J. Plant Physiol. Vol. 140. pp. 673-680 (1992)

Introduction

Changes of the Laser-Induced Blue, Green and Red Fluorescence Signatures during Greening of Etiolated Leaves of Wheat

FRED STOBER and HARTMUT K. LICHTENTHALER

Botanisches Institut II, Universitat Karlsruhe, Kaiserstr. 12, D-7S00 Karlsruhe, Germany

Received May 30, 1992 . Accepted June 24, 1992

Summary

The UV-Iaser-induced blue, green and red fluorescence-emission spectra were used to characterize the pigment status of etiolated leaves of wheat (Triticum aestivum L.) during a 48 h greening period under white light conditions. Upon UV-light excitation (337 nm) leaves not only show a fluorescence emission in the red spectral region between 650 and 800nm (chlorophyll fluorescence with maxima near 690nm and 735 nm), but also in the blue and green regions between 400 to 570 nm with maxima or shoulders near 450 nm (blue) and 530 nm (green). During greening of etiolated leaves the chlorophyll-fluorescence ratio F690/F735 strongly correlated with the total chlorophyll content and the ratio of the chlorophylls to the carotenoids (a+b/x+c). The ratio of the blue to the green fluorescence F450/F530 was also cor­related with the total chlorophyll content and the ratio of chlorophylls to total carotenoids (a+b/x+c). Consequently, there also existed a correlation between the chlorophyll-fluorescence ratio F690/F735 and the ratio of the blue to green fluorescence F450/F530. In contrast, the ratios of the blue to red fluo­rescences F450/F690 and F450/F735 did not show clear relations to the pigment content of the investi­gated plants. The particular shape of the UV-Iaser-induced-fluorescence emission spectra of wheat leaves as well as the dependencies of the fluorescence ratios on the pigment content are due to a partial and dif­ferential reabsorption of the emitted fluorescences by the photosynthetic pigments.

Key words: Blue-green fluorescence, carotenoids, chlorophyll fluorescence, etiolement, fluorescence emission, greening, pigment ratio, wheat.

Abbreviations: a+b = chlorophyll a and b; x+c = total carotenoids (xanthophylls x and carotenes c); F690/F735 = ratio of the chlorophyll fluorescence intensity at 690nm and 735nm; F450/F530 = ratio of the fluorescence intensity at 450nm and 530nm (blue/green); F450/F690 = ratio of the fluorescence intensity at 450nm and 690nm (blue/red); alb = ratio of chlorophyll a to chlorophyll b; a+b/x+c =

ratio of chlorophylls a + b to total carotenoids x + c.

The major part of the visible light absorbed in leaves by the pigments of the photosynthetic apparatus (chlorophyll a and b and the carotenoids) is used to drive the photo­synthetic electron-transport chain, thus generating NADPH and ATP for the reduction of atmospherical CO2• Only a minor part of the absorbed energy is dissipated as heat (Buschmann et al., 1984) or is emitted as red chlorophyll

fluorescence (Kautsky and Hirsch, 1934; Krause and Weis, 1984; Lichtenthaler and Pfister, 1978) with two maxima near 690 nm (F 690) and 735 nm (F735) (Lichtenthaler and Busch­mann, 1987; Lichtenthaler and Rinderle, 1988). Green leaves illuminated with UV-light (e.g. by a nitrogen laser: 337 nm) not only show fluorescence emission in the red spectral range (chlorophyll fluorescence), but also in the blue and green regions with a maximum near 450 nm (F450) and a shoulder near 530 nm (F530).

© 1992 by Gustav Fischer Verlag, Stuttgart

674 FRED STOBER and HARTMUT K. LICHTENTHALER

A blue fluorescence was observed early on in some particu­lar plant tissues (Kohnen, 1908), but was never studied in its spectral properties or recognized as a regular feature of green plant leaves. Kautsky, who detected the red chlorophyll­fluorescence induction kinetics (Kautsky effect), also noticed the blue fluorescence of leaves and stated that it was constant and did not exhibit a light-induced transient (Kautsky and Hirsch, 1934). The blue fluorescence of leaves redetected by Chappelle (Chappelle et al., 1984; Chappelle et al., 1985) is not emitted by chlorophylls or carotenoids (Lang et aI., 1991; Lichtenthaler and Stober, 1990). The origin of the blue and green fluorescence is not yet fully understood, yet var­ious phenolic plant constituents in the vacuoles and! or cell walls such as the coumarins aesculetin and scopoletin as well as caffeic and chlorogenic acid, sinapic acid and catechin are the most likely candidates (Lang et al., 1991; Lichtenthaler et aI., 1991 b; Goulas et aI., 1990; Goulas et aI., 1991).

In the last few years the red chlorophyll fluorescence has been established as a stress indicator of plants (Krause and Weis, 1984; Lichtenthaler and Rinderle, 1988; Hak et al., 1990); this also may apply to the blue and green fluorescence (Chappelle et al., 1991; Lichtenthaler et aI., 1991 a; Theisen, 1988). The new fluorescence ratios F450/F530 (blue/green) and F450/F690 (blue/red) may contain valid information on changes in the pigment content of leaves. If so, the blue/ green fluorescence, in addition to the red chlorophyll fluo­rescence F690/F735, could possibly be used as a tool in the future remote sensing of the state of health of terrestrial veg­etation (Gunther et al., 1991; Lichtenthaler et al., 1991 a). This, however, requires a thorough study of the relationship between pigment content and fluorescence emission charac­teristics of plants. The variation of the blue, green and red fluorescence-emission and fluorescence ratios with increas­ing pigment content during greening of etiolated wheat leaves is therefore a matter of this investigation.

Materials and Methods

Plants

Wheat seedlings (Triticum aestivum L., var. Rector) were grown for 8 days in the dark in a green house on peat-soil with full comple­ments of nutrition for growth (TKS 2). After 8 days the etiolated plants were exposed to moderate continuous white light (180 /lmol m-2 S-I) in a phyto chamber at 22°C and a relative humidity of ca. 50 to 60 %. The first fluorescence-emission spectra were taken 2 min after onset of illumination. In the following 2 days of greening, the UV-laser-induced fluorescence-emission spectra as well as the pigment content and pigment ratios of primary leaves (ca. 2-4cm from the top) were determined every 2h.

Pigment determination

The pigments of the photosynthetic apparatus (chlorophyll a+b and the total carotenoids x + c) were extracted in 100 % acetone and determined by a spectrophotometer (Shimadzu UV 200) using the redetermined extinction coefficients and equations of Lichtenthaler (1987), which allow the simultaneous determination of chlorophyll a and b as well as the total carotenoids in the same pigment extract solution. The pigment values given represent the mean of 3 deter-

minations per condition. Maximum deviation amounted to 6 % or less for the same leaf condition.

Fluorescence·emission spectra

Fluorescence-emission spectra were taken from the upper adaxial side of intact wheat leaves every 2 h after the induction of chloro­phyll synthesis by white-light illumination during a total time course of 48 h. A pulsed nitrogen laser (UV-12, Laser Photonics) was used to excite the blue and green fluorescence as well as the red chlorophyll fluorescence. The fluorescence was sensed by an optical multichannel analyzer (OMA-III, EG & G) containing a diode array with 512 intensified, red-enhanced detection elements (Fig. 1). It en­ables the simultaneous measurement of the emitted fluorescence be­tween 400 nm and 800 nm via a polychromator containing a grating with 150 lines per mm and an entrance slit width of 25 /lm (resolu­tion: ca. 1 nm). The UV-Iaser, emitting at 337 nm, was operated at 10 Hz with a pulse width of 10 ns and a pulse energy of 2.5 mJ. The integration time of the gateable detector was 100 ms per scan with a gating time of ca. 160 ns. The optical multichannel analyzer trig­gered the laser and was used to determine the fluorescence ratios blue/red (F450/F690, blue/green (F450/F530) and the chloropbyll. fluorescence ratio (F690/F735) from the blue, green and red emission bands with bandwidths of 10 nm. Leaf fluorescence was excited and sensed in an angle of 45° to the leaf plain. For one fluorescence­emission spectrum of a leaf, 50 scans (the result of the fluorescence induced by 50 laser pulses) were accumulated. Each ratio is the mean of 6 different leaf measurements. The fluorescence intensity of the spectra given in the figures is expressed as counts per one lasershot (cts ·lasershoc l

). The fluorescence emission spectra presented were not corrected. The standard deviation in the intensity of the fluo­rescence emission of primary leaves with the same greening time amounted to 11 % or less. The variation was much lower in the later than the earlier hours of greening.

Absorbance spectra

Absorbance spectra were taken from etiolated and fully green leaves of wheat using the VlRAF single-beam spectrometer as de­scribed by Buschmann and Lichtenthaler, 1988. The VlRAF spec­trometer enables consecutive measurement of the absorbance, re­flectance and chlorophyll-fluorescence spectra in the visible and near-infrared region of intact leaves without changing the leaf posi­tion. The presented spectra are the mean of 6 different leaf measure­ments. Standard deviation 7 % or less.

filter

blue and red \\ f fluorescence *'

- ~;ciiaiio~: ii;l n~) - - .. s~~~'e

triggersignal

OMA 111-system

Fig. 1: Setup for measuring UV-laser-induced fluorescence-emission spectra of intact leaves.

1500..-------------------,

>-'OJ c ~ 1000

CI> () C CI> () ., CI>

o ~

TrifiCClm aesfivClm

2 min 12h 48h

Fig. 2: Fluorescence-emission spectra of etiolated primary leaves of wheat illuminated for 2 min, 12 hand 48 h with white light. The fluorescence intensity is given in signal counts per one laser pulse (see Methods). The chlorophyll content amounted to 0.5 (2 min light), 12.9 (12h light) and 30.1Ilga+bcm-2 (48h light) and the carotenoid content of 2.4 (2 min light), 4.1 (12h light) and 6.1 Ilg x +ccm-2(48hlight).

Triticum aestivum ~ '§ ... / ................ .

etiolated green

~ CI> /\

11\/ / \\ 400 450 500 550 600 650 700 750 800

wavelength (nml

Fig. 3: Absorbance spectra of an etiolated (2 min light) and a green leaf (48 h light) of wheat. The chlorophyll content of the etiolated leaf was 0.5 and that of the green leaf 30.1 Ilga+bcm-2. The carote­noid content was 2.4 (etiolated leaf) and 6.1 Ilgx+ccm-2 (green leaf).

Results

Fluorescence-emission spectra

The fluorescence-emission spectra of wheat leaves which were illuminated for 2 min, 12 hand 48 h are shown in Fig. 2. The leaves exhibited a strong emission in the blue, green and red regions. The etiolated leaves possessed the strongest blue and the weakest red fluorescence but in addition also a quite distinct shoulder in the green region around 530 nm. The red chlorophyll fluorescence only consisted of one peak near 684 nm indicating the presence of low amounts of chloro­phyllide and/or chlorophyll a (2 min white light). With in­creasing illumination time and pigment accumulation the green fluorescence shoulder decreased and finally disap­peared, whereas the blue fluorescence (F450) remained, though at a lower intensity. After 48 h the shoulder of the green fluorescence around 530 nm was no longer seen. Upon prolonged illumination the red chlorophyll-fluorescence signal near 690 nm initially increased (12 h light) and then de­creased (48 h light), whereas the original shoulder at 735 nm increased to a new fluorescence peak (Fig. 2).

Fluorescence signatures during leaf greening 675

,...., 40.,...----------------,10.0 0

N A. .......... corotenoids: x+c C I ~ E .-. chlorophylls: a+b B.O g ~ 30 .......... _ .... -. a

~ 20 /f:~::~~:~:.···· .... 6.0 [

c .~ m o ..• ..• .. ::.:4' 4.0 ~ o ," ..... ....... ~ ..... ~/-. ]' >,

10 ..c a. 0

.Q

..c () 0

12.0

!l.D

6.0

3.0

~ ~ ~ ~ I ~ ~

+-~~-·~~_+-~-+_-;-_r-+_~~-+O

B. .-. alb • a+b/x+c

\ ............ -..... . .\ ... · ···16··

/~

.•..•......•..•..• .. . .... . . 1> ••

~ ~:-.-." .... -.-....... -.-.... -.... -.... -.-.-.-.... -.

6.0

o + c-

4.0"­x

2.0

+ n

0.0 4----1 ........ -. --+-+--~_+--+--+_-;-_r-+O.O o 6 12 18 24 30 36 42 48

time [h]

Fig. 4: Development of A. the chlorophyll and carotenoid content as well as B. the pigment ratios alb and a + bl x + c of etiolated leaves of wheat during greening under white light (n = 3; maximum devia­tion ± 6%).

Absorbance spectra

The absorbance spectrum of the green leaf showed two broad maxima in the blue and in the red spectral range, whereas that of the etiolated leaf consisted of one broad band in the blue region with three minor peaks near 430, 450 and 480 nm, as primarily caused by the absorption bands of the carotenoids (Fig. 3). In the red region of the absorbance spectrum of the etiolated leaf there exists a very low ab­sorbance peak near 670 nm due to the low amounts of chlo­rophyllide/ chlorophyll a (0.5Ilg cm -2). The absorbance of the green wheat leaf was much higher in the blue and green regions than that of the etiolated leaf due to the presence of chlorophyll a and b and the higher level of carotenoids.

Pigment content and pigment ratio

Both the content of chlorophylls and total carotenoids in­creased during greening, but the pigments exhibited different rates of accumulation (Fig. 4 A). The chlorophyll content of the etiolated leaves started from ca. 0.5Ilga+bcm-2 and ended at ca. 30 Ilg a + b cm -2 after 48 h of white light in the fully green leaves. The total carotenoid content of the etio­lated leaves was 2.4llg x + c cm -2 and increased only 2.9 fold to ca. 6.1Ilgx+ccm-2 after 2d of light. The ratio of chloro­phyll alb started from 12 (etiolated leaves) and reached con­stant values of ca. 3.1 after 6 h of illumination and was then constant (Fig. 4 B). The ratio of chlorophylls to carotenoids (a+b/x+c) increased continuously from 0.23 (etiolated leaves) to values of 5 (green leaf) and exhibited a saturation­like curve.

676

6.0

5.0

Ll) 4.0 I"'} I' LL.

3.0 "-0 OJ tD 2.0 LL.

1.0

0.0

FRED STOBER and HARTMUT K. LICHTENTHALER

!',.T .. 1:

'r .. r ......

! .. -·· •.. A ...•

.•. ~ •...•... o\' ........... ... .................. ,." ...................... ~

o 6 12 18 24 30 36 42 48

time [h]

Fig. 5: Temporal decrease of the chlorophyll-fluorescence ratio F690/F735 during greening of etiolated leaves of wheat.

6.0..-------------------r

5.0

~ 4.0

c:: "- 3.0 o OJ ~ 2.0

1.0

!r .. 1

T .. 1

T

!

0.0 +---!---+---+--I---+--+---+---+ o 5 10 15 20 25 30 35

chlorophyll content [J.l.g a+b cm-2]

Fig. 6: Curvilinear relationship between the chlorophyll-fluo­rescence ratio F690/F735 and the total chlorophyll content a+ b of leaves of wheat during greening.

Chlorophyll-fluorescence ratio F690lF735

The chlorophyll-fluorescence ratio F690/F735 consid­erably decreased within the first 10 h of illumination (Fig. 5) parallel to the increasing chlorophyll content from values of 5 to 1.5. Thereafter the ratio F690/F735 declined more slowly to nearly constant values of about 1.2. The chloro­phyll-fluorescence ratio F690/F735 was inversely correlated with the total chlorophyll content a+ b of the leaf (Fig. 6). There e~isted a curvilinear, hyperbolic correlation which can be expressed by a power function: F690/F735 = 4.56· (a+ b)-O,426 j r2 = 0.971.

There also existed an inverse correlation between the fluo­rescence ratio F690/F735 and the pigment ratio of chloro­phylls to carotenoids (a+b/x+c) (Fig. 7). The dependence can be expressed by an exponential function: F690/F735 = 4.941' e-O,508(a+b/x+c)j r2 = 0.988.

Ratio of the blue to green fluorescence F450lF530

The time course of the blue to green fluorescence F4501 F530 showed the characteristics of a saturation curve (Fig. 8).

Fig. 7: Relationship between the chlorophyll-fluorescence ratio F690/F735 and the ratio of chlorophylls to carotenoids (a+b/x+c) during greening of etiolated leaves of wheat (exponential depend­ence).

3.0..-----------------::---, T ••••• iI .. i ..•..•. ·!··, .. ,··,

o 2.0 I"'}

t:'2 "­o Ll)

i1: 1.0

•. T:.l ..•..•.. , ..•. · •..•..• ··, .L - ..

T .... '1' .,''I'',1Ii

T.'·

·'T

0.0 +---1--+--+---1--+--+---1--+--+--+ o 6 12 18 24 30 36 42 48

time [h]

Fig. 8: Increase of the ratio of the blue to green fluorescence F4501 F530 during greening of etiolated leaves of wheat.

3.0 .,------------------"T

g 2.0 Ll) LL. "-o Ll)

i1: 1.0

• •

~ • • 1! ~~ Iii • . "

0.0 +--+---+---+---I--t---+---t---+ o 5 10 15 20 25 30 35

chlorophyll content [J.l.g a+b cm-2]

Fig. 9: Increase of the ratio of the blue to green fluorescence F450/ F530 with increasing chlorophyll a+b content of leaves of wheat during greening (linear relationship).

Starting from a value of 1.75 the ratio rose with increasing il­lumination time to a value of 2.8 after 48 h of light. A linear dependence of the ratio F450/F530 and the total chlorophyll

3.0

T ,ti-I # , .!, 0 2.0 •

, • I"') T I!} .! u... """-0 I!} '<t 1.0 u...

0.0 +--+-+---+-+---+-+---+-+---+--t----+ o 234

a+b/x+c 5 6

Fig. 10: Dependence of the ratio of the blue to the green fluo­rescence F450/F530 on the ratio of the chlorophylls to the carote­noids (a+b/x+c) during greening of etiolated leaves of wheat. The correlation of F450/F530 on a+b/x+c is linear.

3.0,-------------------.-Ii Tii ..... • I '! .

o 2.0 I"') I!} u... """-o I!}

i1 1.0

T i· .. ~, 1 '!'

0.0 +---+---+---+---+----t----+ 1 23456

carotenoid content [tL9 x+c cm-2] 7

Fig. 11: Increase of the ratio of the blue to green fluorescence F450/ F 530 with increasing carotenoid content x + c of leaves of wheat dur­ing greening (curvilinear dependence).

content a + b was observed (Fig. 9). The ratio increased with increasing chlorophyll content and can be described with a linear regression: F450/F530 0.031·(a+b)+1.908; r2 = 0.930.

A linear relationship also existed between the ratio F 4501 F530 and the pigment ratio of chlorophylls to carotenoids (a+b/x+c) (Fig. 10) which can be described by F450/F530 = 0.211·(a+b/x+c)+ 1.703; r2 = 0.970.

The ratio F450/F530 also correlated to the carotenoid con­tent, however, in a curvilinear relationship (Fig. 11), which can be expressed by a power function: F450/F530 = 1.478· (x + C)O.J~9; r2 = 0.895.

Ratios of the blue to red fluorescences: F450lF690 and F4501 F735

F4501F690: The ratio of the blue to red fluorescence F4501 F690 started from a high value of 5 (etiolated leaf, 2 min light) and declined during the first 2 h of illumination to a value below 3 and then varied between 2 and 3 in the next 2 d of illumination (Fig. 12). The same behavior was observed

6.0

5.0

0 Ol

4.0 <D lL. ........ 3.0 0 l!) v 2.0 lL.

1.0

0.0

Fluorescence signatures during leaf greening 677

,-----------------,30

.; .. ! .. ~.'! ..•.. ~ ................................................ ~.

25

20 ~ ()1 o

15 ........

" -.,J

10 ~

5

+--+--t--~-+-+-~--r-+--+--+O o 6 12 18 24 30 36 42 48

time [h]

Fig. 12: Temporal behavior of the ratios of the blue to the red fluo­rescences F450/F690 and F450/F735 during greening of etiolated primary leaves of wheat.

6.0

T 5.0 !

0 4.0 OJ to u... T T

~t """- 3.0 • • T

U 0 1 1 • T T T TT I!) i •• i! fr '<t 2.0 ! 1· u... T • •

1.0

O.O+---r--+--~-+---r--+--~-+ o 5 10 15 20 25 30 35

chlorophyll content ["'9 a+b cm-2]

Fig. 13: Relationship between the fluorescence ratio F450/F690 and the to.tal chlorophyll content a + b of primary leaves of wheat during greemng.

6.0

T 5.0 • 1

0 4.0 OJ to u... """- 3.0 0 I!} '<t 2.0 u...

1.0

0.0 0

T • 1

T • 1

2

T • •

345

a+b/x+c 6

Fig. 14: Relationship between F450/F690 and the ratio of chloro­phylls to carotenoids (a + b/x + c) of leaves of wheat during greening. With the exception of the first measuring point the ratio F 450/F 690 consisted of constant values between 2 and 3.

between the relationship of F450/F690 to the total chloro­phyll content a+b (Fig. 13) and the pigment ratio (a+bl x+c) (Fig. 14). There are no correlations beween the ratio

678 FRED STOBER and lliRTMUT K. LICHTENTHALER

Table 1: Relationship of the chlorophyll-fluorescence ratio F690/ F735 and the fluorescence ratios F450/F530 and F450/F690 on the total chlorophyll a + b content and on the ratio of chlorophylls to carotenoids (a + b/x + c).

Fluorescence ratio chlorophyll a + b content a + b/x + c F690/F735 F450/F530 F450/F690

6.0

5.0

Lf) 4.0 I"") r-.. lL..

3.0 '-.. 0 O"l CD 2.0 lL..

1.0

0.0 1.5

curvilinear; r2 = 0.971 linear; r2 = 0.930 no; r2 = 0.325

•

exponential; r2 - 0.988 linear; r2 = 0.970 no; r2 = 0.436

2.0 2.5 3.0

F450/F530

Fig. 15: Dependence of the chlorophyll-fluorescence ratio F690/ F735 on the blue/green fluorescence ratio F450/F530 (curvilinear relationship).

F450/F690, and the pigment content or the pigment ratio (r2 < 0.5, s. Table 1), because all values of F450/F690 with the exception of the first measuring point (2 min light), varied around a mean value of 2.5 ± 0.28.

F4501F735: The ratio of the blue to the far-red fluo­rescence F450/F735 exhibited in the shortly illuminated leaves (2 min light) high values around 26, due to the fact that the initially very low chlorophyll content only yielded a very low chlorophyll fluorescence near 735 nm. With in­creasing illumination time and pigment content the ratio F450/F735 decreased to values of 4 after 8 h light and then declined to values of 3 (Fig. 12). A similar decrease-kinetic as for the ratio F450/F735 was also found for the chlorophyll ratio alb (Fig. 4 B). This may indicate that the decrease in the ratio F450/F735 could be due to the increasing content of chlorophyll b, the absorption bands of which overlap with the emitted blue fluorescence. The kinetics of the decrease of the ratio F450/F735 are clearly different than those of the ratio F450/F690 (Fig. 12).

Relationship between F450lF530 and F6901F735

Due to the fact that the chlorophyll-fluorescence ratio F690/F735 and the blue/green fluorescence ratio F450/ F530 showed a clear correlation to the same pigment param­eters (a+b content and the pigment ratio a+b/x+c) (s. Table 1) they also exhibit a curvilinear relationship to one another (Fig. 15) which can be expressed as F690/F735 = 31.7· (F450/F530)-3.384; r2 = 0.969.

Discussion

The complete fluorescence-emission spectra of wheat leaves and the relations between the UV-Iaser-induced (337 nm) fluorescence ratios F690/F735, F450/F690 and F450/F530 and the pigment content and pigment ratios were studied during greening of etiolated wheat leaves. That the blue and green fluorescences do not derive from the photosynthetic pigments, but are emitted by phenolic plant constituents in vacuoles and cell walls had been pointed out before (Goulas et al., 1991; Lang et al., 1991; Lichtenthaler and Stober, 1990). During greening of etiolated wheat leaves several changes in particular regions of the fluorescence­emission spectra occurred which can be explained by a par­tial reabsorption of the emitted fluorescence by the various photosynthetic pigments. This particularly applies to the de­crease in the intensity of the blue-green fluorescence with in­creasing chlorophyll and carotenoid content of the leaf (Fig. 2). The decrease of the fluorescence emission near 450 to 460 nm seems to be due to a partial reabsorption of the emitted fluorescence by the photosynthetic pigments, chlo­rophylls a and b as well as carotenoids, all of which show ab­sorption bands in this spectral region. In turn, the disap­pearance of the green fluorescence emission shoulder between 500 and 540 nm during greening appears to be main­ly caused by a partial reabsorption of the emitted fluo­rescence by chlorophyll band carotenoids which, in contrast to chlorophyll a, absorb light in this spectral region.

The chlorophyll-fluorescence emission spectrum of the etiolated leaf illuminated for 2 min only exhibited a fluo­rescence maximum at 684 nm and a very faint shoulder near 735 nm (Fig. 2). Reabsorption processes of the emitted chlo­rophyll-fluorescence by chlorophylls can be excluded in this case and the chlorophyll-fluorescence ratio F690/F735 ex­hibited correspondingly a high value of 5. That the ratio F 690/F735 decreases with increasing chlorophyll a + b content, as shown here for wheat, can be explained with reabsorption processes of the fluorescence at 690 nm region. In the 690 nm region the absorbance spectrum of the leaf chlorophyll overlaps with the chlorophyll-fluorescence emission (Lichtenthaler et al., 1986; Lichtenthaler and Rin­derle, 1988).

Consequently, increasing chlorophyll content results in an increasing reabsorbance of the shorter-wavelength red chlo­rophyll-fluorescence near 690 nm and thus in decreased val­ues of the chlorophyll-fluorescence ratio F690/F735 (Hak et al., 1990; Szabo et al., 1992). The far-red chlorophyll fluo­rescence in the 735 nm region is effected very little or not at all by reabsorption processes and develops with increasing chlorophyll content from an emission shoulder to a new fluorescence maximum, the position of which varies be­tween 730 to 740 nm depending on the chlorophyll content. There exists a curvilinear correlation between the chloro­phyll-fluorescence ratio F690/F735 and the total chloro­phyll content a+b of primary wheat leaves (Fig. 6). This had also been described before for green leaves of trees (Hak et al., 1990) and during the autumnal chlorophyll breakdown (D'Ambrosio et al., 1992).

That there also exists a correlation between the ratio F690/F735 and the pigment ratio of chlorophylls to carote-

noids (a + bl x + c) is described here for the first time. The in­creasing ratio of chlorophylls to carotenoids (a+b/x+c) dur­ing greening is caused by the fact that the etiolated leaves already possess carotenoids and that the carotenoids exhibit a rather linear accumulation curve, whereas the chlorophyll show a saturation type accumulation (Fig. 4). Since the leaf carotenoids do not absorb light in the chlorophyll-fluo­rescence region, the dependence of the ratio F690/F735 on the pigment ratio (a+b/x+c) seems not to have a causal connection; it is rather an accidental relationship. It only shows up due to the fact that the ratio chlorophyllsl carote­noids increases with increasing chlorophyll content in the formerly etiolated wheat leaf. Whether this relationship can be used e.g. under stress conditions to estimate from a given ratio F690/F735 an approximate value (a+b/x+c) and thus give information on the relative carotenoid content is ques­tionable and has yet to be investigated.

As a consequence of the disappearance of the green fluo­rescence-emission shoulder near 530 nm during greening of wheat leaves, the blue/green fluorescence F450/F530 in­creased during greening. The ratio F450/F530 was linear not only to the chlorophyll content (Fig. 9) but also to the ratio of the chlorophylls to the carotenoids (a+b/x+c) (Fig. 10). The observed dependencies are the result of differential reab­sorbance effects of the blue and the green fluorescence by the pigment apparatus. The green fluorescence is particularly reabsorbed by the accessory pigments (chlorophyll band carotenoids). As in the case of the chlorophyll-fluorescence ratio F690/F735, the linear correlation of F450/F530 with the ratio (a + bl x + c) seems to be casual and not to stand in a causal connection. In contrast the ratio F450/F530 exhibited a curvilinear relationship (Fig. 11) with the increasing caro­tenoid content x + c. This is a causative dependency, since the carotenoids seem to be responsible for the partial reabsorp­tion of the green fluorescence.

In contrast to the fluorescence ratios F690/F735 and F450/F530, no correlations were found between the bluel red (F450/F690) and the bluelfar-red fluorescence ratio (F450/F735) and the pigment content of wheat leaves. After 10 h of greening at a chlorophyll content of 151lg a+ b cm-2

the two ratios F450/F690 and F450/F735 appear to be fairly constant with values near 2.5 and 0.6, respectively and seem then to reflect the normal fluorescence ratios of intact wheat plants. The ratios of blue to red fluorescence can, however, change due to stress conditions, as had been found in the pre­liminary studies of various kinds of stresses (Chappelle et al., 1984; Gunther et aI., 1991; Lichtenthaler et al., 1991 a). In which way the fluorescence ratios bluel red can change by either new accumulation of blue-emitting substances and/or by changes in the content of photosynthetic pigments has yet to be determined under defined stress conditions.

In summary, due to the established relationships between fluorescence ratios and pigment data, the different fluo­rescence ratios studied provide information on the photo­synthetic pigments and on changes in the pigments status of plants. In addition to the red chlorophyll fluorescence (Hak et al., 1990; Lichtenthaler and Rinderle, 1988), the blue and green fluorescence signatures to be suitable tools to in­vestigate the development and state of health of plants as well as to detect stress events.

Fluorescence signatures during leaf greening 679

Acknowledgements

This work was sponsored by a grant of the BMFT Bonn within the EUREKA Programme No. 380 LASFLEUR, which is gratefully acknowledged. We wish to thank Ms. Ursula Prenzel for excellent assistance.

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