Planta
Planta 146, 551-557 (1979)
9 by Springer-Verlag 1979
The Intensification of Absorbanee Changes in Leaves by Light-Dispersion Differences between High-Light and Low-Light Leaves W. Riihle and A. Wild Institut fiir Allgemeine Botanik der Universitfit Mainz, SaarstraBe 21, D-6500 Mainz, Federal Republic of Germany
Abstract. In dispersive samples, like leaves, the absorbance of pigments is intensified. The intensification is due to a longer optical path through the dispersive sample. However, in chloroplast suspensions the optical path is not much longer than in clear solutions. The factor of intensification fi (= the lengthening of the optical path) is calculated by comparing the absorbance of leaves and the absorbance of chloroplast suspensions with equal pigment-content. This method also includes the influence of possible sieve effects which could decrease absorbance. The measurements are carried out with high- and low-light leaves of different thickness and pigment content. The intensification of absorbance was 2 2.5 fold. In highlight leaves it was somewhat less than in low-light leaves. The factor fi is better correlated to the pigment content than to the thickness of the leaves. The plot of absorbance versus the pigment content of the leaves shows that fi decreases with increasing pigment content. In contrast, chloroplast suspensions show a linear dependence as expected from Lambert-Beer's law. Thus, in leaves with very low pigment content the absorbance is intensified up to 6 fold while the intensification decreases with increasing absorbance. These results are in good agreement with measurements of Tsel'niker (1975) and with the theoretical predictions of Butler's formula (1960). Absorbance changes due to photooxidation of P-700 and cytochrome f in intact leaves are measured, and fi is used to calculate the amount of the oxidized components. Without correction for p the values would be much greater than the amount actually present. The corrected data show that between 70 and 90% of A-absorbance; fl-factor of intensification= lengthening of the optical path; Chl=chlorophyll a + b content; DCMU = 3-(3,4-dichlorophenyl)- 1,1-dimethylurea; FW-fresh weight; HL=high-light; LA~leaf area; LL=low-light; PhAR photosynthetically active radiation Abbreviations."
the present P-700 and cytochrome f can be photooxidized in the intact leaf.
Key words: Cytochrome f - High-light plants light plants - Light-dispersion - P-700.
Low-
Introduction Spectrophotometric measurements of a substance in flat samples are often simplified if the amount (c') of the substance on an area vertical to the measuring beam is calculated instead of the concentration c based on the volume of the sample. In this case c' is independent of the sample's thickness. LambertBeer's law changes to A = c'.e for nondispersive samples. In dispersive samples, however, the measuring beam has to pass a longer way than in nondispersive samples. Thus, absorption is enhanced by the factor fi by which the optical path is lengthened. To estimate c' in dispersive samples, it is therefore necessary to know fi and take it into account in Lambert Beer's law: A=c'.e.[3. Several papers are engaged in the influence of dispersion on absorbance measurements. Butler (1964) summarizes the possible effects and their influence. Selective dispersion (Rayleigh-dispersion and anomalous dispersion) is low in multiple-dispersive samples (Butler and Norris, 1960), especially when the photomultiplier covers a large angle of the transmitted light (Murchio and Allen, 1962). So the major part of dispersion in a leaf is non-selective dispersion. Butler (1962) proposes to determine the lengthening of the optical path by adding a dye to the dispersive sample and to a clear solution. Comparison of the absorbance caused by the dye in the sample with that in the solution shows the lengthening of the optical path. Refraction between media with dif-
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w. Riihle and A. Wild: Intensification of Absorbance Changes
f e r e n t r e f r a c t i o n indices c a n also l e n g t h e n t h e o p t i c a l path. T h i s is o f special i n t e r e s t in leaves w h e r e t h e l i g h t h a s t o p a s s cells a n d i n t e r c e l l u l a r s . H e r e a n o t h e r c o m p l i c a t i o n m a y o c c u r b e c a u s e t h e p i g m e n t s are n o t d i s t r i b u t e d h o m o g e n e o u s l y b u t o n l y in a p a r t o f t h e s a m p l e ' s v o l u m e . T h i s so c a l l e d sieve effect r e d u c e s the a b s o r b a n c e ( D u y s e n s , 1956). W i t h l o w a b s o r b a n c e t h e i n f l u e n c e o f t h e sieve effect is o n l y s m a l l b u t the effect increases with increasing absorbance. In this p a p e r w e try to e s t i m a t e the i n f l u e n c e o f t h e s e effects by d e t e r m i n a t i o n o f fl at v a r i o u s w a v e l e n g t h s . By the use o f h i g h - l i g h t a n d l o w - l i g h t leaves w e e x p e c t to see an i n f l u e n c e o f t h i c k n e s s or p i g m e n t c o n t e n t o f t h e l e a f o n ft. S i m u l t a n e o u s l y , an a p p l i c a t i o n is s h o w n b y c o m p a r i n g the a b s o r b a n c e c h a n g e s d u e to p h o t o o x i d a t i o n o f P-700 a n d c y t o c h r o m e f in i n t a c t leaves w i t h t h e t o t a l a m o u n t o f t h e s e c o m p o n e n t s .
pension was determined by adding small amounts of a dye solution to the chloroplast suspension and to the same medium without chloroplasts. The absorbance change caused by the dye in both cases was measured and compared. At 554 nm phenol red was used as the dye and CuC12 was used at 700 nm. The chloroplasts were prepared according to Wild et aI. (1975) and suspended in a medium containing tricine (50 lnmol 1- 1) pH 7.8, NaCl (50 mmol 1 i) and MgC12 (5 mmol i- 1). Then the absorbance caused by the pigments in the leaf was compared to the absorbance caused by the same pigment content (on an area vertical to the measuring beam) of a chloroplast-suspension. Leaf and chloroplast-suspensions are samples with different dispersions, therefore, the absorbance cansed by dispersion alone was measured at 750 nm. In the case of multiple dispersion this value is nearly the same for all wavelengths (Fig. 1) and, therefore, it can be subtracted to yield the absorbance caused by the pigments alone. In this way the absorbance Aa caused by the pigments in the leaf and the absorbance Ac caused by the pigments in the chloroplast suspension were determined and the ratio 13=Al:Ac was calculated. Care was taken that the leaves did not contain substantial amounts of water-soluble pigments, such as anthocyane, which would cause a large error in the determinations.
Material and Methods
Results
Growth Conditions
Fresh Weight, L e a f Area and Pigment Content
Plants of Helianthus annuus, Vicia faba, Brassica rapa, Raphanus sat&us, Sinapis alba, Calendula officinalis, and Zea mays were grown in Lecaton (Fa. Luwasa, Wiesbaden) with Hoagland solution. They were illuminated in a 16:8 h light-dark schedule by fluorescent tubes (Osram 40 W 25) with 6 W m 2 PhAR. Highlight conditions were achieved by 50-80 W m-2 PhAR from HQI 2,000 W lamps (Fa. Osram). Temperature was 21 ~ C, the relative humidity variedbetween65 % and 75%. Primary leaves were taken froln 17 25 daysoldlow-lightplantsor 11-16days oldhigh-light plants, depending on the growth of the species.
Leaf Area (LA), Fresh Weight (FW) and Pigment Content (Chl) A segment was cut out of the measured leaf, its fresh weight and leaf area was determined, and then the pigments were extracted with acetone. The chlorophyll content was calculated according to Ziegler and Egle (1965).
T a b l e 1 s h o w s the a d a p t a t i o n o f the p l a n t s to the g r o w t h c o n d i t i o n s . I n h i g h light the m a t u r e leaves o f all species a r e t h i c k e r , w h i c h is e x p r e s s e d in t h e ratio FW:LA. Some plants exhibit xeromorphical s t r u c t u r e s , e.g,, h a i r s (Sinapis, Helianthus, Raphanus) o r w a x - l a y e r s (Brassica). T h e i n c r e a s e o f F W : L A u n d e r h i g h - l i g h t c o n d i t i o n s is s i g n i f i c a n t f o r all species (p
Planta 146, 551-557 (1979)
9 by Springer-Verlag 1979
The Intensification of Absorbanee Changes in Leaves by Light-Dispersion Differences between High-Light and Low-Light Leaves W. Riihle and A. Wild Institut fiir Allgemeine Botanik der Universitfit Mainz, SaarstraBe 21, D-6500 Mainz, Federal Republic of Germany
Abstract. In dispersive samples, like leaves, the absorbance of pigments is intensified. The intensification is due to a longer optical path through the dispersive sample. However, in chloroplast suspensions the optical path is not much longer than in clear solutions. The factor of intensification fi (= the lengthening of the optical path) is calculated by comparing the absorbance of leaves and the absorbance of chloroplast suspensions with equal pigment-content. This method also includes the influence of possible sieve effects which could decrease absorbance. The measurements are carried out with high- and low-light leaves of different thickness and pigment content. The intensification of absorbance was 2 2.5 fold. In highlight leaves it was somewhat less than in low-light leaves. The factor fi is better correlated to the pigment content than to the thickness of the leaves. The plot of absorbance versus the pigment content of the leaves shows that fi decreases with increasing pigment content. In contrast, chloroplast suspensions show a linear dependence as expected from Lambert-Beer's law. Thus, in leaves with very low pigment content the absorbance is intensified up to 6 fold while the intensification decreases with increasing absorbance. These results are in good agreement with measurements of Tsel'niker (1975) and with the theoretical predictions of Butler's formula (1960). Absorbance changes due to photooxidation of P-700 and cytochrome f in intact leaves are measured, and fi is used to calculate the amount of the oxidized components. Without correction for p the values would be much greater than the amount actually present. The corrected data show that between 70 and 90% of A-absorbance; fl-factor of intensification= lengthening of the optical path; Chl=chlorophyll a + b content; DCMU = 3-(3,4-dichlorophenyl)- 1,1-dimethylurea; FW-fresh weight; HL=high-light; LA~leaf area; LL=low-light; PhAR photosynthetically active radiation Abbreviations."
the present P-700 and cytochrome f can be photooxidized in the intact leaf.
Key words: Cytochrome f - High-light plants light plants - Light-dispersion - P-700.
Low-
Introduction Spectrophotometric measurements of a substance in flat samples are often simplified if the amount (c') of the substance on an area vertical to the measuring beam is calculated instead of the concentration c based on the volume of the sample. In this case c' is independent of the sample's thickness. LambertBeer's law changes to A = c'.e for nondispersive samples. In dispersive samples, however, the measuring beam has to pass a longer way than in nondispersive samples. Thus, absorption is enhanced by the factor fi by which the optical path is lengthened. To estimate c' in dispersive samples, it is therefore necessary to know fi and take it into account in Lambert Beer's law: A=c'.e.[3. Several papers are engaged in the influence of dispersion on absorbance measurements. Butler (1964) summarizes the possible effects and their influence. Selective dispersion (Rayleigh-dispersion and anomalous dispersion) is low in multiple-dispersive samples (Butler and Norris, 1960), especially when the photomultiplier covers a large angle of the transmitted light (Murchio and Allen, 1962). So the major part of dispersion in a leaf is non-selective dispersion. Butler (1962) proposes to determine the lengthening of the optical path by adding a dye to the dispersive sample and to a clear solution. Comparison of the absorbance caused by the dye in the sample with that in the solution shows the lengthening of the optical path. Refraction between media with dif-
0032-0935/79/0146/0551/$01.40
552
w. Riihle and A. Wild: Intensification of Absorbance Changes
f e r e n t r e f r a c t i o n indices c a n also l e n g t h e n t h e o p t i c a l path. T h i s is o f special i n t e r e s t in leaves w h e r e t h e l i g h t h a s t o p a s s cells a n d i n t e r c e l l u l a r s . H e r e a n o t h e r c o m p l i c a t i o n m a y o c c u r b e c a u s e t h e p i g m e n t s are n o t d i s t r i b u t e d h o m o g e n e o u s l y b u t o n l y in a p a r t o f t h e s a m p l e ' s v o l u m e . T h i s so c a l l e d sieve effect r e d u c e s the a b s o r b a n c e ( D u y s e n s , 1956). W i t h l o w a b s o r b a n c e t h e i n f l u e n c e o f t h e sieve effect is o n l y s m a l l b u t the effect increases with increasing absorbance. In this p a p e r w e try to e s t i m a t e the i n f l u e n c e o f t h e s e effects by d e t e r m i n a t i o n o f fl at v a r i o u s w a v e l e n g t h s . By the use o f h i g h - l i g h t a n d l o w - l i g h t leaves w e e x p e c t to see an i n f l u e n c e o f t h i c k n e s s or p i g m e n t c o n t e n t o f t h e l e a f o n ft. S i m u l t a n e o u s l y , an a p p l i c a t i o n is s h o w n b y c o m p a r i n g the a b s o r b a n c e c h a n g e s d u e to p h o t o o x i d a t i o n o f P-700 a n d c y t o c h r o m e f in i n t a c t leaves w i t h t h e t o t a l a m o u n t o f t h e s e c o m p o n e n t s .
pension was determined by adding small amounts of a dye solution to the chloroplast suspension and to the same medium without chloroplasts. The absorbance change caused by the dye in both cases was measured and compared. At 554 nm phenol red was used as the dye and CuC12 was used at 700 nm. The chloroplasts were prepared according to Wild et aI. (1975) and suspended in a medium containing tricine (50 lnmol 1- 1) pH 7.8, NaCl (50 mmol 1 i) and MgC12 (5 mmol i- 1). Then the absorbance caused by the pigments in the leaf was compared to the absorbance caused by the same pigment content (on an area vertical to the measuring beam) of a chloroplast-suspension. Leaf and chloroplast-suspensions are samples with different dispersions, therefore, the absorbance cansed by dispersion alone was measured at 750 nm. In the case of multiple dispersion this value is nearly the same for all wavelengths (Fig. 1) and, therefore, it can be subtracted to yield the absorbance caused by the pigments alone. In this way the absorbance Aa caused by the pigments in the leaf and the absorbance Ac caused by the pigments in the chloroplast suspension were determined and the ratio 13=Al:Ac was calculated. Care was taken that the leaves did not contain substantial amounts of water-soluble pigments, such as anthocyane, which would cause a large error in the determinations.
Material and Methods
Results
Growth Conditions
Fresh Weight, L e a f Area and Pigment Content
Plants of Helianthus annuus, Vicia faba, Brassica rapa, Raphanus sat&us, Sinapis alba, Calendula officinalis, and Zea mays were grown in Lecaton (Fa. Luwasa, Wiesbaden) with Hoagland solution. They were illuminated in a 16:8 h light-dark schedule by fluorescent tubes (Osram 40 W 25) with 6 W m 2 PhAR. Highlight conditions were achieved by 50-80 W m-2 PhAR from HQI 2,000 W lamps (Fa. Osram). Temperature was 21 ~ C, the relative humidity variedbetween65 % and 75%. Primary leaves were taken froln 17 25 daysoldlow-lightplantsor 11-16days oldhigh-light plants, depending on the growth of the species.
Leaf Area (LA), Fresh Weight (FW) and Pigment Content (Chl) A segment was cut out of the measured leaf, its fresh weight and leaf area was determined, and then the pigments were extracted with acetone. The chlorophyll content was calculated according to Ziegler and Egle (1965).
T a b l e 1 s h o w s the a d a p t a t i o n o f the p l a n t s to the g r o w t h c o n d i t i o n s . I n h i g h light the m a t u r e leaves o f all species a r e t h i c k e r , w h i c h is e x p r e s s e d in t h e ratio FW:LA. Some plants exhibit xeromorphical s t r u c t u r e s , e.g,, h a i r s (Sinapis, Helianthus, Raphanus) o r w a x - l a y e r s (Brassica). T h e i n c r e a s e o f F W : L A u n d e r h i g h - l i g h t c o n d i t i o n s is s i g n i f i c a n t f o r all species (p
