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American Society of Plant Biologists
A Role for a Light-Harvesting Antenna Complex of Photosystem II in PhotoprotectionUniversity of Illinois at Urbana-Champaign, Urbana, IL 61801-3707
gov{at}uiuc.edu
High light (beyond what is needed for maximum photosynthesis) is a major plant stress. Under extreme high-light conditions, the photosynthetic apparatus can be damaged irreversibly, but plants and algae have devised various strategies to protect themselves (photoprotection) (Björkman and Demmig-Adams, 1994
In this issue of The Plant Cell, Elrad et al. (pages 1801–1816) characterize a mutant of Chlamydomonas reinhardtii, designated npq5, that is defective in its ability to establish rapid, reversible NPQ of chlorophyll a fluorescence. This mutant lacks a major light-harvesting polypeptide (Lhcbm1), suggesting that Lhcbm1 is required for the cells to elicit NPQ. The relationship between specific antenna complexes and NPQ has been, and still is, hotly debated. The article by Elrad et al. (2002)
The first act of photosynthesis is the absorption of photons by antenna molecules (within femtoseconds), leading to the formation of excited chlorophyll molecules (Chl*). These Chl* decay to ground state by (1) excitation energy transfer to reaction centers, leading to photochemistry, (2) light emission as fluorescence, and (3) heat loss. The production of oxygen, NADPH, and ATP requires the four major multiprotein complexes of PSI and PSII, the cytochrome b6/f complex (Cyt bf), and ATP synthase, plus a multitude of antenna complexes whose major function is to transfer excitation energy to the photosynthetic reaction centers. Another crucial function of antenna complexes is to serve as a safety valve for the thermal dissipation of excess absorbed light energy. The primary charge separations of photosynthesis occur simultaneously in the reaction center chlorophylls P700 (of PSI) and P680 (of PSII). Photochemistry is over within picoseconds, and all further reactions can proceed in darkness. The positive charges produced by PSII oxidize water to molecular O2, and the negative charges reduce plastoquinone to plastoquinol. Protons of water are released into the lumen. Plastoquinol also releases protons into the lumen of the thylakoids as it transfers electrons to Cyt bf. The positive charges produced by PSI ultimately are reduced by electrons available from Cyt bf, and the negative charges reduce NADP+ to NADPH. The energy stored in the proton gradient across the thylakoid membranes is used to synthesize ATP (by the ATP synthase complex). However, when photosynthesis becomes saturated and excess light is absorbed, NADPH and ATP are not made rapidly enough to dissipate the proton gradient, and the pH of the thylakoid lumen decreases. Acidification of the lumen initiates reactions leading to photoprotection events that involve changes in the deexcitation of Chl*. One path for this deexcitation is fluorescence.
Chlorophyll fluorescence of antenna molecules has been used over the years as one of the most powerful noninvasive tools to probe photosynthesis and the ways in which excitation energy is dissipated within the light-harvesting and reaction center complexes (Govindjee, 1995
Errors in interpretation are possible, for example, if light-harvesting complexes move from the strongly fluorescent PSII region of the photosynthetic apparatus to the weakly fluorescent PSI regions. This movement of antennae between PSII and PSI is designated "state changes" (Allen and Forsberg, 2001 To guard against misinterpretations of fluorescence measurements, both the lifetimes of fluorescence, which directly reflect quantum yields, and the state changes must be measured. A convenient way to measure state changes is through low-temperature (77K) fluorescence spectra in which both PSII and PSI are strongly fluorescent, but with emission peaks at specific wavelengths. In state II, the ratio of fluorescent bands at 685 and 695 nm (both from PSII) to those at 720 and 735 nm (both from PSI) are much lower than in state I.
A role of xanthophylls in photoprotective heat dissipation was demonstrated by Demmig et al. (1988)  (reviewed by Demmig- Adams et al., 1996). The xanthophyll cycle (Yamamoto et al., 1999) involves the conversion of violaxanthin (V) to zeaxanthin (Z) via antheraxanthin (A). Thermal dissipation is correlated with the disappearance of V and the appearance of A and/or Z, and the cycle is reversed when excess light is removed. The hypothesized molecular mechanism can be summarized as follows. Excess protons that accumulate in the lumen (see above) can (1) stimulate the deepoxidation of V, and (2) protonate one or more of the antenna complexes of PSII. Process 2 may (a) promote a close association of Z with Chl*, or (b) cause a conformational change in the antenna complexes that leads to increased heat loss from Chl*. In mechanism a, excitation energy would be transferred from Chl* to Z, and a dissipation of energy as heat would take place in Z. There is a great deal of debate regarding whether mechanism a or b is correct, but it is possible that both mechanisms operate, depending on the species and/or environ-mental conditions.
When plants are exposed to excess light, the quantum yield of chlorophyll a fluorescence (from the antenna of PSII) is decreased (i.e., there is a quenching of chlorophyll a fluorescence). The possibility of a photochemical component of fluorescence quenching at saturating light, involving a photoprotective role for O2 as an electron sink, has been discussed recently by Ort and Baker (2002) . However, the nonphotochemical character of this process is demonstrated by the observation that it occurs even when photochemistry is saturated and all QA molecules (the first quinone electron acceptor of PSII) are in the reduced state (QA-). Gilmore et al. (1995)(1998) have shown by measuring the lifetime of chlorophyll a fluorescence in several higher plants that as V is converted to Z during thermal dissipation and NPQ, the fraction of a 2-ns component of the chlorophyll fluorescence lifetime decreases, along with a concomitant increase in a 0.4-ns component. This is a reversible phenomenon that has been referred to as the "dimmer switch." It represents not only reduced fluorescence but also a means of reducing the excitation energy that is directed from the antennae to the reaction center, because the energy stored in Chl* is lost rapidly via processes that promote thermal dissipation within the antennae. As fluorescence yield decreases, the yield of thermal dissipation increases. Figure 1 is a generalized but all-inclusive scheme of some of the possibilities involved in the chemistry and components of the dimmer switch. Excess light leads to overacidification of the thylakoid lumen that promotes the conversion of V to Z and also the protonation of lumenal polypeptides, perhaps the minor chlorophyll–protein complexes (CP26 and CP29) and/or PsbS. Alternatively, conformation changes may occur in LHCIIb trimers. On the other hand, binding of Z to PsbS, to minor antenna (CP26 and CP29), or to LHCIIb trimers may lead to NPQ, either directly by heat loss in Z or by "self-quenching" by chlorophyll a itself. More research is needed to clearly define the mechanistic aspects of this process.
There is general agreement that the xanthophyll cycle–dependent photoprotection mechanism involves the thermal dissipation of excitation in the antenna of PSII. The debate that is highlighted by Elrad et al. (2002) concerns the nature of the antenna complexes that are critical for this dissipation. Is it LHCIIb, the major peripheral antenna, the minor antenna (e.g., CP26 and CP29), or another component? There are 10 genes in Chlamydomonas that encode the major Lhcb polypeptides that form the trimeric domain of LHCII. There are also minor, monomeric Lhcb polypeptides that may link the trimeric domain of the complex to the core of PSII.
One hypothesis is that the peripheral LHCIIb complex is involved through structural/conformational changes (reviewed by Horton et al., 1999
Bassi et al. (1993)
Gilmore et al. (1996)
Higher plants are closely related to green algae; thus, it is natural to use single-celled green algae as model systems for higher plants. In the early days of Otto Warburg and Robert Emerson, Chlorella was the chosen green alga. However, the current alga of choice is Chlamydomonas, because it can grow heterotrophically (using acetate as a source of fixed carbon in the absence of photosynthesis) and is amenable to manipulation using sophisticated molecular techniques (Rochaix et al., 1998 ; Grossman, 2000).
Niyogi et al. (1997)
Similarly, research on Arabidopsis mutants has established a central role of the xanthophyll cycle in NPQ and thus in photoprotection (Niyogi et al., 1998
The npq5 mutant isolated by Elrad et al. (2002) clearly establishes a relationship between the generation of NPQ and trimeric LHCIIb. First, there is a correlation between reduced NPQ and the absence of the Lhcbm1 gene in the mutant. Elrad et al. (2002) show that the lesion in Lhcbm1 is responsible for the decrease in NPQ, because transformation of the mutant strain with the Lhcbm1 gene led to the restoration of normal NPQ. Furthermore, by measuring fluorescence intensities and ratios of fluorescence emitted at 680 and 710 nm (which reflect the ratio of antenna chlorophyll associated with PSII relative to PSI) after experimentally induced state changes, they show convincingly that there are no differences in the state changes between the wild type and the mutant strain. Because mobile LHCIIb is responsible for state changes, these results suggest that the mobile components of LHCIIb are not affected by the loss of Lhcbm1. This demonstrates that there is at least some functional distinction between the components needed for NPQ and those needed for LHCIIb mobility. Further research is needed to determine whether Lhcbm1 is or is not also part of the mobile species. Figure 2 shows false-color images of NPQ in colonies of wild-type Chlamydomonas and a mutant defective for thermal dissipation (A) and in normal Arabidopsis plants and an npq mutant (B). Figure 2C shows two colonies each of wild-type Chlamydomonas cells, the npq5 mutant (which has an incomplete pJD67 insert in the first exon of the Lhcbm1 gene; the insertion event led to a deletion of 320 bp), and a complemented strain (a transformant harboring an ectopic Lhcbm1 gene). These results beautifully depict the differences in NPQ among these strains.
The npq5 mutant showed the following differences from wild type cells:
(1) A much lower ( (2) Fifty percent far-red light reversal of NPQ in the mutant compared with 85% in wild-type cells.
(3) Reversal of NPQ of only 10% after treatment with 10 µM nigericin, compared with 75% in wild-type cells. This shows that the proton gradient across the thylakoid membranes is important for eliciting NPQ. Although small differences in the extent of this gradient between mutant and wild-type cells could cause differences in the final level of NPQ (Govindjee and Spilotro, 2002 (4) Lower NPQ in the mutant at all light intensities relative to wild-type cells, suggesting that there are intensity-dependent differences in the level of NPQ between the npq5 mutant and wild-type cells.
(5) A significantly greater degree of photoinhibition after 10 min of exposure with (6) Decreases in the pigments in the mutant strain: chlorophyll a (by 18%); chlorophyll b (by 27%); xanthophyll pool (by 15%); and neoxanthin and loroxanthin (together by 34%). The chlorophyll a/b ratio is slightly higher (10%) in the mutant, which could be ascribed to decreases in an LHCIIb component. These changes in pigments need to be understood more fully through the isolation, characterization, and quantification of pigment protein complexes in mutant and wild-type cells.
Nevertheless, the npq5 mutant and wild-type cells are similar in many respects. The ratio of Z+A/Z+A+V (deepoxidation state of the cells) at various light intensities and the kinetics of Z formation in high light are essentially identical in mutant and wild-type cells. Elrad et al. (2002) One very important observation is that the "state transitions" are not changed in the mutant. Furthermore, the maximum photosynthetic rate, measured as Fv/Fm (maximum photochemical efficiency of PSII in the dark-adapted state), and the doubling time are the same for the mutant and the wild-type strains. Thus, under standard growth conditions, Lhcbm1 is not of crucial importance, although a more pronounced phenotype may be observed if the mutant is exposed to higher intensity light and/or other stressful conditions (e.g., desiccation and nutrient deprivation).
Future research also should focus on the role of PsbS (Li et al., 2000
I end this essay with a quotation from Vauvenargues: "Those who cannot manage to look from many viewpoints sometimes attribute to one entire object what actually belongs only to the little they are aware of. The neatness of their ideas hinders them from being suspicious." I would add "suspicious of their own ideas." I admit that this may apply to me as it may apply to others involved in the debate regarding which antenna is more important in protecting against damage by excess light. I believe that we have an elephant before us, and different scientists are looking at different parts of this elephant. The key is to keep working hard but also to engage in rational discussions and focused collaborations with those who think differently than us. I thank Nancy Eckardt, Arthur Grossman, Adam Gilmore, and Krishna Niyogi for their various suggestions. The arguments presented in published papers and even those presented here are not "watertight"; thus, this field of research has a tremendous future. The views expressed here are solely mine, and I take full responsibility for all errors.
Allen, J.F., and Forsberg, J. (2001). Molecular recognition in thylakoid structure and function. Trends Plant Sci. 6, 317–326.[CrossRef][Web of Science][Medline]
Andersson, J., Walters, R.G., Horton, P., and Jansson, S. (2001). Antisense inhibition of the photosynthetic antenna proteins CP29 and CP26: Implications for the mechanism of photoprotective energy dissipation. Plant Cell 13, 1193–1204. Bassi, R., Pineau, B., Dainese, P., and Marquard, J. (1993). Carotenoid-binding proteins of photosystem II. Eur. J. Biochem. 212, 297–303.[Web of Science][Medline] Björkman, O., and Demmig-Adams, B. (1994). Regulation of photosynthetic light energy capture, conversion and dissipation in leaves of higher plants. In Ecological Studies, Vol. 100, E.-D. Schulze and M. Caldwell, eds (Berlin: Springer Verlag), pp. 17–47. Chow, W.S., Funk, C., Hope, A.B., and Govindjee (2000). Greening of intermittent-light-grown bean plants in continuous light: Thylakoid components in relation to photosynthetic performance and capacity for photoprotection. Indian J. Biochem. Biophys. 37, 395–404.[Medline] Crimi, M., Dorra, D., Bösinger, C.S., Giuffra, E., Holzwarth, A.R., and Bassi, R. (2001). Time-resolved fluorescence analysis of the recombinant photosystem II antenna complex CP29: Effects of zeaxanthin, pH and phosphorylation. Eur. J. Biochem. 268, 260–267.[Web of Science][Medline] Crofts, A.R., and Yerkes, C.T. (1994). A molecular mechanism for QE quenching. FEBS Lett. 352, 265–270.[CrossRef][Web of Science][Medline]
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Elrad, D., Niyogi, K.K., and Grossman, A. (2002). A major light harvesting polypeptide of photosystem II functions in thermal dissipation. Plant Cell 14, 1801–1816. Frank, H.A., Das, S.K., Bautista, J.A., Bruce, D., Vasil'ev, S., Crimi, M., Croce, R., and Bassi, R. (2001). Photochemical behavior of xanthophylls in the recombinant photosystem II antenna complex, CP26. Biochemistry 40, 1220–1225.[CrossRef][Medline] Gilmore, A.M., Hazlett, T.L., Debrunner, P.G., and Govindjee (1996). Photosystem II chlorophyll a fluorescence lifetimes and intensity are independent of the antenna size differences between barley wild type and chlorina mutants: Photochemical quenching and xanthophyll cycle-dependent non-photochemical quenching of fluorescence. Photosynth. Res. 48, 171–187.[CrossRef]
Gilmore, A.M., Hazlett, T.L., and Govindjee (1995). Xanthophyll cycle dependent quenching of photosystem II chlorophyll a fluorescence: Formation of a quenching complex with a short lifetime. Proc. Natl. Acad. Sci. USA 92, 2273–2277. Gilmore, A.M., Shinkarev, V.P., Hazlett, T.L., and Govindjee (1998). Quantitative analysis of the effects of intrathylakoid pH and xanthophyll cycle pigments on chlorophyll a lifetime distributions and intensity in thylakoids. Biochemistry 37, 13582–13593.[CrossRef][Medline] Govindjee (1995). Sixty-three years since Kautsky: Chlorophyll a fluorescence. Aust. J. Plant Physiol. 22, 131–160. Govindjee, and Seufferheld, M. (2002). Non-photochemical quenching of chlorophyll a fluorescence: Early history and characterization of two xanthophyll cycle mutants of Chlamydomonas reinhardtii. Funct. Plant Biol., in press.
Govindjee, and Spilotro, P. (2002). An Arabidopsis thaliana mutant, altered in the Grossman, A.R. (2000). Chlamydomonas reinhardtii and photosynthesis: Genetics to genomics. Curr. Opin. Plant Biol. 3, 132–137.[CrossRef][Medline] Holub, O., Seufferheld, M.J., Gohlke, C., Govindjee, and Clegg, R.M. (2000). Fluorescence lifetime imaging (FLI) in real time: A new technique in photosynthesis research. Photosynthetica 38, 581–599.[CrossRef] Horton, P., Ruban, A.V., and Young, A.J. (1999). Regulation of the structure and function of the light harvesting complexes of photosystem II by the xanthophyll cycle. In The Photochemistry of Carotenoids, H.A. Frank, A.J. Young, G. Britton, and R.J. Cogdell, eds (Dordrecht, The Netherlands: Kluwer Academic Publishers), pp. 271–291. Li, X.I., Björkman, O., Shih, C., Grossman, A.R., Rosenquist, M., Jansson, S., and Niyogi, K.K. (2000). A pigment-binding protein essential for regulation of pho-tosynthetic light harvesting. Nature 403, 391–395.[CrossRef][Medline] Niyogi, K.K. (1999). Photoprotection revisited: Genetic and molecular approaches. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50, 333–359.[CrossRef][Web of Science] Niyogi, K.K., Björkman, O., and Grossman, A.R. (1997). Chlamydomonas xanthophyll cycle mutants identified by video imaging of chlorophyll fluorescence quenching. Plant Cell 9, 1369–1380.[Abstract]
Niyogi, K.K., Grossman, A.R., and Björkman, O. (1998). Arabidopsis mutants define a central role for the xanthophyll cycle in the regulation of photosynthetic energy conversion. Plant Cell 10, 1121–1134. Ort, D., and Baker, N.R. (2002). A photoprotective role for O2 as an electron sink in photosynthesis? Curr. Opin. Plant Biol. 5, 193–198.[CrossRef][Web of Science][Medline] Rochaix, J.-D., Goldschmidt-Clermont, M., and Merchant, S., eds (1998). The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas. (Dordrecht, The Netherlands: Kluwer Academic Publishers).
Swiatek, M., Kuras, R., Sokolenko, A., Higgs, D., Olive, J., Cinque, G., Müller, B., Eichacker, L.A., Stern, D.B., Bassi, R., Herrman, R.G., and Wollman, F.-A. (2001). The chloroplast gene ycf9 encodes a photosystem II (PSII) core subunit, PsbZ, that participates in PSII supramolecular architecture. Plant Cell 13, 1347–1367. Yamamoto, H.Y., Bugos, R.C., and Hieber, A.D. (1999). Biochemistry and molecular biology of the xanthophyll cycle. In The Photochemistry of Carotenoids, H.A. Frank, A.J. Young, G. Britton, and R.J. Cogdell, eds (Dordrecht, The Netherlands: Kluwer Academic Publishers), pp. 293–303. Related articles in Plant Cell:
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PHOTOSYNTHESIS AND CHLOROPHYLL...
PHOTOPROTECTION: THE ROLE OF...
30% of the Chlamydomonas NPQ is xanthophyll cycle dependent. Thus, caution is advised when extrapolating data from one organism to the other. 
subunit of ATP synthase, has a different pattern of intensity-dependent changes in non-photochemical quenching and kinetics of the P-to-S fluorescence decay. Funct. Plant Biol. 29, 425–434.