for...Comparison to spectrophotometric...The limits of the...Relationship to accumulated...Measurements on attached leaves...ConclusionReferences |
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Recently, a new method for estimating epidermal transmission of UV radiation in higher plants has been proposed. The empirical evidence for the usefulness of this method is reviewed here. Direct comparison with spectroscopically determined epidermal transmission yielded equivalent results. A linear correlation to the concentration of epidermal screening compounds has been shown. Relating UV-A and UV-B absorbance allowed some preliminary conclusions about the chemical nature of the screening compounds. A new portable apparatus is presented for the first time, which allows the non-destructive assessment of UV-A screening even under field conditions. Repeated measurements on identical leaves over a time-course of 6 d demonstrated a strong age-dependence in the capacity for the synthesis of UV-A screening compounds upon exposure to UV-B radiation. It is concluded that the new method may provide a valuable tool for the investigation of the acclimation of plants to UV-B radiation and, when accompanied by HPLC analysis, of the reaction of phenolic metabolism to environmental stimuli.
Key words: Acclimation, epidermal transmission, flavonoids, leaf age, UV-B radiation.
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Introduction |
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TopAbstractIntroductionThe fluorescence method for...Comparison to spectrophotometric...The limits of the...Relationship to accumulated...Measurements on attached leaves...ConclusionReferences |
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The reduction in stratospheric ozone layer and the resulting increase in UV-B radiation (280–320 nm) on earth has induced widespread interest for the acclimation and adaptation of plants to UV-B radiation (Madronich et al., 1995
; Jordan, 1996; Jansen et al., 1998). UV-B can damage proteins and DNA (Britt, 1999), reduce genome stability (Ries et al., 2000) and inhibit photosynthesis (Vass, 1996). Since UV-B radiation is an integral part of the solar spectrum, terrestrial organisms have had to cope with its harmful effects throughout their evolutionary history (Rozema et al., 1997). In principle, two different tolerance strategies occur in vascular plants: repair of inflicted damage and screening of the internal tissues against the radiation (Bornman et al., 1997; Jansen et al., 1998). Both mechanisms complement each other and both are apparently indispensable. Arabidopsis thaliana mutants defective in one of these protective mechanisms became hypersensitive to UV-B (Li et al., 1993; Landry et al., 1997).
Screening of the plant tissues is achieved by the accumulation of UV-B absorbing compounds, mostly flavonoids or hydroxycinnamic acid derivatives (HCAs) in the epidermis, where they can be located in the cuticle, in the cell wall or in the vacuole (Caldwell et al., 1983; Schnitzler et al., 1996; Krauss et al., 1997; Hutzler et al., 1998). For the determination of the extent of epidermal screening, the extraction of soluble compounds from leaves may give a first indication. However, this information may be incomplete since the screening compounds may be insolubly bound to cell wall or cuticle and, furthermore, extractable UV-B absorbing compounds may not provide protection if they are located in the mesophyll (Grammatikopoulos et al., 1999). Therefore, direct measurements of epidermal transmission are necessary. Until recently, two different methods have been employed: measurement of transmission through peeled epidermal tissue (Lautenschlager-Fleury, 1955; Robberecht and Caldwell, 1978) and measurement of leaf internal radiation using fibre-optic microprobes (Bornman and Vogelmann, 1988; Day et al., 1992). Both methods have their drawbacks. In the vast majority of plant species, it is not possible to remove the epidermis intact, confining direct measurement of epidermal transmission largely to mesophytic plants. Equally, where it is possible to remove the epidermis, this can easily be damaged and even an apparently intact epidermis may contain a large number of destroyed cells which open windows for the transmission of radiation. The use of optical fibres is much more widely applicable and always measures transmission through an intact epidermis. An additional advantage of this method is that UV-B irradiance can be determined at any depth within a leaf. However, the technique is rather complicated and requires elaborate and expensive apparatus. In addition, irradiance is not directly accessible but has to be calculated from several independent measurements at different angles (Vogelmann and Björn, 1984).
Recently, a new technique based on chlorophyll (chl) fluorescence measurements has been introduced (Bilger et al., 1997), which may overcome some of the problems associated with the other methods. In the meantime, this technique has been refined and a couple of publications from our and other groups have appeared. In the first part of this report, the present state of this new technique is reviewed based on measurements with the XE-PAM fluorometer. Subsequently, an outlook on new developments is given which will allow an even wider use of the method.
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The fluorescence method for the assessment of epidermal transmission |
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TopAbstractIntroductionThe fluorescence method for...Comparison to spectrophotometric...The limits of the...Relationship to accumulated...Measurements on attached leaves...ConclusionReferences |
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The rationale of the fluorescence method is demonstrated in Fig. 1
. Light absorbed by chloroplasts in the mesophyll will excite fluorescence emission. While a large part of the UV-B excitation beam is already absorbed in the epidermis, depending on the relative amount of UV-B absorbing substances located there (indicated by the yellow colour), a blue-green (BG) excitation beam will reach the chloroplasts largely undiminished. The fluorescence intensity emitted by the chloroplasts is dependent on the amount of light absorbed by them. A comparison of the fluorescence intensities induced by the two different beams provides a relative measure of how much UV-B radiation is reaching the uppermost chloroplasts.
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The method is based on the following principles, which ideally should be satisfied experimentally.
- (1) The absorption spectra of chlorophylls a and b extend well into the UV-B spectral region.
(2) The fluorescence intensity emitted by chlorophyll is proportional to the intensity of the excitation beam, as long as the latter does not induce variable fluorescence.
(3) The relative amount of excitation light reaching the chloroplasts in the mesophyll depends on its absorption within the epidermis and the upper cell wall of the mesophyll cells.
(4) Epidermal absorption of the blue-green reference beam is negligible.
(5) There is no spectral difference between fluorescence excited by UV and blue-green radiation. Accordingly, any modulation of the fluorescence emission by the mesophyll and the epidermis due to scattering and reabsorption is identical for any type of reference beam.
Hence, accumulation of UV-B absorbing compounds in the epidermis will reduce the fluorescence excited by the UV-B beam, F(UV-B), or generally speaking, F(UV), without or only negligibly affecting the fluorescence excited by the blue-green reference beam, F(BG). This results in a low F(UV)/F(BG) ratio. A maximal ratio is observed in the complete absence of UV-screening compounds or after removal of the epidermis. (In some species, for example, Cucumis sativus L., identical F(UV)/F(BG) ratios were found on the abaxial side in the presence and after removal of the epidermis. However, there was no observation that the ratio was lowered by the removal of the epidermis.) The F(UV)/F(BG) ratio of epidermis-free leaves can be taken as a reference to calculate epidermal transmission.
This model provides a robust basis for calculating epidermal transmission from fluorescence measurements and can be experimentally tested.
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Comparison to spectrophotometric measurements in isolated epidermal tissue |
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TopAbstractIntroductionThe fluorescence method for...Comparison to spectrophotometric...The limits of the...Relationship to accumulated...Measurements on attached leaves...ConclusionReferences |
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While theoretical considerations strongly indicate the usefulness of the outlined fluorescence method, it is also necessary to check its functionality experimentally. This was done by comparison with direct measurements of the transmission of epidermal peels in two independent investigations (Barnes et al., 2000
; Markstädter et al., 2001). Figure 2 shows the results of a study by Markstädter et al. for UV-A and UV-B transmission in Vicia faba L. leaves (Markstädter et al., 2001). In both cases, there was a linear correlation between transmission calculated from fluorescence measurements (using epidermis-free leaves as reference), and transmission directly measured in epidermal peels. The regression coefficient is relatively large and the regression passes the ordinate through the origin (UV-B) or close to the origin (UV-A). The slope of the regression was slightly larger than 1. Similar results were obtained by Barnes et al. with the same species (Barnes et al., 2000). There, however, the regression for the UV-B data had an intercept of 20% transmission, as calculated from the fluorescence data. Slightly different filter combinations were used in these studies for the definition of the measuring beams. In the study by Barnes et al. some spectral overlap between the excitation beam and the wavelength range for fluorescence detection might have caused the relatively high offset of the regression (Barnes et al., 2000). In any case, both studies confirm that the fluorescence method is able to determine epidermal transmission over a wide range of transmissions.
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The limits of the method |
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TopAbstractIntroductionThe fluorescence method for...Comparison to spectrophotometric...The limits of the...Relationship to accumulated...Measurements on attached leaves...ConclusionReferences |
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After having confirmed the principal applicability of the fluorescence method for the determination of epidermal transmission, its limits with respect to attainable precision and general applicability also have to be considered. First of all, the above-mentioned principles should not be violated. However, point 4 is not true of all plant material, i.e. epidermal absorption of the BG reference beam is not negligible in some cases. Some plants contain blue light-absorbing anthocyanins in their epidermis. Furthermore, hairs or waxes on the surface of the leaf may reflect and absorb blue light. If this is not taken into account, erroneously high transmissions are obtained. Barnes et al. observed an apparent UV-B-induced increase in F(UV-B)/F(BG) in leaves of Gunnera magellanica which could not be explained by changes in UV-B absorbing compounds (Barnes et al., 2000
). Instead, they found UV-B-induced formation of blue-green-absorbing pigments, most probably anthocyanins.
Epidermal transmission can be calculated in a straightforward manner from fluorescence measurements, when the epidermis can be removed from the leaves. However, in the majority of leaves and other tissues, this is not possible and either only F(UV)/F(BG) ratios can be used or transmission has to be calculated using another reference. One might speculate that F(UV)/F(BG) ratios are similar for epidermis-free leaves from differently treated leaves of the same species and even for leaves from different species. A prerequisite for this is that the chloroplasts of different leaves have the same or at least sufficiently similar excitation spectra. This has been discussed in some detail earlier (Bilger et al., 1997). In some cases, however, considerable intra- and interspecific variation of F(UV)/F(BG) from epidermis-free leaves was observed (Barnes et al., 2000; Markstädter et al., 2001). There is a tendency for decreasing F(UV)/ F(BG) values when the values measured with the intact leaf also decreased, i.e. epidermal transmission decreased (Markstädter et al., 2001; W Bilger, unpublished results). On the other hand, fluorescence ratios from leaves of different species converged to a similar value when epidermal transmission was high (Bilger et al., 1997; W Bilger, unpublished results). This could be interpreted as an indication that UV-B screening compounds may not only be located within the leaf epidermis, but also directly below it, i.e. most probably in the outer cell wall of the mesophyll cells. This hypothesis has to be further investigated.
As long as general information on fluorescence ratios from chloroplasts and epidermis-free leaves is not available, one should normalize the data to another stable reference to obtain a relative indicator for epidermal transmission. As a suitable reference a commercially available blue plastic foil may be recommended which happens to display roughly similar UV/BG excitation and fluorescence emission properties as the mesophyll (Barnes et al., 2000; Markstädter et al., 2001).
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Relationship to accumulated epidermal UV-B screening compounds |
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TopAbstractIntroductionThe fluorescence method for...Comparison to spectrophotometric...The limits of the...Relationship to accumulated...Measurements on attached leaves...ConclusionReferences |
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Another way to assess the validity of the fluorescence method is to relate the transmission results to epidermal pigment contents. A correlation of epidermal transmission and UV-B absorbance in total leaf extracts cannot be expected if there are also UV-absorbing compounds in the mesophyll. However, if compounds located in the epidermis and the mesophyll are chemically different, as is the case for rye, they can be determined separately after HPLC analysis of the extract (Schulz and Weissenböck, 1986
). Using primary leaves of rye as the experimental object and leaf age as a variable to obtain samples with different amounts of UV-screening compounds, Burchard et al. demonstrated a linear relationship between transmission determined via the fluorescence method and epidermally located phenolic compounds (Fig. 3) (Burchard et al., 2000). When both, epidermal hydroxycinnamic acid derivatives (HCAs) and epidermal flavonoids were included in the analysis, the data could explain virtually all epidermal absorbance by the presence of soluble epidermal pigments. In the UV-A region (data not shown) epidermal flavonoids alone were sufficient to account for the observed epidermal absorbance. As most of the measured chlorophyll fluorescence originates from the uppermost mesophyll cell layer, flavonoids located in the majority of the mesophyll, i.e. adjacent to or even below the fluorescing chloroplasts, cannot contribute much to the screening assessed via fluorescence (Fig. 3). The presence of UV-B absorbing compounds in the mesophyll might have caused the relatively poor correlation betweeen F(UV)/F(BG) and UV-B absorbance in crude leaf extracts in the study of Barnes et al. (Barnes et al., 2000). On the other hand, a good correlation has been observed between UV-B absorbance in extracts and the UV-B-induced fluorescence intensity from the abaxial side of soybean leaves, thus confirming the applicability of the fluorescence method to assess the contents of epidermally located UV-B absorbing compounds and to track changes in this content (Mazza et al., 2000).
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Such compounds consist of two chemically different classes: flavonoids and HCAs. The latter have absorbance maxima at 227–245 nm and at 310–332 nm, but very little absorbance at longer wavelengths in the UV-A spectral region (Ribéreau-Gayon, 1972
), whereas flavones and flavonols usually display a strong absorbance band in the UV-A (Mabry et al., 1970). The differential absorbance at 366 nm of the two different classes can be used to detect variation in their relative contents in epidermal tissue by the fluorescence method. In a plot of UV-A absorbance versus UV-B absorbance for Vicia faba, which contains mainly kaempferol and quercetin glucosides and very little HCAs, the slope of the regression is close to 1 (Fig. 4). A proportional contribution of HCAs to epidermal screening would have caused a reduction of the slope due to their preferred UV-B absorption. Similarly, the presence of other flavonoids with a UV-A absorption band located at shorter wavelengths would influence the slope in this plot negatively. Rye is an example where HCAs contribute to epidermal screening. HCAs were constitutively present in the leaves, whereas epidermal flavonoids were virtually absent in the youngest leaves and increased with increasing leaf age (Burchard et al., 2000). This caused a negative intercept of the regression line (Fig. 4). These data demonstrate that parallel measurements with UV-A and UV-B excitation can give an indication of the absorption properties of the screening compounds. Appropriate calibration of the ratio between UV-A and UV-B transmission to a ratio between UV-A and UV-B absorbing compounds as determined by HPLC analysis may allow fluorescence analysis to be used as a simple procedure for roughly estimating the ratio between HCAs and flavonoids.
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Measurements on attached leaves using a new portable device |
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TopAbstractIntroductionThe fluorescence method for...Comparison to spectrophotometric...The limits of the...Relationship to accumulated...Measurements on attached leaves...ConclusionReferences |
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The measurements of F(UV)/F(BG) described so far were all carried out with the so-called XE-PAM Fluorometer (Heinz Walz GmbH, Effeltrich, Germany) which constitutes a typical laboratory research instrument. This device features a Xe-discharge lamp as the source of pulse-modulated measuring light, the wavelength of which can be varied between the UV-B and near infrared, using appropriate filters. For the assessment of epidermal transmission, leaves have to be detached and placed inside the sample compartment. Hence, such measurements with the XE-PAM may not be considered non-invasive. Very recently, however, a battery-driven portable device, the so-called UV-A-PAM (Gademann Instruments, Würzburg, Germany) became available which employs a flexible UV transmitting light-guide with leaf clip, such that the same leaf can be studied in situ in a non-invasive way over extended periods of time. While a detailed description of the UV-A-PAM will be published elsewhere (U Schreiber and Gademann, unpublished results), only some of the most important features shall be outlined here; pulse modulated measuring light is obtained from an array of UV-A (peak 370 nm; UV-A) and blue (peak 470 nm; B) LEDs, with the two wavelengths rapidly alternating, such that F(UV-A) and F(B) are monitored quasi-simultaneously. A measurement involves sampling of F(UV-A) and F(B) as well as calculation of the F(UV-A)/F(B) ratio. The intensity of the blue measuring light can be adjusted such that F(UV)/F(B)=1 with the blue plastic fluorescence-standard mentioned above. The standard deviation caused by instrumental errors is in the order of 0.001 which is much less than the natural heterogeneity of F(UV)/F(B) found on a single leaf.
A distinct advantage of non-invasive measurements with the UV-A-PAM is the possibility of repeated measurements on the same spot of a leaf under changing environmental conditions or during development. In this way, potential dynamic changes in UV screening can be assessed. Seasonal accumulation of UV-B screening pigments in Norway spruce has been reported (Fischbach et al., 1999), while a considerable diurnal variation of flavonoid contents in response to UV-B radiation in the fern Cryptogramma crispa R. Gr. ex Hook has been described (Veit et al., 1996). Indications for similar variations were reported for the tropical tree Anacardium excelsum (Bertero & Balb.) Skeels in the same study. The UV-A-PAM fluorometer was used to follow the kinetics of flavonoid formation in leaves of V. faba which were suddenly subjected to UV-B radiation in a growth room. In Fig. 5 the time-dependent changes of F(UV-A)/F(B) over the course of 6 d after start of the UV-B treatment is shown for three leaf age classes. Leaf numbers 1 and 4 were fully mature at the beginning of the treatment, while number 7 was the youngest leaf. On day 1, i.e. approximately 20 h after the end of the first UV-B irradiation period and directly before the second 4 h treatment, no changes were apparent. However, on day 2 the youngest leaf showed a considerable decline of F(UV-A)/F(B), indicating the ongoing synthesis of flavonoids. This apparently continued over the next 4 d, while the mature leaves did not show any change.
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The fact that the four oldest leaves did not respond to UV-B irradiation is even more evident from Fig. 6
where F(UV-A)/F(B) ratios from the last day of the treatment are shown for all leaf age classes. The first leaf showing a distinct response was leaf 5, which was almost fully developed when the treatment started. Leaves 5 to 9 showed significant growth over the treatment period. Leaves 8 and 9, which unfolded during the treatment, were exposed for almost their whole life to UV-B radiation and showed even lower values than the leaves which received the first treatment at a later developmental stage. The induction of flavonoid synthesis by UV-B irradiation and an ensuing decrease in epidermal transmission for UV-B radiation is a commonly observed response (Cen and Bornman, 1993).
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The age dependence of adjustment of epidermal screening in response to enhanced UV-B appears interesting. It was reported that mature Arabidopsis thaliana leaves were unable to accumulate flavonoids when suddenly exposed to enhanced UV-B irradiation, whereas young leaves showed a significant induction of flavonoids (Lois, 1994
). A strong decline with increasing leaf age in the activity of phenylalanine ammonia lyase, a key enzyme of flavonoid metabolism, was reported for barley (Blume and McClure, 1979; Liu et al., 1995). Young leaves have a very active metabolism and may be especially susceptible to UV-B-induced DNA damage. Furthermore, being at the top of the plant, they are most exposed and concomitantly shade the older leaves. Hence, the inability to adjust UV protection in mature leaves may not be considered a disadvantage for a plant. Under natural conditions, UV-B irradiation can change rapidly, for instance during variable weather conditions, or during the unpredictable movements of the polar ozone hole (Pyle, 1997; Rousseaux et al., 1999). Then, slow kinetics or the absence of the formation of UV-B screening substances may necessitate the presence of other protective mechanisms such as DNA repair, which might have different induction behaviour.
The fact that only data on UV-A transmission are available with the new apparatus may be considered as a drawback at first sight, as UV-B transmission is ecologically more important. However, UV-A may also cause damage, in particular to the photosynthetic apparatus (Karsten et al., 1999; Turcsányi and Vass, 2000). Furthermore, UV-A detection will pick up most of the flavonoids which are prominent UV-B screening compounds, in some plants such as V. faba, even the sole ones. In the latter cases, there is a straight relationship between UV-A and UV-B protection (Fig. 4
). In plants where HCAs also play an additional role, linear relationships between screening in both wavelength ranges is common (Fig. 4; W Bilger, M Rolland and L Nybakken, unpublished data). Therefore, in many practical cases the advantage of non-intrusively monitoring time-dependent changes in UV-A screening may be considered distinctly more important than the lack of specific information on UV-B screening. It may be foreseen that with the ongoing and rapid progress in optoelectronics UV-B LEDs or laserdiodes will become commercially available in the near future, so that the fluorescence method may be extended to a comparative assessment of UV-A and UV-B screening.
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Conclusion |
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TopAbstractIntroductionThe fluorescence method for...Comparison to spectrophotometric...The limits of the...Relationship to accumulated...Measurements on attached leaves...ConclusionReferences |
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The results presented above have validated the fluorescence method for estimating epidermal transmission in higher plants. It may now be used for assessing this important component of plant photoprotection in various contexts. The rapidity and the non-intrusive nature of the fluorescence measurements allow a large number of parallel measurements to be conducted with minimal sample preparation. The introduction of a new fully portable battery-operated apparatus makes real field investigations possible.
A large variability of epidermal transmission has been shown which was not only interspecific, but also strongly dependent on growth conditions and the age of the plant (Bilger et al., 1997; Fischbach et al., 1999; Grammatikopoulos et al., 1999; Burchard et al., 2000). The variation in epidermal screening is to a very large extent due to different contents in screening compounds in the epidermis. When followed by HPLC analysis, the fluorescence method may provide a means to monitor the accumulation of screening compounds under a wide variety of environmental conditions, even in the field. Due to its non-destructiveness, the fluorescence method opens new perspectives for the investigation not only of the acclimation of plants to UV-B radiation but also on the reaction of the metabolism of epidermal phenolic compounds to other environmental stimuli such as wounding, drought or unfavourable temperatures.
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Acknowledgments |
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Financial support by the Deutsche Forschungsgemeinschaft (SFB 251) is gratefully acknowledged. We thank Drs Petra Burchard, Claus Markstädter, Erhard Pfündel, Markus Riederer, Markus Veit, and Gottfried Weissenböck for stimulating discussions.
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Notes |
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3 Present address and to whom correspondence should be sent: Botanisches Institut, University of Kiel, Olshausenstr. 40, D-24098 Kiel, Germany.
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References |
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TopAbstractIntroductionThe fluorescence method for...Comparison to spectrophotometric...The limits of the...Relationship to accumulated...Measurements on attached leaves...ConclusionReferences |
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T. Hura, S. Grzesiak, K. Hura, E. Thiemt, K. Tokarz, and M. Wedzony Physiological and Biochemical Tools Useful in Drought-Tolerance Detection in Genotypes of Winter Triticale: Accumulation of Ferulic Acid Correlates with Drought Tolerance Ann. Bot., October 1, 2007; 100(4): 767 - 775. [Abstract] [Full Text] [PDF]
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S. Barthod, Z. Cerovic, and D. Epron Can dual chlorophyll fluorescence excitation be used to assess the variation in the content of UV-absorbing phenolic compounds in leaves of temperate tree species along a light gradient? J. Exp. Bot., May 1, 2007; 58(7): 1753 - 1760. [Abstract] [Full Text] [PDF]
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 Y. Yang, R. Sulpice, A. Himmelbach, M. Meinhard, A. Christmann, and E. Grill Fibrillin expression is regulated by abscisic acid response regulators and is involved in abscisic acid-mediated photoprotection PNAS, April 11, 2006; 103(15): 6061 - 6066. [Abstract] [Full Text] [PDF]
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