Protochlorophyllide reduction and greening in angiosperms: an evolutionary perspective

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Journal of Photl~helni~,t."y u."" Phatohioh*gy B: Biohl~) 41 ( I tP.i7 i 2!11-221

New Trends in Photobiology ( Invited Review)

Protochlorophyllide reduction and greening in angiosperms: an evolutionary perspective H.Y. Adamson ~,.h.. R.G. Hiller", J. Walmsley ~' ~.$'chool of lliolo~it td Science.~ J,ltlcqtt,,rlc Univer.sit.v. Sv, lm'v. 210~I. Attstrali,, " School of lIiologh'al Scienc¢.~, &.'niler.~itv ol Svdncv. S~dm'v. 2006. Att~tralta R¢cei*.ed 18 April ItPg-/: accepted 1 October It)~)7

Abstract Synthesis of chlorophyll involves the conversitm of a porphyrin-type molecule m the .Tlagnesium branch of the tetrapyrrole path~,va~to a dihydroporphyrin or chlorin. This is achieved in vivo mainly, if not exclusively, by the reduction of protochlorophyllide a to chhlnlphyllide a. At least two types of enzyme are involved, one which requires light I NADPH-prtltochlorophyl!ide oxidoreductase or POR-type enzyme l and the tither which dries not I chlL. N. B-type enzyme ). Both types have been identified in cyanobacteria, green algae and land plants up to and including gynmosperms. There is. however, no evidence i~f the h~rmer in anoxygenic purple photosynthetic bacteria or of the lauer in angiosperms. It has been commonly accepted that angiosperms are incapable of synthesizing, chlorophyll in darknes,, because they have lost the chloroplast-encoded chlL. N. B genes during the course of evolution. Thi,, review I~vcusestin the biochemical and genetic attributes of the light-dependent and light-independent retluctases and evidence I'~lrlight-independent chlorophyll synthesis in angiosperms. In it we argue that because angiosperms which are synthesizing chlorophyll in light frequently continue to do so Ihr hour~ or even days when light is ~,ithbeld. angiosperms have t',e capacity for light-independent protochloropkyllide reduction ! or st,me functional equivalent I and a mechanism for it needs to be t'ountl. ~.~ 1997 Elsevier Science S.A.

Keywordx: Prolochlorophyllidereduction:Chlorophylls)nthesis: Light: Dark: Angiosperm:Evoluti~m

1. Introduction Protochlorophyllide (Pchlide) is air intermediate in the chlorophyll (Chl) and bacteriochlorophyll ( Bchl ) biosynthetic pathways of photosynthetic organism.,, and its reduction to chlorophyllide (Chlide) is a key regulatory slep in :.,ngiosperms and other taxa. Two xtructurally different types of enzyme have been implicated in Pchlide reduction. Catalytic activity has been demonstrated in one type. exemplified by NADPH-Pchlide oxidoreductase [ 1.21. and interred in the other, a putative three-subuni! enzyme c~xled tbr by homoIogues of the bacteriochlorophyll genes, bchL, N and B 13 I. The NADPH-Pch!ide oxidoreductase i or POR-type enzyme j is light-dependent, the bchL. N, B-type is light independent. To date there is no evidence for the POR-type enzyme in anoxygenic purple photosynthetic bacteria. Nor is there any evidence for the bchL. N. B-type enzyme in angiosperms. However, both types have been identified in representative * Correspondingauthor. 1011-1344/97/$17.00 © 1997 Elsevier Science S.A. All rights rescr.'ed P I I S I O I I- 1 3 4 4 ( 9 7 J 0 0 1 I J 5 - X

specie.,, of ~.),aitobactcria. green algae :,nd land plants, up to and including gymnosperms 14.5 I. The simplest interpretation of this pattern of distribution of light-dependent and light-independent Pchlide reducta~s is that: l I ) u n l i k e oxygenic photosynthetic bacteria which evolved both forms of the enzyme, anoxygenic photosynthetic bacteria evolved only the light-independent bchL. N. B tbrm. and ( 2 } angiosperms, whose ancestors possessed both forms of the enzyme, have lost the capacity to synthesize the lightindepenue:Lt bchL. N. B form. It is the second proposition that is central to this review. If angiosperms have lost the bchL, N. B-type enzyme during the course of evolution, they should also have lost the ability to synthesize Chl in darkness, unless of course the lightindependent reduction of Pchlide can be achieved by some other means. Have angiosperms lost the ability to synthesize Chl in darkness? The generally accepted view is that they have. An

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alternative, minority view is that angiosperms have not lost the ability to synthesize Chl in darkness but, unlike gymnosperms and lower plants, can do so only if they have been expo~d to light during development. Recently there have been a number of reviews dealing with Chl biosynthesis and Pchlide reduction [ 2.5- ! 01; however. only one [6] has drawn attention :o the body of literat~re underpinning the minority view. There is an obvious need to evaluate the evidence for light-independent Chl synthesis in angiosperms and consider its implications. This article reviews the literature on light-dependent and light-independent Pchlide reduction and greening in angiosperms against a backdrop of other organisms, in it we emphasize the role of light in hzitiath~g the greening process of angiosperms and suggest ways in which this most highly evolved group of plants might be able to accomplish Pchlide reduction in darkness in the absence of chloroplast-encoded bchL. N. B-type genes.

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The nomenclature of tetrapyrroles is confusing because there is more than one numbering system in use. This review uses the IUPAC I I I I s~,stem in which C atoms in the tetrapyrrole moiety are numbered consecutively from I to 20 and employs the same terminology as Scbeer 1121. Scheer defines chlorophylls biologically as cyclic tetrapyrrDles of the porphyrin, dihydroporphyrin (chlorin) and tetrahydroporphyrin ( bactefiochlorin ) type. They are characterized by a filth (isocyclic) ring derived from the CI3 propionic acid side chain of protoporphyrin and are active in photosynthesis. Whilst most contain a central Mg atom, phaeophytins, which play an important role in photosynthetic electron uansport. do not. We use the same trivial names for the substrate(s) and product(s) of the Pchlide reduction reaction as G:iffiths 121. specifically protochlorophyll(ide) and chlorophyll ( ide ). These terms, abbreviated to Pchl (ide) and Chl( ide ) encompass both mono and divinyl forms of protochlorophyllide ( Pchlide. without brackets ) and protochlorophyll ( Pchl ) and mono and divinyl forms of chlorophyllide (Chlide, without brackets) and chlorophyll ( Chl ). 2.2. Background Chl exists in a variety of forms in all oxygenic photosynthetic organisms. Bchl also exists in a variety of forms and is present in anoxygenic photosynthetic bacteria. Chl a and Bchl a are essentially ubiquitous in their respective groups. Chl b is pre~nt in some prokaryotes, green algae and all land plants: Bchl b is pre~nt in purple bacteria [ 121. The main steps in the Chl and Echl pathways, summarized in Fig. I, were worked out several decades ago using chemical and biochemical techniques applied to a small but diverse group of wild-

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4, BACI'ERIOCHLOROPHYLL a Fig. I. Summary of the classical linear chlorophyll u and bacleriochlorophyll r: biosynthetic pathways from amin~)lcvulinic acid ( ALA ).

tyl)C a,d lnutal:t photosynthetic bacteria, cyanobacteria, green algae and land piants. The first aim of this work was to isolate and characterize intermediates 113-171. Early work revealed a very close relationship between the two pathways. This is borne out by the thct that intermediates in one pathway can act as substrates for enzymes of the other [ 18,19 I. Both Chl and Bchl pathways begin with aminolevulinic acid (ALA) and proceed via the same route to Chlide. Chlide is a dihydroporphyrin (i.e.. a chlorin) having one of its four pyrroles (ring D) reduced by the addition of 2H with the consequent loss of a double bond. Esterification of the C17 propionic acid side chain of ring D produces Chl a. Bchl is a tetrahydroporphyrin (i.e., a bacteriochlorin) having two of its four pyrroles (rings D and B) similarly reduced with the consequent loss of two double bonds. Following reduction of ring B, the CI7 propionic acid side chain is esterified, as in the Chl branch of the pathway, and Bchl a is formed. These final steps in the pathways are illustrated in Fig. 2. Once a chemical framework was established, the focus of work on the Chl pathway switched to analyses of the genetic loci of the enzymes involved. Progress was particularly rapid

H. ¥. Ada, xon et oL /Journal of Photochemi.~tO and Photohiolo.gy B: Biolog3 41 t 1997~ 201-221

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H~C ~C~-~--C~ ~~CH1 cl.~ o=c o C~ Fig. 2. Final steps of chlorophyll c~and bac;eriochh)rophylla biosynthetic pathways from protochloroph)llide. on the bacteriochlorophyll front. It soon became evident that in R h o d o b a c t e r c a p s u l a t u s all o f the loci known to be implicated in Bchl a synthesis, as well as most o f the genes essential

for photosynthetic growth, were tightly linked in a 45 kbp region o f the c h r o m o s o m e termed the photosynthesis gene cluster [ 20-22 ]. It also emerged that the genes involw:d were

211-1

it. K A~htm.~on et ttL / Jourmd c~' Pholochemisto" and Photohioh~gy B: BiohJgy 41 ( I t~71201-221

tightly compacted and arranged in a precise order, flanked in some cases by light-harvesting and reaction centre genes coding h~r the proteins to which the pigments bind 123-301. According to Yildiz et al. 131 I it is likely that there has been selective pressure to retain the linkage order in the photosynthesis gene cluster because ( i ) the same arrangement of photosynthesis genes has been observed in diverse species of photosynthetic bacteria 1321 and (ii) there appears to be "functionally significant transcriptional coupling of lightharvesting and reaction centre genes and bacteriochlorophyll biosynthesis genes" in R h o d o b a c t e r Ctlpslllttlll.~'. In contrast to prokaryotes, genes coding for components of the photosynthetic apparatus in eukaryotes are present in two compartments. The chloroplast genome codes lot some of the genes inwflved in synthesis of photosystems I and 2, light-independent Pchl(ide) reduction, nabisco, ATP syntbetase and cytochrome complex 133 I. It also encodes probably all of its own tRNA and rRNA [ 34 I. The nuclear genome codes for the rest 134]. This arrangement calls for a high level of coordination of the ac,ivities of the two genomes and depends on protein-specific transport mechanisms between cellular and sub-cellular compartments [35,361, as well as signalling mechanisms between nucleus and chloroplast 137 I. As with prokaryotes, the chloroplast genomes of land plants exhibit consensus sequences suggesting that they have also evolved under functional constraint [ 38.39 I2.3. C h l a bio.~.vnthetic r o u t e ( s )

ALA. the first committed precursor in the Chl and Bchl pathways, is synthesized via two quite distinct routes. In one. ALA is formed from the intact carbon skeleton of glutamic acid (the C5 pathway): in the other it is produced by the condensation of succinyl Co-A and glycine ( the Shemin pathway ). The C5 pathway from glutamate is widely distributed among phototrophic and non-phototrophic bacterial groups 140 I. and according to Bealc and Weinstein I 17 l. is probably the sole source of ALA in angiosperms and other land plants. green and red algae and cyanobacteria. The Shemin pathway is much more restricted in its distribution. It has been reported in purple non-sulfur bacteria, the non-phototrophic genus R h i z o b i u m and several strains of bacteria that form Bchl under aerobic conditions. It has also been reported in a few land plants, but the evidence for this has been questioned by Bealc and Weinstein I 17 I. According to these reviewers, the pattern of distribution of the two pathways suggests that the C5 route is the older and more primitive of the two. Chl synthesis begins with the condensation of two molecules of ALA to lk)rm a pyrrole ring ( porphobilinogen ). Four pyrrole rings join to form a tetrapyrrole chain ( l-hydroxybilane), which closes to form a tetrapyrrole ring (uroporphyrin.ogen I11 ). The acetic acid s;de chains and two of the pmpionic acid side chains are convu~ed to methyl and vinyl groups respectively, forming protop:~rphyrinogen, which in turn is oxidized to protoporphyrin IX with its characteristic conjugated double-bond system. The next step. which

involves insertion of Mg into the ring. is the first step specific to the lormation of Chl (and Bchl). Interpretation of the pathway from this point onwards is controversial. The debate revolves around the question of whether the pathway is essentially linear 141-431 with individual reactions catalysed by enzymes with wide substrate specificity [21, or multibranched with a number of discrete and parallel biosynthetic routes 144-47 I. In the classical linear scheme addition of a fifth ring to Mg protoporphyrin IX, following its conversion to the monomethyl ester, produces divinyl ( DV ) Pchlide. DV Pchlide is converted to monovinyl (MV) Pchlide by an eight-vinyl reductase. In the penultimate step which is the subject of this review, MV Pchlide is reduced to MV Chlide by the addition of hydrogens to CI7 and CI8 and, in the final step, the CI7 propionic acid side chain is esterilied by the addition of a long-chain alcoholic residue i usually phytol) to lorm MV Chl a. T,) account lot the variety of Chl a chemical species that have been identified in angiosperms. Rebeiz et al. 1461 proposed five additional pathways from MV and DV protoporphyrinogen IX. These. together with the classical pathway outlined above, constitute the six branches of their multibranch scheme. They suggest that most of the Chl in green plants in synthesized via separate MV and DV routes through Pchlide with minor amounts coming from two fully esterified routes through Pchl. The remaining branches are hypothetical and are included to accommodate geometric isomers of the major MV and DV Pchlide routes. The evidence for interconnected MV and DV pathways utilizing dicarboxylic and monocarboxylic intermediates has been summarized by Rebeiz el al. [ 7 I. It comes from two main sources; identilicatkin and characterization of intermediates by electronic. field-desorption and NMR spectroscopy, together with TLC and spectrolluorimetry and precursor-product relationships. The following reactions Ilave been identified in vivo and in organello: DV protoporphyrin --* Mg-DV protoporphyrin

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Reactions matching ( I ) - ( 4 ) but involving MV precursors and products have also been documented. Evidence that the two main pathways to MV and DV Chl a via MV and DV Pchlide are interconnected is comparatively meagre. Cross-over of intermediates from the DV to MV route would presumably depend on the activity of substratespecific eight-vinyl reductases that convert a vinyl group at C8 to a methyl. Eight-vinyl Pchlide reductase activity has been demonstrated in organello 1481 but the enzyme has not yet been isolated and i~.s properties contirmed. Eight-vinyl

/L Y. Adamstm et aL /Jounud of Photochemistry and PhotohuJbJgy B: BiohJ~ty 41 ¢1<,~17~201-221

Chlide and eight-vinyl Chl a reductases have been inferred from physiological studies by Duggan and Rebeiz 1491 and Belanger and Rebeiz 1441. Whilst more work is needed to confirm the possibility of conversion of DV Pchlide and DV Chl to MV forms, in the context of this review it is the fact of Chl heterogeneity and the heterogeneity of its precursors that is important. Structural diversity among intermediates and end products of Chl biosynthesis increases the number of theoretically possible biosynthetic routes, which in tutti increases the likelihood that one or more may turn out to be light independent. The reservations of Gdfliths concerning the multibranched scheme of Rebeiz and co-workers stem from the fact that many of the intermediates in the various pathways have only been identified by spectrolluori,netry and have not been isolated and structurally characterized. In addition, some might be procedural artefacts. According to Grifliths. heterogeneity of Chl a and its precursors is best explained by a single pathway with individual reactions catalysed by enzymes showing broad substrate speciticity in the manner of the light-dependent Pchlide reductase, which accepts both MV and DV Pchlide. Broad substrate specilicity of this type in enzymes catalysing different steps in the pathway could obviously give rise to a variety of de facto biosynthetic routes. Again, in the conte^t of this review, it is not the precise nature of these routes that is important: it is the need to postulate either a large number of highly substrate-specific enzymes or a small number ofenzymes with broad substrate specificity to account for the observed heterogeneity of intermediates and end p¢oducts of the Chl pathway. This dichotomy highlights how much work still needs to be done on the Mg branch in general and, from our point of view, the Pchlide reduction step in angiosperms in particular. 2.4. C h l b b i o s y n t h e t i c r o u t e ( s )

Chl b differs from Chl a in having a formyl instead of a methyl group at C7. Until recently the biosynthetic pathway of Chl b was completely unknown, but presumed to be via Chl a. One of the mechanisms envisaged was the conversion of the seven methyl to a formyl, catalysed by a mixed-|unction oxidase (monooxygenase). Evidence for this has now been obtained for a green alga [ 50 ] and an angiosperm [ 51 I. In each case. the oxygen in the tormyl group was derived from molecular oxygen. Porra et al. [ 51 ] obtained extremely high (93%) isotopic enrichment of the seven lormyl oxygen. They concluded from this that there is only a single pathway lor the formation of the formyl group of Chl b and a single precursor ( molecular oxygen). They nG'o,d, in addition, that since +monooxygenases generally yield alcohol as products, either a single enzyme with unusual properties leading directly to a carbonyl group is required or an additional dehydrogenase'. Whilst the experiments of Porra 151 I and Schneegurt and Beale [50] are consistent with the presumed direction of Chi b formation ( from Chl a), they do not reveal the substrate for oxygen incorporation at C7. Pchlide a,

205

Chlide a and Chl a are all potential candidates given the pre.~nce of Pchlide b. Chlide b and Chl b in higher plants 1491. The formation of h-type from a-type tetrapyrroles does not rule out reactions in the opposite direction, i.e.. the reduction of the C7 formyl group of Chl(ide) b to a ~lethyl gronp. forming Chl(ide) a. However. until very, recently the conversion of Chl b to Chl a seemed intrinsically unlikely because of the difficulty of reducing a formyl group to a methyl group without also, reducing the carbonyl at the isocyclic ring. h~ 1993, Ito and coworkers ob.~rved that when Chlide b was incubated with cucumber etioplasts in the dark. Chl a was formed 1521. ~/hen the experiment was repeated with t4C Chlide b, Chl a became labelled a.s well [53]. Because Chl b accumulated ahead ofChl a. Ito et al. inlerred that Chl a was derived from Chl b. No mechanism was su.~gested. However, it was noted in these and other experiments that 7-hydroxymethyl Chl was an intermediate in the Chlide b to Chl a conversion [ 541. Interestingly. the end product of the reaction
3. Genetic analysis of Chl biosynthesis Enzymes involved in the synthesis of ALA via the C5 pathway have been well characterized genetically [ 17,57 l. as have the enzymes catalysing reactions in the early part of the pathway from ALA to protoporphyrin IX [ 58 I. The Mg branch from Mg-protoporphyrin IX to Chl a is less well known, particularly in cyanobacteria and green plants. although valuable insights are being obtained from photosynti:etic bacteria.

2116

H. I" A~htm.~onet ¢tLIJounud of Photot'hcmi.~tr3am/Photohh~h~gyB: Bioh~gy41 q19971201-221

The entire photosynthesis gene cluster containing loci involved in the synthesis of Bchl a has been mapped and sequenced in the pu..'ple bacterium Rhodobacler cdpsttlttltts and defined sets of insertion mutations constructed within each of the open reading trame:~ ( ORFs ). As a result individual ORFs can be matched to specific steps in the Bchl/Chl biosynthetic pathway and the information used to construct probes for homologous genes in other photosynthetic organisms. As reviewed in Bauer et al. 151. Bollivar et al. 1591 and yon Wettstein el al. 18 ]. nine loci in the photosynthetic gone cluster o f RhothJbacter capsttltttlts have been implicated in the live steps of the Bchl pathway between protoporphyrin IX and Chlide a as follows: ( I ) hchD. I, H: insertion of Mg into the macrocycle of protoporphyrin IX to h~nn Mg protoporphyrin IX, catalysed by Mg chelatase: (2) bchM: addition of a methyl group to the propionic acid side chain of ring C of Mg protoporphyrin IX. catalysed by Mg protoporphyrin IX methyl transferase: 13) bChE: formation of the filth (isocyclic) ring from the methyl propionate side chain of ring C of Mg protoporphyrin monomethyl ester, to form DV Pchlide(4) bchJ: conversion of DV Pchlide to MV Pchlide. catalysed by eight-vinyl reductase: (5) b~'lzL. N. B: reduction of ring D of Pchlide to form Chlide. catalysed by Pchlide reductase. Homologues of most of these genes have been identified in oxygenic photosynthetic organisms. The subsequent steps in the bacterial pathway are specific to Bchl. Chlide is converted via two intermediates to Bchlide a which, in turn, is esterified to Bchl a. An additional seven to eight loci are involved 15.59 I. Three of these, bchX, Y. Z. are implicated in a Chlide ring B red,orion reaction which follows and mirrors the reduction of ring D of Pchlide. The enzyme which catalyses the reduction of Chlide to Bchlide is called chlorin reductase. Since the chemistry of the ring D and B reduction reactions is similar, it was anticipated that the Pchlide and cnlorin reductase enzymes would also be similar. Sequence analysis has provided evidence consistent with this hypothesis [ 3,601. B~'hL. N and B arc homologous with their chlorin reductase counterparts, b~'hX, Y and Z. BuhL and X share approximately 32ch amino acid sequence identity. The corresponding values for bchN and Y and bchB and Z are approximately 22 and 24c~. respectively. These two sets of photosynthetic pigment genes are thought to code for two structurally as well as functionally similar enzyme complexes, both with three subunits essential for reductase activity and both catalysing the addition of 2H to a specific pyrrole ring in the macrocycle. Signilicantly. both Pchlide and chlorin bacterial genes also bear a striking resemblance to genes involved in nitrogen fixation. In Azotobtwter rinehmdii these genes are designated n~/H. n~/K and m./D. BchL and bchX share 30-37% amino acid sequence identity with nifl-I 13.4.61--63 I. Nt'~ encodes a nitrogenous Fe protein that functions as a unique electron donor for the nitrogenase complex where catalysis occurs.

The nitrogenase complex has two subunits coded by m.'/Kand n~ID. BchN is homologous with m'~. the gene encoding the MoFe protein of nitrogenase 164.65 I: bchB is homologous with m.'/D, the gene encoding the Vd-Fe alternative subunit of nitrogenase 18 I. One of several importar- conclusions that l~urke and coworkers derive from their comparative analysis of Pchl(ide). chlorin and nitrogenase reductase genes is that "all three processes are related mechanistically, structurally and evolutionarily'. Another is that "because the residues which are conserved among all three classes of proteins are those required for electron transfer I661, ATP binding and hydrolysis, or structural integrity of n(/H 1671, it seems likely that the pigment synthesis proteins, bchL and X, serve as the unique electron donors into their respective catalytic subunits ( bc.hN-bc.hB and bc'hY-bchZ ) and that both ATP hydrolysis and transfer of electrons throu~,h a ( 4Fe-4S ) cluster serve as integral components of their reaction mechanisms" 13 ]. p. 2412. Although there is. as yet, no direct evidence that the polypeptides encoded by bc'hL. N, B or bchX, Y, Z have a catalytic function, it is obvious frum the amino acid sequence data referred to above that this is the simplest and most likely explanation of their role. It is also consistent with the observation that mutations in Pchl(ide) and chlorin reductase genes inhibit Bchl synthesis and result in the accumulation of either Pchlide or 2-desacetyl-2 vinyl Bchlide a. Unlike the Chl biosynthetic pathway of oxygenic photosynthetic organisms, the Bchl pathway is not regulated by light. The Pchl(ide) reductase step is light independent and there are no homologues of the light-dependent Pchl(ide) reductase of angiosperms, lower plants, algae and cyanobacteria in bacteria.

4. The Pchlide reduction step in cyanobacteria and oxygenic eukaryotes 4. I. Ligh~-imlependent P¢'hlich, reth,'thm

Homologues of the light-independent Pchl(ide) reductase genes of purple bacteria (bchL, N, B) have been identilied in cyanobacteria and the chloroplast genomes of green algae and land plants, up to and including gymnosperms. In some species homologues of all three have been identified: e.g., in the cyanobacterium P/ectonema boryamon 165,68,691: the green alga Chhmzydomomls remhardtii 14.70-731 and the liverwort Mart'hantia polymorpha 161.74,75 I. In others, one or two h,'wc been described. Lidholme and Gustafsson 1761 reported strong hybridization signals from total DNA of two conifers, Pinus ctmtorta and Pit'ea abiex, in a Southern blot analysis using a chiN (g/dA) probe derived from Chhlmydomonas rehahardtii. Sequence analysis of the hybridizing pine chloroplast DNA region revealed a sequence highly homologous to that deduced from the Chh,nydomonas gene and to ORF465 of

H.Y. Adum.w,net aL /Jounud .~fPhottn'he,:ai,try ,rod Ph,,ttd,ioh~gy B: Bh,h~gy41 11997~201-221 Marchantht chloroplast DNA. Upstream of the chiN sequence there was a trnN(GUU) gene and an 291 codon sequence that was 78ch identical to the chlL (fraC) gene of Mart'hantia. When the same technique ( Southern blot analysis of restriction digests of total DNA) was applied to tobacco, there was no sign of .my hybridization with the t'hlN (gidA) probe. This could not.he attributed to inappo~priate experimental conditions for tobacco, at least with respect to the presence of detectable chloroplast DNA fragments in the tobacco lanes, because rehybridizing the lilter with a prohe specilic for the chloroplast-encoded large subunit of ribulose bisphosphate carboxylase ( rbcL ) produced strong signal., of equal intensity in all three species. Nor was the failure of tobacco to hybridize with the ch/N probe from Chlamvdomonas unexpected given the absence of this and the chlL homologue in the chloroplast genome of tobacco 177 I. A similar result was obtained by Suzuki and Bauer 141. who kinked for evidence of hybridization between DNA gel blots of total DNA from two cyanobacteria, a bryophyte ( liverwort), live pteridophytes ( ferns or fern allies), five gymnosperms and four angiosperms and a chlL probe from Chhmo'thmumas. Cross-hybridizing bands were obtained with the Chhmlydomonas control. Anacy.vtis and Svnechoco,'t',s (cyanobacterial, Marchantia (liverwort). Pellaea. Polystich.tm, Cystopteris. Athvrium (ferns), Equi,wtum (feru-ally). Ginkgo. Psetuhltsuga (Douglas li,'), T~trus (yewl, Junipertts (red cedar) and Ar:mt'arix ( gymnosperms). There was however no sign of cross hybridization in Psihmm~ Ca fern-ally) even with a three- to lour-fold increase in exposure time or in Zea Imaize), Nit'otiatla (tobacco), Arabidopsis or Bougainri/lea ( angiosperms 1. In the two papers cited above, Lidholme and Gustat'sson and Suzuki and Bauer also refer to unpublished observations indicating cross-hybridization in Em'ephahtrtos Ca primitive gymnosperm) and Seh~ghaelht Ca fern-ally) but not pea tan angiosperm). Taken overall, these are very striking results. With the exception of Psihmmt. all of the non-flowering plants tested by Lidholme, Suzuki and co-workers reacted positively to light-independent Pchlide reductase probes from Chhmo'domonas: none of the flowering plants did so. A wider range of fern-allies and gymnosperms was examined for the presence ofc/dB by Boivin et al. 178 I. In addition to Psihmm~. Gnemm was also lacking chIB as judged by negative Southern blots and PCR reaction. Curiously, although Nep/mdepis contained a chlB homologue, the coding sequence was interrupted by two in frame stop codons. The data tbr Psih~tmn, Gnetum and Nephrolepis may indicate that the light-independent Chl reductase has been lost independently several times. It is doubtful if any of the above studies would have unfailingly detected such a gene(s) if it had been transferred to the nucleus, especially if it had acquired introns. We note in passing that other chloroplast genes (e.g.. secA and Y) that have been transferred to the nucleus in the angiosperm line were discovered by processing/uptake protein experiments rather than by Southern blotting, There have been no reports of PCR-based searches based

207

on totally degenerate primers made to conserved regi;ms of the primary sequence of chlB, chiN and c h l L a p~siblc approach in the absence of a complete genome ~cqucnce for any angiosperm. Our own initial experiments in this direction using a ~ery limited set of primers have. however, been negative. Nevertheless. the absence of an)' evidence of hybridization between c/dl.. N or B genc probes from cyanobac~.cria.

green algae or non-flowering land plants and total DNA from flowering plants is certainly striking. While Suzuki and Bauer 141 make it clear that they "cannot exclude the possibility that absence of ch/L cross-hybridization is due to gene sequence divergence or gene transfer to the nucleus', the:r clearly favour the hyputh.,~sis that "structural genes involved in light-independent protochlomphyllide reduction may have simply been lost in the angiosperm lineage'. We agree with Lidholme and Gustaf.,,son 1761 that "whether or not the gidA ( chiN ) and/k'xC ( chlL ) genes have been re,~ued a.s nuclear genes in angiosperms is an interesting question which remains to be answered'. So far. the enzyme that catalyses light-independentPchlide reduction in bacteria, cyanobacteria and chloroplasts of algae. bryophytes, ptcridophytes and gymnosperms is known almost entirely from DNA sequencing. It has not been isolated and purified .and its molecular weight, structure, mechanism of action and manner of regulation '.'re all unknown. Nor has catalytic activity of the beh/cldL. N and B gene prtvducts been dentonstrated in vitro. What is known is that the enzyme in green plants is encoded by the chloroplast genome: 'hat at least three separate loci are involved: that the organization of these loci is similar in different species: that mutations in any of the loci result in the accumulation of Pchlide in darkness (the so-called yellow-in-the-dark syndrome ): that homology hetween bacterial, cyanobacterial and chloroplast L. N, B-type genes is mainly due to con.~rvation of a small number of ATP and 4Fe--4S binding sites: that homology between chlL. N or B genes in organisms from different taxonomic groups is mainly due to con~rvation of the same sites: and that these ge,es are absent from the chloroplast genome of flowering plants. In the ab~nce of biochemical evidence, the genetic evidence points to a lightindependent Pchlide reductase consisting of three subunits encoded by separate genes with each subunit bearing a re,semblance to an homologous counterpart in a nitrogenase-type enzyme. One of the subunits, encoded by t'h/L, is thought to be analagous to the nitrogenase iron protein encoded by n//K in Azotobacter. The function of the nitrogenase iron protein in Azotobacter is to donate electrons to the ancilliary catalytic subunits encoded by n/]K and n0'D. Again, by analogy, subunits encoded by ch/N and crib are thought likely to have a catalytic role, Since ATP hydrolysis and transfer of electror, s through a 4Fe-.4S cluster are es~ntial components of the nitrogenase reduction mechanism, they are also presumed to be important in the light-independent reduction of Pchl(ide). There is very. good circumstantial evidence for this hypothesis, i.e., striking amino acid ,~quence identity between homologous Pchl(ide). chlorin and nitrogenase

208

H.E Adam.~¢met al. / Joun~al of Photochemi.~'tryaotd PhotohiohJgy B: Biology 41 t 1997) 201-221

reductases in bacteria and oxygenic photosynthetic organisms ( see Ref. 1631 for details). Obviously. physiological and biochemical studies of lightindependent Chl synthesis in cyanobacteria, algae and land plants are needed. The light-independent Pchl ( ide) reductase enzyme is relatively easy to assay in vitro using eyanobacteria 179.801 and higher plants 1811 and it is already known, particularly from the studies of Peschek and co-workers, that the light-independent enzyme, like its light-dependent counterpart, is membrane associated and uses NADPH as a cofactor. There is also evidence that the Pcblide to Chlide reaction in Anacvstis is driven in the reverse direction by NADP and is specifically stimulated by calcium ions 180 I. 4.2. Light-dependent P~ hlide rednction

In contrast to the light-independent Pchlide reductase whose physical properties at this stage can only be inferred from genetic information, the light-dependent enzyme is well known biocbemically as well as genetically. It exists in two forms, known as P e R A and P e R B. P e R A is synonymous with NADPH:Pchlide oxidoreductase EC 1.3.1.33 (originally classified as EC 1.6.99.1 ) I 1,82 I. P e R B is a closely related enzyme encoded by a separate gene. its activity in vitro closely resembles that of P e R A but there are ,;ignificant differences between the two enzymes with respect to gene expression, requirements for import of their precursor (pP e R A and p-PeR B) into the chloroplast and stability in light (lor detailed review, see Reinbothe et al. 1101 ). In a landmark paper Forreiter and Apel 1831 identified two cDNAs from Phm.~ rouge which encoded two distinct but related P e R polypeptides. These shared an amino acid sequence identity of approximately 85c,I with P e R sequences from barley 184-87 I. The overall homology of the nucleotide sequence of the two pine ~-DNAs within the ORF was 94%, but much less ( < 79%) in the 3' non-translated regions of the corresponding transcripts. Using an anti-serum against a well-characterized lightdependent Pchlide reduetase of barley that recognised two bands in Western blots of prolamellar bodies, prothylakoids and membrane fractions of pine plastids. Forreiter and Apel 1831 observed that the subcellular location of the two P e R enzymes in Pinus tnugo was different. One was predominantly present in prolamellar bodies of etiochloroplasts, the other was mainly present in thylakoids. The enzyme present in prolamellar bodies was very abundant but broke down repidly when dark-grown seedlings were transferred to light. In contrast, the enzyme in thylakoids was only present in small amounts but did not break down in the light. The molecular weights of these enzymes were also different: 36 kD and 38 kD, respectively. Although Forreiter and Apel were not able to match the cDNAs and immunoreactive polypeptide bands, they deduced that the "~6kD polypeptide corresponded to the well-characterized NADPH:Pchlide oxidoreductase of angiosperms. When this paper was published no polypeptide equivalent to the second 38 kD immunoreactive P e R poly-

peptide of P h m s inugo ?lad been described in angiosperms. Shortly afterwards one was identified in barley by Holtoff et al. 1881. This second Pchlide reductase, designated P e R B, like its P h m s rouge counterpart, was only present in small amounts that did not change on illumination. P e R A and P e R B mRNA patterns paralleled those of their respective enzymes. P e R A message was present in relatively large amounts in dark-grown seedlings and declined when they were transferred to light; P e R B message was present in approximately equal amounts in both dark- and light-grown seedlings. When tested in vitro, precursors of both enzymes, p-PeR A and p-PeR B, reduced Pchlide to Chlide when supplied with NADPH in the light, but not in the dark. When PeR-related cDNA fragments were amplified and sequenced the amino acid sequence identity of the two immunoreactive polypeptides turned out to be 75c~. Highly conserved light-dependent Pchlide reductases are present in cyanobacteria, algae and all major taxonomic groups of land plants and like their light-independent counterpart are prokaryotic in origin 189 I. Fig. 2 of Suzuki and Bauer 1891 shows the amino acid sequence alignment of a genomic DNA fragment from the cyanobacterium Synechocystis with deduced amino acid sequences of light-dependent Pchlide reductases from seven species of seed plants: thrze monocots ( barley 1841, oats 185 I, wheat 1901 ), two dicots (Arabidopis I861, pea 1871) and two pines (Pitltts taeda 1831 and Phtns rouge 1831 ). Table 2 of the same paper gives estimates of the percentage sequence identity among various light-dependent Pchlide reductase homologues. Comparing Synec'hocvstis with each of the seven higher plants, sequence identity ranged from 52 to 56%. Sequence ide,,tity among the more closely related seed plants varied from 79 to 98%. As Suzuki and Bauer [ 891 observe, alignment oftbe deduced 5~vnec'hocx'.~'tis light-dependent Pchlide reductase peptide sequence with the land plant sequences demonstrates "a high degree of sequence conservation with very few gaps along the entire length of the enzyme'. They also note that inclusion of the cyanobacterial sequence in the comparison di rides the reductase into "modular domains of identically conserved residues" and point to strong conservation of residues 5 through 34 at the N-terminal end of the protein ( provisionally identified as an NADPH binding site by Darrah et al. 1851 ) and three cysteine residues (thought to be involved in the active site). For practical reasons, most of the pioneering work on Pchlide reduction in angiosperms was carried out using darkgrown seedlings. The molecular weight of the predominant reductase in such plants ( P e R A) is commonly given as 3637 kD but molecular weights ranging from 33 to 38 kDa have been reported [ 61 and at least four different forms have been resolved by isoelectric focusing 191,921. The build up of substrate (Pchlide) that occurs in darkness is accompanied by a build up of both the enzyme ( P e R A) and co-factor (NADPH) required for its phototransformation to Chlide. These reactants form stable enzyme/substrate/eo-factor complexes which aggregate in prolamellar bodies [931.

H. Y. Adamson et uL I Jountal ,~ Photm'hemistryand Phou~bioh~gyB: 8u,h,gy 41 q1997~201-221

Whereas most enzymes are present in very low concentration. POR A is present in abundance in dark-grown angiosperm seedlings and. in the continued absence of light, serves a structural rather than a catalytic purpose. As soon as light is supplied to dark-grown seedlings, excised leaves or etioplasts, the block in the Chl biosynthetic pathway is overcome. Pchlide is reduced to Chlide: prolamellar bodies start to disintegrate: the activity as well as ~mlouat of POR A protein and its mRNA fall rapidly and synthesis of thylakoids begins. These changes are spectacular at+d can he quantilied without difficulty because dark-grown angiosperms lack both Chlide and Chl and as a consequence, light-induced synthesis of even trace amounts of these pigments is easily observed. The kinetics of Pchlide photoreduction can also be lbllowed readily because both reactants and products have distinctly different spectral properties and. although there are a number of interconvertible forms of each. these can be identified by characteristic absorbance and fluorescence maxima. These attributes, together with the availability of relatively large amounts of comparatively pure ['OR A protein and excellent methods Ibr assaying its activity in vitro, explain why photoconversion of Pchlide is by far the most widely studied reaction of chlorophyll biosynthesis in higher plants, why far more is known about light-dependent rather than light-independent Pchlide reductases and why, until recently..°OR B. which constitutes only a very small fraction of the total Pchlide reductase in dark-grown angiosperms, went unrecognized. The reaction catalysed b:' NADPH:Pchlide oxidoreducrose/POR A in dark-grown angiosperms in vivo and etioplast membrane and Pchlide holochrome prepar'~.tions in vitro has been reviewed in depvh by Griffiths 121 and Schulz and Senger 161. In summary, it invokes the elimination of the double bond between C ; 7 and C 18 of ring D of the po|phyrin macrocycle of Pchlide by the addition of two hydrogen atoms 12e- and 2H ~) to form Chllide). NADPH serves as the reductant and light is essential for the reaction, which is extremely rapid ( T~/_,= 6--9 p.s I. Pchl(ida), which is the primary light-absorbing pigment 194 I. exists in vivo and in etioplast membrane preparations in different light-absorbing lbrms: Pchl(ide) 630 and Pc'hl(ide) 638/650. The former, which absorbs maximally at 630 nm. is construed as free pigment because it is not converted to Chl(ide) when exposed to light ( i.e.. is photoinactire). The latter, which absorbs maximally at 638 and 650 nm. is photoactive and is converted within subseconds to Chl(ide) 678 when exposed to light. It is construed as either one or two Pchl(ide) species bound in a ternary complex with NADPH and its light-dependent reductase. The characteristic feature of this enzyme/substrate/cofactorcomplex is that it is stable in darkness and accumulates in the prolamellar bodies of etioplasts of dark-grown angiosperms. The first stable product of Pchl(ide) photoreduction is Chl(ide) 678. This is interpreted as bound pigment in the transformed temary complex, i.e., Chl(ide) complexed with the redt:ctase and the now-oxidized cofactor. NADP. Chl(ide) 684.

Cht.lide) 672 and Chl(idej 67~ are later products. Within

seconds the absorbance maximum changes to 684 nm: then. within minutes drops to 672 nm. in a temperature and agerelated manner, before fina!ly stabilizing at 678 nm. ChlOde) 672 is interpreted as the end product of the photoreduction. i.e.. free Chl(ida). When assayed in vitro. NADPH:Pchlide oxidoreductase utilizes Pchlide as a substrate and is unable to reduce its Cl7 ester. Pchl [ ! 8.95 ]. According to Grifliths [ 21 this "strongly suggests that, at least in vitro, a free CO2H group at CI7 is essential for activity as a substrate for the reductase', in support of this conclusion he cites an unpublished observation that "neither Chl c~ or c_,. which are essentially identical to Pchlide except for an unesterified acrylate side chain at C 17. are uti!izcd as subst:ates by the reducta.se'. However. the situation may he different in vivo. Schulz and Scnger [ 6 ] list seven papers that provide evidence of phototransformation of Pchl in vivo in Chlorella [961. Scenedesmu.~ [971. Euglena [98 ]. Cucumi.~ sativus [14.99 I. Hordeum vulgate [ 1001 and Phaseolus vtdgari,~ [ 1011. Taken together, tbe~ findings strongly suggest that, in vivo if not in vitro, the presence of a long-chain alcoholic residue on C17 does not interfere with the attachment of Pchl to the enzyme. In support of this conclusion. Schulz and Senger cite an unpublished finding made in collaboration with R. Knaust. namely, that when Pchl extracted from the C-2A' mutant o f Scenedesmu.~. NADPH and hybrid p~tein of Pchlide reducta.se were combined in vitro and illuminated with dim white light, Chl was formed. It is interesting to note that whereas P,.hlide reductase discriminates in vitro against certain substituents at CI7 of the macrocycle, it appears to be relatively unaffected by differences at C8. i.e., it accepts both DV Pchlide which has a vinyl group at this position and MV Pchlide which has an ethyl group 12 I. With respect to co-factor specility. NADPH is essential for photoreduction: all other reductants tested have been without effect I I I. It has been suggested that a florin may a i ~ be involved. Walker and Griffiths [ 1021 noted that large quantities of FAD copurified with Pchlide reductase extracted from dark-grown seedlings and the flavin inhibitor, quinacrin, was exceptionally effective as an inhibitor of photoreduction of Pchlide in vitro. Citing these findings as evidence of the presence of flavins in the native complexes of Pchlide and Pchlide reductase, lgnatov et al. [ 1031 interpreted changes in the tow-temperature fluorescence excitation and emission spectra of dark-grown maize leaves irradiated at 77 K in terms of energy wansfer from flavin to Pchlide to Chlide. However, in view of the recent comment by Griftiths and co-workers ! 104] that they have a paper in preparation which demonstrates "reductase activity in a protein purified from an E.coli ,t-ansformant expressing the higher plant reductase gene in the absence of flavin', some of the spectral changes routinely observed during photoreduction may need to be reinterpreted. A number of observations point to highly organized and specific binding of Pchlide and the nucleotide at the active

2 I0

H.Y.Adums¢me' al. IJourmd of Photochemistry and Phomhudogy B: Biology 41 (1997) 201-221

site of the enzyme. Phototransformation of Pchlide is inhib ited by thiol reagents. This suggests that one or more c/steine{s) in the enzyme are essential for activity and, since NADPH affords some protection, implies that they are associated with the NADPH-binding site 1105 ]. In addition, the reduction reaction is stereospecific, as evidenced by the fact ,hat the introduced hydrogens tn naturally occurring Chl are always trans. According to Begley and Young [ 106], the hydride transferred trom NADPH is derived from the pro-S face of the nicotinamide ring and added stereospecifically to CI7 of the Pchlide ring and the hydrogen added to CI8 is obtained lrom the medium. This model has been refined by Wilks and Timko [ 19] following experiments in which they muzated conserved residues Tyr-275 and Lys-279 within the proposed active site ef pea Pchlide. In their opinion, the proton at the CI8 position is derived from Tyr-275. They attributed the close proximity of the other conserved residue Lys-279 to the need to facilitate the deprotonation of the phenolic group of Tyr 275. Oddly enough, the most remarkable feature of the enzyme. its light dependence, is the least well characterized. A key question is whether the transfer of the two H atoms ( 2e " and 2H ~) needed to reduce Pcbl(ide) to Chl(idc) is accomplished by a single photochemical event or two such events. In a recent paper 11041 Griffiths and co-workers note that evidence for both scenarios is present in the literature and acknowledge that the free energy available from a single quantum of red ( 650 nm ) light and the oxidation of NADPH may not be enough to drive the reduction of Pchl(ide) to Chl (ide). At the same time they report that quantitative measurements of Pchl(ide) reduction in etioplast membranes obtained from dark-grown :vheat subjected to a wide range of light intensities are best described by a theoretical curve modelling a single rather than a two-step process. This interpretation is supported by the results of an ingenious experiment in which etioplast membranes were subjected to a series of femtosecond flashes i.8 ms apart. They reasoned that since a photon of light is absorbed within 10- ~5 s, femtosecond pulses of light should be capable of converting Pchl(ide) to Chl(iae). However, if two successive quanta cf light are required to reduce a single Pchl(ide) molecule, they further reasoned that the product of the first light reaction would not survive the relatively long time between successive !ight pulses. Since small amounts of Chl(ide) were detected in membranes treated in the manner described, Griffiths et al. concluded that "the absorbed quantum must effect the direct transfer of two electrons (as hydride?) from the co-enz~ me to ,.he pigment'.

incapable of greening in the dark because light is essential for Pchlide reduction. This is reflected in the quotes below. 'One of the more extensively characterised steps in the chlorophyll biosynthetic pathway involves reduction of the fourth ring of the Mg-tetrapyrrole intermediate, protochlorophyllide. Interest in this step of the pathway owes to the

dependence of angiosperms on light for protochlorophyllide reduction. Protochlorophyllide reduction has been thought to play a regulatory role in angiosperm development since it functionally acts as a gate in the biosynthetic pathway that allows chlorophyll synthesis only when the plant is illuminated." ( Suzuki and Bauer [ 89] ( p. 3749, emphasis added). The belief that light is essential for Pchlide reuuction in angiosperms rests on two observations: first, that angiosperm seeds germinated and grown in darkness accumulate Pchlide, not Chl. and secondly, that the enzyme known to catalyse the transformation of the po:'phyrin precursor (Pchlide) to a chlorin (Chlide) in such seedlings is unable, in vitro, to bring about the reaction without light. "It is well known that angiosperms when germinated in darkness produce etiolated chlorophyll-free plants in marked contrast Io the green tissues enriched with chlorophyll p,oduced in the light. The reason for this difference is that the biosynthesis of chlorophyll by angiosperms involves a reaction that is obligatorily light dependent i.e. the reduction of protochlorophyllide (pehlide) to chlorophyilide (chlide). Gymnosperms and certain algae however can presumably achieve this reduction without light since they can synthesise chlorophyll and become green in ~omplete darkness." (Grifliths et a,. [ 107] (p. 19, emphasis added). The presumption that gymnosperms and certain algae have a mechanism whereby protochlorophyllide can be reduced in darkness has since been substantiated. Genes homologous with the light-independent Pchlide reductt~se genes of ano×ygenic photosynthetic bacteria (bchL, N, B ) have beea demonstrated in all major plant groups except angiosperms. This has led to the suggestion that in the course of evolution

"angiosperms have simply lost the capability of synthesising the light-independent enzyme' ( Bauer et al., [ 5 ] ). At first sight the argument is compelling: enzymes implicated in light-independentChl synthesis in gymnosperms and lower plant,~ are absent in angiosperms, therefore, angiosperms are anable to syn',hesize Chl in darkness. What then are we to make of the small body of evidence which indicates that angiosperms can, in fact, synthesise Chl in darkness? Logically, there are only two possibilities: either, the reasoning that rules out dark Chl synthesis in angiosperms is false or. the evidence itself is flawed.

5. Light-independent Chl synthesis in angiosperms

5.2. Reasonblg and evidence

5. I. The maiorit3" view

Referring to the second quote above: it is valid to compare angiosperms grown in darkness with angiosperms grown in light; different growth conditions produce obvious differences in phenotype. However, it is not valid to conclude that

Light-independent Chl synthesis in angiosperms is a contentious issue. The majority view is that angiosperms are

H.Y. Adams+m et aL / Jtmnud of Plumst'hemistry and Phou~hh~logy B: Bioh~gy 41 11997~ 201-221

*the reason for this differev.ce is that the biosynthesis of chlorophyll by angiosperms involves a reaction that is obligatorily light dependent'. There is no scientific justification for generalizing from dark-grown angiosperms to all angiosperms. The evidence only allows that "the reason for this difference is that the biosynthesis of chlorophyll by darkgrown angiosperms involves a reaction that is obligatorily light dependent" (which is, incidentally, a sufficient explanation). The first quote is open to the same criticism as the second: generalizations that are appropriately applied to dark-grown angiosperms because they are supported by evidence are presumed to apply to all angiosperms ( i.e., light as well as dark grown). If the presumption is correct, i.e., if all angiosperms behave in the same way as dark-grown angiosperms, then anomalies should not arise. If angiosperms are "obligatorily light dependent" alld "protochlorophyllide reduction

allows chlorophyll synthesis only when the plant is illuminated', evidence that angiosperms are able to synthesize Chl in darkness is clearly anomalous. The question is. is it reliable or, is it seriously flawed? Table I lists papers which either claim, or appear to provide, evidence consistent with light-indt,pendent Chl synthesis in angiosperms. This evidence is of two types: ( i ) net gains in either total Chl content or Chl a content of seedlings, leaves or tissues and associated ultrastructural and biochemical observations following transfer of plants from light to darkness: (ii) incorporation of labelled chlorophyll precursors into Chl in darkness, and specifically into the tetrapyrrole moiety. Both monocots and dicots are represented. In some cases the plants examined had been grown under natural day/night conditions prior to transfer to darkness, in others, dark-grown seedling:; were exposed to light for short periods to initiate Chl synthesis and then returned to darkness. In three instances trace amounts of Chl were reported in seedlings or tissue cultures raised in total darkness. One of the earliest reports of dark Chl synthesis by angiosperms v,as by Seybold and Egle in 1938 [1081. These researchers grew a variety of angiosperm species from seed in darkness, greened them for a few hours in light and then observed what happened when they were returned to darkness. In general, Chl a continued to accumulate while Chl b decreased and in several instances, aet gains in total Chl were also obse~ed. As Table I indicates, these two patterns of response have since been observed quite frequently. One pattern, net gains in total Chl in darkness, can only he interpreted in terms of de novo Chl synthesis. The other pattern, net gains in Chl a in the absence of net gains in total Chl, is I'.arder to interpret. Two explanations are feasible. Either, Chl a is formed from pre-existing Chl b, or, Chl a is synthesised de novo while Chl b is degraded. The first explanation, favoured by Kupke and Huntingdon [ 109 l, Wieckowski and Ficek I 1101 and Tanaka and Tsuji [ I I I l, provides no support for light-independentChl synthesis in the sense in which the term is used in this review: the second, favoured by Popov and

21 I

Dilova I I 12 I, Oelze-Karow and Mohr I I 131 and Waimsley and Adamson I 1141. requires it. The question of which explanation is more likely is considered in Section 5.5 below. Since net gains m total Chl content of seedlings, leaves-and leaf pieces following transfer from light to darkness have been reported over six decades in a variety of angiosperms. it is pertinent to ask why they have. for the most part, been disregarded. Or. looking at it another way. if the majority view is correct and chlorophyll synthesis in angiosperms only occurs when plants are illuminated, how does one account for the coherent body of information to the contrary contained in the papers listed in Table I? In our opinion, the .~epticism which presumably underlies dismis~l of all existing evidence for dark Chl synthesis in angiosperms or. alternatively, the fault leading to false evidence for su~,h a concept must originate in real or perceived problems related to the conduct of the cited experiments. There are only two possibilities. Either the Chl measurements are false or thought to he false or the plants have been exposed or are believed to have been exposed to light during dark treatment. The former seems unlikely. Provided care is taken both to minimize and deal appropriately with variation between ~mples, there is nothing intrinsically difficult about measuring the quantities of Chl a and b gained or lost when green plants are transferred to darkness. The possibility that plants may have in',glvertently been exposed to light during dark treatment is a far greater c a u ~ for concern. The credibility of the evidence for light-independent Chl synthesis in angiosperms depends on the str.ngency of the "dark" conditions employed. 5.3. D a r k C h l s w l t h e s i s in b a r l e y

One of the key papers in the early literature on Chl synthesis in angiosperm,; +s that of Popov and Dilova [ 112l. who followed "the changes of chlorophyll a and b in etiolated barley seedlings in the course of greening when illuminated and at repeated darkening'. They observed that when darkgrown seedlings were illuminated for varying lengths of time and then returned to darkness, increases in Chl a and in .some cases total Chl content of leaves occurred during dark treatment. The changes were related to the length of the light pretreatment. When it was short (3 h). Chl a ned b were unstable and broke down in the subsequent dark period ( 20 h). With 6 h light pretreatment, a small net gain in Chl a was accompanied by some loss of Chl b. resulting in essentially no change overall. However. when the period of prior illumination was extended to 8 h. there were net gains in both Chl a and b in darkness and a net gain of 22% in Chl a + b. They concluded that these data provided ".some evidence for the possibility that the terminal reactions of chlorophyll biosynthesis in barley leaves may take place in the d',u'k" (p. 6O8) Popov and Dilova also observed qualitative differences in the ease with which pigments could he extracted after different lengths of both light and light plus -dark exposure. They defined, and successively extracted, three fractions. Fraction

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214

H.Y. Adamson et aL / Journal qf Photorhemi.~try and Photohiology B: Bioh~k,y41 (1997) 201-221

I was the most easily extracted, and fraction 3 was the most difficult. The proportion ,,ff fraction I decreased with time of exposure to light, while the proportion of fraction 3 increased. The same pattern was obserJed when light-pretreated plants were returned to darkness: fraction I pigments decreased and fraction 3 increased. They suggested that changes in the extractability of chlorophyll and especially the decrease in the percentage of Chl a in fraction I under prolonged illumination resulted from "a gradual stabilization of the chlorophyll molecules within the pigment-lipoprotein complex" ( p. 609) and inferred that processes of pigment-protein complex stabilization also took place in darkness. Popov's and Dilova's experiments were carefully conducted. Photosynthetic pigments were extracted in "full darkness" (i.e.. a "safelight" was not used): "the quantity of p'~gments was determined spectrophotometrically alter paper chromatographic separation" and all results were analysed statistically. We have confirmed their findings. In a series of experiments with either dark-grown seedlings illuminated for different lengths of time prior to transfer to darkness or seedlings raised in a day/night environment, we have observed that: ( I ) light-independent Chl synthesis in barley, evidenced by a net gain in Chl per leaf. is promoted by ALA I 1151 and inhibited by gabaculine [ 1161 and further, that the inhibitory effect of gabaculine on light-independent Chl synthesis i:~ overcome by the simultaneous application of ALA I I 161 : (2) immature barley chloroplasts developing normally from proplastids continue to increase in volume and form thylakoids when transferred to darkness I I 15, I 171 : (3) the ability to accumulate Chl in darkness is strongly inguenced by (i) daylength during growth and the length of irradiation immediately prior to dark treatment [ 1181, (ii) seedling age I 1191 and (iii) tissue age 1 I 15 I. The experiments described in ( I )-( 3 ) above were carded out under stringent dark conditions. Seedlings were raised in a speciall,.! constructed dark growth room separated from the general sl:ace by a dark laboratory and two intervening light locks. The required emergency exit is light tight and can only be operated from the inside. As previously described [ 1201. estimates of the intensity of ambient actinic light and pointsource leaks in the dark growth and dark laboratory rooms were made by means of an ( S I ) Photoemissive Device ( ITT. Fort Wayne. IN. USA) amplified through a Rikadenki chart recorder. The device was calibrated against a Li-Cor Integrating Quantum/Radiometer Photometer. The proportion of actinic I~ght was estimated as a fraction of red light in the ambient light outside the dark-room complex. Stray actinic light in the dark growth room was estimated as 10 --'t' mol m --" s- : and in the dark laboratory. 10- "' tool m - -~s - a. The amount of Chl(ide) that could be formed as a result of photoreduction by this stray light is five orders of magnitude less than the amount of Chl(idc) we have detected in five-dayold wheat seedlings ( 5 pmol per shoot ). germinated, grown, harvested and extracted under these conditions [1201 and negligible in relation to the changes in Chl a and b contents

that we observe in green plants returned to darkness. Although some of our experiments have been carded out in total dari,,~ess, others have involved the use of a dim green safe light (40 W green Osram light globe and Ilford filter 909) whose effectiveness we judge by its failure to convert Pchlide to Chlide in etiolated test plants. We attribute our success in measuring small changes in Chl content of already bright green leaves to selection of uniform starting material (seedlings/leaflets/tissue pieces), adequate replication and choice of an invariant (such as seedling/leaf/leaflet/ginitia/ fresh weight), as the basis for expression of results (for discussion, see Ref. 181 I ). Dark Chl synthesis in barley has also been confirmed by labelling studies [12 I-I 23l but not without difficulty. The first report was negative 11241. Apel and coworkers were unable to obtain any evidence of light-independent Chl synthesis in dark-grown barley exposed to light tor 6 to 72 h as seedlings and returned to darkness as excised shoots. They used J4C-ALA as the precursor and supplied it via the transpiration stream in darkness. Pchlide became highly labe!led: Chlide was very slightly labelled: and Chl a. not at all. This indicated that the pathway was blocked at the Pchlide reduction step. We repeated and extended this experiment to include two extra treatments: ( 1 ) barley seedlings grown under glasshouse conditions, transferred to darkness and supplied with *4C-ALA via cut roots, and ( 2 ) dark-grown barley seedlings illuminated for 18 h. transferred to darkness and supplied with *~C-ALA 1121 I. Our findings with excised leaves mirrored those of Apel et al. although, in both cases, the seedlings incorporated label into Chl as well as Pchlide. In our experiments Chl a and b were extracted and purified to constant radioactivity by HPLC and TLC of their magnesium-free derivatives, phaeophytin a and b. To dete:'mine whether label was present in the tetrapyrrole ring of each compound, the phytyl and methyl esters were removed by alkaline hydrolysis and the resulting c;:rboxyl groups methylated using diazomethane. The products, chlorin or, from phaeophytin a and rhodin g7 from phaeophytin b. were purified by TLC and found to contain 74 and 60%, respectively. of the total radioactivity of the parent compounds from which they were derived. These experiments confirmed the existence of a light-independent Chl biosynthetic pathway in intact, live- to six-day-old barley seedlings which had been exposed to light during development. However. they seriously underestimated its activity I 125 I- When we compared rates of Chl accumulation in the light, prior to transfer to darkness, with rates of Chl accumulation after transfer, the ratio was 7: I. This seven-fold difference was ver) similar to that observed in etiolated greening plants by Popov and Dilova [ I 12 I. When we compared rates of incorporation of label into Chl in light with rates of incorporation into Chl in darkness, the ratio was 800:1. in other words, although the plants in darkness accumulated 18 times more Chl than Pchlide, 95% of the label incorporated into the two pigments combined was in Pchlide and only 5% in Chl.

H. Y Adamsnn ~'taL /Jounud ¢~ Pho;t~'hemtstry cuul Phntohioh,~y B: ltinh,;z~ 4 i ~I i~Tt 201 -221

Rudoi and Chkanikova 11261 ran into the same problem. In a very similar experiment they found that only I-3% of the radioactivity was in Chl: the rest was in Pchlide. Not surprisingly, they concluded from their results that the process of dark Chl formation "is almost non-existent in barley'. In fact, the problem lies with the method. The first indication of this came from the work of Tripathy and Rebei," [1221. who demonstrated that exogenous glutamate and exogenous ALA are not equivalent precursors of MV and DV Pchlide and of Chl in greening barley leaves transferred to darkness. When we supplied I-"~C glutamic acid to fiveto six-day-old dark-grown greeni~lg seedlings in the light and then transferred them to darkness several hours la;er, the results were quite different [1231 from those ~,e have obtained previously with ALA 11251. Alter 2 h exposure to light, followed by a further 6 h illumination in the prese,~ce of I-J4C glutamic acid. seedlings had accumulated 4-7 mmol Chl leaf-~ and had incorporated 900-1350 Bq (g fresh weight) - ~of radioactive l~hel into the Chl pool. When seedlings were trat;sferred to darkness, there was no net gain in Chl content but label continued to be incorporated. After 18 h the radioactivity of the Chl pool had increased by 300700 Bo.. ( g fresh weight ) This is a very significant result. As a consequence of labelling with 1-14Cglutamic acid there was no measurable activity in the phytyl esters of Chl a and b. all the radioactivity being present in the macrocycles. The increase in radioactivity of the Chl pool in darkness was not only substantial ( about 40%), it was entirely due to de novo Chl synthes;s via the tetrapyrrole pathway and, most importantly, it occurred in the absence of Chl accumulation. A similar result was obtained with the Chlorina 2 (b-less) mutant of barley [ 1271. These experiments point to Chl turnover and have important implications. When Chl is turning over. Chl accumulation will inevitably underestimate its synthesis, it therefore follows that failure to observe a net gain in Chl content in darkness cannot be equated with the absence of a functional dark pathway. It might simply mean. as is often the case in the light, that the rate of Chl synthesis is equal to or less than the rate of Chl breakdown. In view of our own findings with barley we believe that the res~dts of Popov and Dilova [ I 12 ] were reliable and their interpr.~tation correct. 5.4. D a r k Chl synthesis in other angh~spenns

In the introduction ;o another important early paper or. dark Chl synthesis in angiosperms. Godnev, Shlyk and Rotfarb [ 1281 noted that "it is inadequate to study this process by the usual methods of periodical determination of pigment content in living tissue'. Citing Chl turnover as the reason, they opted for an isotopic method involving exposure of lightgrown plants to "~CO2. or. in the case of aquatic plants, Na,'4CO.~. Their paper provides convincing evidence of incorporation of radioactivity into exhaustively purified Chl a and b from four angiosperm species. However, the fact that

215

they did not establish that label was pre.~nt in the tetrapyrrole nucleus, only that it was present in "the chlon~)hyll molecule" was a serious weakness, as v, as their failure to provide any information about the "dark" conditions employed. We repeated their experiment and found ;.hat when the seagrass Zo.~tera caprit'oraii was transferred from daylight to darkness and supplied with Na_,"sCO. in seawater, label v,as incorporated into Chl a and b. 8'~; of the label ,~as in the tetrapyrrole ring. 34c/~ in the methyl group on the i~xcyclic ring and 58% in the phytyl ester I 129 I. The small prolx)rtion of label in the tetrapyrrole ring was predictable, given its source. However. as Godnev et al. noted. "it is not the quantitative assessment of chlorophyll biosynthesis in the dark which is of primau, importance, but the adduction ofcv;dence that it takes place at all" [ 1281. p.M. Since we had already established that substantial amounts of Chl a and h continued to accumulate in Zostera in darkness in immature and mature tissue, in the same ratio as in the light, and were incorporated into Chl-protein complexes in the thylakoids I 130 I. we were satislied with the result. Again referring to early work. we have recently verified the conclusion of R6bbelen I 131 I that normal Arahidop.~i.~ thaliana plants fi)rm Chl in complete darkness 11321 and Rudoi and Chkanikova I 1261 have confirmed our obsenation that Tradexcanth~ alb(/tora has a pronounced capacity for light-independent Chl synthesis I 133.134 ITwo of the papers cited in Table I deal with angiosperms in tissue culture. Ikegami and co-workers [ 135 ] ob~rved the regeneration, in darkness, of fluorescence emission peaks at 628 and 675 nm in dark-grown and subsequently pbotobleached tobacco callus cultures. They attributed t h e ~ peaks to Pchl(ide) and Chl(ide). respectively. The net gain in Chllide) was extremely :~mall. 0.0037 Ixg Ig fresh weight) - E compared with 0.016 p.g (g fresh weight) ~ l't,r Pchltide). Hendrich and Bereza [ 1361 reported that they extracted plastids from carnation ( D k t n t h u s c'ar3"oph)lhts) callus tissue and incubated them with Pchlide and NADPH at room temperature in darkness for I0 rain. The spectrum at the end of dark treatment was consistent with the reduction of Pchlide to Chlide. althot,gh no quantitative treatment of the data was attempted. In some papers findings relevant to dark Chl synthesis in angiosperms came out of experiments designed to answer c'her questions. The angiosperms were Pelargtmhlm 11371. milo 11131, radish 11381, Fexntca 11391 and cucumber [ II 1.1401. We have confirmed and extended the findings with Festut'a 1141 ] and cucumber [ 120 I- As tar a:~ we are aware, findings consistent with dark Chl synthesis in Pelargonhtm. rice. milo and radish have not been pursued. 5.5. The evidence overall

The most obvious, and he~ce most strdightforward, evidence for light-independent Chl synthesis in angiosperms

comes from experiments that demonstrate significant net gains in total Chl of seedlings, leaves and tissu~ s following

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their transfer from light to darkness and/or incorporation of labelled precursors into the tetrapyrrole moiety of Chl in darkness. The first has been reported in most if not all of the angiosperms listed in Table I. Both have been demonstrated in barley and Zostera. Net gains in Chl a in darkness in the ab.~nce of net gains in total Chl are harder io interpret. They could be the result of either ( i ) conversion of Chl b to Chl a, or (ii) de novo synthesis of Chl a and simultaneous breakdown ofChl b, or both. There are three line:: of evidence which indicate that de novo synthesis of Chl t:. involving a light-independent porphyrin ( Pchl( ide ) ) to chlorin ( Chl( ide ) reduction step. must be involved in many instances when fhere is no net gain in tuia.] Chl overall. 1 I ) Response q/light-gr,~wn/greeniotg leaves to gabacuIohze supplied in dorkness. Gabaculine. which is a specific

inhibitor of ALA and hence Chl synthesis 11421. ha the potential to discriminate between Chl t1 synthesized via the classic linear (Pchlide reductase) route and Chl a derived from pre-existing Chl b. It should inhibit the former and have no effect on the latter. In experiments with very young lightgrown barley seedlings transferred to darkness in the presence and absence ofgabaculine 1143 I. we observed, in the absence of gabaculine, a very small increase in the total Chl content of the seedlings 14"h. P = 0 . 0 5 ) . a net gain in Chl a ( 10%. P<0.01 ) ~md a net loss of Chl h ( 15%, P<0.01 ). In the presence of :._.,abaculine. there was a slight drop ir~ the total Chl content of the seedlings (4%. P < 0.05). no change in Chl a and an even more pronounced drop in Chl b than in the control 126%. P < 0.01 ). These findings are consistent with de novo synthesis of Chl a and simultaneous degradation of Chl h in darkness in the experiments reported and are indicalive of Chl turnover. ( 29 Behaviour o f dark-grown se~ dlohzgs exposed to light .h~r tl short time and then returned to dttrkness I 123.127 I.

Because Chl turns over very rapidly in such seedlings, incorporation of labelled precursors into the tctrapyrrole moiety e f C h l in darkness can be observed even when there is a net loss of Chl overall. ( 3 ) Abili O" o.flthe Chh~rina 2 (oh-less) mutant o f barley to accumulate Chl ill dorktzess [ 12"/I This confirms that accu-

mulation of Chl a in darkness by barley, and by implication other angiosperm species, does not depend on the conversion of pre-existing Chl b or Chlide b to Chl t, or Chlide a. Returning to the central quesqon of this review: is the evidence for light-independent Cl-+lsynthesis in angiosperms seriously flawed? We do not believe that it is. In seeking to explain why the paper by Seyboid and Egle 11081 did not arouse comment at the time of its publication. Godnev eL al. 11281 noted that although in a number of cases the amount of Chl in the green leaves of plants returned to darkness did r.ol decrease, but even increased quite markedly. "these workers did not establish clear-cut patterns and the chlorophyll content of plants transferred to the dark in many cases lirst declined, then once more rose. sometimes quite sharply'. They also noted that "the authors themselves did not attempt

a theoretical interpretation of their results'. The small body of literature reviewed above is not subject to the same criticism. Clear patterns have emerged which are explicable in terms of simultaneous Chl synthesis and breakdown in light and dark .-,rid are now predictable. There is evidence that Chl synthesis via the light-independent pathway is promoted by ALA and inhibited by gabaeuline in the same way as in the light-dependent pathway. There is also evidence that Chl synthesized via the light-independent pathway is stabilized by incorporation into Chl-protein complexes and that thylakoids are assembled in darkness in the usual manner. Overall. the findings are consistent with the hypothesis that angiosperms which have been exposed to light during development h,'.we the capacity to synthesize Chl via two routes, one light dependent and the other light independent. We attribute the very small number of reports of dark Chl synthesis in angiosperms to the pervasiveness of the view that angiosperms lack a light-independent Chl biosynthetic pathway, the prefcrem.e of mo:+t researchers for dark-grown seedlings as starting material for their experiments attd the widespread practice of equating "dark-grown angiosperms" with "all angiosperms'.

6. Discu,~sion h is universally recognized that light is essential to initiate chloroplast development in angiosperms, whereas in gymnosperms and lower plants this is not necessarily (or even usually) the case. What is not generally recognized is that angiosperms which have been exposed to light during development do pot stop synthesizing Chl as soon as light is withheld. In some plants such as barley. Chi synthesis can continue in darkness for up to 24 h: in others such as Tradest'alztia it can continue for a week. How do angiosperms make Chl in darkness when they lack toe chloroplast-encoded Pchl(ide) rcductase genes ( t.hlL, N, B) present in gymnosperms and lower plants? What is the route and. in particular~ how is the porphyrin to chlorin transformation achieved'? We can only speculate. As far as the enzyme is concerned, there are three possibilites: ( I ) a reductase coded by recognizable chlL, N. B-type genes which have been transferred to the nucleus in the course of evolution: (2) a reductase of the POR type thm is able to effect the chemical reduction of an appropriate substrate in vivo( 3 ) a reductase of a different type. coded by a completely new gene. Because the Chlorina 2 (h-less) mutant of barley is able to accumulate Chl a in darkness, the simplest hypothesis is that light-independent Chl synthesis occurs via the classical pathway or one of the other routes to Chl a identnlied by Rebeiz and co-workers 1461. However, we do not rule out the possibility that Chl a might also be synthesized in darkness via Chl b in wild-type barley and other angiosperms. Recent findings of lto and co-workers 152-541 and Scheu-

tl. ): A+htm~on,'l aL I Jotmml t?l l'hotochcmistrv am/I~h,m,l,ioh,~:v B: Bi,,u,'.,x 41 r I t~7 , 201 221

m a n n et al. 156] point to the existence o f a C7 formyl reductase capable o f transforming h-typ.: tetrapyrroles to a-types. This increases the number o f potential Chl a biosynthetic routes that need to be i~v,estigated. It also increases the chances o f detecting one that is light independent. Two recent lindings are significant it, this regard. The first is the widespread tv,:currence o f Pchlide h and Pchl h in lower and higher plants 11441. Pchl(ide) b pigments differ from their Pchl(ide) a counterparts in having a fi~rmyl instead o f a methyl group at C / o f the ntacrocycle. Reduction o f the C 1718 double bond would convert them to chlorins (chl( ide ) b ) in the usual manner. According to Shedbalkar et al. 11441 "the ca:u o f reEaction o f the 7 - 8 ( i.e.. 17-18 ) double bond in 2-MV Pchl(ide) b as compared with 2-MV Pchl(ide) a suggests that the possible conversion o f these tetrapymfles to 2-MV C h l ( i d e ) b may exhibit a different biochemistry than the 2-MV pa~tochlorophyllide a analog, which requires light to drive the reduction o f the 7 - 8 ( 1 7 - 1 8 ) double bond'. In other words, the reduction o f P c h l ( i d e ) h might not require light. The second linding o f interest is the ability o f a PORtype reductase to reduce a Chl h related compound, zinc protophaeophorbide h. to zinc phaeophorbide h in the light. These zinc compounds are analogues o f Phlide h and Chlide b. respectively 1145 I. If it can hc shown that u different PORtype reductase can convert a m t t t l r o l l y o c c u r r h l g h-type porphyrin into a chlorin h~ the a b s e m ' e +~]"light then. in the presence o f an appropriate C7 formyl reductase, this would provide a mechanism for light-iPdependent Chl synthesis in angiosperms (and an additienal ntechanism for c h l L N. Btype organisms ).

7. Conclusions Angiosperms can synthesize Chl v i a a light-independent route: this is a functional attribute they share with algae, lower plants and gymnosperms. Where they difl~'r is in It) the overproduclio,~ o f Pchlide when seeds are germinated and raised, or organs initiated and grown in darkness and ( ii ) the need for light to activate Chl synthesis via the light-independent mute. We attribute overproduclkm o f Pchlide in darkness to the loss o f c h l L N, B genes from the angiosperm chloroplast genomc during evolution, the consequent reduction in capacity for conversion of Pchl(ide) to Chl(ide) and the accumulation o f Pchl(ide) in stable complexes in prolamellar bodies and primary thylakoids. The need for light to activate the dark Chl biosynthetic pathway presumably reflects both the general need for light to drive pnytochromemediated developmental processes and the specilic need for light to break up prolamellar bodies that accumulate in angiosperms in darkness. Our impression is that the unknown enzyme which catalyses the light-independent reducuon o f Pchlide in angiosperms is extremely susceptible to inhibition by its substrate and when it is inhibited in this way. light is needed to remove the block. According to this scenario, the I:.andamental difference between angiosperms and other plan;

217

taxa is that angit,.',.perms ha,,e brought their capacity f o r lightindependent Chl synthsis under light control. Intuitively it seems that they must also have reduced their dependence ~m light-independent pathways, but this may he misleading. Chl turnover studies in angio+;perms and lower plants are urgently needed to quantify the relative contrihutions o f the lightdependent and light-independent pathways to the total Chl r~,+l under normal ( d a y / n i g h t ) conditions

Acknowledgements We wish to dedicate this r e v i e w to Sophia I)ilova. ,,~hose insights into :hlorophyll synthesis in angiosperms were far ahead o f their time and whose observations in collal~ration with K. Popov were so easily conlirmed. We gratefully acknowledge the funding provided by the Australian Research Council I ARC ) and Macquarie University thr,.mgh its Research Grant Scheme I M U R G ) mid Outside Studies Pt~+gram t fI.Y.A. ).

References I I I ~,V.I.(;rillflh,.Recon,,lilUliUi!ol'¢hh~r,~ph)llkleformation h) i~fated ¢liopla~l mcmhrmle~. Bh~;hem L. 174. 1197.~ t 6,~1-692. 121 W.T. Grinith~. Prot~hh~rophxllid¢ phuh,redu,:ti,m, in H..~In:cr . ~ L "Fh¢ t .:hm;ph~ll',.C R C Pro,,,. B~.'a Raton. H.. H~H. pp. 4) I. -149

1"I! [).Ft. Burke. M...\ll~.-rtuJ.E. Flear',t. The/+h,,d.l,ac tcr Cal.+ul.m', ,=hlorm reducta~e-cno,~|hl~ h~us. I~'hA. cou,q.tx of three ged,.:.,.

~'hX. l'x:hY,and bcbZ. J. Bacterhq.. 175 I 109.11 24<)7-2413. 141 J.Y. Suzuki. CI'. Bauer. I.i~ht-indepcnt;entchhm~phyllhio,,)nth¢,,i,: in: oh ement ol the chhwop+a~,tgone chll. +Ir\C }. Plant ('ell. 4 I I'.)02} 9++.9-94n 151 CI +. Bauer. DAV. ltollivar. J.~. Suzuki. Geuetic aual.',sis ill"phoI~q',i~mcntbi+~s',nthe,,i,,in I'.':dul<'lcrh+:a ~uiding light lbr algae and plant,,, J. Bacteritfl.. 175 I It.~93) 3919-39"~5. 161 R. Schulz. H. Sengcr. Prtlt~:hhm~l+h.vllidereductase:a ke~ en"Ynle in the .,.'reeningpn~:e',',, in C. Sundquist. M. rytx:rg reds. ). Pitinem-pn~teincomplexe,, in plastids: synthesis and a~,m:ml,ly..&caderek" Prc,s. Sau Diego. CA. 1093. pp. I,";()-2IS. 171 C.A. Retx'iz. R. Parham. D.A. Fa,,tmla.l..".l,hmnide,~.Chhm~phyll a hi~n)nthetic heterogeneity.Ciba FoundationS~rap. 180.TI~ Bio,,ynthesis uf the Tetrapyrrole Pigments. Wile,.. C'hichester. 1~,~;4. pp. 177-195. IS] D. Von Welts!On. S. Gough. C.G Kannan~ara. Chh,roph',ll biosynthesis. Plan! Cell. 7 I It~.L~) 1039-1057. It; I S. Reinl'~+the.(.'. Reinl'~+the.The regulation ol'¢nz) lues in,,ohed in chlorophyll ,,. nthe',is. Eur. J. Bitmhem.._.237t I~.~ ~323-3..i.3. IOI S. Reinhoth¢. C Rein~the. N. l.cl'~dcv. K. Apcl. PORA and PORB. IV.o li~ht-der~.'ndentprott~:hlorophyllidc-reducingenz.,,me,, or an,_'~ospcrmchlorophyll bios', nthesi,.. Plant Cell. X I It~J~ ) 763769. I I I IUI'.-\C-IUB

Joint Commission I+i~.'hcmical Nomenclature ( ICBN ~. Tctrap)rroles. Pure Appl. Chem.. 51 ~1979) 2251-231bk 121 H. Scheer. Structure and t~.-,..um:nceof uhh,roph.~I1,,. in H..~heer I ed. ~. "FheChhwoph.',11~,.CRC Prc,,s.Bt~a Raton. FI.. I~,~01.pp. 327. ll31 S. Granick. S.I. Be;fie.Hem..'n,chh+rnph.xII, ;rodrelatedcomt~mnds: hios>nthesis and metabolic r¢gulatiou. Ad~. lin].,,tool.. 46 ( ]t~7SI 33-2(13.

218

1t. E Adamson et aL I Jountal of Photochemistry and Ph,m~biology B: Biology 41 (19971201-221

1141 C.A. Rt:beiz. P.A. Castelfranco. Prott~:hh)rophyll and chlorophyll biosynthesis in cell-free systems from higher plants. Ann. Rev. Plant Physiol., 24 ( 19731 129-172. 1151 O.T.C. Jones, Biosynthesis or porphyrins, heroes, and chlorophylls, in R.K. Clayton, W.R. Sistrom ( eds. I. The Photosynth,:tic Bacteria. Plenum Press. New York. 1978. pp. 751-777. l U61 K.M. Smith. The structure and b;osyntht:sis of bacterkvdhh)rophylls. in P.M. Jordan I ed. ). Biosynthesis of Tdrapyrro[c~,. New Comprehensi'.'e Bitx.'hemiswy. Vol. 19. Elsevier. Amsterdam 1991, pp. 237-256. 1171 S.I. Be;de, J.D. Weinstein. "li,~:ht:mistr2, and regulation of photosynthetic pigment fi)rmation in plants and alguc, in P.M. Jordan (ed.I. Biosynthesis of Tctrapyrrolt:s. New Compreht:n:;ive Bioch~-mistry. Vol. ' 9 l.-'b;cvier.Amsterdam. 1991. pp. 155-236. I 181 W.T. Griflithn. Magne.;ium 2.4-divinylpha,:oporphyrin a5 as a subsir'ate fi)r chlorephyll hit)synthesis in vitro. FEBS Lett.. 50 ( 1975 ) 355-358. ll91 H.M. Wilkes. M.P. Timkc,. A light-dependem complemen:ation system fi)r analysis of NADPH:protta:hlorophyllld.: oxidor,:duclane: idt:ntitication and mulagenesis of two const:rvt:d residues that are essential fi)r enzymt: activity. Pr(a:. Natl. Acad. Sci.. USA. 92 ( 1995 ) 724-728. 1201 H.C. Yen. B.l.. Marrs. Map of gent:s for carotemfid and bacteriochIorophyll biosynthe~;i,, in Rht~dop.wudonumas cupsulata. J. Bacteriol.. 126 ( 19761 619-629. 1211 B.L Marrs. Mobilisation of lht: gent:s f~)t photosynthesis from Rh~,dop.wtuhmtonas eap.~ulata by a promiscuous pin)mid. J. Bactt:riol.. 146 ( 1981 ) I(X13-1012. J221 D.P. Taylor. S.N. Cohen. W.G. Clark, B.I,. Marrs. Alignmenl of genetic and restriction maps of the photosynthetic region of the Rhodopsetuhmuma.s cap.snlata chromosome by a conjugaUonmr:dinted marker rt:cut: technique. J. Bacleriol., 1.54 ( 19831 58051~). 1231 K.M Zst:bo. J.l'. Hearst. Genetic physical mapping of a photosynIht:tic gt:ne cluster from R. ('ap.~'ulata, Cell. 37 119841 937-947. 1241 G.A. Armstrong. M. AlberU. F. Leach. J.E. Hearst. Nuclcotidt: sequence, organisation, and nature of protein products of the carol entfid biosynthesis gene cluster of Rh,dohucter eapxuhm:s. Mol. Gen. Goner.. 216 119801 254-268. 1251 D.W. Bollivat. C.E. Bauer. Ass(v.:iation of tetrapyrrole intermediates in the hacteritg'hlorophyll a biosynthetic pathway with the major outer-mt:mbrane prolt:in of Rhodohat'ier Biochcm J.. 282 I lt,~2) 471-476.

C(lpMIhllnA'.

1261 C.L. Wellington. J.T. Beatty. Promott:r mapping and nucleotide Nequcnct: of the bchC bacteritv.:hlorophyll biosynthesis gent from Rhodobactercap.s'ulatus. Gene. 83 ( 19891 251-261. 1271 Z. Yang. C.E. Bauer. ICht)dobucter t'aps,lhltus genes inwflvcd in early steps of the bactt:rit~hloruphyll pathway. J. Bactcriol.. 172 1 I~,H) ~ 51X)I-5010. 1281 D.C_'. Youvan. E . L Bylina. A.M. A]berti. H. Bt:gush. J.E. Hearst.

Nucleotidc and deduced polypeplidt: sequences of the photosyntht:tic reaction centre. B7(}Oantenna and Ilanking sequences from Rho~b~p~euthmumas ,'up.~uluta. Cell. 37 ( 19841 949-957. 129] G.D. Giuliano. D. Pollock. H. Stapp. P.A. Scolnik. A geneticphysical map of the I~htnlohtlcter t'llpMdltttL~ carotenoid bit)synthesis gent: cluster. Mol. Gen. Gcnt:t.. 213 119881 78-83. 13111 D.A. Young. C.E. Bauer. J.C. Williams. B.L Man's. Genetic t:vidr:net: l'tw superopcronal organisation of genes ti)r photosynthetic pigments and pigment binding prott:ins in Rho+h~h,t'tercapsnlatus. Mol. Gt:n. Genet.. 218 119891 1-12. [ 31 I F.H. Yildiz. H. Gest. C.E. Bauer. ConNervation in the photosynthesis gone cluster in Rhodospirilhml ('entenlnn. Mol. Micmbiol.. 6 119921 2683-2691. 1321 S.A. Ctmmber. M. Chaudhri. G. Conner. G. Britton. ('.N. Hunter. Ltv,:alized transposon Tn5 mutagenesis of tht: phott~synlhetic gent: clusterol'Rhodohacler.~pheroidex. Mid. Microbiol.. 4 ~ Itr-/t)) '47 ;989.

1331 L Mullet, Chloroplast developmt:nt and gene expression. Ann. Rev. Plant Physiol, Plant Mol. Biol.. 39 ( 19881 472-502. 1341 M. Posno, M. van Noort. R. Debise. G.S.P. Groot. Isolation, characlerisation, phosphorylation and site of synthesis of Spinuciu chloroplast ribosomal proteins, Cuff. Genet.. 8 (1984) 147-154. 135 ] C. Dean, R. Schmidt. Plant gcnomt:s: a current molecular de,scrip tion. Ann. Rev. Plant Physiol. Plant Mol. Biol.. 46 (1995) 395418. 136] K. Kecgstra. L.J. Olsen, S.M. Theg, Chloroplastic precursors and their transport across the envelope membranes. Ann. Rev. Plant Physiol. Plant Mol. Biol.. 40 (1989) 471-501. 1371 R.E. Sust:k. J. Chory. A tale of two gcnomcs: role of a chloroplast signal in coordinating nuclear and plastid genome expression. Aust. J. Plant Physiol., 19 (1992) 387-399. 138[ JD. Palmer, Comparative organisation of chloroplast genomes. Annu. Rev. Genet.. 19 (1985) 325-354. 1391 J.D. Palmer, D.B. Stein, Conser,'ation of chloroplast genome structure among vascular plants, Cuff. Genet.. IO (1986) 823-833. [401 Y.J. Avissar. J.G. Onner~u.I.S.I. Beale. Distribution of &aminolevuiinic acid biosynthetic pathways among photosynthetic bacteria and related organisms. Arch. Micrubiol.. 151 ( 19891 513-519. 141 I S. Granick. Magnesium vinyl phcoporphyrin a5. another intermediate in the biological synthesis of chlorophyll. J. Biol. Chem.. 183 ( 19501 713-730. 1421 J.B. Wolff. I.. Price. Terminal steps of chlorophyll a biosynthesis in higher plants. Arch. Bitx:hem. Biophys.. 72 ( 1957 ) 293-301. 1431 O.T.G Jones. Magnesium 2,4-divinylphat:oporphyrin a5 monomethyl ester a protochk)rophyllide-like pigment prc,duced by Rlnnlopseudommu+s )phaeroides, Bi~vdhem. J.. 89 (19631 182189. 144[ F.C. Belanger. C.A. Rebeiz. Chlorophyll biogenesis: Detection of divinyl prott~:hlorophyllide in higher plants, .I. Biol. Chem., 255 ( 19801 1266--1272. 1451 S.A. McCarthy. J.R Mauht:is. C.A. Rt:beiz. Chloroplast biogenesis: biosyntht:sisof prott~hlorophylll ide ) via acidic and fully esterilied biosynthetic branches in higher plants. B itx:hem.. 21 ( 19821 242247. 1461 C.A. Rebeiz, S.M. Wu. M. Kuhadja. H. Daniell. E.J. Perkins. Chlorophyll a bio..~ynthcticroutes and chlorophyll a chemical heterogeneity in plants, Mol. Cell. Bit~hem...58 ( 19831 97-125. [47l B.C. Tripathy. C.A. Rebeiz. Chloroplast biogenesis: demonstration of the monovinyl and divinyl mom~arboxylic routes of chlorophyll biosynthesis in higher plants, J. Biol. Chem., 261 (1986) 13 55613 564. [ 48 [ B.C. Tripathy. C.A. Rcbeiz. Chloroplast biogcnt:sis: conversion of divinyl protochlorophyllidc to m(mavinylprott:chk)rophyllide in green(lag) barley, a dark monovinyl/light divinyl plant species, Plant Physiol.. 87 ( 19881 89-94. 1491 J.X. Duggan. C.A. Rt:beiz. Chloroplast biogenesis: Conversion of divinyl chh)rophyllidc a to monochloruphyllide a in vivo and in vitro. Plant Sci. Left.. 27 ( 19821 i37-145. 1501 M.A. Sehnt:egurt. S.I. Beale. Origin of th- chlorophyll b formyl oxygen in Ch/ore/la vnlgari.s. Bka:hemistry. 31 119921 II 677I I 683. [ 51 I RJ. Porra. W. Schafcr, E. Cmiel. 1. Kathcdcr. H. Scheer. Derivation of the [brmyl-group oxygen of chlorophyll b from molecular oxygen in greening leaves of a higher plant ( Zea mays). FF+BS Letts.. 323 ( 19931 31-34. 1521 H. Ito, Y. Tanaka, H. Tsuji. A. Tannka, Conversion of chlorophyll b to chh)rophyll a by isolated cucumber t:tioplasts. Arch. Biochem Bit)phys., 3(~5 ( 19931 148-151. 1531 H. ho, S. Takaichi. H. Tsuji. A. Tanaka. Properties of synthesis of chlorophyll a from chlorophyll b m cucumber ctiuplasts. J. Biol. Chem., 269 ( 19941 22 034-22 038. 1541 H Ito. T. Ohlsuka. A. Tanaka. Conversion of chlorophyll b to chlorophyll a via 7-hydroxymethyl chlorophyll, J. Biol. Chem., 271 ( 19961 1475-1479.

H. Y. Adam.~'on el oL I Jounzal of Photoehemi.~t~" , n d Photohi,h~gy B: Bioh~gy 41 ¢1997j 201-221 1551 T. Ohtsuka. H. ho. A. Tanaka. Converston of chlon)phyll b to chlorc.phyll a and the at,;.~mbly of chlorophyll with apoprotcinr, by isolated chloroplasts, Plant Physiol.. I 13 I 1997 ~ 137-147. 1561 V. Scheumann. M. Helfrich. S. Schoch. W. Rudiger. Reduction of the tormyl group of zinc pheophorbide b in vitro and in virtu, a model for the chlorophyll b to a transformation, Z. Naturfi)r~ch.. 51e ( 19961 185-194. 1571 A.M. Kumar. U. Schanb, D. Soil. M.L. Ujwal. Glutamyl-transfcr RNA: at the crossroad between chlorophyll and protein biosynthesis. Trends in Plant Science. I 119961 371-376. 1581 P.M. Jordan, B.I. Mgheje. The genes of tetrapy~xol~: biosynthesi:,, in P.M. Jordan ( ed. t. Biosynthesis of Tctrapyrroles. New Comprehen:;ive Biochemistry. Vol. 19. El~e,,'ier. Amsterdam. 199 I. pp. 67I00. 1591 D.W. Bollivar. J.Y. Suzuki, J.T. Beatty. J.M. I~hrowolski. C.E. Bauer, Directed mutational analysis of bacterit~hlorophyll a biosynthesis in Rho&~hacter ('ttpxuhltus. J. Mol. Biol.. 237 (ITS4) 622--640. 1601 D.H. Burke. M. Alheni, LE. tlearst, BchFNBH bactcri~v.:hlorophyll synthesis genes, of Rhod:~ha~'ler ('up.~ldattt~ and identilication of the third subunit of light-independent prott~=hlor~)phyllide reduetar, c in bacteria and plants. J. Bacteriol.. 175 ( 1993 ) 2414-2422. 161 I T. Kohchi. H. Shirai. H. Fukuzawa. T. Sano, T. Komuno. K, Umesono. H. Inokuchi. H. Ozeki. K. Ohyama. Structure and organi,,ation of Mczr('hantia polymorpha chloroplast genome IV. Inverted repeat and small single copy rcgtons. J. Mol. Biol.. 203 119881 353-372. 1621 Y+ Fujita. Y. Takahashi. F. Shonai. Y. Ogura. H. Mat..,uhara. Cloning. nucleotidc sequences and dift~erential expression of the nitH and nitH-like 11"rxCl genes from the lilamentou~ nilrogen-lixing cyanobacterium Plec'tonennz h++,ryanun+, Plant Cell Phyr, it)l.. 32 ( 1991 ) 1093-11~}6. 1631 D.H. Burke, J.E. Hearst. A. Sidow. Early evolution of photosynthesis: clues from nitrogenasc and chlorophyll iron protcinr,. Proc. Natl. Acad. Sci- USA, 90 I It,~931 7134-7138. 1641 Y- Ogura. M. Takemura. K. Oda, K. Yamata. E. Ohta, H. Fukuzawa. K. Ohyama. Cloning and nuclcotidc ~,equcncc of a fi'xC-ORF469 gene cluster of Syneehtwystis PCC68113: conservation with liverwort chloroplar,t frxC-ORF4G.I and nil+ operon. Bio~ci. Biotech. Bio~hem., 56119921 788-793. 1651 Y. Fujita, H. Matsumoto. Y. Takahashi. I-1. Mal~,ubara, Identilication of a nifDK-like gene ( ORF 467 ) involved in the biosynthesis of chlorophyll in the cyanot,,actcrium Plec'tonl'nnt bor)'attnnl. Plant Cell Physiol., 34 ( 1993 ) 305-314. 1661 J.B. Howard, R. Davis, B. Moldenhancr. V.L. Cash. D. I~an, FeS cluster ligands are the only cysteines ~equired for nitrogenusc Fe-protein activity, J. Biol. Chem.. 262 ( 1989 ) I I 271~-I I 274. 1671 M.R. Georgiadis, H. Komiya. P. Chakrabarti, D. Woo, J.J. i~.omuc, D. Rees, Crystallographic structure of the nilrogena.',e iron protein from A=.ou~bacter vb~elandii. Science. 257 ( 19921 1653-1659. 1681 Y. Fujita, Y. Takaha~,hi. M. Chuganji. H. Marsubara. The nilH-like (frxC) gene is involved in the bio~,nthesis of chlorophyll in the lilamentous cyanobacterium Ple('tonema horyanum, Plant Cell Physiol.. 33 119921 81-92. 1691 Y. Fujita, H. Takagi, T. Hase. Identilication of the chlB gent and gene product essential for light-independent chlorophyll biosynthesis in the eyannbacterium Ph'cumema hory(znnm, Plant Cell Physiol., 37 ( 19961 313--323. 1701 C. Roitgrund. L.J. Mets. Ltx:alization of two novel chloroplu~,t genome functions: trans-splieiag of RNA and prottx:hlorophyllide reduction, CulT. Genet., 17 119901 147-153. 1711 Y. Ch~luet. M. Rahire. H. Girurd-Ba,seou. J. Erickson, J.-D. Rochaix. A chloroplast gene is required for the light-independent accumulation of chlorophyll in Chhzm.~lomonas rebzhurdtii. EMBOJ.. II 119921 1697-1704. 1721 X-Q, Liu, H. Xu, C. Huang. Chloroplast chlB gene is required fi)r

219

~ight-indcpend¢'at chlorophyll ac~:umulathm in ('hh~mydo,:,na.s r¢inh, rdtii. Plant Mol. Biol.. 23 ( It~L~ ) 297-308. 1731 J. Li, M. Gold~¢hmidt-Clerm~mt.MP. Timk~:. Chh)ropl~t.cncoded chlB i~. required ftrr light-independent Pchlide rcut~ta.,.e activity in C'hhm~x'donnma.~ reblhardzii. Plant Cell. 5 ( 1993 t 1817-1829. ]74l K. Ohyama et al., Chloropla.,,t gent organi,,ation dedu,=cd from complete ,,rdrecht, 1987. Vol. IV, No. 8. p p 483-486. [ 80l G.A. Pe~, and tv.'o di,,tin¢t NADPHprott~:hloH)phyllide oxidoreducta.,,e polypeptides in mountain pine ( Pinu.~ mugo). Planta. t91111993 ) 536.-545. 1841 R. Schulz et at., Nu~:!,:'otide ~equence tbr a eDNA c~v,ling lot the NADPH prott~chlorophyllide-ox;doreductase (PCRI of barley ( Honlez,n rulgare L. ) and it~,expre.,,r,:en in E~t'heri('hiu ('oil Mol. Cert. Genet.. 17 ( 1989} 355-'~61. 1851 P.M. Darrah. S.A, Kay, G.h. Teakle. W.T. Gri~'liths. Cloning and ~quencing of N A DPH -protovhlorophy !lidea)xidor~.~ucta,,,e, Bio'.:hem., J.. 265 ( 199111789-798. 1861 M. Bcnli. R. Schulz, K. Apel, Effect ~ff light on the NADPHprotochlon)phyllide oxidoredu,'la~ ofAnzhidop.~i.~" thaliana. Plant Mol. 13io1., 16 ( Iq,~l ) 615-625. 1871 A. ~ Spare), Z. He. H. Michel, D.F. Hunt. M.P. Timko. Mo!ecular cloning, nuclear gene ~tructure, and de,.elopmental expres,.ion of NADPH-protochlorophyllide-oxidorcducta>e in pea 1Pisum satiYtntl L+), Plant Mol. Biol.. 18 [ 1992~ 967-97? 1881 H. Hohorf S. Reinbothe. C. Reinoothe, B. Bercza. K. Apel, Tv.o route~ of chlorophyllide synthesis, that are differentially regn+at,.at by light in b~'ley (Hordenm v,lgare L.), Prt.~:. Natl. Acad. Sci. USA, 92 ( 1995> 3254-3258. [ 891 J.Y. Suzuki. C.E. Bauer, A prokaryotic origin for light--dependent chlorophyll bio.~ynthes,s of plants, Proc. Natl. Aead. Sci. USA. 92 ( 1995 ) 3749--3753. 1901 G.R. Teakle, ~'.T. Grifliths. Cloning. characterization and import

210

H. Y. A~htm~on ct ~d. / Jrmrnal oJ l~hotocl,emist£v
ntudie,, ,tlu prolochloruph) ]lid.e reducta,e frolll ',vh.eat ( 7)4tiium ai'stil'u,i I. Bh~,:hern. 1.. 206 ( 10931 225-2311. 191 I M. Ikeuchi. S. Murakami. Behaviour of the 36 (XIO-dalton prol.ein iu the inlernal nielnbrane,, of squa,,h eliopla,t, during greening. Planl Cell Phy',i.I.. 23 119821 574-583. I U21 K. Dehe`,h. M. Klaa`,. I. Haur,er. K. AI~-'I. Lighl-induced .change`, in the di,lribution of Ih.e 36 IXIO Mr i~llypeplide ill" NADPHpro0.~hh~roph) Ilid.e oxid~,~rcducla`,¢ 'l~,iihin dill~.'r.ent cellnlar cl.Ullparlincni, of barlc} (Hor¢h,um i'lllt,,tu~' L . I . I . [ ,x;.ili~ath)n b) inlnlunilgilkl labelling in uhraihhl n.ection,. Plania. 160 1198£11 172-11¢,3. 1931 M. R}berg. ('. ~Ulldq',isl. The r.egular ultra~iructur¢ of i~olalcd prolani.ellar I~dic~ d: Fend,* ~l, th.e ple,cncc of liiCllihrail.e-hound NAI)l~H-l~rOlochhlrophyllid.e oMd#.u.educla,'J. I~ll)~iol. Plant.. 73 i 1981¢,) 2 I,"1-22fl. IO41 V.M. Ko,,ki. C.S. l'rcnch. J.H.('. Sinilh, Th.e a.etion ~l~clr!JIn Itlr th.e transtbrmafilm ill" pr,tochb.~rtlph.', II to chhtr.lph,.ll a ill normal and all'tint, corn ,...'cdling,. Arch. Bu~:henl. Bil~phy,.. 31 ( 1051 I I 17. It~51 W.T. Grillilh,,. Subnirate ,,pc.eili.eiL', ,tudie, on protol:hloruphvlide r.educta,,e in barle.', f~lltt'¢h,ttln I'lll.k't/l'l') eliopla,t ulenlhruncr,. Bile'heal. J.. 1861 198111 267-278. IUfll T. Sa,a. K. Sugahara. Phol.,,Icon',. ¢r:,ion ~ff proto.eh hirophyll hi .ehhlrilph} II a in a inulanl .~ll"(gih,i'lht re.t,uhtrii. Plant ('.ell Phy`,iol.. 17 I 19761 273-279. t971 K. Kotzaba,,i,. M P Schuriug. II. Sengcr. Occurrence of prlltllchhlroph.'. II and il~ phohllran~lilrula.ion hi .ehhlrophyll ill ululanl C-2a' ill .~'ll'ltl'di'~nill% ¢~]~liqllu>%. Phy`,iol. Planl.. 75 ! 1989) 221-226. 1081 ('.1-. ('ohen. J A . Schi ff. I-',.enl~, , u r r , unding Ihc early developnlenl ol I:u~h,,a chhnopla,,l`,. XI Pr.~lhl.chlorliph)I1(id¢) and it`, plullllcon,.cr,,ion. Pholochenl. Pholobiol.. 24 ( 19761 555-566. 1001 IL('. IJelangcr. ('.,%. Rebel/. ('hhlrllpla,,t bio.,Jcne,l,: detecli,n of di', in.', I prot~chloroph 3 Ilid.e e~ler in higher plaut,. B i,.~heou`,ir). 10 i Itj1411141475~',w,h3. I11t11 C. Liljcnbcr,_'. ('haracleri~,ati~ln and pn~perticr, o i , prllhlchl.~lroph} Ilidc c,.ler in lea',.u, ol dark gro',', n barl.ey v<,ith gclauylgeraui,.ll a', e,,leril) ing alcilhol. Ph.~ ,,Jill. Plant.. 32 ( I tJ74 ~ 2118-213. IIII I H.A.I.anc.er. C.I!. ('tlllcll. J.A. Schifl. Changing ratios of photoIran,f, lrmablc i'~rot,.~chll~roph)ll and prottl,..'ill,lro.nhyilide ,ill b,:an ,c.edliug, dc,.cloping ill Ihe dark. Plala Phy,ild.. 57 I Itl7fl) 3~'10374. IO21 ('.J. \Valkcl. W.I'. (;rillith,,. Pr~.ltilchloroph)Ilide rcducla~,.e: a l]il',oprc,tcin?. FI!BS l.ell.. 231; I 19881 250-262. II)]I I N.V. lgnato',. ().B.B.el',a¢',a. F.F.I.it',iI: P,.~,',iblc r,.dc ol Ila',.in .eolnponenl, ill" prlll~K:hhlr~lph,,liide-prolein conlph.'~.e, in prinlary pro.ce,~,e,, of probo.chl.~ln,~ph,,Ilid¢ pholorcduction in elilllated planl Iea',c'~. Phl~io',3 nth¢lica. 29 110031 235-241. II1-1.I W.T. (;rillith,. T. Mcl-hlgh. R.I'. Bhink<.'n,,hip. The lighl-inteu',il 3 dcpcndt'ncc ill pr,ttlchl,.ir.~lpl'lyllide phot~lcon',er,,i,n aud it, niguilicanc.e I¢1lh¢ cat al.', lic tncchani~.in ol pro~,och hln'~ph', I lid.e r.educta`,¢. FI'BS Left.. 30:'; I It.1061 235-231"; 111151 R.P. Oli',er. W . T (;rlflith',. lden:ilication of Ihe plllypcplid¢,, ol NADPH-prolocllloroph)llide oxidorcducta`,e Biochcm. J.. 191 11914111 277-2811. I IC161 "IP. Bogle',. H. Young. I~r,toclil.~ln~plL,,llide rcducla',e. I. Dcl.erUliUalitln uf Ihe rcgit~hcmi,.tr,, alld the MercocheiniMr) of the rcduclilm ~1' prll"~,:hh~rl~phyllitl¢ Io chhlruph,,llid¢. J. Am. (_'he,n. Silo.. III ( 10891 31Y.J5-31196. 1071 W.T. Griltilh',. R.P. Olin.or. S.A. Ka 3, A .critical appraixal ~ll Ihe role a,ld regulation ll|" NADPI I-pr.tochhlrllph~ Ilidc i.lx;dorcdu¢tan¢ in gr¢.:'niug planl`,, ill C. Siron',al. M. Br,.It;er~, I cd`,. I. I"rotllchhlrol"lh~ Ilid¢ Rcducti, m arid Greening. Marlin.J, Nijhoff/Dr W. Junk Puhl ,her,. The Hague. 1984. p[~. 1'.)-20. 1:!81 A. Scybldd. K Egl.e. Li.ehll~:ld und Blatt|arbntolfi: II. Planla. 28 119381 87-123. 11191 D.W. Kupk.e.J.I.. Huntingdlm, Chhlroph)ll a appearance in the dark ill higher plant,,. Science. 1411 { 19631 49-.51.

I 11111 S. Wi.ekow`,ki. S. Fleck. On Ihe chlorophyll metah,li`,m of greccn ,.eedlings in darkness,. Bull. A.ead. Polon. Sci.. 18 (1971)) 47-51. I I I I I A. Tai~aka. H T',uji. Changes in chlorophyll a and b content itl d;!,'k-incubaled co|yledon, excb, ed I'rolll illuminated cucumber ~,.e.edling`,. Plant Ph)niol.. 68 11981 ) 5f)7-570. II12i K. Popov. S. Dilova. On 1h¢ dark `,)nihe`,is and niabilization nf chhlroph.',ll, in H. M¢l/ner l e d I. Progress in Photor, ynthcsis Rc,ear,.:h. Int. Union Bitll. Sci.. Tiihiug.en. 1069. Vol. II. pp. 6 . 1 ~ fi09. fl. Ocl/e-Karow. II. Mollr. Siabilil) ,~f chhlrophyll during gre.ening, IIlll I,ra.ei J. Bot.. 33 I 1984 ) 211-223. 11141 J. Walm~ley. H. /~dam`,on. C'llanges in tile chloroph)ll-prolein complcxe, of grecuing b:lrl.ey ,eedlingn tran,li:n'.ed to darkne`,~,, in N. Murata led. 1. Re`,eureh in Phot+.l,)nlh.e`,in, Klu'.~,er. Dtlrdr.e.ellt+ 1992. Vol. I11. pp. 79-82. 1151 II. Adam~,.u.W.T. Griflilhs. N. Packer. ,%,1.Sutherhlnd, I.ight-independent accculntllalion of.ehhlroph)l! a and b and prlllochlorophylhd¢ ill gre¢ll barley i Hordl,ulll Itt/gllrl, ). I~h)'`,iol. Plant.. 64 ( 1985 ) 345-352. I. Wahnsley. H. Adamson. (;abaculine inhibition of chloruph)ll `,) nlhenb, ill light and darkne,s ill intact barley 1Ilorth'mn I'ul.~are ) `,eedling`,. Plant Sci.. 68 119Ulil 65-711. H. Adanlson, Chh)roplasl developluent in green hurley lea; es lransletted to darkne,s, in G. Ako)uuoghlu. A.E. I-vangclopouhl,. J. Gcorgaiml`,. (;. I'alaiolgos. A. Trakai.ellis. C. Tsiganos (ed`,. i. Pr,gre,,, in Clinical and Biological Re`,earch: Cell Function and DifI~.,renliation, Alan R. I.b,n. New Y,.~rk. Vol. 1112B. 101'12,pp. 189109. ! i l 8 1 7. Wahn~l¢). (_'hlorol~h)ll accuinulalion in phololl,.'riodicall)'~ro'it, n barle) ,¢.edling~ IranM'~rred Ill darkll¢`,~: efli:ci of lime of Irall>l~:r and daylenglh, llh(ihl,ynlh¢liC;.i. -.25 11991 i 41i94 18. I l i 9 t J. Walnlslcy. tl. Adanlson. Chlorol'lh).ll acCtlliitll;llion and breakdll',~,il in lighl-~r(iwn barle) Irau,l'elred It) darklic~;~,: efl¢¢l o1" ,cedlinL,, age. I>h)'`,illl. Plani.. 77 I 19801 312-310. 112i)1 II. Adaul,llll. M. Leunon. K. Ou. N. Packer. J. ~iVahll`,l¢). 1"]',id.ence Ior a lighl-indelxrndenl chhlroph) ll l,ionynlheti.e path¢,;.ly in agionperni ,ecdn gernlinalt'd hi darkne,,, in M. Baltscheff~,ky led. l. ('nlr¢lll Re,eareh in l~hllhln) nlheni~. Klu~ver. Dordr.echl. 1991), Viii. III. pp. 61~7-6911. 11211 N. Packer. 11. Adaul,,on. Inc~,Jrl'soration of 5-anfinolevulinic acid into chl~.~r.~ph',ll in darkue,, in barlc}. Phy',iol. Planl.. 61'1 119861 222-2311. 11221 H.C. "l'ripalh). C.A. Rchci#. Noncqui',idcu,,.~ of glatauli.e acid dud ~$-aluhl,.llc',ulinic acid a, ~,uhr,trale,, Ibr protochlorophyllidc and chlorophyll bior,)nthc,i, iu darknc:,,, hi J. Biggins led. 1. Progress in Photo,ynlhc~.is Re,catch. Marilnu`, Nijhoff. Dordlecht. 1987. Viii. IV. No. 8. pp. 439-..143. 1231 J. Vti'allllMey. H. Adani`,on. Chl.~lroflh', II turn,.iv.er hi etkdaled greening barley Iranr, lL.rred t,.i darkne~,~,: b,,.itopic ( I J ~C glulanlic a.eidl .e', idene.e ill"dark chlorophyll '..',nlhc,i`, in Ihe ab`,enc¢ ,.ff chloo.lph', II accuinulali.~ln. Phyniol. Planl.. 93 119941 435-444. 1241 K. Apel. M. Motzku~,. K. Dehc,h. The bio,ynthesis lll'.ehlorophyll in greening barl¢) I ttordeunl I'ldgllr¢ f. Is there a light-indel~ndellt proiochlor,.iflhyllide reducta~c?. Planla. 161 ( 19841 551;--554. 125i N. Pack.er. tt. Adam~,ou. J. Walnl~,lc.',. Colnpari',,.m ol .ehl~.lrophyll ac.euululaiion and HC-AI.A incorporali,.lu inlo chlilo.lpllyll in dark and liglu in green hurley, in J. Biggin,, I ed. I. Progrens iu Phtli.tl~,ynthe,is Rc~,earch. Marlinu~, Nijhoff. Dordr.echt. 1987, Vt,I. IV. No. 8. pp. 487--1911. A.B. Rudoi, R.A. Rudoi. R.A. Chkaniko~ a, [3ark Ibrmalion ofchlorilpllyll in angio`,perln`,. Fiziol. Rust.. 35 ( 1988i 1092-1098. J. Wahn~ley. fl. Adamson, t'~idence for liglu-independent s)nlhe,b, and turnover of (._'Ill a in a barl,..'y nlutaui lacking (-'hi b. in P. Mathb, led. I. Phoio,ynthesi~,: t'r,anl Lighl tl.I Biosphere. Klu',ver. Dordrecht. 1905. Vol. III. pp. 993-996. 1281 T.N. Godne',. A.A. Shlyk, R.M. Rotfarb, The synthesis of chlorophyll in the dark in angio`,perm.,,. Fiziol. Rast.. 6 ( 19591 33-38.

H. Y. Admen,win et aL IJol#rncd olPlumwhcmi,~try am/I'hn/.hi.h~gx B: Bu,h~'4y 41 ¢1~7~ 201-22!

1129l H. Adamson. N, Packer. Chlorophyll lalxlling pauem~, in Znstera translerrcd to darkness. Photosymhetica, 21 ! 1987 ) 472--48 I. 11301 |1. Adamson. N. Packer. J. (;regory. Chloropla~,t development and the synthesis of chlorophyll and prott~hlorophyllide in Zn~ter. transferred to darkness,. Planta. 165 ( 1985 ) 469-476. 11311 (;. R~ihbelen. Uber die Prott~:hlorophyllreduktion in einer Mutar,t¢ wm Artthidnp.~i,~ lha/ium~ ( 1.. ) Heyn. Planta. 47 ! 1956 ) f,32-546. 11321 H. Adamson, unpublished. ItF)6. 11331 H. Adamson. Evidence I~.lr the accumulation of l'~th chlorophyll a and b in an angio~,perm, in G. Akoyunoglou. J.H. ArgyroudiAkoyunoglou. l eds. ), Chloroplas! Development. Else'.ier- North Holland, Amsterdam, 1078. pp. 135-140. [134[ H.Y. Adamstm. R.(;. Hiller. M. Vesk. Chloroplast de,.eiopment and the synthesis of chhwophyll a and h and cldorophyll protein complexes I and II in tile dark in Tnuh'.~vunth~ ,Ihilh,ra ( Kumh ). Planta. 150 (198(I) 2(~9-274. 11351 I. Ikcgami, A. Kamo'u, l-. tlase. Dark fiwntation of chloroph',ll in cultured tobacco cells. Plant (_'ell Physiol.. 25 ( 1984 t 343-348. 11361 W. Hunrich. B. Bereza, Sp.cctroscopi¢ charactcrisalion of pnltochlorophyllide and its tranM~.~rmation. Photo~.} nthctica. 28 ( 1t)93 I 1-16. ] 1371 T. Bomer. A. Mei,,ler. C'hhwophyll attd car,~tcnoid content el rihosome-delicient plastids. Photosyn'.hctica, 14 119S11) 5,"19-593. 11381 M. Knee, Role of ethylene in chlorophyll degradation m radi',h cotyledons. Plant (;rowth Reg.. I0 ( 1901 ) 157-162. I 139 ] L. Nt~:k. L J. Roger,,. H. Thoma:,. Mctal~di~,m of prot~:m and ~:hlorophyll in leafli,,,.ue in/"e.,ittwup,.'ate,.r'd,,, during chloropla~,t as~,cmbly and senescence. Phylt~:hcm.. 31 ( 1992 ) 146f,- 1471). 11401 A. Tanaka. Y. Tanaka, H. Tsuji, Calcium-induced accumulathm , f light-harvcsti~.,, chlorophyll a/b protein complex, in N. Mvram ted. ). Research in Photo~,ynthesis. Kluv.cr, Dordrecht, 1092. Vol. III. pp. 2t~.I-302. 1141 I H. Adamson. G. Huttle~. A. Wilson. unpubli~&cd. 199f,. 11421 R.R. Rando. Mcdtani~,mt~firrc'.cr~.ihlc inhihition ofaminohut)rie acid ketoglutaric acid transalnhlase by the neuretoxin .~ahac~ilme. Bit~.:hem.. I e, { 1977 ) 46(~ 1-4610. 11431 J. Walmslcy. H. Adatn~on. (.'han~cs in the ¢hloroph)ll-prot¢in complexes of greening harley seedlin[2,, lranM~.'rred to d:.rknc~,,., in N. Murala (cd.), Re,,carch m Photo~,.',nthe~,i~,.Kluv, cr. I)ordrecht, 1992. Vol. II1. pp. 79-82. 11441 V.P. Shedbalkar, I M . hmnide~,. ('.A. Rehei.'. ('hloroplast hi.gen-

221

c~.is. I)cleclion el tmmtnin,,I prol+u,:hloroph)lll idol h in plant+. J. Biol. Chem.. 2f~ I ILF.I'I ) 17 151-17 If,?. 11451 S Scht~:h. M. Heltrich. B. Wiktor,,,on. ('. Sundq,.i+l. W. Rudi~cr, ."d. Rybcr,_,. Photoreduction of/inc pheoph~rhide b with N AI)PI'tproh~hlor=q~hyllid¢ rcducta~.c |torn ctiolatcd '~.h,.:zll ( [.+;.!;.~um ae'.;h'mn [.. ). Eur. J. Bitv,:hem.. 229 t lot)51 2tJ[ -2tl."L 1.161 .'i. Dilov a. K. Polalx. Thc inllucncc . f asc orhic acid ox=thc d} namic,. of chlorophyll ',ynthe,,i~, in bade) plant,, m darkne',',. ('.R. Acad. Bulg. Sci.+ 23 t 19701 73f,-73~. 1471 II. Ad;..m,,on. l:,,.idence for a li';ht.indcptmdent Frott~:hlon+phy llide rcducta,,e in green harle} It:axe,,. ill (;. Ako.,.uno;Iou+ A.I-. h a n gchq~mlo~,. J. (;eorgat,,o,,. (;. Pulah~l._'o,., ..\. Trakatclli-. (;. T,,i~ano', qed',.~. Pn~gre~,,, in ('lhucal and Bh~h~gical Re,,carch. ('ell Function and I)ilferenliation. :\lan R. I.i,,,,. Net. York. V o l 102B. I~X2, pp. 33-41. 14~] K-I.. Ou. J. Walmsle,.. !t. ,Adam~,on.."iu,.ceplihilit'. of da,~. chlorophyll ,,~nthe~,i,, m harlc~ and pine ~,ecdlin~,, to inhibition it'. gabaculme, in J.|l. Ar~3roudL.-\k:.~uno!:lou (ed.). Rcgulali~m of ('hloropla,,t Bi.~2¢ne,,i,,. Plenum Pre,.'.. Nc',s York, 1~..12.pp. 25325,";. 14OI H. Adam~,on. R.(;. tlill"er, t hloroph)ll ,,)nthesi-. m tile dark in arn~io',lx'rnP,, in (;. Akoyunoghm. * ¢d. ). Photo,,.-,nthc,,i,, V. C'hloropla~,t l)e~elopn~en~. Balahan Intcrnatitmal Science Scr',icc~., Philadelphia. P:V 1981. pp. 213-221. 1501 N. Packer. tl. Adam~,on. J. (irene,, ('hloropla',l dc,,eh~pment and as,,*~.iated chan~c', in pr~tt~:hh~roph} Ih ide ) and ch[on~ph} II Ic'.cb, m ~eugra~,~,e~, tran,,tcrred to darkne,,,,, m (.'..~)hesma (ed.}. ..\d',ance,. in Phom~,ynthe,,b, Research..Martmu~, Nijhoff/[h" W. Junk. The Flague, 19,~4. Vol. IV. No. h, pp. 74f,-74~. 1511 H. Adamson. IL Packer. Chh~roph,.ll la.~,,:!lin; pattern,, in Zn~tera tran,,lk:rred to darkne',',. Photo,,ynlhctica. 21 t 19,H7) 472.-.4H I. 1521 H :\dam~.tm, N. Parker. B. Sanders. ('hlonlphyll ,,:.nth¢~.is in th,z dark ill Ix:as. in C. S3besnta led. ). Ad',ances in Photo~,,.ntl~.i,. Research, Mmlinus Nilholf/Dr W. Junk. The Hague. 1984. V e t L pp. 705-708. I.¢.31 P..Maitra. S. Mukherji. Dark con~.er,,ion ot pretty:hi, rophyl]ide to ehloroph}llidc lollov.in~ brief illuminatitm and temperature dependence el photo-reduced carotenoid ,,', rtthc~,i~, in rice ( Ory=a ~.tt~. ~ ,eedlin~.. Bk~hem. Phy~,iol. Pills,,'.. 178 t i983 ) 21)7-21 I. 154] R. (h~d'.vin. O.V.H. O',a.en,,. The fiwmation of chloruphyll a in etiolated oat ~¢edlin~s. Plane Ph3,.iol.. 22 t It~J,7) lt}7-2(X).