Photosynthesis Research 32: 23-35, 1992. © 1992 Kluwer Academic Publishers, Printed in the Netherlands.
Regular paper
Chloroplast movements in leaves: Influence on chlorophyll fluorescence and measurements of light-induced absorbance changes related to ApH and zeaxanthin formation* Enrico Brugnoli I & Olle Bj6rkman
Carnegie Institution of Washington, Department of Plant Biology, 290 Panama Street, Stanford, CA 94305-1297, USA; 1Permanent address: C.N.R., Istituto per l'Agroselvicoltura, Via Marconi 2, Porano (TR) 05010, Italy Received 12 September 1991; accepted in revised form 3 December 1991
Key words: absorptance, blue light, chloroplast movements, fluorescence, photosynthetic light utilization, zeaxanthin formation Abstract
Light-induced chloroplast movements were found to cause changes in chlorophyll fluorescence emission, closely matching those in leaf absorptance, both in terms of the kinetics and the maximum extent of the changes observed in different species. The results demonstrate that chloroplast movements can have a significant effect on the efficiency of light utilization in photosynthesis. They further show that chloroplast movements need to be taken into account in measurements of fluorescence quenching and especially in measurements of light-induced optical changes used to monitor zeaxanthin formation and ApH associated light scattering in leaves. Means of minimizing and of adjusting for the influences of chloroplast movements in such measurements are discussed.
Abbreviations: F-fluorescence emission; P F D - p h o t o n flux density; R-reflectance; T - t r a n s m i t tance; a - absorptance
Introduction
Chloroplasts can change their orientation within the cell in response to the intensity and direction of the incident light. When light is limiting to photosynthesis chloroplasts move to positions yielding maximum light absorption, whereas in saturating light they reorient themselves such that light absorption is minimized (for reviews, see Britz 1979, Haupt and Scheuerlein 1990). In leaves, only wavelengths below 500 nm are effective in bringing about such chloroplast movements and the action spectra for both the low intensity response and the high intensity re*C.I.W.- D.P.B. Publication No. 1116.
sponse appear to be the same with a peak around 450 nm (Zurzycki 1962, Lechowsky 1972, Inoue and Shibata 1973). The photoreceptor pigment has not been identified but is thought to be a flavin (Haupt and Scheuerlein 1990). The effect of chloroplast movements on the absorptance of photosynthetically active radiation by a leaf can be expected to depend on leaf anatomy and would be most pronounced in leaves with a single layer of chloroplast-containing cells and least pronounced in those with a well-developed palisade parenchyma and multiple cell layers. Although one would expect an increased leaf absorptance to result in a proportional increase in the photosynthetic yield per incident photon in limiting light and a decrease in absorptance to
24 result in a reduction in the excess light energy in saturating light, it has been questioned whether chloroplast rearrangements can have a substantial effect on photosynthesis (Britz 1979, Haupt and Scheuerlein 1990). As suggested by Britz (1979)7 further insight might come from techniques which investigate the fate of absorbed light energy, e.g., in vivo chlorophyll fluorescence. In this study we examine the effects of chloroplast rearrangements on spectral leaf transmittance, reflectance and absorptance, and compare these with changes in fluorescence emission in intact leaves of species having different leaf anatomies and varying degrees of light-induced changes in leaf optical properties. In addition, we examine the impact of chloroplast movements on measurements of light-induced absorbance changes used to monitor the deepoxidation of violaxanthin to zeaxanthin (Yamamoto et al. 1972, Bilger et al. 1989, Bilger and Bj6rkman 1990, 1991) and of light-induced scattering changes used to monitor conformational changes in the thylakoid membrane associated with a build-up of trans-membrane proton gradient (Heber 1969, Kobayashi et al. 1982, Heber et al. 1986).
Materials and methods
Plant materials Leaves of Oxalis oregana Nutt. (redwood sorrel) [Oxalidaceae], Marah fabaceus (Naud.) Greene (Western wild cucumber) [Cucurbitaceae], Helianthus annuus L. (sunflower) [Asteraceae] and fronds of Cyrtomium falcatum K. Presl. (holly fern) [Polypodiaceae] were used. Except for sunflower, leaves were taken in the spring from a garden at Stanford, California (37 °N. lat). Sunflower plants were grown in a temperature-controlled greenhouse (25°C day/20°C night) under natural light during the same time period. Daylength varied from 11 to 13 h. Oxalis leaves developed in the shade of a redwood tree. The PFD remained in the range 10-30/zmol m -2 s -1 for most of the daylight hours but sunflecks increased the PFD to 350/xmol m -2 s -1 for about 1 h; the approximate daily photon
receipt was 1.4 mol m -2. Marah and Cyrtomium grew in partial shade in a wooded area; maximum PFD was 1500/zmol m - 2 s -~ and daily photon receipts were 4.5 and 3.9mol m -2 respectively. Maximum PFD and daily photon receipt for the sunflower leaves were 1700/xmol m - 2 s -~ and 43mol m -2. Leaves of Hedera canariensis Willd. (Algerian ivy) and Gossypium hirsutum L. cv. Acala SJC-1 (cotton) were also used in some experiments. These plants were grown as described by Bilger and Bj6rkman (1990, 1991). The photon receipt for ivy and cotton leaves was 5-7 and 42mol m -2 d -~, respectively. Sunflower plants and leaves of the other species were collected in the morning before any exposure to direct sunlight. With sunflower and cotton, intact attached leaves were used; with other species, petioles were cut and kept in water. Plants or leaves were kept in dim fluorescent light (5-15/zmol m -2 s -1) in the laboratory until the start of the experiment. Measurements of PFD and daily photon receipt were made with Li-Cor quantum sensors and a LI1000 data logger (Li-Cor, Lincoln, NE, USA).
Measurements techniques A Li-Cor 1800 portable spectroradiometer and a Li-Cor 1800-12 integrating sphere and illuminator were used to obtain quantitative measurements of leaf absorptance, reflectance and transmittance. Absorptance (A) was calculated from the measured values of transmittance (T) and reflectance (R) as A = 1 - (T + R). Barium sulfate was used as reflectance standard. A filter (No. 80 B, Eastman Kodak, Rochester, NY, USA) which attenuates at wavelengths > 500 nm was inserted in the Li-Cor halogen light source during the period of actinic illumination of the leaf. The total PFD (400-700 nm) reaching the leaf was 940/~mol m -2 s -1 and the PFD at wavelengths
Regular paper
Chloroplast movements in leaves: Influence on chlorophyll fluorescence and measurements of light-induced absorbance changes related to ApH and zeaxanthin formation* Enrico Brugnoli I & Olle Bj6rkman
Carnegie Institution of Washington, Department of Plant Biology, 290 Panama Street, Stanford, CA 94305-1297, USA; 1Permanent address: C.N.R., Istituto per l'Agroselvicoltura, Via Marconi 2, Porano (TR) 05010, Italy Received 12 September 1991; accepted in revised form 3 December 1991
Key words: absorptance, blue light, chloroplast movements, fluorescence, photosynthetic light utilization, zeaxanthin formation Abstract
Light-induced chloroplast movements were found to cause changes in chlorophyll fluorescence emission, closely matching those in leaf absorptance, both in terms of the kinetics and the maximum extent of the changes observed in different species. The results demonstrate that chloroplast movements can have a significant effect on the efficiency of light utilization in photosynthesis. They further show that chloroplast movements need to be taken into account in measurements of fluorescence quenching and especially in measurements of light-induced optical changes used to monitor zeaxanthin formation and ApH associated light scattering in leaves. Means of minimizing and of adjusting for the influences of chloroplast movements in such measurements are discussed.
Abbreviations: F-fluorescence emission; P F D - p h o t o n flux density; R-reflectance; T - t r a n s m i t tance; a - absorptance
Introduction
Chloroplasts can change their orientation within the cell in response to the intensity and direction of the incident light. When light is limiting to photosynthesis chloroplasts move to positions yielding maximum light absorption, whereas in saturating light they reorient themselves such that light absorption is minimized (for reviews, see Britz 1979, Haupt and Scheuerlein 1990). In leaves, only wavelengths below 500 nm are effective in bringing about such chloroplast movements and the action spectra for both the low intensity response and the high intensity re*C.I.W.- D.P.B. Publication No. 1116.
sponse appear to be the same with a peak around 450 nm (Zurzycki 1962, Lechowsky 1972, Inoue and Shibata 1973). The photoreceptor pigment has not been identified but is thought to be a flavin (Haupt and Scheuerlein 1990). The effect of chloroplast movements on the absorptance of photosynthetically active radiation by a leaf can be expected to depend on leaf anatomy and would be most pronounced in leaves with a single layer of chloroplast-containing cells and least pronounced in those with a well-developed palisade parenchyma and multiple cell layers. Although one would expect an increased leaf absorptance to result in a proportional increase in the photosynthetic yield per incident photon in limiting light and a decrease in absorptance to
24 result in a reduction in the excess light energy in saturating light, it has been questioned whether chloroplast rearrangements can have a substantial effect on photosynthesis (Britz 1979, Haupt and Scheuerlein 1990). As suggested by Britz (1979)7 further insight might come from techniques which investigate the fate of absorbed light energy, e.g., in vivo chlorophyll fluorescence. In this study we examine the effects of chloroplast rearrangements on spectral leaf transmittance, reflectance and absorptance, and compare these with changes in fluorescence emission in intact leaves of species having different leaf anatomies and varying degrees of light-induced changes in leaf optical properties. In addition, we examine the impact of chloroplast movements on measurements of light-induced absorbance changes used to monitor the deepoxidation of violaxanthin to zeaxanthin (Yamamoto et al. 1972, Bilger et al. 1989, Bilger and Bj6rkman 1990, 1991) and of light-induced scattering changes used to monitor conformational changes in the thylakoid membrane associated with a build-up of trans-membrane proton gradient (Heber 1969, Kobayashi et al. 1982, Heber et al. 1986).
Materials and methods
Plant materials Leaves of Oxalis oregana Nutt. (redwood sorrel) [Oxalidaceae], Marah fabaceus (Naud.) Greene (Western wild cucumber) [Cucurbitaceae], Helianthus annuus L. (sunflower) [Asteraceae] and fronds of Cyrtomium falcatum K. Presl. (holly fern) [Polypodiaceae] were used. Except for sunflower, leaves were taken in the spring from a garden at Stanford, California (37 °N. lat). Sunflower plants were grown in a temperature-controlled greenhouse (25°C day/20°C night) under natural light during the same time period. Daylength varied from 11 to 13 h. Oxalis leaves developed in the shade of a redwood tree. The PFD remained in the range 10-30/zmol m -2 s -1 for most of the daylight hours but sunflecks increased the PFD to 350/xmol m -2 s -1 for about 1 h; the approximate daily photon
receipt was 1.4 mol m -2. Marah and Cyrtomium grew in partial shade in a wooded area; maximum PFD was 1500/zmol m - 2 s -~ and daily photon receipts were 4.5 and 3.9mol m -2 respectively. Maximum PFD and daily photon receipt for the sunflower leaves were 1700/xmol m - 2 s -~ and 43mol m -2. Leaves of Hedera canariensis Willd. (Algerian ivy) and Gossypium hirsutum L. cv. Acala SJC-1 (cotton) were also used in some experiments. These plants were grown as described by Bilger and Bj6rkman (1990, 1991). The photon receipt for ivy and cotton leaves was 5-7 and 42mol m -2 d -~, respectively. Sunflower plants and leaves of the other species were collected in the morning before any exposure to direct sunlight. With sunflower and cotton, intact attached leaves were used; with other species, petioles were cut and kept in water. Plants or leaves were kept in dim fluorescent light (5-15/zmol m -2 s -1) in the laboratory until the start of the experiment. Measurements of PFD and daily photon receipt were made with Li-Cor quantum sensors and a LI1000 data logger (Li-Cor, Lincoln, NE, USA).
Measurements techniques A Li-Cor 1800 portable spectroradiometer and a Li-Cor 1800-12 integrating sphere and illuminator were used to obtain quantitative measurements of leaf absorptance, reflectance and transmittance. Absorptance (A) was calculated from the measured values of transmittance (T) and reflectance (R) as A = 1 - (T + R). Barium sulfate was used as reflectance standard. A filter (No. 80 B, Eastman Kodak, Rochester, NY, USA) which attenuates at wavelengths > 500 nm was inserted in the Li-Cor halogen light source during the period of actinic illumination of the leaf. The total PFD (400-700 nm) reaching the leaf was 940/~mol m -2 s -1 and the PFD at wavelengths
