Spatial location of photosystem pigment–protein complexes
in thylakoid membranes of chloroplasts of Pisum sativum studied by chlorophyll fluorescence F. Vachaa,b,, F. Adameca,b, J. Valentaa,c, M. Vachad a Institute of Physical Biology, University of South Bohemia, Branisovska 31, 370 05 Ceske Budejovice, Czech Republic b Biological centre, Academy of Sciences of the Czech Republic, UMBR, Branisovska 31, 370 05 Ceske Budejovice, Czech Republic c Department of Chemical Physics and Optics, Faculty of Mathematics and Physics, Charles University, Ke Karlovu 3, 121 16 Praha 2, Czech Republic d Department of Organic and Polymeric Materials, Tokyo Institute of Technology, Ookayama 2-12-1-S8, Meguro-ku, Tokyo, 152-8552 Japan Available online 9 March 2006
Abstract
Ultrastructure of plant chloroplasts was studied by a single-molecule spectroscopy setup at a temperature of 77 K exploring spatial location of photosystems. Two chloroplast thylakoid membrane regions were visualized by fluorescence microscopy and detected at different wavelengths. The size of these regions and the spatial resolution of the microscope allowed us to measure their chlorophyll fluorescence emission spectra of these membrane domains. While the grana regions are characterized by a predominant presence of Photosystem II pigment–protein complexes emitting at 685 nm, Photosystem I complexes are localized in stroma regions and emit at 730 nm. r 2006 Elsevier B.V. All rights reserved.
Keywords: Photosynthesis; Single molecule spectroscopy; Chloroplast ultrastructure; Photosystem; Fluorescence
1. Introduction regions contain mainly photosystem I (PSI) complexes [2].
The presence of chlorophyll molecules in photosynthetic The process of photosynthesis converts the energy of pigment–protein complexes gives to these complexes light radiation into the energy of chemical bonds. Primary distinctive absorption and fluorescence properties. While processes of photosynthesis take place in pigment–protein the PSII emits light in the region of 680–695 nm both at complexes of thylakoid membranes of plants and algae room and low temperature, the emission of PSI at room chloroplasts, cyanobacteria and photosynthetic bacteria. temperature is negligible and can be detected only below Chloroplast thylakoid membranes are organised into two 200 K as a broad emission around 720–730 nm. types of regions, grana and stroma. Stroma regions are free The spatial distribution of PSI and PSII has been floating membranes, grana are formed by several mem- observed by several destructive methods based mainly on branes stacked together [1]. The grana and stroma are electron microscopy or analysis of isolated thylakoid interconnected but the distribution of photosynthetic membrane fragments. The only method that was, so far, pigment–protein complexes in these two regions is differ- used to follow the different PSI and PSII distribution in ent. Whereas cytochromes b6f are distributed equally, native state of chloroplast is a spectrally resolved laser grana regions are predominantly formed by photosystems scanning microscopy [3,4]. With this method the fluores- II (PSII) with their light-harvesting complexes, and stroma cence images of single chloroplast were obtained at different emission wavelengths, and a picture of spatial Corresponding author. Institute of Physical Biology, University of distribution of PSI and PSII was created. In this work, we have used single molecule spectroscopy South Bohemia, Branisovska 31, 370 05 Ceske Budejovice, Czech Republic. Tel.: +420 387775533, fax: +420 385310356. setup to detect different fluorescence emission spectra of E-mail address: [email protected] (F. Vacha). the two major photosynthetic pigment–protein complexes
0022-2313/$ - see front matter r 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.jlumin.2006.01.148 ARTICLE IN PRESS 302 F. Vacha et al. / Journal of Luminescence 122–123 (2007) 301–303
of chloroplast thylakoid membrane, the PSI and PSII, in
their natural environment. This approach allowed us to study non-invasively their spatial distribution.
2. Materials and methods
Chloroplasts were isolated from pea plants (Pisum
sativum) according to Vacha et al. [4]. Chlorophyll a was extracted from pea leaves by 100% acetone and isolated by preparative HPLC. Isolated chlorophyll was transferred from methanol to diethylether and dried under stream of nitrogen gas. The single molecule setup used in this work is based on Olympus IX70 inverted fluorescence microscope, long- working-distance objective (60 , N.A. 0.7), micro-cryo- stat, imaging spectrometer and liquid-nitrogen-cooled back-thinned CCD camera. The excitation was provided by 442 nm line of HeCd laser in the case of chlorophyll molecules and by a 100W mercury lamp when chloroplasts were detected. The scheme of the setup is shown in Fig. 1. The images were recorded using mirror position of the spectrometer’s turret with fully opened entrance slit (7 mm). The emission spectra of spots placed in a central region of the image (defined by the entrance slit closed to 0.5 mm) were measured by the diffraction grating. For low temperature experiments, the chloroplast suspension was placed in a cryoprotecting medium of 70% (v/v) of glycerol prior to cooling to 77 K.
3. Results and discussion
As a test sample, we have used chlorophyll a molecules
deposited on a cover slip by spin coating in 6% (v/v) of poly(methyl methacrylate) in chloroform. Fig. 2a shows
Fig. 2. (A) Microscope image of fluorescence of chlorophyll a molecules
embedded in solid poly(methyl methacrylate) matrice by spin coating and viewed by Olympus 100 objective, N.A. ¼ 0.95. Number of pixels is 100 objective, 100. The molecules were excited by a 442 nm laser line from HeCd laser in a total reflection mode. (B) Cross-section fluorescence intensity profile of the chlorophyll a fluorescence spots (diamonds). The solid line is Gausian fit of cross sections of six chlorophyll a molecules.
the fluorescence image of individual chlorophyll molecules.
The lateral size of the dots is on the average 7.6 pixles in diameter. The Gaussian fit of cross sections of chlorophyll fluorescence dots is in Fig. 2b and gives the microscope experimental resolution (rexp) of 600764 nm.This should be compared with theoretical resolution rth based on the Rayleigh criterion, given by rth ¼ 0.61l/N.A. For the 100 objective with N.A. ¼ 0.95 the rth is 437 nm at 680 nm, the emission wavelength of chlorophyll. Compar- Fig. 1. Scheme of the single-molecule set up based on an Olympus IX70 ing rexp and rth we can conclude that the theoretical and inverted fluorescence microscope attached to a Jobin-Yvon Triax320 experimental values of microscope resolution are in good spectrograph equipped with a liquid-nitrogen-cooled CCD camera. correlation. Similar procedure can be applied to other ARTICLE IN PRESS F. Vacha et al. / Journal of Luminescence 122–123 (2007) 301–303 303
only. Finally, the ‘‘PSI’’ and ‘‘PSII’’ fluorescence images
were overlaid. The resulting image shows the PSI stroma regions in light and the PSII grana regions in dark shades. The size of the chloroplast and its ultrastructure and organisation of grana and stroma membrane domains fit well with many other observation as reviewed recently [5,6]. Chlorophyll fluorescence emission spectra were mea- sured from various sites of the imaged chloroplast. Results are shown in Fig. 3b, with the spectra normalised at 685 nm. Locations 1 and 2 were chosen to demonstrate the emission of PSII from grana membranes, location 3 and 4 to detect the presence of PSI in stroma. Spectra 1 and 2 show typical shape of PSII fluorescence emission at low temperature. They have maximum at 685 nm with a shoulder and 695 nm. The presence of emission of PSI may be explained by several reasons. (i) The spatial resolution of the optical microscope is close to the size of the grana compartments and therefore some overlap can be expected. (ii) In the inner space of the chloroplast, grana membranes and the intergranal stroma regions can occur above each other affecting the resulting spectrum by the fluorescence of PSI located out of the focal plane. (iii) PSI complexes can occur at the sides of grana membranes. Spectra 3 and 4 show the presence of both PSI and PSII complexes. The intergranal stroma membranes are known to be populated mainly by functional PSI but contain also PSII [7]. The heterogeneity in spatial distribution of photosyn- thetic pigment–protein complexes in thylakoid membrane of plants has been observed and reported many years ago. However, the measurements of such heterogeneity using Fig. 3. (A) Fluorescence image of single chloroplast from Pisum sativum showing grana membrane regions in dark and stroma lamellae in light intact systems in vivo or in situ were impossible until the shades. Numbers show positions where the spectra were recorded from. development of appropriate methods. The image data we (B) Fluorescence emission spectra recorded from four different positions presented here are consistent with our previous measure- in the chloroplast. Two positions, 1 and 2, represent areas of grana ments [4], yet this is the first spectral characterisation of the membranes, positions 3 and 4 represents areas of stroma membranes. grana and stroma membrane regions in intact chloroplasts in their natural conditions.
microscope objectives and it was found that the experi-
mental resolution rexp is sufficient to record the chloroplast Acknowledgements ultrastructure and to measure the spectra of different thylakoid membrane domains. This work was supported by grants MSM6007665808 Chloroplasts were carefully isolated to get a high yield of and AV0Z50510513. intactness. Fig. 3a shows the low-temperature chlorophyll fluorescence image of chloroplast processed according to Vacha et al. [4]. First, the overall fluorescence image was References recorded capturing all emitted radiation above 515 nm.The [1] W. Menke, Experientia 16 (1960) 537. image was composed of contributions of both photosys- [2] B. Andersson, J.M. Anderson, Biochim. Biophys. Acta 593 (1980) 427. tems, PSI and PSII. Keeping the low temperature during [3] M. Vacha, H. Yokoyama, T. Tokizaki, M. Furuki, T. Tani, Rev. Sci. the experiment is essential since PSI almost does not emit Instrum. 70 (1999) 2041. light at a room temperature. In the next step, the [4] F. Vacha, M. Vacha, L. Bumba, K. Hashizume, T. Tani, Photo- fluorescence image was recorded using a long pass filter synthetica 38 (2000) 493. [5] L.A. Staehelin, Photosynth. Res. 76 (2003) 185. RG715 to detect only the emission of PSI complexes. Then, [6] S.G. Wildman, A.M. Hirsch, S.J. Kirchanski, D. Spencer, Photosynth. such image was subtracted from the overall fluorescence Res. 80 (2004) 345. image to get the image of areas with fluorescence of PSII [7] J. Barber, B. Andersson, TIBS 17 (1992) 61.
