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The structural basis of non-photochemical quenching is revealed?
Update
TRENDS in Plant Science
9 Hwang, J.U. et al. (2005) GTPase activation leads the oscillatory polarized growth of pollen tubes. Mol. Biol. Cell 16, 5385–5399 10 Tao, L.Z. et al. (2002) Plant Rac-like GTPases are activated by auxin and mediate auxin-responsive gene expression. Plant Cell 14, 2745–2760 11 Berken, A. et al. (2005) A new family of RhoGEFs activates the Rop molecular switch in plants. Nature 436, 1176–1180 12 Cherfils, J. and Chardin, P. (1999) GEFs: structural basis for their activation of small GTP-binding proteins. Trends Biochem. Sci. 24, 306–311 13 Cool, R.H. et al. (1999) The Ras mutant D119N is both dominant negative and activated. Mol. Cell. Biol. 19, 6297–6305
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14 Kaothien, P. et al. (2005) Kinase partner protein interacts with the LePRK1 and LePRK2 receptor kinases and plays a role in polarized pollen tube growth. Plant J. 42, 492–503 15 Wengier, D. et al. (2003) The receptor kinases LePRK1 and LePRK2 associate in pollen and when expressed in yeast, but dissociate in the presence of style extract. Proc. Natl. Acad. Sci. U. S. A. 100, 6860–6865 16 Zimmermann, P. et al. (2004) GENEVESTIGATOR. Arabidopsis microarray database and analysis toolbox. Plant Physiol. 136, 2621–2632 1360-1385/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tplants.2005.12.001
The structural basis of non-photochemical quenching is revealed? Richard J. Cogdell Division of Biochemistry and Molecular Biology, Davidson Building, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, UK G12 8QQ
Light-harvesting complex II (LHCII, the major plant light-harvesting pigment–protein complex, efficiently harvests light-energy. However, if the incident light intensity is too high and photosynthesis becomes saturated, LHCII can switch into a quenching state that prevents photodamage. This important process is called non-photochemical quenching, or NPQ, and represents feedback control. Andrew Pascal et al. have recently proposed a detailed model of NPQ based upon the crystal structure of LHCII from spinach.
The problem There is an old saying that ‘too much of a good thing is bad for you’. This certainly holds true for plants where light is concerned. Photosynthesis requires light but excess light can be harmful and cause extensive photodamage when it becomes saturating. However, the plant antenna system has evolved not only to be efficient at light harvesting but also to be induced to switch into a dissipative state by excess light. This widely studied and physiologically important phenomenon is called non-photochemical quenching, or NPQ. NPQ can be measured in vivo as a reduction in the yield of chlorophyll fluorescence. It is a complex process and operates over a range of different time scales. Induction of this fluorescence quenching has been associated with the lowering of the pH of the thylakoid lumen, induction of the xanthophyll cycle and the conversion of violaxanthin into zeaxanthin, the presence of the PsbS protein and oligomerization of light-harvesting complex II (LHCII) [1–4]. However, the precise molecular mechanisms responsible for NPQ remain controversial and Corresponding author: Cogdell, R.J. ([email protected]). Available online 6 January 2006 www.sciencedirect.com
are a topic of hot debate. This might now be about to change following a recent publication by Andrew Pascal et al. [5], which has provided new insight into probable structural changes in the major light-harvesting complex LHCII that are responsible for switching between efficient lightharvesting and the dissipative state where excess light energy is converted into heat. The model Pascal et al. have studied the state of the LHCII complex from spinach in the same crystals that were used by Zhenfeng Liu et al. [6] to determine its 3-D structure. The packing of these LHCII complexes in the crystal is unusual. They are organized into small proteoliposomes. Within these liposomes, the individual LHCII trimers are ordered forming regular icosahedral structures. The liposomes then come together to form crystals that are reminiscent of those seen when icosahedral viruses crystallize. In the crystal lattice, the protein–protein contacts between the adjacent trimers are minimal, particularly in the regions of their transmembrane domains, and so each trimer is essentially functionally independent. Pascal et al. [5] have found that the LHCII trimers in the crystal are in a quenched state, which appears to be similar to the quenched state seen with LHCII oligomers in vitro, even though the crystals contain no zeaxanthin. They used FLIM (fluorescence lifetime imaging microscopy) to measure the fluorescence of the LHCII trimers in the crystals. This is a type of laser-scanning microscopy combined with confocal imaging. Chromophores within a small domain are excited and their fluorescence decay curves are measured by single photon counting. The beauty of this technique is that the measured lifetimes are independent of chromophore concentration and light scattering. A FLIM analysis
Update
60
TRENDS in Plant Science
(a)
(b)
Figure 1. The two hypothetical quenching domains in LHCII crystals. (a) The chlorophyll a611–a612 dimer is in close association with lut 620, chlorophyll a610 and xat 622 [xat 622 is associated through the phosphatidyl-glycerol (PG) molecule]. (b) Sandwiched interactions form the chlorophyll b606–b607 dimer, involving water molecules 308 and 310 (depicted in blue), and Gln131 of the C helix. Lut 621, as well as nex 623 and xat 622, are also in close contact. Xat 622 is from the adjacent monomer within the trimer – note position of PG tail relative to (a). This figure was prepared using the information in the Protein Data Bank (http://www.rcsb.org/pdb/) file 1RWT. Abbreviations: gln, glutamine; lut, (3R,3 0 R,6S)-4,5-didehydro-5,6-dihydro-b,bcarotene-3,3 0 -diol; nex, (1R,3R)-6-{(3E,5E,7E,9E,11E,13E,15E,17E)-18-[(1S,4R,6R)-4hydroxy-2,2,6-trimethyl-7-oxabicyclo[4.1.0]hept1-yl]-3,7,12,16-tetramethyloctadeca1,3,5,7,9,11,13,15,17-nonaenylidene}-1,5,5-trimethylcyclohexane-1,3-diol; xat, (3S,5R,6S,3 0 S,5 0 R,6 0 S)-5,6,5 0 ,6 0 -diepoxy-5,6,5 0 ,6 0 -tetrahydro-b,b-carotene-3,3 0 -diol.
reveals that the crystals are homogeneous with respect to fluorescence lifetime and this has been determined to be 0.89 ns. The fluorescence lifetime of LHCII trimers in their efficient light-harvesting state [7,8] is w4 ns; this drops to 0.1–1.5 ns in the quenched state. Pascal et al. then used fluorescence emission and resonance Raman spectroscopy to look for possible differences in chromophore interactions, comparing the LHCII trimers in the crystals (i.e. in the quenched state) with trimers in solution (i.e. in the non-quenched state). The fluorescence emission spectrum in the crystal was broader and more red-shifted than for the LHCII trimers in solution. This is suggestive of the formation of chlorophyll aggregates, which tend to be red-shifted and are possible quenching centres [9]. This model must then be contrasted to those of Nancy Holt et al. [10] and Jorg Standfuss et al. [11] who
www.sciencedirect.com
Vol.11 No.2 February 2006
suggest that quenching results from either electron transfer or reverse energy transfer between the chlorophylls and zeaxanthin. The resonance Raman spectra showed that neoxanthin is significantly more twisted in the crystal (seen as enhancement of signature vibrations at 951 and 955 cmK1) and that the formyl-group of a chlorophyll b molecule becomes hydrogen bonded in the crystal. Given that these pigments are isolated, well within the body of each trimer, Pascal et al. concluded that they indicate conformational changes in the protein and that these changes are not just due to protein–protein interactions caused by the crystal packing. Now, armed with their spectroscopic data, they looked carefully at the crystal structure to see if they could determine where the changes in chromophore organization reflecting the quenched state are located. Although they do not yet have the benefit of structures for both the quenched and unquenched states, they focused attention on three pigment clusters where chlorophyll pairs can be seen that could act as quenching centres. The two main quenching centres are illustrated in Figure 1. Now, for the first time, we have a possible structural picture for understanding the mechanism of NPQ. In this model, pH, PsbS, induction of the xanthophyll cycle and conversion of violaxanthin to zeaxanthin all act by promoting or stabilizing the formation of the quenched state of LHCII (i.e. chlorophyll aggregates), moreover, the main features of the in vivo quenched state are reflected in its crystal structure. This is exciting and the stage is now set for this well defined hypothesis to be rigorously tested in vivo. References 1 Horton, P. et al. (1996) Regulation of light-harvesting in green plants. Annu. Rev. Plant Physiol. 47, 655–684 2 Niyogi, K.K. (1999) Photoprotection revisited. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50, 333–359 3 Demmig-Adams, B. (1990) Carotenoids and photoprotection: a role for the xanthophyll zeaxanthin. Biochim. Biophys. Acta 1020, 1–24 4 Li, X.P. et al. (2000) A pigment-binding protein essential for regulation of photosynthetic light-harvesting. Nature 403, 391–395 5 Pascal, A.A. et al. (2005) Molecular basis of photoprotection and control of photosynthetic light-harvesting. Nature 436, 134–137 6 Liu, Z. et al. (2004) Crystal structure of spinach major light-harvesting ˚ resolution. Nature 428, 287–292 complex at 2.72A 7 Moya, I. et al. (2001) Time-resolved fluorescence analysis of photosystem II antenna proteins in detergent micelles and liposomes. Biochemistry 40, 12552–12561 8 Mullineaux, C.W. et al. (1993) Excitation-energy quenching in aggregates of the LHCII chlorophyll–protein complex: a time-resolved fluorescence study. Biochim. Biophys. Acta 1141, 23–28 9 Beddard, G.S. and Porter, G. (1976) Concentration quenching in chlorophyll. Nature 260, 366–367 10 Holt, N.E. et al. (2005) Carotenoid cation formation and the regulation of photosynthetic light-harvesting. Science 307, 433–436 11 Standfuss, J. et al. (2005) Mechanisms of photoprotection and nonphotochemical quenching in pea light-harvesting complex at ˚ resolution. EMBO J. 24, 919–928 2.5 A 1360-1385/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tplants.2005.12.002
TRENDS in Plant Science
9 Hwang, J.U. et al. (2005) GTPase activation leads the oscillatory polarized growth of pollen tubes. Mol. Biol. Cell 16, 5385–5399 10 Tao, L.Z. et al. (2002) Plant Rac-like GTPases are activated by auxin and mediate auxin-responsive gene expression. Plant Cell 14, 2745–2760 11 Berken, A. et al. (2005) A new family of RhoGEFs activates the Rop molecular switch in plants. Nature 436, 1176–1180 12 Cherfils, J. and Chardin, P. (1999) GEFs: structural basis for their activation of small GTP-binding proteins. Trends Biochem. Sci. 24, 306–311 13 Cool, R.H. et al. (1999) The Ras mutant D119N is both dominant negative and activated. Mol. Cell. Biol. 19, 6297–6305
Vol.11 No.2 February 2006
59
14 Kaothien, P. et al. (2005) Kinase partner protein interacts with the LePRK1 and LePRK2 receptor kinases and plays a role in polarized pollen tube growth. Plant J. 42, 492–503 15 Wengier, D. et al. (2003) The receptor kinases LePRK1 and LePRK2 associate in pollen and when expressed in yeast, but dissociate in the presence of style extract. Proc. Natl. Acad. Sci. U. S. A. 100, 6860–6865 16 Zimmermann, P. et al. (2004) GENEVESTIGATOR. Arabidopsis microarray database and analysis toolbox. Plant Physiol. 136, 2621–2632 1360-1385/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tplants.2005.12.001
The structural basis of non-photochemical quenching is revealed? Richard J. Cogdell Division of Biochemistry and Molecular Biology, Davidson Building, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, UK G12 8QQ
Light-harvesting complex II (LHCII, the major plant light-harvesting pigment–protein complex, efficiently harvests light-energy. However, if the incident light intensity is too high and photosynthesis becomes saturated, LHCII can switch into a quenching state that prevents photodamage. This important process is called non-photochemical quenching, or NPQ, and represents feedback control. Andrew Pascal et al. have recently proposed a detailed model of NPQ based upon the crystal structure of LHCII from spinach.
The problem There is an old saying that ‘too much of a good thing is bad for you’. This certainly holds true for plants where light is concerned. Photosynthesis requires light but excess light can be harmful and cause extensive photodamage when it becomes saturating. However, the plant antenna system has evolved not only to be efficient at light harvesting but also to be induced to switch into a dissipative state by excess light. This widely studied and physiologically important phenomenon is called non-photochemical quenching, or NPQ. NPQ can be measured in vivo as a reduction in the yield of chlorophyll fluorescence. It is a complex process and operates over a range of different time scales. Induction of this fluorescence quenching has been associated with the lowering of the pH of the thylakoid lumen, induction of the xanthophyll cycle and the conversion of violaxanthin into zeaxanthin, the presence of the PsbS protein and oligomerization of light-harvesting complex II (LHCII) [1–4]. However, the precise molecular mechanisms responsible for NPQ remain controversial and Corresponding author: Cogdell, R.J. ([email protected]). Available online 6 January 2006 www.sciencedirect.com
are a topic of hot debate. This might now be about to change following a recent publication by Andrew Pascal et al. [5], which has provided new insight into probable structural changes in the major light-harvesting complex LHCII that are responsible for switching between efficient lightharvesting and the dissipative state where excess light energy is converted into heat. The model Pascal et al. have studied the state of the LHCII complex from spinach in the same crystals that were used by Zhenfeng Liu et al. [6] to determine its 3-D structure. The packing of these LHCII complexes in the crystal is unusual. They are organized into small proteoliposomes. Within these liposomes, the individual LHCII trimers are ordered forming regular icosahedral structures. The liposomes then come together to form crystals that are reminiscent of those seen when icosahedral viruses crystallize. In the crystal lattice, the protein–protein contacts between the adjacent trimers are minimal, particularly in the regions of their transmembrane domains, and so each trimer is essentially functionally independent. Pascal et al. [5] have found that the LHCII trimers in the crystal are in a quenched state, which appears to be similar to the quenched state seen with LHCII oligomers in vitro, even though the crystals contain no zeaxanthin. They used FLIM (fluorescence lifetime imaging microscopy) to measure the fluorescence of the LHCII trimers in the crystals. This is a type of laser-scanning microscopy combined with confocal imaging. Chromophores within a small domain are excited and their fluorescence decay curves are measured by single photon counting. The beauty of this technique is that the measured lifetimes are independent of chromophore concentration and light scattering. A FLIM analysis
Update
60
TRENDS in Plant Science
(a)
(b)
Figure 1. The two hypothetical quenching domains in LHCII crystals. (a) The chlorophyll a611–a612 dimer is in close association with lut 620, chlorophyll a610 and xat 622 [xat 622 is associated through the phosphatidyl-glycerol (PG) molecule]. (b) Sandwiched interactions form the chlorophyll b606–b607 dimer, involving water molecules 308 and 310 (depicted in blue), and Gln131 of the C helix. Lut 621, as well as nex 623 and xat 622, are also in close contact. Xat 622 is from the adjacent monomer within the trimer – note position of PG tail relative to (a). This figure was prepared using the information in the Protein Data Bank (http://www.rcsb.org/pdb/) file 1RWT. Abbreviations: gln, glutamine; lut, (3R,3 0 R,6S)-4,5-didehydro-5,6-dihydro-b,bcarotene-3,3 0 -diol; nex, (1R,3R)-6-{(3E,5E,7E,9E,11E,13E,15E,17E)-18-[(1S,4R,6R)-4hydroxy-2,2,6-trimethyl-7-oxabicyclo[4.1.0]hept1-yl]-3,7,12,16-tetramethyloctadeca1,3,5,7,9,11,13,15,17-nonaenylidene}-1,5,5-trimethylcyclohexane-1,3-diol; xat, (3S,5R,6S,3 0 S,5 0 R,6 0 S)-5,6,5 0 ,6 0 -diepoxy-5,6,5 0 ,6 0 -tetrahydro-b,b-carotene-3,3 0 -diol.
reveals that the crystals are homogeneous with respect to fluorescence lifetime and this has been determined to be 0.89 ns. The fluorescence lifetime of LHCII trimers in their efficient light-harvesting state [7,8] is w4 ns; this drops to 0.1–1.5 ns in the quenched state. Pascal et al. then used fluorescence emission and resonance Raman spectroscopy to look for possible differences in chromophore interactions, comparing the LHCII trimers in the crystals (i.e. in the quenched state) with trimers in solution (i.e. in the non-quenched state). The fluorescence emission spectrum in the crystal was broader and more red-shifted than for the LHCII trimers in solution. This is suggestive of the formation of chlorophyll aggregates, which tend to be red-shifted and are possible quenching centres [9]. This model must then be contrasted to those of Nancy Holt et al. [10] and Jorg Standfuss et al. [11] who
www.sciencedirect.com
Vol.11 No.2 February 2006
suggest that quenching results from either electron transfer or reverse energy transfer between the chlorophylls and zeaxanthin. The resonance Raman spectra showed that neoxanthin is significantly more twisted in the crystal (seen as enhancement of signature vibrations at 951 and 955 cmK1) and that the formyl-group of a chlorophyll b molecule becomes hydrogen bonded in the crystal. Given that these pigments are isolated, well within the body of each trimer, Pascal et al. concluded that they indicate conformational changes in the protein and that these changes are not just due to protein–protein interactions caused by the crystal packing. Now, armed with their spectroscopic data, they looked carefully at the crystal structure to see if they could determine where the changes in chromophore organization reflecting the quenched state are located. Although they do not yet have the benefit of structures for both the quenched and unquenched states, they focused attention on three pigment clusters where chlorophyll pairs can be seen that could act as quenching centres. The two main quenching centres are illustrated in Figure 1. Now, for the first time, we have a possible structural picture for understanding the mechanism of NPQ. In this model, pH, PsbS, induction of the xanthophyll cycle and conversion of violaxanthin to zeaxanthin all act by promoting or stabilizing the formation of the quenched state of LHCII (i.e. chlorophyll aggregates), moreover, the main features of the in vivo quenched state are reflected in its crystal structure. This is exciting and the stage is now set for this well defined hypothesis to be rigorously tested in vivo. References 1 Horton, P. et al. (1996) Regulation of light-harvesting in green plants. Annu. Rev. Plant Physiol. 47, 655–684 2 Niyogi, K.K. (1999) Photoprotection revisited. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50, 333–359 3 Demmig-Adams, B. (1990) Carotenoids and photoprotection: a role for the xanthophyll zeaxanthin. Biochim. Biophys. Acta 1020, 1–24 4 Li, X.P. et al. (2000) A pigment-binding protein essential for regulation of photosynthetic light-harvesting. Nature 403, 391–395 5 Pascal, A.A. et al. (2005) Molecular basis of photoprotection and control of photosynthetic light-harvesting. Nature 436, 134–137 6 Liu, Z. et al. (2004) Crystal structure of spinach major light-harvesting ˚ resolution. Nature 428, 287–292 complex at 2.72A 7 Moya, I. et al. (2001) Time-resolved fluorescence analysis of photosystem II antenna proteins in detergent micelles and liposomes. Biochemistry 40, 12552–12561 8 Mullineaux, C.W. et al. (1993) Excitation-energy quenching in aggregates of the LHCII chlorophyll–protein complex: a time-resolved fluorescence study. Biochim. Biophys. Acta 1141, 23–28 9 Beddard, G.S. and Porter, G. (1976) Concentration quenching in chlorophyll. Nature 260, 366–367 10 Holt, N.E. et al. (2005) Carotenoid cation formation and the regulation of photosynthetic light-harvesting. Science 307, 433–436 11 Standfuss, J. et al. (2005) Mechanisms of photoprotection and nonphotochemical quenching in pea light-harvesting complex at ˚ resolution. EMBO J. 24, 919–928 2.5 A 1360-1385/$ - see front matter Q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.tplants.2005.12.002