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Evolution of Chlorophyll Biosynthesis—The Challenge to Survive Photooxidation

1996; Cell Press; Volume: 86; Issue: 5 Linguagem: Inglês

10.1016/s0092-8674(00)80144-0

1097-4172

Steffen Reinbothe, Christiane Reinbothe, Klaus Apel, Nikolai Lebedev,

Plant Stress Responses and Tolerance

Light is of fundamental importance for life on earth. Through photosynthesis, light ultimately provides the sole source of energy for the growth and development of almost all living organisms. The ability to harvest and to convert the sunlight into chemical energy to be conserved in ATP and NADPH2 is one of the most striking characteristics of plant organisms. It is based on the evolution of sophisticated light absorption and energy transduction mechanisms. In higher plants, chlorophylls (chlorophyll a plus chlorophyll b) are involved in these processes. Most other photosynthetically active organisms, such as the Chloroflexaceae, many purple bacteria (proteobacteria), including Rhodobacter capsulatus, and the green sulfur bacteria, such as Chlorobiaceae, all contain a different porphyrin compound, bacteriochlorophyll a, in their reaction centers. Chlorophyll and bacteriochlorophyll are structurally related porphyrin compounds (Figure 1). Most of the early steps in their biosynthetic pathways appear to be identical. However, at the stage of protochlorophyllide, the two pathways use different enzymes to convert protochlorophyllide to chlorin (Figure 1). In place of a three-subunit enzyme in bacteriochlorophyll biosynthesis, encoded by bchB, bchL, and bchN, two proteins encoded by por are used in chlorophyll synthesis (references can be found in13von Wettstein D Gough S Kannangara C.G Plant Cell. 1995; 7: 1039-1057PubMed Google Scholar). Therefore, the important question emerges as to why chlorophyll- and bacteriochlorophyll-containing organisms use different enzymes to catalyze a virtually identical reaction. Did these enzymes appear under the constraint to catalyze the same reaction under different environmental conditions? For example, was their evolution a consequence of the transition from the anaerobic Archaean Earth's atmosphere to an aerobic atmosphere similar to that existing today? In the presence of light and oxygen, porphyrins such as protochlorophyllide cause photooxidative damage of cellular and subcellular structures. Therefore, one possible reason for the different enzyme systems is that these porphyrins were not damaging under the anaerobic conditions of the Archaen Earth's atmosphere but had to be protected from the later aerobic atmosphere. In other words, photoprotection of porphyrin biosynthesis was specifically required only when the Earth's atmosphere became aerobic. Understanding the evolution and operation of mechanisms that confer photoprotection on plants is particularly important because it may provide the basis for the construction of new, porphyrin-based photobleaching herbicides to be exploited in modern weed control. It is the aim of this review to summarize current ideas on the evolution of chlorophyll biosynthesis in a quest to explain how early photosynthetic organisms acquired the capability to solve the problem of photooxidation. Photosynthetically active organisms are continuously forced to cope with the problem of photooxidation under the aerobic conditions of our current atmosphere on Earth. Not only the end products, chlorophylls a and b, but also many intermediates in the biosynthetic pathway of porphyrins, such as protoporphyrin IX and its various Mg2+ derivatives, including protochlorophyllide, tend to interact after light absorption in an uncontrolled manner with other macromolecules in their environment. Instead of transferring their excitation energy onto their ultimate targets, the excited porphyrin pigments, by triplet–triplet interchange, interact with oxygen to produce singlet oxygen. This compound is potentially harmful to the plant because it causes membrane peroxidation, protein denaturation, and pigment bleaching, effects that collectively lead to photooxidative damage of the chloroplast, the site of porphyrin synthesis, and ultimately to cell death. In higher plants such as angiosperms, several different photoprotection mechanisms operate. During photosynthesis, excited chlorophyll molecules contained in the light harvesting complexes transfer their excitation energy to the reaction centers. Excess energy not to be used for this purpose is normally dissipated by transfer onto accompanying pigments, such as carotenoids, which quench triplet states of chlorophyll and, if spin–spin interchange has already happened, also quench singlet oxygen. Within the reaction centers, for example, of photosystem II, the "special pair" of chlorophyll a molecules (chlorophyll P680 and pheophytin, a Mg2+-free chlorophyll a derivative) uses light for charge separation and subsequent electron flow. However, if the energy of the incoming light exceeds that which can be used during photosynthesis, normal electron flow slows and then finally stops. As a result of this "photoinhibition," one of the proteins in the reaction center of photosystem II, the D1 protein, can no longer operate in its normal way. After charge separation, its chlorophyll P680+ recombines with pheophytin− to form triplet chlorophyll that then dissipates its excitation energy by interacting with ground-state oxygen to form singlet oxygen. In turn, photobleaching of chlorophyll and carotenoids is induced, and photooxidative damage occurs to the D1 protein (5De Las Rivas J.D.L Shipton C Ponticos M Barber J Biochemistry. 1993; 32: 6944-6950Crossref PubMed Scopus (47) Google Scholar). However, at a time when the D1 protein undergoes proteolytic degradation, a specific class of proteins with presumed photoprotective function accumulates at the thylakoids (1Adamska I Ohad I Kloppstech K Proc. Natl. Acad. Sci USA. 1992; 89: 2610-2613Crossref PubMed Scopus (120) Google Scholar). These "early light induced proteins" (ELIPs; 11Meyer G Kloppstech K Eur. J. Biochem. 1984; 138: 201-207Crossref PubMed Scopus (107) Google Scholar) are thought to bind carotenoids (10Lers A Levy H Zamir A J. Biol. Chem. 1991; 266: 13698-13705Abstract Full Text PDF PubMed Google Scholar) and thus could be able to quench triplet states of chlorophyll as well as singlet oxygen. The risk that intermediates in the biosynthetic pathway of chlorophyll cause photooxidative damage of the plant is low for several reasons (for references, see13von Wettstein D Gough S Kannangara C.G Plant Cell. 1995; 7: 1039-1057PubMed Google Scholar). First, the rate of synthesis of the porphyrin pigment precursors is tightly controlled to reflect the requirements of the plant for chlorophyll in the light. Furthermore, an as yet undetermined control mechanism restricts the production of δ-aminolevulinic acid, the first committed precursor of all porphyrins pigments, in dark-grown angiosperm plants. The only intermediate that accumulates to detectable levels is protochlorophyllide. But the major part of this pigment is located in a specialized subcellular structure, termed the prolamellar body, and is bound to NADPH:protochlorophyllide oxidoreductase (POR). Together with protochlorophyllide and NADPH2, POR forms a photoactive ternary complex. Once exposed to light, this complex converts its protochlorophyllide to chlorin. Efficient energy transfer from non-reducable protochlorophyllide to the ternary complex in etiolated plants and also to chlorin after its formation in light-exposed plants prevents accumulation of photodestructive porphyrin species in the bulk of the pigment. Furthermore, the product of catalysis is not dissociated from POR. Proteolysis seems to be required for the liberation of chlorin. The POR-degrading protease is not present in the dark but appears in the light. At a time when the POR-degrading protease has reached its full activity, ELIPs accumulate (11Meyer G Kloppstech K Eur. J. Biochem. 1984; 138: 201-207Crossref PubMed Scopus (107) Google Scholar) and might thus bind chlorin produced during the proteolytic degradation of POR. Chlorin is then channelled into subsequent steps of chlorophyll biosynthesis. The final products, chlorophyll a and chlorophyll b, in turn bind to the nuclear-encoded, posttranslationally-imported, and the plastid-encoded chlorophyll-binding proteins (for review, see13von Wettstein D Gough S Kannangara C.G Plant Cell. 1995; 7: 1039-1057PubMed Google Scholar). Sequential expression of POR, ELIPs, and the nuclear-encoded chlorophyll-binding proteins may ensure that the actual levels of free intermediates and products of porphyrin biosynthesis are kept very low during the early stages of the light-induced greening of dark-grown, etiolated angiosperm plants. The problem of photooxidation is a rather old one that dates back at least 2.5 gigayears (Ga) to a time when oxygenic photosynthesis had already begun and had increased the partial oxygen pressure to a level presumably similar to that present in the atmosphere of the Earth today. Primitive types of photosynthesis, however, existed even before this and may have occurred at least 3.5 Ga ago, when the atmosphere of the Archaen Earth was largely anaerobic (references can be found in8Larcum, A.W. D. (1991). In Chlorophylls, H. Scheer, ed. (Boca Raton: CRC Press), pp. 367–383.Google Scholar). Relics of this early type of photosynthesis live to this day. For example, one such relic could be Rhodobacter capsulatus, a facultative anaerobic purple bacterium that utilizes bacteriochlorophyll for photosynthesis, but cannot do so in the presence of oxygen, because porphyrin biosynthesis is blocked. If one studies the predicted amino acid sequences of bchL, bchN, and bchB, which encode putative components of the protochlorophyllide reductase of Rhodobacter, significant homologies can be seen to bchX, bchY, and bchZ, respectively, which encode putative components of chlorin reductase, another enzyme of bacteriochlorophyll biosynthesis (Figure 1). Even more exciting, however, is the observation that bchX and bchL, bchY and bchN, and bchZ and bchB each share notable similarity to nifH, nifK, and nifD, respectively, which encode components of extant type I nitrogenases (4Burke D.H Hearst J.E Sidow A Proc. Natl. Acad. Sci. USA. 1993; 90: 7134-7138Crossref PubMed Scopus (124) Google Scholar). These enzymes are known to be sensitive to oxygen (6Fay P Microbiol. Rev. 1992; 56: 340-369Crossref PubMed Google Scholarreferences therein). It is therefore tempting to speculate that it could have been this oxygen sensitivity which required POR to appear. Presumably because the bchLNB-encoded protochlorophyllide reductase and the bchXYZ-encoded chlorin reductase could no longer operate under the aerobic conditions of the Earth's atmosphere, free excited protochlorophyllide and/or chlorin molecules could accumulate and cause photooxidation of early photosynthetic structures in the O2-rich atmosphere. Therefore, a novel protochlorophyllide-reducing enzyme was likely to have been required. Evolution solved this problem in an elegant way: an oxygen-insensitive POR protein was created that tightly bound protochlorophyllide and used light and NADPH2 for the reduction of protochlorophyllide to chlorin. This extraordinary behaviour presumably distinguished POR not only from the bchLNB-encoded, light-independent protochlorophyllide-reducing protein but presumably also from nearly all other enzymes existing at that time. If this ancestral POR protein retained the product of catalysis, as modern POR proteins do, a first scavenger of porphyrin pigment precursors thus could have existed in the Archean aerobic Earth's atmosphere. The capability to perform protochlorophyllide reduction under aerobic conditions and thereby to avoid the danger of photooxidation was certainly a landmark in the evolution of porphyrin biosynthesis. Such a scenario, however, rests on the assumption that POR already occurred at the roots of all plant organisms. In fact, por-related gene sequences have been shown to occur in cyanobacteria (14Suzuki J.Y Bauer C.E Proc. Natl. Acad. Sci. USA. 1995; 92: 3749-3753Crossref PubMed Scopus (77) Google Scholar) and thus can be traced back to the presumed endosymbiotic origin of chloroplasts. In ancestral cyanobacteria and presumably also in early forms of vascular plants, such as the rhyniophytes, both types of protochlorophyllide-reducing enzymes, BCHLNB and POR, were likely to coexist. However, during the evolution of angiosperms, bchL, bchB, and bchN were lost. Molecular evidence based on the analysis of chloroplast DNA structure and organization of the major extant lineages of vascular land plants suggests that presumably as a result of a DNA inversion in a particular 30-kb region, the chlB, chlL, and chlN genes, which are the counterparts of bchB, bchL, and bchN of Rhodobacter and chlB, chlL, and chlN of extant cyanobacteria, were lost at the divergence point between gymnosperms and angiosperms (12Raubeson L.A Jansen R.K Science. 1992; 255: 1697-1699Crossref PubMed Scopus (203) Google Scholar). By contrast, the gene encoding POR was preserved and relocated to the nucleus, where it duplicated and diverged into two distinct por genes, termed porA and porB (3Armstrong G.A Runge S Frick G Sperling U Apel K Plant Physiol. 1995; 108: 1505-1517Crossref PubMed Scopus (218) Google Scholar, 7Holtorf H Reinbothe S Reinbothe C Bereza B Apel K Proc. Natl. Acad. Sci. USA. 1995; 92: 3254-3258Crossref PubMed Scopus (163) Google Scholar). This diversification is likely to have occurred even before speciation of gymnosperms and angiosperms. Reasons that evolution invented and maintained two POR enzymes may be deduced from our studies of the expression patterns of the PORA and PORB polypeptides (3Armstrong G.A Runge S Frick G Sperling U Apel K Plant Physiol. 1995; 108: 1505-1517Crossref PubMed Scopus (218) Google Scholar, 7Holtorf H Reinbothe S Reinbothe C Bereza B Apel K Proc. Natl. Acad. Sci. USA. 1995; 92: 3254-3258Crossref PubMed Scopus (163) Google Scholar). In both gymnosperms and angiosperms, PORA and possibly PORB are active during the transitory stage from dark to light growth (9Lebedev N van Cleve B Armstrong G.A Apel K Plant Cell. 1995; 7: 2081-2090Crossref PubMed Google Scholar) when protochlorophyllide and chlorin must be shielded from interacting with O2 in the atmosphere. PORB is present also in light-adapted plants, where it may serve housekeeping functions in chlorophyll synthesis (3Armstrong G.A Runge S Frick G Sperling U Apel K Plant Physiol. 1995; 108: 1505-1517Crossref PubMed Scopus (218) Google Scholar, 7Holtorf H Reinbothe S Reinbothe C Bereza B Apel K Proc. Natl. Acad. Sci. USA. 1995; 92: 3254-3258Crossref PubMed Scopus (163) Google Scholar). Due to the operation of PORA and PORB, the risk is kept very low that porphyrins, such as protochlorophyllide and chlorin, will cause photooxidative damage to cellular and subcellular structures during the entire plant life cycle. The proposed rooting of POR is based on the hidden assumption that the last common ancestor of all photosynthetic eubacteria contained bacteriochlorophyll, not chlorophyll, in its reaction center. In such a "bacteriochlorophyll-first" scenario, chlorophyll-based photosynthesis would be a late invention unique to the cyanobacteria/chloroplast lineage. In the alternative, "chlorophyll-first" scenario, however, extant bacteriochlorophyll-containing purple bacteria would represent "deposed monarchs," waiting, in specialized environments such as thermoclines of lakes, until "aerobic republicans" have gone out of fashion (2Allen J.F Nature. 1995; 376: 26Crossref Scopus (2) Google Scholar). The lower number of steps required for chlorophyll biosynthesis, as compared to bacteriochlorophyll synthesis, would be consistent with such a model. However, the "chlorophyll-first" hypothesis does not account for the fact that primitive cyanobacteria, by analogy to those existing today, already contained both, the oxygen-insensitive POR and the bchLNB-encoded protochlorophyllide reductase. We therefore prefer the "bacteriochlorophyll-first" hypothesis, which could explain why anoxygenic purple bacteria, which synthesize a photosystem only under anaerobic conditions, contain only a light-independent form and why cyanobacteria, which produce oxygen as a consequence of photosynthesis, have evolved an additional protochlorophyllide-reducing enzyme. Presumably the need to cope with the problem of photooxidation in the O2-rich atmosphere led to the evolution of POR.