Download - Genetic Analysis of Chlorophyll Biosynthesis

P1: ARS

October 9, 1997 15:24 Annual Reviews AR044-03

Annu. Rev. Genet. 1997. 31:61–89Copyright c© 1997 by Annual Reviews Inc. All rights reserved

GENETIC ANALYSIS OFCHLOROPHYLL BIOSYNTHESIS

Jon Y. SuzukiCenter for Gene Research, Nagoya University, Nagoya 464-01, Japan

David W. BollivarDepartment of Biology, Illinois Wesleyan University, Bloomington, Illinois 61702

Carl E. BauerDepartment of Biology, Indiana University, Bloomington, Indiana 47405

KEY WORDS: Mg-tetrapyrrole, chlorophyll, bacteriochlorophyll, photosynthesis

ABSTRACT

During this decade, there have been major advancements in the understandingof genetic loci involved in synthesis of the family of Mg-tetrapyrroles known aschlorophylls and bacteriochlorophylls. Molecular genetic analysis of Mg-tetra-pyrrole biosynthesis was initiated by the performance of detailed sequence andmutational analysis of the photosynthesis gene cluster fromRhodobacter capsu-latus. These studies provided the first detailed understanding of genes involved inbacteriochlorophylla biosynthesis. In the short time since these studies were ini-tiated, most of the chlorophyll biosynthesis genes have been identified by virtueof their ability to complement bacteriochlorophylla biosynthesis mutants as wellas by sequence homology comparisons. This review is centered on a discus-sion of our current understanding of bacterial, algal, and plant genes that codefor enzymes in the Mg-branch of the tetrapyrrole biosynthetic pathway that areresponsible for synthesis of chlorophylls and bacteriochlorophylls.

CONTENTS

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

GENETIC DISSECTION OF THE BIOSYNTHETIC PATHWAY. . . . . . . . . . . . . . . . . . . . . 62Bacterial Genetics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63Algal Genetics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66Plant Genetics. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

610066-4197/97/1215-0061$08.00

Ann

u. R

ev. G

enet

. 199

7.31

:61-

89. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

Acc

ess

prov

ided

by

AL

I: A

cade

mic

Lib

rari

es o

f In

dian

a on

08/

18/1

5. F

or p

erso

nal u

se o

nly.

P1: ARS


62 SUZUKI, BOLLIVAR & BAUER

COMMON STEPS IN Mg-TETRAPYRROLE PATHWAYS. . . . . . . . . . . . . . . . . . . . . . . . . . 68Mg-Chelation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68Methyl Transferase. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72Isocyclic Ring Formation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73Vinyl Reductase(s). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73Protochlorophyllide Reduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74Phytol Addition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

DIVERGENT BRANCHES OF THE PATHWAY. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80Bacteriochlorophyll a Specific Steps. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80Other Bacteriochlorophylls. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81Chlorophylls b, c, and d. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81

CONCLUDING REMARKS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82

INTRODUCTION

Photosynthetic organisms ranging from eubacteria to plants synthesize a vari-ety of Mg-tetrapyrroles used for light harvesting and energy-generating chargeseparation. Cyanobacteria and chloroplasts of algae and plants, which evolveoxygen as a byproduct of photosynthesis, typically synthesize chlorophylla orb (8). Purple and green photosynthetic bacteria, which do not evolve oxygen,synthesize a variety of related tetrapyrroles, termed bacteriochlorophylls (103).All of these Mg-tetrapyrroles contain a similar five-membered ring structure,with variations in side chains and/or hydration states of the ring structure. Al-terations in the ring structure allow photosynthetic organisms to harvest light atdifferent wavelengths, depending on the type of chlorophylls that are synthe-sized.

The two types of Mg-tetrapyrroles discussed in detail in this review are bac-teriochlorophylla and chlorophylla for which thorough genetic understandingof their biosynthetic pathways exists (Figure 1). As shown in Figure 1, bothpathways utilize common intermediates from Mg-protoporphyrin IX throughchlorophyllidea. Indeed, most of the various bacteriochlorophylls and chloro-phylls that are synthesized by photosynthetic organisms appear to utilize sim-ilar early metabolic intermediates, which suggests that the various endprod-ucts arose as variants of an evolutionarily conserved biosynthetic pathway(6, 8, 103). Common ancestry of these pathways has been convincingly demon-strated by the presence of extensive sequence homology among enzymes thatcatalyze similar steps in both chlorophylla and bacteriochlorophylla biosyn-thetic pathways; details of these are discussed below.

GENETIC DISSECTION OF THE BIOSYNTHETICPATHWAY

The first genetic analyses of the Mg-tetrapyrrole branch were studies by Granickin 1948 on chlorophylla biosynthesis in the green algaChlorella(41–45). This

Ann

u. R

ev. G

enet

. 199

7.31

:61-

89. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

Acc

ess

prov

ided

by

AL

I: A

cade

mic

Lib

rari

es o

f In

dian

a on

08/

18/1

5. F

or p

erso

nal u

se o

nly.

P1: ARS


CHLOROPHYLL BIOSYNTHESIS 63

initial work was followed in the early 1960s by genetic and biochemical analysesof bacteriochlorophylla biosynthesis with the purple bacteriumRhodobactersphaeroides(see Reference 55 for a review of these initial studies). These earlystudies demonstrated that the bacteriochlorophyllaand chlorophyllapathwaysutilized a common set of intermediates from Mg-protoporphyrin IX throughchlorophyllidea. These studies were followed by the cloning, sequencing anddirected mutational analyses of genes for the bacteriochlorophylla biosyn-thetic pathway in the bacteriumRhodobacter capsulatus. TheRb. capsulatusstudies have significantly advanced our understanding of bacteriochlorophyllabiosynthesis genes, which in many cases exhibit an ancestral relationship withchlorophylla biosynthesis genes that catalyze similar reactions. The sectionsbelow discuss what is known about individual steps of the Mg-tetrapyrrolepathway in bacterial, algal, and plant systems that have been studied.

Bacterial GeneticsMolecular genetic analysis of the Mg-branch of the tetrapyrrole biosyntheticpathway was initiated by Marrs and coworkers with the bacteriumRb. capsu-latus (73). A combination of generalized transduction (124), R′ mobilization(73), plasmid-based complementation/marker rescue (91), and transposon mu-tagenesis (10, 128) techniques demonstrated that all of the known loci essentialfor bacteriochlorophylla biosynthesis were tightly linked to a 45-kb region ofthe chromosome termed the “photosynthesis gene cluster.” Sequence analysisof the entire photosynthesis gene cluster (2, 3, 7, 12, 16, 120, 127), coupled withthe construction of defined sets of insertion mutations within each of the openreading frames (14, 15, 123, 125), provided the first comprehensive molecularunderstanding of genes involved in specific steps in the biosynthetic pathway(Figure 1).

Genetic analysis of the chlorophylla biosynthetic pathway in cyanobacte-ria was advanced first by cloningnifH-like genes fromPlectonema boryanum,Synechococcussp. PCC 7002, andSynechocystissp. PCC 6803 (18, 33, 35, 81).Subsequent mutational analysis indicated that thenif-like gene was actuallyinvolved in protochlorophyllide reduction (34). In a functional approach, sev-eral chlorophyll biosynthesis genes fromSynechocystis6803 were obtainedby complementation of bacteriochlorophyll biosynthesis mutants ofRb. cap-sulatus(102, 109). A major advance in this field was obtained by completesequencing of theSynechocystis6803 genome from which numerous chloro-phyll biosynthesis genes have been identified based on sequence homology toRb. capsulatusbacteriochlorophyll biosynthesis genes (56, 57). Because of thehigh degree of sequence identity that exists between the cyanobacterial andplant homologs, it should be possible to obtain many plant genes by directhybridization using cyanobacterial chlorophyll (chl) genes as probes (18, 108).

Ann

u. R

ev. G

enet

. 199

7.31

:61-

89. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

Acc

ess

prov

ided

by

AL

I: A

cade

mic

Lib

rari

es o

f In

dian

a on

08/

18/1

5. F

or p

erso

nal u

se o

nly.

P1: ARS



N

CH 2

CH 2

CH 2

CH 2

COOH

3C CH 3

Mg-protoporphyrin

H

N N

N

CH

CH 2

CH 3

CH CH 2H 3C

Mg

COOH

bchE

bchJbchHDI Mg

NH N

N HN

CH

CH 2

CH 3

CH CH 2

CH 3

CH 2

CH 2

COOH

CH 2

CH 2

COOH

H 3C

H 3C

Protoporphyrin IX

chlE

chlHDI

N N

N N

CH

CH 2

CH 3

CH CH 2

CH 3

CH 2

CH 2

COOCH3

CH 2

CH 2

COOH

Mg

Mg-Protoporphyrinmonomethyl ester

H 3C

H 3C

N

COOH

N N

CH

CH 2

CH 3

CH CH 2

CH 3

C

COOCH 3

CH 2

CH 2

H 3C

Mg

HC O

DV-Protochlorophyllide

NH 3C

COOH

CH 3

N N

N N

CH

CH 2

CH

CH CH3

C

COOCH 3

CH 2

CH 2

H 3C

H 3C

Mg

HC O

MV-Protochlorophyllide

2

3

bchMchlM

Figure 1 (Continued)

Ann

u. R

ev. G

enet

. 199

7.31

:61-

89. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

Acc

ess

prov

ided

by

AL

I: A

cade

mic

Lib

rari

es o

f In

dian

a on

08/

18/1

5. F

or p

erso

nal u

se o

nly.

P1: ARS



bchBLNbchWXYZ

bchF

bchC

bchG

PP-Phytyl chlG

chlBLNpor

CH

CH 2

–CH– –CH

N N

N NCH

3

C

COOCH3

CH2

CH2

C–O–CH2–CH=C–(CH2)3 (CH2)3 (CH2)3

H3

C

Mg

HCO

CH3 CH3 CH3 CH3

CH3

–CH–

CH

H3CH

CH

H

Chlorophyll a

2 3

CH3

–CH

N N

N NCH

3

C

COOCH3

CH2

CH2

C–O–CH2–CH=C–(CH2)3–CH–(CH2)3–CH–(CH2)3

H3

C

Mg

HCO

CH3 CH3 3 CH3

CH3

CH

H3CH

H

Bacteriochlorophyll a

C

CH

O

3

CH CH

H

H

H3

C

32

N N

N N

CH 3

CH CH

CH 3

C

COOCH 3

CH 2

CH 2

COOH

H 3C

H 3C

Mg

HCO

H

H

Chlorophyllide a

32

CH

CH 2

PP-Phytyl

Figure 1 Mg-branch of the bacteriochlorophylla and chlorophylla biosynthetic pathways. Bothpathways share common intermediates up to the synthesis of chlorophyllide, at which point thetetrapyrrole ring in the bacteriochlorophylla pathway undergoes additional modification. Modifi-cations of the tetrapyrrole ring at various stages of the pathway are highlighted with a black box.Genetic loci that affect individual steps of the pathway are also indicated above the arrows.

Ann

u. R

ev. G

enet

. 199

7.31

:61-

89. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

Acc

ess

prov

ided

by

AL

I: A

cade

mic

Lib

rari

es o

f In

dian

a on

08/

18/1

5. F

or p

erso

nal u

se o

nly.

P1: ARS



Alternatively, polymerase chain reaction can be used to amplify plant genesby designing primers that hybridize to regions that are conserved amongRb.capsulatusandSynechocystis6803 homologs (18).

Algal GeneticsGenetic analysis of the chlorophyllabiosynthetic pathway in algae was initiatedin the mid-1940s by Granick, who isolated chlorophyll-deficient strains ofChlorella (41–45). These studies were complemented by the characterizationof additionalChlorella mutants by Ellsworth & Arnoff in the late 1960s (20–23). Work with the green alga,Chlamydomonas reinhardtii,began in 1955(95), and it has become the model organism for genetic analysis of chlorophylla biosynthesis in algae.

One of the first detailed studies of a chlorophyll biosynthesis mutant ofC. reinhardtiiwas performed by Ruth Sager, who used the mutanty-1 to helpdefine the nuclear inheritance patterns of this organism (95, 96).y-1 mutantsexhibit a “yellow in the dark” phenotype as a result of dark accumulationof protochlorophyllide, although these cells are still capable of synthesizingchlorophyll in the light. Despite many attempts over the years, no additionalloci with a similar phenotype were identified, until Ford & Wang described aseries of temperature-sensitive mutants that were generated by UV mutagenesis(25–27). Their work led to the identification of six more nuclear loci withsimilar “yellow in the dark” phenotypes, as described fory-1. A set of brownmutants (br), also isolated by Wang et al (117), accumulate protoporphyrin IXthat presumably contains a defect in Mg-chelatase.

The development of mutagenesis techniques that allow the creation of nuclearinsertion mutations inC. reinhardtii should facilitate the cloning of some ofthese nuclear-encodedyandbr loci in the future. The utility of such an approachis highlighted by the cloning of nuclear genes fromC. reinhardtii that areinvolved in flagellar biosynthesis (111).

Plant GeneticsMutants characterized for chlorophyll deficiency have been isolated from bar-ley, wheat, rice, maize, and Arabidopsis. Mutant classes described in theseplants includealbina, xan, chlorina, vir, andchlb. albinamutants lack de-tectable tetrapyrrole intermediates, whereasxan, chlorina, andvir are defectiveat biosynthetic steps from magnesium insertion to the synthesis of protochloro-phyllide. chlorina mutants, which are defective in Mg-chelation, are usuallyrecessive lethal.vir mutants, which are a phenotype that describes variouspigment types, typically appear conditionally such as under low temperatures.

Pigment-deficient seedling lethal mutants of grasses attain a relatively ad-vanced level of growth morphologically, even in the absence of light. This

Ann

u. R

ev. G

enet

. 199

7.31

:61-

89. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

Acc

ess

prov

ided

by

AL

I: A

cade

mic

Lib

rari

es o

f In

dian

a on

08/

18/1

5. F

or p

erso

nal u

se o

nly.

P1: ARS



property allows for easy visual identification of mutants as well as providing forsufficient plant material for biochemical analysis. To date, systematic screensfor chlorophyll biosynthesis mutants of barley have yielded five loci thought toencode structural genes for chlorophyll biosynthesis at two biosynthetic steps.The loci xan-f, xan-h,and xan-g are mutants in subunits of the enzyme forprotoporphyrin IX Mg-chelatase, whereasxan-l and vir-k are thought to becomponents of the cyclase reaction (discussed in detail below).

Runge et al (93) recently attempted to isolate mutants in structural genes forchlorophyll biosynthesis on a systematic basis in Arabidopsis. Three classesof mutants were screened for: (a) mutants for steps prior to protoporphyrin IX,(b) mutants for steps between magnesium chelation and protochlorophyllidereduction, and (c), mutants at steps following protochlorophyllide reduction.In their screen for mutants, the authors utilized the fact that loss of chlorophyllis often a consequence of chloroplast dysfunction correlating with nutritionaldeficiencies rather than the cause of chloroplast dysfunction. A fraction ofmutants initially isolated as chlorophyll deficient could be eliminated based ontheir ability to produce chlorophyll when sucrose was added to the medium.As observed in other systems, no mutants in the first class were isolated.

A study by Kruse et al (63) addressed why this and other studies in barleyfailed to result in mutants at biosynthetic steps prior to protoporphyrin IX. Intheir analysis, they assayed the effect of reducing expression of enzymes early inthe pathway using antisense inhibition. Using an antisense coproporphyrinogenoxidase gene behind the strong constitutive cauliflower mosaic virus promoterin tobacco, the authors found growth-inhibition phenotypes similar to thosein plants treated with the diphenyl ether–based herbicides, which disrupt thesame enzyme. Thus depression of the activities of certain enzymes can resultin the accumulation of photoactive tetrapyrrole intermediates that cause pho-tooxidative damage. Similar experiments using a glutamate-1-semialdehydeaminotransferase antisense gene (an enzyme involved in synthesis of ALA)yielded plants with varying degrees of pigment loss, which coincided with theeffectiveness of the antisense gene at inhibiting expression (48). Completelyinviable seeds and white plantlets, which could not be kept alive, were some-times obtained and presumably represent clones containing complete repressionof enzyme synthesis. Based on these results, it appears that mutants that ac-cumulate intermediates prior to protoporphyrin IX have pleiotropic effects ongermination and growth that may explain why this class of mutants is difficultto obtain. Pleiotropic effects, such as lethality caused by defects at enzymaticsteps prior to protoporphyrin IX formation, would also be expected if heme andchlorophyll are indeed derived from the same pool of intermediates.

The tagging of genes by random insertion ofAgrobacteriumT-DNA, orwith transposable mobile elements, has proven useful in isolating at least two

Ann

u. R

ev. G

enet

. 199

7.31

:61-

89. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

Acc

ess

prov

ided

by

AL

I: A

cade

mic

Lib

rari

es o

f In

dian

a on

08/

18/1

5. F

or p

erso

nal u

se o

nly.

P1: ARS



chlorophyll genes in plants (50, 61). Coincidentally, they are both mutantsthat disrupt the Mg-chelatase step. T-DNA can be utilized in any plant thatAgrobacteriumcan infect (62). Pigment mutants are found among T-DNAtransformed lines of Arabidopsis that are maintained at the Ohio BiologicalResource Center, USA, but most have not been characterized as to their primarydefect. The use of transposon mutagenesis has the advantage that the mobileelements can be found in a wide variety of model plants. For example, theAcandDs transposons of maize are mobile in a number of other plants such asNicotiana tabacco, Lycopersicon esculentum(tomato), and Arabidopsis (40),which widens the use of this mutagenesis technique. On the other hand, manymutants involved in chloroplast development and biogenesis have defects inchlorophyll synthesis, which makes it difficult to determine the primary lesion.

Random sequence analysis of expressed sequence tagged (EST) cDNAsfrom dicots Arabidopsis,Brassica campestris,and Castor bean; from monocotsmaize and rice; and the coniferPinus taedahas recently yielded putative chloro-phyll biosynthesis homologs of genetically characterized bacteriochlorophyllgenes. Isolates ofchlG(chlorophyll synthetase) andchlPfrom Arabidopsis andrice have been detected (69, 100). As shown in Table 1, four of the seven locilisted yielded plant homologs. The various cDNA sequencing projects are thususeful clone pools for identifying chlorophyll biosynthesis genes from plants.As the plant genome sequencing databases become more complete, most if notall of the higher plant chlorophyll biosynthesis loci will likely be identified byhomology searches.

COMMON STEPS IN Mg-TETRAPYRROLE PATHWAYS

Mg-ChelationMutational analysis of theRb. capsulatusphotosynthesis gene cluster initiallyrevealed that three sequenced genes,bchH, bchD, andbchI, are involved inMg-chelation (12, 14, 123, 128). Identification of BchH as the probable mag-nesium binding subunit of the Mg-chelatase was reasoned based on homology tothe divalent cation binding subunit of the cobalto-chelatase enzyme fromPseu-domonas denitrificans(50). Subsequent expression of theRb. sphaeroidesandSynechocystis6803 homologs inEscherichia colihas definitively proven therequirement for all three subunits for catalytic activity (39, 53). The reactionis shown in vitro to involve the formation of an ATP-binding BchI homodimerthat interacts with the 550-kDa BchD aggregate. Mg insertion appears to occuron the 140-kDa BchH subunit that binds protoporphyrin IX on a 1:1 molar ratio.ATP activation is consistent with studies by Walker & Weinstein (118).

Brown mutations ofC. reinhardtii,described by Wang et al (117), accumu-late elevated protoporphyrin IX, which suggests that they affect Mg-chelatase.

Ann

u. R

ev. G

enet

. 199

7.31

:61-

89. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

Acc

ess

prov

ided

by

AL

I: A

cade

mic

Lib

rari

es o

f In

dian

a on

08/

18/1

5. F

or p

erso

nal u

se o

nly.

P1: ARS



These authors isolated mutants representing two different loci: Strains bear-ing mutations in thebrc locus were able to make chlorophyll in the light, butnot in the dark, whereas strains having mutations in thebrs, locus were unableto synthesize chlorophyll regardless of the light conditions. The mutanty-yhas been described that accumulates protoporphyrin IX at much lower levelsthan are observed with thebr mutants (78, 79). The reason for this differenceas well as the relationship between these genetic loci and theRhodobactersubunits remain unclear. Sequence analysis of chloroplasts from a number ofalga (Porphyra purpurea, Cyanophora paradoxa, Olisthodiscus luteus, Cryp-tomonas phi, Odontella sinensis; see Table 1) has revealed the presence ofbchIhomologs (chlI ). Homologs ofbchI in plant chloroplasts are, however, absent.Homologs ofbchHandbchDare also absent in sequenced chloroplasts.

Plant homologs of theRhodobacterMg-chelatase complex have been iden-tified in several species (Table 1). T-DNA tagging resulted in the disruptionof a homolog ofbchI (ch-42) in Arabidopsis (61), whereas Tam transpositionmutagenesis resulted in the disruption of abchHhomolog (oli) of Antirrhinummajus(50). So far,oli andch-42are the only examples of tagged genes thathave been identified as being involved in chlorophyll biosynthesis.xanmutantsof barley, which are defective in Mg-chelation, were evaluated to ascertain thebasis of their mutant phenotype using antibody probes to CH42 of Arabidopsisand OLI ofA. majus(54). Some, but not all,xan-f alleles showed a loss of OLIantibody crossreactivity, indicating a defect in synthesis of a BchH homolog(Xan-H or ChlH), whereasxan-hmutants appeared defective in synthesis ofa BchI homolog (Xan-I or ChlI) (54). Loss of accumulation of ChlI did notnecessarily affect the accumulation of ChlH and vice versa. Accumulation ofboth ChlI and ChlH could be observed inxan-gmutants purported to be de-fective in a homolog of BchD (ChlD) (54, 58). These studies showed that thedifferent mutants in Mg-chelation did not necessarily prevent the accumulationof other subunit proteins involved in the same step. Although thexanmutantshad defects in Mg-chelation, all could perform the subsequent enzymatic stepof methylation in vitro when Mg-protophorphyrin IX was included in the reac-tion mix, indicating that loss of chelatase activity did not abolish activity of alater step.

xan-h and xan-f were cloned through the use of degenerate PCR primersthat were designed to hybridize to conserved regions of known sequences (54).Templates for the PCR amplification primers were single-strand cDNA fromgreening barley leaves. The deduced XAN-H (ChlI) protein sequence of barleywas found to be 85% identical to Ch42 of Arabidopsis, 49–69% identical tothe algal andEuglenasequences, and 49% identical to BchI ofRb. capsulatus.The XAN-F protein (ChlH) was found to be 82% identical to theA. majusOLI,66% to ChlH ofSynechocystis6803, and 34% to BchH ofRb. capsulatus.

Ann

u. R

ev. G

enet

. 199

7.31

:61-

89. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

Acc

ess

prov

ided

by

AL

I: A

cade

mic

Lib

rari

es o

f In

dian

a on

08/

18/1

5. F

or p

erso

nal u

se o

nly.

P1: ARS



Table 1 Genbank submitted genes common to the bacteriochlorphylla and chlorophylla biosynthetic pathways

Enzyme gene Organism Alternate name Accession no.

Mg-ChelatasebchH/chlH Rhodobacter capsulatus Z11165, M74001

Synechocystis D90902, X96599Antirrhinum majus oli(olive) X73144Arabidopsis thaliana chlH D68495, Z68495Hordeum vulgare xan-f U26916Arabidopsis thaliana H76104Oryza sativa D47916

bchI/chlI Rhodobacter capsulatus Z11165Synechocystis D90904Anabaena variabilis D49426Porphyra purpurea U38804Cyanophora paradoxa U30821Olisthodiscus luteus Z21959Cryptomonas phi Z21976Odontella sinensis Z67753Arabidopsis thaliana ch-42(chlorata) Z11165Glycine max D45857Hordeum vulgare xan-h U26545Arabidopsis thaliana AA042226Zea maize W49410

bchD/chlD Rhodobacter capsulatus Z11165Synechocystis X96599Anabaena variabilis D49426

Mg-Protoporphyrin methyl transferasebchlM/chlM Rhodobacter capsulatus Z11165

Synechocystis D64006, L47125

CyclasebchlE/chlE Rhodobacter capsulatus Z11165

Rhodobacter sphaeroides L37197Synechocystis D64003, L47125

Protochlorophyllide reductase (light-dependent)por Synechocystis L37783, D64004

Chlamydomonas reinhardtii U36752Hordeum vulgare porA, porB X15869, 84738Avena sativa X17067Oryza sativa D46584Zea maize T27547, W49454Arabidopsis thaliana porA, porB U29699,U29785,

AA042730Pisum sativum pcr X63060Cucumis sativa npr S78381Pinus strobus lpcrPinus taeda lpcr X66727, H75261Pinus mugo porA?,porB? S63824, S63825

(Continued)

Ann

u. R

ev. G

enet

. 199

7.31

:61-

89. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

Acc

ess

prov

ided

by

AL

I: A

cade

mic

Lib

rari

es o

f In

dian

a on

08/

18/1

5. F

or p

erso

nal u

se o

nly.

P1: ARS



Table 1 (Continued)

Enzyme gene Organism Alternate name Accession no.

Protochlorophyllide reductase (light-independent)bchL /chlL Rhodobacter capsulatus Z11165

Synechocystis D90916Synechococcus X67694Plectonema boryanum D00665Chlamydomonas reinhardtii frxc X60490Porphyra purpurea U38804Marchantia polymorpha frxC X04465Pinus thunbergii chlL D17510Pinus contorta frxC X56200

bchB/chlB Rhodobacter capsulatus Z11165Synechocystis U36144, D64000Plectonema boryanum D78208Chlamydomonas reinhardtii U02526Porphyra purpurea U38804Marchantia polymorpha ORF513 X04465Ginkgo biloba U01531, U04440Pinus thunbergii D17510Pinus strobus U02533Larix eurolepis X98680, X98681Pinus sylvestris X98683, X98684Picea abies X98686, X98687

bchN/chlN Rhodobacter capsulatus Z11165Synechocystis D90916Synechococcus X67694Plectonema boryanum D12973Chlamydomonas reinhardtiiPorphyra purpurea U38804Marchantia polymorpha ORF465 X04465Pinus thunbergii D17510Pinus contorta gidA X56200

8-vinyl reductase activitybchlJ Rhodobacter capsulatus Z11165

Chlorophyll synthetasebchG/chlG Rhodobacter capsulatus Z11165

Chloroflexus aurantiacus U43963Synechocystis U36144Arabidopsis thaliana g4 U19382, Z34566Oryza sativa D48639

Phytol synthesis (from HMG-CoA)bchP/chlP Rhodobacter capsulatus Z11165

Synechocystis X97972Arabidopsis thaliana Gghyl T13808Oryza sativa Gghyl D47484

Ann

u. R

ev. G

enet

. 199

7.31

:61-

89. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

Acc

ess

prov

ided

by

AL

I: A

cade

mic

Lib

rari

es o

f In

dian

a on

08/

18/1

5. F

or p

erso

nal u

se o

nly.

P1: ARS



Cloning of thebchDhomolog thought to correspond toxan-ghas not yet beenreported.

Probes toxan-fandxan-hwere used to monitor expression patterns duringgreening of etiolated seedlings (54). Results indicated that the steady statelevel of xan-hmRNA was tenfold lower than that observed forxan-fmRNA.During greening, bothxan-handxan-f mRNA levels increased to maximumlevels, withxan-f increasing 2–3-fold andxan-f increasing 18–20-fold at 4 h.Mg-chelatase activity showed a 3- to 4-fold induction during this period, witha peak at 6 h and a drop to dark levels at 24 h. These levels were maintainedup to 20 h in continuous light forxan-f, but declined at 8 h and to the darklevel at 24 h. xan-f also showed a diurnal pattern of induction with plantsgrown on a 8:00–24:00-h light period.xan-fexpression peaked at 11:00, withlight-harvesting chlorophyll binding gene expression (lhcb2) exhibiting a peakat 13:00. Subsequent growth of plants in continuous light showed that bothxan-fandlhcb2maintained a circadian rhythm in transcript accumulation thatwas not reflected by changes in chelatase activity.

A cDNA coding for a homolog ofbchH(CHL H) has also been cloned andsequenced from Arabidopsis (38). When grown in a light /dark cycleCHL Htranscripts exhibited a diurnal variation with maximal levels at the end of thedark cycle, increasing slightly during the onset of light and then decreasing tominimal levels until the end of the light period. In situ hybridization experimentsreveal that cytosolic messages forCHL H andch-42(a homolog ofbchI) arefound associated with the surface of the chloroplast (38, 72).

The availability of thechlI gene from Arabidopsis has led to the isolationof a plant homolog from soybean by cross species hybridization with the Ara-bidopsis gene (77). Among the structural features conserved in the plant genesis an ATP binding motif, which is consistent with the requirement of ATP forin vitro activity. In cell cultures of soybean, increased transcript accumula-tion is observed in response to light stimulation similar to the observation inArabidopsis and barley (77).

Methyl TransferaseAfter some initial confusion about the nature of the intermediate accumulated bybchMmutants ofRb. capsulatus, it was demonstrated that this locus most likelycodes for the enzymeS-adenosyl-methionine:Mg-protoporphyrin IX methyltransferase, which is responsible for methylating a propionate side chain onring 3 in protoporphyrin IX (14). The BchM polypeptide was unequivocallyshown to catalyze this reaction by demonstrating thatE. coliextracts containingheterologous expressed BchM could undertake this reaction in vitro (13, 37).

A homolog of BchM (ChlM) was cloned fromSynechocystis6803 by func-tional complementation of abchMmutant ofRb. capsulatuswith aSynechocys-tis expression library (102) (Table 1). Sequence analysis indicates that the

Ann

u. R

ev. G

enet

. 199

7.31

:61-

89. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

Acc

ess

prov

ided

by

AL

I: A

cade

mic

Lib

rari

es o

f In

dian

a on

08/

18/1

5. F

or p

erso

nal u

se o

nly.

P1: ARS



SynechocystisChlM polypeptide exhibits 29% sequence identity with that ofRb. capsulatusBchM. As yet, no plant gene for this step has been isolated.

Isocyclic Ring FormationA fifth ring of the tetrapyrrole is synthesized by a complex multistep processinvolving the 6-methyl propionate group (the biochemistry of this reaction is re-viewed in Reference 8). To date, only thebchEgene ofRb. capsulatushas beengenetically demonstrated to be involved in this reaction (14, 128). TheSyne-chocystis6803 genome sequencing project has demonstrated that this speciescontains a homolog ofbchE (56) (Table 1). The existence of an identifiablechlorophyll biosynthesis homolog ofbchE is notable, given that the cycliza-tion reaction is fundamentally different in these two pathways. Specifically,the addition of a hydroxyl during the closure reaction in chlorophyll biosyn-thesis involves a mixed function oxidase reaction that utilizes dioxygen (O2)as a substrate (119). In contrast, the hydroxyl group in the anaerobic reactionthat occurs during bacteriochlorophyll biosynthesis is obtained from H2O (84).This would indicate either that two BchE homologs have evolved to use separatesubstrates or that there are unrelated polypeptide components of this reactionthat are yet to be identified.

In barley, thexan-l and vir-k mutants may contain defects in the cyclaseactivity since these mutants exhibit both Mg-chelatase and methyltransferaseactivity but do not produce protochlorophyllide. It is not known whether theseloci code for structural components of this enzyme or for regulatory compo-nents. There have been no reports of the cloning and sequence analysis of thesegenes, nor of any other plant or algal genes in this step of the pathway.

Vinyl Reductase(s)Most of the early common intermediates in bacteriochlorophyll and chlorophyllbiosynthesis (from protoporphyrin IX through Mg-protoporphyrin IX) containvinyl groups at the 2 and 4 positions of the ring and are thus appropriatelycalled divinyl intermediates (8). However, the 4-vinyl group is reduced toan ethyl group in both pathways, giving rise to monovinyl chlorophyll andbacteriochlorophyll as the final product. Even though the final product inthe pathway is exclusively monovinyl, reduction of the 4-vinyl group occursincompletely at several locations of the pathway. As a result, there are mixedpools of monovinyl and divinyl intermediates. A number of studies show thatprotochlorophyllide and chlorophyllide are found in varying monovinyl/divinylratios depending on species, environmental condition, or plant age (discussedin Reference 110). The significance of these observations is not known.

A mixed pool of monovinyl and divinyl intermediates indicates either thatthere is a single enzyme with a reduced substrate specificity or that there aremultiple 4-vinyl reductases that are responsible for reducing the 4-vinyl group

Ann

u. R

ev. G

enet

. 199

7.31

:61-

89. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

Acc

ess

prov

ided

by

AL

I: A

cade

mic

Lib

rari

es o

f In

dian

a on

08/

18/1

5. F

or p

erso

nal u

se o

nly.

P1: ARS



Light-Independent Enzyme

Light-Dependent Enzyme

CyanobacteriaAlgae

Non-Flowering Land PlantsAngiosperms

Purple BacteriaHeliobateria

CyanobacteriaAlgae

Non-Flowering Land Plants

N N

N N

CH

CH 2

CH 3

R

CH 3

C

COOCH 3

CH 2

CH 2

COOH

H 3C

Mg

HCO

Protochlorophyllide

3H C

N N

N N

CH

CH 2

CH 3

R

CH 3

C

COOCH 3

CH 2

CH 2

COOH

H 3C

H 3C

Mg

HCO

H

H

Chlorophyllide

Figure 2 Light-dependent versus light-independent protochlorophyllide reduction.

on different intermediates. The existence of a nonenzymatic route or possiblymultiple enzymes is supported by genetic studies ofRb. capsulatusin which itwas observed that disruption ofbchJresulted in the accumulation of a largerpool of divinyl protochlorophyllide (110). However, there still remained a sig-nificant fraction of monovinyl protochlorophyllide inbchJmutants indicatingthat the cells were still capable of 4-vinyl reduction at some level. No homologof bchJ has been observed in any other photosynthetic organism includingSynechocystis. Thus chlorophyll biosynthesis either has an alternative mecha-nism of reducing the 4-vinyl group or a chlorophyll biosynthesis homolog ofbchJhas diverged to a point where no readily identifiable sequence similarityremains.

Protochlorophyllide ReductionThere are two mechanisms for reducing the double bond in the fourth ring ofprotochlorophyllide (Figure 2) (recently reviewed in 31, 85, 86). One enzymecomplex functions irrespective of the presence or absence of light and is thustermed “light-independent protochlorophyllide reductase.” The second is alight-dependent reaction that utilizes the enzyme NADPH-protochlorophyllideoxidoreductase (POR). Until recently, the POR enzyme was thought to beunique to the angiosperm lineage of plants, which require light for chloro-phyll synthesis. However, we now know that angiosperms have simply lostthe light-independent enzyme complexes and that all other photosynthetic or-ganisms ranging from gymnosperms to cyanobacteria, as well as some speciesof anoxygenic photosynthetic bacteria, contain both light-dependent and light-independent protochlorophyllide reductase enzyme complexes. Below is adiscussion of these two enzymes.

Ann

u. R

ev. G

enet

. 199

7.31

:61-

89. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

Acc

ess

prov

ided

by

AL

I: A

cade

mic

Lib

rari

es o

f In

dian

a on

08/

18/1

5. F

or p

erso

nal u

se o

nly.

P1: ARS



THE LIGHT-INDEPENDENT REACTION Our understanding of light-indepen-dent protochlorophyllide reductase stems from genetic studies initiated inRb.capsulatus, where it was demonstrated that three genes,bchL, bchB, andbchN,were involved in protochlorophyllide reduction (7, 105, 123, 128) (Table 1).Subsequent sequence analysis revealed the surprising finding that the three openreading frames exhibit significant sequence similarity to the three subunits ofnitrogenase (32, 33, 35, 108). It has thus been proposed that light-independentprotochlorophyllide reductase and nitrogenase share a common evolutionaryancestor (17, 31, 33, 108). In vitro light-independent activity has, however,been difficult to observe. Thus, biochemical proof that these open readingframes code for catalytic subunits awaits future studies. Based on similari-ties of this enzyme to nitrogenase, which has oxygen-labile metal centers, ithas been proposed that the light-independent enzyme might similarly be sen-sitive to oxygen (71, 108). Purification and assays for activity under anaerobicconditions may be a fruitful approach to this problem.

Algal and plant homologs of light-independent protochlorophyllide reductasewere identified by piecing together clues from sequence data of plant and algalchloroplasts that contained open reading frames with sequence similarity to thebacterial subunits (Table 1). A study withC. reinhardtii initially uncovered alocus that caused the loss of protochlorophyllide reduction in the dark whichwas appropriately termedgidA (green in the dark) (90). In 1992, thegidAlocus (subsequently been renamedchlN) was revealed to be a homolog ofthe bchNgene ofRb. capsulatus(19). Sequence analysis of the chloroplastgenome ofMarchantia polymorpharevealed that the chloroplast contained agene, termedfrxC, with a high degree of sequence identity to thenifH geneof nitrogenase, as well as to thebchLgene present in the photosynthesis genecluster ofRb. capsulatus(60). Clues to the function of this open reading framecame from work by Yang & Bauer (123), who showed that mutations in thebchLgene resulted in the loss of light-independent protochlorophyllide reduction.Suzuki & Bauer (108) confirmed that the chloroplast genefrxC (renamedchlL)was involved in light-independent protochlorophyllide reduction by isolating ahomologous gene fromC. reinhardtiiusing theM. polymorphagene as probe.Subsequent disruption ofchlLby particle-gun mediated transformation resultedin a “yellow in the dark phenotype” caused by the inability of the transformantsto reduce protochlorophyllide in the dark. Similarly, directed mutations inchlN confirmed earlier evidence for the role of this gene in light-independentprotochlorophyllide reduction inC. reinhardtii (19). Li et al (66) and Liu et al(68) discovered and disrupted a third chloroplast-encoded gene inC. reinhardtiithat is homologous tobchBin Rb. capsulatus.

One or more of the protochlorophyllide reductase subunit genes have beendetected in the chloroplast genome of all algae and nonflowering land plants

Ann

u. R

ev. G

enet

. 199

7.31

:61-

89. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

Acc

ess

prov

ided

by

AL

I: A

cade

mic

Lib

rari

es o

f In

dian

a on

08/

18/1

5. F

or p

erso

nal u

se o

nly.

P1: ARS



tested with the exception ofPsilotumandWelwitchia(18, 108). The sequencesare notably absent from the sequenced chloroplasts of Euglena (46), rice (47),tobacco (107), maize (70), and beechdrop (122), which lack light-independentchlorophyll synthesis. Thus, the distribution of these genes appears to correlatewell with the ability of nonflowering plants, algae, and bacteria to synthesizechlorophyll in the dark. More recently,chlBhas been utilized to study the phylo-genetic relationship among nonflowering plants based on structural differencesin thechlB gene from various phyla (11). Interestingly,chlB, chlL,andchlNare unlinked in theC. reinhardtii chloroplast (19, 66, 68, 108), whereaschlNandchlL are linked in all other chloroplast genomes of nonflowering plants(60, 67) and nongreen algae (87) so far sequenced, as well as in the genome ofSynechocystis6803 (81) andPlectonema boryanum(33–35). ThechlBgene isunlinked to the other loci in all cases so far investigated.

Except for the initial studies, expression/function of genes for light-independ-ent protochlorophyllide reduction has not been extensively examined in vivo. Inone study, transcripts forchlBin synchronously grown cells ofChlamydomonasmoewusiiwere observed mainly between the tenth hour of light and fourth hourof darkness when grown on a 12-h light /dark cycle (92), a finding that correlateswith a function for this gene in dark chlorophyll synthesis.

Genetic selection for strains ofC. reinhardtiidefective in light-independentprotochlorophyllide reduction has resulted in the isolation by Sager, Wang, andcoworkers (25–27, 95, 97) of mutations in seven different nuclear loci,y-1, y-5,y-6, y-7, y-8, y-9, andy-10. Most of these loci were represented by temperature-sensitive mutations, indicating that they encode polypeptides. This leaves inquestion the role that the nuclear loci play in the synthesis or assembly of thelight-independent protochlorophyllide enzyme: several reasonable hypothesesbear consideration. The “yellow in the dark” mutants have an unusual chloro-plast morphology (82, 83, 97), suggesting that somey loci may encode genesinvolved in regulation of chloroplast development. Another reasonable hypoth-esis is that these loci are involved in coordinating gene expression between thechloroplast and the nucleus, either as nuclear-encoded chloroplast transcriptionfactors or as translational regulators. Recently, RNA editing has been reportedfor the chlB gene of conifers (59), although it is not known how widespreadthis phenomenon is, or whether other genes such aschlL andchlNare affectedor if editing affects gene function. In the pinePinus thunbergii, editing ofchloroplast genes has the potential of creating start and stop codons (116).

THE LIGHT-DEPENDENT REACTION The biochemistry of the light-dependentprotochlorophyllide reductase enzyme (POR) is interesting in that it is oneof only two enzymatic activities requiring light for catalysis (reviewed in

Ann

u. R

ev. G

enet

. 199

7.31

:61-

89. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

Acc

ess

prov

ided

by

AL

I: A

cade

mic

Lib

rari

es o

f In

dian

a on

08/

18/1

5. F

or p

erso

nal u

se o

nly.

P1: ARS



Reference 8); the other activity is a reaction carried out by photolyase. Thepolypeptide binds both protochlorophyllide and NADPH. Reduction of the dou-ble bond does not occur until protochlorophyllide absorbs light at 628–630 nm.The enzyme reaction then undergoes several intermediate states that can be ob-served by spectral shifts in the tetrapyrrole, ultimately resulting in the releaseof chlorophyllide and NADP.

The first eukaryotic chlorophyll biosynthesis gene successfully cloned wasthat of POR. Isolation of theporgene was achieved by purification of the enzymefrom barley in quantities large enough to allow the production of antibodies forcloning por by immunodetection from cDNA expression libraries (99). Thepor gene has subsequently been isolated and sequenced from a wide varietyof plants such as barley, wheat, oat, pea, Arabidopsis, and pine (see 85 andreferences therein) as well as fromC. reinhardtii (65) andSynechocystis6803(109) (Table 1). Molecular analysis of POR expression has recently beendiscussed in several reviews (31, 85, 114).

It was initially thought that the light-dependent enzyme may have evolvedto fulfill a functional role for light-dependent chlorophyll synthesis in an-giosperms. However, genetic analysis of the light-independent enzyme re-vealed that algal and cyanobacterial cells also contain a gene that codes fora copy of the light-dependent POR enzyme (27, 34, 95–97). The presence ofPOR in cyanobacteria was confirmed by cloning theSynechocystis porgene byfunctional complementation of light-independent protochlorophyllide reduc-tase mutants ofRb. capsulatus(109). Sequence analysis demonstrated that theSynechocystisPOR polypeptide exhibited 53–56% sequence identity with de-duced mature peptides of plant homologs. This clearly demonstrates a direct an-cestral relationship of the bacterial and plant enzymes. Thus, it appears that bothenzymes are of bacterial origin and that their co-presence is a widely dissemi-nated feature among photosynthetic organisms. The exceptions are purple bac-teria, which appear to have retained only the light-independent enzyme, and an-giosperms and Euglena, which have retained only the light-dependent enzyme.

The activity and expression pattern of the light-dependent enzyme have beenstudied in a variety of land plants. Twopor genes with distinct expressionpatterns,porA andporB, have recently been discerned in both the monocotbarley and the dicot Arabidopsis (4, 49). Noticeable differences exist betweenthe predicted protein precursors of PORA and PORB in barley (75% overalla.a. identity; 388 a.a., 395 a.a., respectively), particularly in the putative transitpeptide region (46% a.a. identity) (49) but less so in Arabidopsis (88% overalla.a. identity; 405 a.a., 401 a.a., respectively) (4). In barley, PORA (36 kDa)is abundant in dark-grown tissue and disappears upon exposure of seedlings tolight, whereas the minor form PORB persists in equal abundance throughout

Ann

u. R

ev. G

enet

. 199

7.31

:61-

89. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

Acc

ess

prov

ided

by

AL

I: A

cade

mic

Lib

rari

es o

f In

dian

a on

08/

18/1

5. F

or p

erso

nal u

se o

nly.

P1: ARS



(49). In Arabidopsis, a 36-kDa form of POR (which corresponds to PORB;Reference 94) is both more abundant and persistent in the light compared toa 37-kDa form (PORA). TheporA gene is expressed specifically in etiolatedtissue and its transcript levels decrease after exposure to white light, whereasporB is expressed more or less constitutively irrespective of light conditionsin both types of plants (4). Germination of Arabidopsis seedlings under redlight causes a light-grown phenotype, and the specific depletion of PORA,but not PORB. These seedlings when transferred to white light photoreduceprotochlorophyllide precursors with PORB. However, while light-harvestingcomplexes are formed, there are defects in reaction center formation demon-strating one distinguishable unique function of PORA in germinating seeds inArabidopsis (94).

A transient increase in transcripts corresponding topor genes in response toexposure of dark-grown seedlings to light has been reported inCucumis sativa(cucumber) andCurcurbita maximacv, Houkouaokawaguri (pumpkin), whichare both members of theCurcurbitaceae(64, 126). In pea, no effect of lightwas observed (105). (However, as the studies in barley and Arabidopsis show,analysis of individual members ofporA or porB-like genes, if they exist, is aprerequisite before any conclusion can be drawn from those studies.) At leastin the case of pea, levels of the enzyme decrease upon exposure of etiolatedseedlings to light, indicating that proteolytic mechanisms to control enzymelevels in pea are also present.

In dark greening conifers,Pinus strobus(104) andPinus mugo, there isalso evidence for at least two genes for light-dependent protochlorophyllidereductases (28). The relevant genes inP. mugoexhibit expression propertiessimilar to barleyporAandporB. In addition, proteins of 38 kDa and 36 kDa arerecognized (similar to the two forms found in flowering plants), with the latterbeing most abundant in dark-grown cotyledons, but disappearing upon exposureto light, whereas the larger peptide persists at unchanged levels similar to thesituation observed in barley. Only one light-dependent protochlorophyllidereductase gene is found in the algaC. reinhardtii(65), and its expression patternhas not yet been thoroughly investigated.

Regarding the mechanism of regulation ofpor expression, phytochrome me-diates negative light regulation of expression ofporAin monocots (5, 75). SinceporAandporBgenes are regulated differently, it would be interesting to knowhow their promoter regions differ. However, to date, genomic sequence analysisonpor has been reported only forPinus taedaandPisum sativum(104).

Structural differences in the barley enzymes apparently account for differ-ences in transport properties of POR into the organelle. For translocation intoetioplasts or chloroplasts, the barley PORA protein requires ATP for initial

Ann

u. R

ev. G

enet

. 199

7.31

:61-

89. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

Acc

ess

prov

ided

by

AL

I: A

cade

mic

Lib

rari

es o

f In

dian

a on

08/

18/1

5. F

or p

erso

nal u

se o

nly.

P1: ARS



binding to a protease-sensitive component in addition to its substrate pro-tochlorophyllide; both of these are required at the surface of the organelle(87, 88). Detectable levels of protochlorophyllide are normally associated withthe cotyledons of dicots or the primary leaf blades of grasses of germinatedseedlings in the period prior to greening, coinciding with the high expressionof the gene for PORA. By contrast, the barley PORB protein can be transportedinto organelles without protochlorophyllide, thereby allowing it to function inthe entire period following greening during which protochlorophyllide does notaccumulate to significant levels at the surface of the organelle (87, 88).

PORA and PORB levels are also regulated by a light-activated protease(s)that is nuclear encoded and synthesized in the chloroplast, but not in etioplasts(89). Studies have indicated that the PORA apoprotein, as well as PORA boundto any combination of substrates (NADPH and/or protochlorophyllide), is re-sistant to degradation. In contrast, POR noncovalently bound to chlorophyllidewas sensitive to digestion. Western analysis indicates that the POR proteinlevels show no fluctuations during light/dark cycles during the greening period.However, diurnal fluctuations of the barleyporB gene in plants grown on a 12h light /12 h dark cycle occurred with a maximum at 4 h into the light cycleand at 16 h (4 h into the dark cycle) similar to that of the chlorophyll bindingprotein genelhcb1. This fluctuation in levels is proposed to compensate fordecreased levels of protein that would result from proteolysis. Similar diurnalfluctuations in expression of theporB gene of Arabidopsis are also observed(4). These findings help to explain the long-debated paradox of decreased ex-pression of this gene in a variety of land plants in the light during the periodwhen its function is required (29, 71, 106).

Recently, Wilks & Timko (121) devised a simple method to identify residuescritical for enzyme function of plant POR proteins by heterologous complemen-tation of protochlorophyllide reductase mutants ofRb. capsulatus. By virtueof homology to members of the short-chain alcohol dehydrogenase family, tworesidues, Tyr-275 and Lys-279, of the pea enzyme that are universally con-served among members of this family were mutated to Phe or Cys and Ile orArg, respectively. These mutant forms of the enzyme were found to be defec-tive in light-dependent complementation ofRb. capsulatusprotochlorophyllidereductase mutants.

Phytol AdditionThe phytol tail of chlorophyllaand bacteriochlorophylla is derived from an es-terification reaction that utilizes phytyl diphosphate as a substrate. Mutationalanalysis ofRb. capsulatusinitially indicated thatbchGcoded for the enzymebacteriochlorophyll synthase that catalyzed this reaction (14, 15, 112, 128). A

Ann

u. R

ev. G

enet

. 199

7.31

:61-

89. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

Acc

ess

prov

ided

by

AL

I: A

cade

mic

Lib

rari

es o

f In

dian

a on

08/

18/1

5. F

or p

erso

nal u

se o

nly.

P1: ARS



chlorophyll synthase homolog ofbchG(chlG) has been identified in theSyne-chocystisgenome sequence database (36% sequence identity tobchG) (57, 59),as well as in the EST database from Arabidopsis and rice (36, 69) (Table 1). Re-cently, bacteriochlorophyll synthase fromRb. capsulatusand chlorophyll syn-thase fromSynechocystishave been overexpressed inE. coli. Cell-free extractsdemonstrated enzymatic activity with remarkable substrate specificity (113).Specifically, bacteriochlorophyll synthase would esterify bacteriochlorophyl-lide a but not chlorophyllidea, whereas chlorophyll synthase would esterifychlorophyllideabut not bacteriochlorophyllidea. Both enzymes also exhibiteda marked preference for phytol diphosphate over geranylgeraniol diphosphate.TwobchGhomologs have also been cloned and sequenced from the anoxygenicbacteriumChloroflexus aurantiacus,which synthesizes bacteriochlorophyllaas well as bacteriochlorophyllc (69). It has been proposed that one of thebchGhomologs is involved in synthesis of bacteriochlorophylla, whereas the otherin synthesis of bacteriochlorophyllc.

There is an interesting dependence of chlorophyll biosynthesis on the carote-noid biosynthetic pathway, which is involved in providing intermediates for thesynthesis of phytyl diphosphate. The carotenoid pathway generates severalisoprenoid intermediates such as isopentylpyrophosphate, farnesyl pyrophos-phate, and geranylgeraniol pyrophosphate (100). Phytyl diphosphate is thoughtto be produced from geranylgeranyl pyrophosphate via multiple step reductioncatalyzed by a reductase that is coded by thebchPgene, which was identi-fied by mutational analysis of theRb. capsulatusphotosynthesis gene cluster(15, 113). Recently, a homolog ofbchPfrom Synechocystis6803 was isolatedand demonstrated to complementbchPmutants inRb. sphaeroides(1). Usingthe cyanobacterial amino acid sequence as a query, EST homologs ofbchPhave also been identified in both rice and Arabidopsis. Reduction of the pool ofgeranylgeraniol pyrophosphate in tomato, by overexpressing a gene involved incarotenoid synthesis, results in significant reduction in chlorophyll content aswell as the phytohormone gibberellin (30). This indicates that there is a delicatepartitioning of early isoprenoid intermediates into divergent pathways and thatthis partitioning can be dramatically altered by subtle changes in expression ofan enzyme in one of these pathways.

DIVERGENT BRANCHES OF THE PATHWAY

Bacteriochlorophyll a Specific StepsDetailed interposon mutagenesis of the sequencedRb. capsulatusphotosynthe-sis gene cluster has given a fairly complete understanding of genes unique to thispathway (See Reference 14). Products of thebchCandbchFgenes are thoughtto be involved in conversion of the 2-vinyl group to 2-acetyl. ThebchX, bchY,

Ann

u. R

ev. G

enet

. 199

7.31

:61-

89. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

Acc

ess

prov

ided

by

AL

I: A

cade

mic

Lib

rari

es o

f In

dian

a on

08/

18/1

5. F

or p

erso

nal u

se o

nly.

P1: ARS



andbchZgenes are responsible for reducing ring 2 in a reaction that appears tobe similar to reduction of ring 4 by the light-independent POR (16). Because ofa high degree of sequence similarity between BchX and BchL (34% identity),it has been proposed that BchL and BchX both function as electron donors toBchB-N and BchY-Z catalytic subunits, respectively (17). BchB-N and BchY-Z are presumed to have specificities for different areas of the tetrapyrrole ringthat they reduce (ring 4 versus ring 2). As is the case for BchBNL, biochemicalactivity for BchXYZ polypeptides has not been demonstrated.

Other BacteriochlorophyllsVery little is known about the genes involved in the synthesis of alternative bac-teriochlorophylls such as bacteriochlorophyllsb, c, d, e, andg. Each of thesepathways most likely utilizes common intermediates from Mg-protoporphyrinIX through chlorophyllide (103). The only definitive information is that bacte-riochlorophyllc synthesis appears to utilize abchGgene product that is distinctfrom that utilized in bacteriochlorophylla production (noted above).

Recently, we have cloned numerous bacteriochlorophyll biosynthesis genesfrom the bacteriochlorophyllg synthesizing speciesH. mobilisand the bacte-riochlorophyll c synthesizing organismChlorobium tepidumby complemen-tation of Rb. capsulatusmutants (K Inoue & C Bauer, unpublished results).Preliminary studies indicate that bacteriochlorophyll biosynthesis genes areclustered inH. mobilis, which should facilitate sequence analysis of genesinvolved in this pathway.

There has also been a report of a Zn-containing variant of bacteriochlorophylla (115). Characterization of the Zn-chelatase would be of interest to see if it isstructurally related to Mg-chelatase.

Chlorophylls b, c, and dThe structure of chlorophyllb differs from that of chlorophylla by the substitu-tion of a formyl group for a methyl group in ring 2. The pathway for chlorophyllbsynthesis is thought to occur by direct modification of chlorophylla to chloro-phyll b via a hydroxymethyl intermediate (80, 98).

Enzymatic studies in cucumber and barley have characterized an activityconverting chlorophyllb to chlorophylla via a hydroxymethyl intermediate(51, 52). Conventional thought is that the reaction is unidirectional from chloro-phyll a to b. This new finding provides an elegant scenario on how the chloro-phyll a /b ratios can be altered in response to environmental changes. Theinterconverting enzyme(s) would thus play a major role in the regulation ofphotosynthesis efficiency by altering light-harvesting and reaction center com-plex formation. This interconversion process has been dubbed the “chloro-phyll cycle.” No genetic models are available among the chlorophylla only

Ann

u. R

ev. G

enet

. 199

7.31

:61-

89. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

Acc

ess

prov

ided

by

AL

I: A

cade

mic

Lib

rari

es o

f In

dian

a on

08/

18/1

5. F

or p

erso

nal u

se o

nly.

P1: ARS



cyanophytes, nor have any organisms been reported that only accumulate chloro-phyll b. In light of the possible physiological relevance of activities at thisstep, further characterization and purification of this enzyme should be of highpriority.

Chlorophyllb minus mutants such aschlorina f 2mutants of barley (9) andch1 of Arabidopsis (76) have major defects in the formation of their light-harvesting complexes due to the instability of their chlorophylla /b bindingproteins (Lhcb) in the absence of pigments. Ten independently isolated chloro-phyll b-deficient mutants of barley were found to belong to a single comple-mentation group. This suggests that only a single locus may be involved inits synthesis (101). Studies with otherchlorina mutants of wheat and barleyshow that mutants generally characterized as chlorophyllb-deficient are actu-ally defective in overall chlorophyll biosynthesis due to partial blocks at themagnesium chelation step (23, 24). It is hypothesized that chlorophyllb issynthesized only from chlorophylla that is leftover after formation of reactioncenter complexes.

Chlorophyllsc1, c2, andc3 are used as accessory light-harvesting pigmentsin many algae. They differ from chlorophylla and b in that ring 4 is notreduced, which indicates that they are most likely derived from protochloro-phyllide. Chlorophyllc2 appears to be derived from divinyl protochlorophyl-lide, whereasc1 is derived from monovinyl protochlorophyllide (8). Chloro-phyll d has recently been identified as a major light-harvesting pigment of aprochlorophyte-like (oxychlorobacteria-like) prokaryoteAcarychloris marinaMiyashita et Chihara gen. et sp. Nov. (74). This pigment, which differs fromchlorophylla by substitution of a 2-formyl group for 2-vinyl, shows an inter-esting absorbance in the red region (716 nm). No mutants or genes involved inchlorophyllc or d production have been identified.

CONCLUDING REMARKS

Since the first chlorophyll biosynthesis gene was cloned and sequenced in 1989(99), sequence information has been obtained for most of the enzymes in theMg-branch of the tetrapyrrole biosynthetic pathway that give rise to chlorophylla and bacteriochlorophylla. Given the rate at which this field has developedof late, most of the “missing genes” in chlorophylla biosynthesis in plants willlikely be identified based on sequence homology to the bacterial genes. Areasstill needing to be addressed include the rigorous establishment that geneticallyidentified genes in specific steps of the pathway actually code for catalyticsubunits of enzymes. This hypothesis is currently being addressed in severallaboratories by the heterologous expression of genes inE. coli coupled with invitro assays for enzyme activity. The issue of regulation of bacteriochlorophyll

Ann

u. R

ev. G

enet

. 199

7.31

:61-

89. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

Acc

ess

prov

ided

by

AL

I: A

cade

mic

Lib

rari

es o

f In

dian

a on

08/

18/1

5. F

or p

erso

nal u

se o

nly.

P1: ARS



and chlorophyll biosynthesis at the transcriptional and posttranscriptional levelsalso needs to be expanded beyond the few experimental systems, and steps ofthe pathway, that have been studied to date. Genes involved in synthesis of addi-tional bacteriochlorophyll and chlorophyll end products must also be identifiedand characterized. A thorough understanding of these additional pathways,particularly in deeply divergent green sulfur and nonsulfur bacteria, could givesignificant insight into the evolutionary relationships of these pathways.

ACKNOWLEDGMENTS

We thank Jin Xiong and Sylvie Elsen for comments regarding the manuscript.We also apologize to those whose work was not directly cited owing to spacelimitations. Research in this area is supported by National Institutes of Healthgrants GM 539040 and GM 00618 to CEB.

Visit the Annual Reviews home pageathttp://www.annurev.org.

Literature Cited

1. Addlesee HA, Gibson LCD, Jensen PE,Hunter CN. 1996. Cloning, sequencingand functional assignment of the chloro-phyll biosynthesis gene,chlP, of Syne-chocystis sp. PCC 6803.FEBS Lett.389:126–30

2. Alberti M, Burke D, Hearst JE. 1995.Structure and sequence of the photosyn-thesis gene cluster. InAnoxygenic Photo-synthetic Bacteria, ed. RE Blankenship,M Madigan, C Bauer. pp. 1083–106. Dor-drecht: Kluwer

3. Armstrong GA, Alberti M, Leach F,Hearst J. 1989. Nucleotide sequence, or-ganization, and nature of the protein prod-ucts of the carotenoid biosynthesis genecluster ofRhodobacter capsulatus. Mol.Gen. Genet.216:254–68

4. Armstrong GA, Runge S, Frick G, Sper-ling G, Apel K. 1995. Identificationof NADPH:protochlorophyllide oxidore-ductases A and B: a branched pathway forlight-dependent chlorophyll biosynthesisin Arabidopsis thaliana. Plant Physiol.108:1505–17

5. Batschschauer A, Apel K. 1984. An in-verse control by phytochrome of the ex-pression of two nuclear genes in barley.Eur. J. Biochem143:593–97

6. Bauer CE, Bollivar DW, Suzuki J. 1993.Genetic analysis of photopigment biosyn-thesis in eubacteria: a guiding light for al-gae and plants.J. Bacteriol.175:3919–25

7. Bauer CE, Young DA, Marrs BL.1988. Analysis of theRhodobacter cap-sulatus puf operon. Location of theoxygen-regulated promoter region andthe identification of an additionalpuf-encoded gene.J. Biol. Chem.263:4820–27

8. Beale SI, Weinstein JD. 1991. Biochem-istry and regulation of photosynthetic pig-ment formation in plants and algae. InBiosynthesis of Tetrapyrroles,ed. PMJordan, 19:155–236. Amsterdam: NewCompr. Biochem.

9. Bellemare G, Bartlett SG, Chua NH.1982. Biosynthesis of chlorophylla/b-binding polypeptides in wild type andthechlorina f 2mutant of barley.J. Biol.Chem.257:7762–67

10. Biel AJ, Marrs BL. 1983. Transcriptionalregulation of several genes for bacte-riochlorophyll synthesis inRhodopseu-domonas capsulatain response to oxy-gen.J. Bacteriol.156:686–94

11. Boivin R, Beauseigle MRD, Bousquet J,Bellemare G. 1996. Phylogenetic infer-ences from chloroplastchlB sequencesof Nephrolepis exaltata(Filicopsida),Ephedra altissima(Gnetopsida), and di-verse land plants.Mol. Phylogenet. Evol.6:19–29

12. Bollivar DW, Bauer CE. 1992. Nucleo-tide sequence ofS-adenosyl-methionine:magnesium protoporphyrin methyltrans-

Ann

u. R

ev. G

enet

. 199

7.31

:61-

89. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

Acc

ess

prov

ided

by

AL

I: A

cade

mic

Lib

rari

es o

f In

dian

a on

08/

18/1

5. F

or p

erso

nal u

se o

nly.

P1: ARS



ferase from Rhodobacter capsulatus.Plant Physiol.98:408–10

13. Bollivar DW, Jiang Z-Y, Bauer CE, BealeSI. 1994. Heterologous overexpression ofthebchMgene product fromRhodobactercapsulatusand demonstration that it en-codes forS-adenosyl-L-methionine:Mg-protoporphyrin methyltransferase.J. Bac-teriol. 176:5290–96

14. Bollivar DW, Suzuki JY, Beatty JT, Do-browlski J, Bauer CE. 1994. Directed mu-tational analysis of bacteriochlorophyllabiosynthesis inRhodobacter capsulatus.J. Mol. Biol.237:622–40

15. Bollivar DW, Wang S, Allen JP, BauerCE. 1993. Molecular genetic analysisof terminal steps in bacteriochlorophylla biosynthesis: characterization of aRhodobacter capsulatusstrain that syn-thesizes geranylgeranyl esterified bacteri-ochlorophylla. Biochemistry33:12763–68

16. Burke D, Alberti M, Hearst JE. 1993.The Rhodobacter capsulatuschlorinreductase-encoding locus,bchA, consistsof three genes,bchX, bchY, andbchZ. J.Bacteriol.175:2407–13

17. Burke D, Hearst JE, Sidow A. 1993. Earlyevolution of photosynthesis: clues fromnitrogenase and chlorophyll iron proteins.Proc. Natl. Acad. Sci. USA90:7134–38

18. Burke DH, Raubeson LA, Alberti M,Hearst J, Jordan ET, et al. 1993. ThechlL( frxC) gene: phylogenetic distribution invascular plants and DNA sequence fromPolysticum acrosticoides(Pteridophyta)andSynechococcussp. 7002 (Cyanobac-teria).Plant Syst. Evol.187:89–102

19. Choquet Y, Rahire M, Girard-BascouJ, Erickson J, Rochaix J-D. 1992. Achloroplast gene is required for the light-independent accumulation of chlorophyllin Chlamydomonas reinhardtii. EMBO J.11:1697–704

20. Ellsworth RK, Aronoff S. 1968. Inves-tigations on the biogenesis of chloro-phyll a: I. purification and mass spec-tra of maleimides from the oxidation ofchlorophyll and related compounds.Arch.Biochem. Biophys.124:358–64

21. Ellsworth RK, Aronoff S. 1968. Investi-gations on the biogenesis of chlorophylla: II. chlorophyllide a accumulation bya Chlorella mutant.Arch. Biochem. Bio-phys.125:35–39

22. Ellsworth RK, Aronoff S. 1969. Inve-stigations on the biogenesis of chloro-phyll a: IV. isolation and partial char-acterization of some biosynthetic inter-mediates between Mg-protoporphyrinIX monomethyl ester and Mg-vinyl-

pheoporphinea5, obtained fromChlo-rella mutants.Arch. Biochem. Biophys.130:374–83

23. Falbel TG, Staehlin LA. 1994. Characteri-zation of a family of chlorophyll-deficientwheat (Triticum) and barley (Hordeumvulgare) mutants with defects in themagnesium-insertion step of chlorophyllbiosynthesis.Plant Physiol.104:639–48

24. Falbel TG, Meehl JB, Staehlin LA. 1996.Severity of mutant phenotype in a series ofchlorophyll-deficient wheat mutants de-pends on light intensity and the severity ofthe block in chlorophyll synthesis.PlantPhysiol.112:821–32

25. Ford C, Wang W-Y. 1980. Three new yel-low loci in Chlamydomonas reinhardtii.Mol. Gen. Genet.179:259–63

26. Ford C, Wang W-Y. 1980. Temperature-sensitive yellow mutants ofChlamy-domonas reinhardtii. Mol. Gen. Genet.180:5–10

27. Ford C, Mitchell S, Wang W-Y. 1981. Pro-tochlorophyllide photoconversion mu-tants ofChlamydomonas reinhardtii.Mol.Gen. Genet.184:460–64

28. Forreiter C, Apel K. 1993. Light-in-dependent and light-dependent protoch-lorophyllide-reducing activities and twodistinct NADPH-protochlorophyllide ox-idoreductase polypeptides in mountainpine (Pinus mugo). Planta190:536–45

29. Forreiter C, van Cleve B, Schmidt A, ApelK. 1990. Evidence for a general light-dependent negative control of NADPH-protochlorophyllide oxidoreductase inangiosperms.Planta183:126–32

30. Fray RG, Wallace A, Fraser PD, ValeroD, Hedden P, et al. 1995. Constitutive ex-pression of a fruit phytoene synthase genein transgenic tomatoes causes dwarfismby redirecting metabolites from the gib-berellin pathway.Plant J.8:693–701

31. Fujita Y. 1996. Protochlorophyllide re-duction: a key step in the greeningof plants. Plant Cell Physiol.37:411–21

32. Fujita Y, Takagi H, Hase T. 1996. Identifi-cation of thechlBgene and the gene prod-uct essential for the light-independentchlorophyll biosynthesis in the cyanobac-teriumPlectonema boryanum. Plant CellPhysiol.37:313–23

33. Fujita Y, Matsumoto H, Takahashi Y,Matsubara H. 1993. Identification of anifDK-like gene (ORF467) involved inthe biosynthesis of chlorophyll in thecyanobacteriumPlectonema boryanum.Plant Cell Physiol.34:305–14

34. Fujita Y, Takahashi Y, Chuganji M, Mat-subara H. 1992. ThenifH-like (frxC) gene

Ann

u. R

ev. G

enet

. 199

7.31

:61-

89. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

Acc

ess

prov

ided

by

AL

I: A

cade

mic

Lib

rari

es o

f In

dian

a on

08/

18/1

5. F

or p

erso

nal u

se o

nly.

P1: ARS



is involved in the biosynthesis of chloro-phyll in the filamentous cyanobacteriumPlectonema boryanum. Plant Cell Phys-iol. 33:81–92

35. Fujita Y, Takahashi Y, Shonai F, Ogura Y,Matsubara H. 1991. Cloning, nucleotidesequences and differential expression ofthenifH andnifH-like ( frxC) genes fromthe filamentous nitrogen-fixing cyanobac-teriumPlectonema boryanum. Plant CellPhysiol.32:1093–106

36. Gaubier PH, Wu HJ, Laudie M, DelsenyM, Grellet F. 1995. A chlorophyll syn-thetase gene fromArabidopsis thaliana.Mol. Gen. Genet.249:58–64

37. Gibson LCD, Hunter CN. 1994. Thebacteriochlorophyll biosynthesis gene,bchM, of Rhodobacter sphaeroidesen-codesS-adenosyl-L-methionine:Mg-pro-toporphyrin IX methyltransferase.FEBSLett.352:127–30

38. Gibson LCD, Marrison JL, Leech RM,Jensen PE, Bassham DC, et al. 1996. Aputative Mg chelatase and transcript anal-ysis of the gene, import of the protein intochloroplasts, and in situ localization ofthe transcript and protein.Plant Physiol.111:61–71

39. Gibson LCD, Willows RD, KannangaraCG, von Wettstein D, Hunter CN. 1995.Magnesium-protoporphyrin chelatase ofRhodobacter sphaeroides: reconstitutionof activity by combining the products ofbchH, I and D genes expressed inEs-cherichia coli. Proc. Natl. Acad. Sci. USA92:1941–44

40. Gierl A, Saedler H. 1992. Plant trans-posable elements and gene tagging.PlantMol. Biol. 19:39–49

41. Granick S. 1948. Protoporphyrin 9 as aprecursor of chlorophyll.J. Biol. Chem.172:717–27

42. Granick S. 1948. Magnesium protopor-phyrin as a precursor of chlorophyll inChlorella. J. Biol. Chem.175:333–42

43. Granick S. 1950. The structural and func-tional relationships between heme andchlorophyll.Harvey Lect.44:220–45

44. Granick S. 1953. Magnesium vinyl pheo-porphyrina5, another intermediate in thebiological synthesis of chlorophyll.J.Biol. Chem.183:713–30

45. Granick S. 1961. Magnesium protopor-phyrin monoester and protoporphyrinmonomethyl ester in chlorophyll biosyn-thesis.J. Biol. Chem.236:1168–72

46. Hallick RB, Hong L, Drager RG, FavreauMR, Monfort A, et al. 1993. Completesequence ofEuglena gracilischloroplastDNA. Nucleic Acids Res.21:3537–44

47. Hiratsuka J, Shimada H, Whitter R,

Ishibashi T, Sakamoto M, et al. 1989.The complete sequence of the rice (Oryzasativa) chloroplast genome: intermolecu-lar recombination between distinct tRNAgenes accounts for a major plastid DNAinversion during the evolution of the ce-reals.Mol. Gen. Genet.217:185–94

48. Hofgren R, Axelsen KB, KannangaraCG, Schuttke I, Pohlenz H-D, et al.1994. A visible marker for antisensemRNA expression in plants: inhibition ofchlorophyll synthesis with a glutamate-1-semialdehyde aminotransferase anti-sense gene.Proc. Natl. Acad. Sci. USA91:1726–30

49. Holtorf H, Reinbothe S, Reinbothe C,Bereza B, Apel K. 1995. Two routesof chlorophyll synthesis that are dif-ferentially regulated by light in barley(Hordeum vulgareL.). Proc. Natl. Acad.Sci. USA92:3254–58

50. Hudson A, Carpenter R, Doyle S, CoenES. 1993. Olive: a key gene required forchlorophyll biosynthesis inAntirrhinummajus. EMBO J.12:3711–19

51. Ito H, Ohtsuka T, Tanaka A. 1996. Con-version of chlorophyllb to chlorophyllavia 7–hydroxymethyl chlorophyll.J. Biol.Chem.271:1475–79

52. Ito H, Takaichi S, Tsuji H, Tanaka A.1994. Properties of synthesis of chloro-phyll a from chlorophyllb in cucumberetioplasts.J. Biol. Chem.269:22034–38

53. Jensen PE, Gibson LCD, HenningsenKW, Hunter CN. 1996. Expression ofthe chlI, chlD and chlH genes from thecyanobacteriumSynechocystisPCC6803in Escherichia coliand demonstration thatthe three cognate proteins are required formagnesium-protoporphyrin chelatase ac-tivity. J. Biol. Chem.271:16662–67

54. Jensen PE, Willows RD, Petersen BL,Vothknecht UC, Stummann BM, et al.1996. Structural genes for Mg-chelatasesubunits in barley:Xantha-f, -gand -h.Mol. Gen. Genet.250:383–94

55. Jones OTG. 1978. Biosynthesis of por-phyrins, hemes and chlorophylls. InThePhotosynthetic Bacteria, ed. RK Clay-ton, WR Sistrom, pp. 751–77. New York:Plenum

56. Kaneko T, Sato S, Kotani H, Tanaka A,Asamizu E, et al. 1996. Sequence anal-ysis of the genome of the unicellularcyanobacteriumSynechocystissp. strainPCC6803. II. Sequence determination ofthe entire genome and assignment of po-tential protein-coding regions.DNA Res.3:109–36

57. Kaneko T, Tanaka A, Sato S, Kotani H,Sazuka T, et al. 1995. Sequence anal-

Ann

u. R

ev. G

enet

. 199

7.31

:61-

89. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

Acc

ess

prov

ided

by

AL

I: A

cade

mic

Lib

rari

es o

f In

dian

a on

08/

18/1

5. F

or p

erso

nal u

se o

nly.

P1: ARS



ysis of the genome of the unicellularcyanobacteriumSynechocystissp. strainPCC 6803. I. Sequence features in the 1Mb region from map positions 64% to92% of the genome.DNA Res.2:153–66

58. Kannangara CG, Vothknecht UC, Hans-son M, von Wettstein D. 1996. Magne-sium chelatase: association with ribo-somes and mutant complementation stud-ies identify barley subunit Xantha-G asa functional counterpart ofRhodobactersubunit BchD.Mol. Gen. Genet.254:85–92

59. Karpinska B, Karpinski S, Hilgren JE.1997. ThechlB gene encoding a subunitof light-independent protochlorophyllidereductase is edited in chloroplasts ofconifers.Curr. Genet.31:343–47

60. Kohchi T, Shirai H, Fukuzawa H, SanoT, Komano T, et al. 1988. Structure andorganization ofMarchantia polymorphachloroplast genome IV. Inverted repeatand small single copy regions.J. Mol.Biol. 203:353–72

61. Koncz C, Mayerhofer R, Koncz-KalmanZ, Nawrath C, Reiss B, et al. 1990. Iso-lation of a gene encoding a novel chloro-plast protein by T-DNA tagging inAra-bidopsis thaliana. EMBO J.9:1337–46

62. Koncz C, Martini N, Mayerhofer R,Koncz-Kalman Z, Korber H, et al. 1989.High frequency T-DNA-mediated genetagging in plants.Proc. Natl. Acad. Sci.USA86:8467–71

63. Kruse E, Mock H-P, Grimm B. 1995. Re-duction of coproporphyrinogen oxidaselevel by antisense RNA synthesis leadsto deregulated gene expression of plastidproteins and affects the oxidative defensesystem.EMBO J.14:3712–20

64. Kuroda H, Masuda T, Ohta H, Shioi Y,Takamiya K. 1995. Light-enhanced ex-pression of NADPH-protochlorophyllideoxidoreductase in cucumber.Biochem.Biophys. Res. Commun.210:310–16

65. Li J, Timko MP. 1996. Thepc-1 phen-otype of Chlamydomonas reinhardtiiresults from a deletion mutation inthe nuclear gene for NADPH:protochlo-rophyllide oxidoreductase.Plant Mol.Biol. 30:15–37

66. Li J, Goldschmidt-Clermont M, TimkoMP. 1993. Chloroplast-encodedchlBis required for light-independent pro-tochlorophyllide reductase activity inChlamydomonas. Plant Cell5:1817–29

67. Lidholm J, Gustafsson P. 1991. Homo-logues of the green algalgidA gene andthe liverwort frxC gene are present onthe chloroplast genomes of conifers.PlantMol. Bio.17:787–98

68. Liu X-Q, Xu H, Huang C. 1993. Chloro-plast chlB gene is required for light-independent chlorophyll accumulation inChlamydomonas reinhardtii. Plant Mol.Biol. 23:297–308

69. Lopez JC, Ryan S, Blankenship RE.1996. Sequence of thebchG genefrom Chloroflexus aurantiacus: relation-ship between chlorophyll synthase andother polyprenyltransferases.J. Bacte-riol. 178:3363–73

70. Maier RM, Neckermann K, Igloi GL,Kossel H. 1995. Complete sequence of themaize chloroplast genome: gene content,hotspots of divergence and fine tuning ofgenetic information by transcript editing.J. Mol. Biol.251:614–28

71. Mapleston ER, Griffiths WT. 1980.Light modulation of the activity ofthe protochlorophyllide oxidoreductase.Biochem. J.189:125–33

72. Marrison JL, Leech RM. 1994. The sub-cellular and intra-organelle recognitionof nuclear and chloroplast transcripts indeveloping leaf cells.Plant J. 6:605–14

73. Marrs BL. 1981. Mobilization of thegenes for photosynthesis fromRhodop-seudomonas capsulataby a promiscuousplasmid.J. Bacteriol.146:1003–12

74. Miyashita H, Ikemoto H, Kurano N,Abachi K, Chihara M, Miyachi S. 1996.Chlorophylld as a major pigment.Nature383:402

75. Mosinger E, Batschauer A, Schäfer E,Apel K. 1985. Phytochrome control ofinvitro transcription of specific genes in iso-lated nuclei from barley (Hordeum vul-gare). Eur. J. Biochem.147:137–42

76. Murray DL, Kohorn BD. 1991. Chloro-plasts ofArabidopsis thalianahomozy-gous for thech-1 locus lack chlorophyllb, lack stable LHCPII and have stackedthylakoids. Plant Mol. Biol. 16:71–80

77. Nakayama M, Masuda T, Sato N, Yam-agata H, Bowler C, et al. 1995. Cloning,subcellular localization and expression ofchlI, a subunit of magnesium-chelatase insoybean.Biochem. Biophys. Res. Com-mun.215:422–28

78. Nicholson-Guthrie CS. 1983. Chloro-phyll inheritance in Chlamydomonasmasking ofy-1gene byy-ygene.J. Hered.74:16–18

79. Nicholson-Guthrie CS, Guthrie GD.1987. Accumulation of protoporphyrinIX by the chlorophyll-lessy-y mutantof Chlamydomonas reinhardtii. Arch.Biochem. Biophys.252:570–73

80. Porra RJ, Schäfer W, Cmiel E, Katheder

Ann

u. R

ev. G

enet

. 199

7.31

:61-

89. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

Acc

ess

prov

ided

by

AL

I: A

cade

mic

Lib

rari

es o

f In

dian

a on

08/

18/1

5. F

or p

erso

nal u

se o

nly.

P1: ARS



I, Scheer H. 1993. Derivation of theformyl-group oxygen of chlorophyllbfrom molecular oxygen in greening leavesof a higher plant (Zea mays). FEBS Lett.323:31–34

81. Ogura Y, Takemura M, Oda K, YamataK, Ohta E, et al. 1992. Cloning and nu-cleotide sequence of afrxC-ORF469genecluster ofSynechocystis PCC6803: con-servation with liverwort chloroplastfrxC-ORF465andnif operon.Biosci. Biotech.Biochem.56:788–93

82. Ohad I, Siekevitz P, Palade GE. 1967. Bio-genesis of chloroplast membranes I. Plas-tid differentiation in a dark-grown algalmutant (Chlamydomonas reinhardtii). J.Cell. Biol.35:521–52

83. Ohad I, Siekevitz P, Palade GE. 1967.Biogenesis of chloroplast membranes II.Plastid differentiation during greeningin a dark-grown algal mutant (Chlamy-domonas reinhardtii). J. Cell. Biol.35:553–84

84. Porra RJ, Schäfer W, Katheder I, ScheerH. 1995. The derivation of the oxygenatoms of the 131--oxo and 3–acetyl groupsof bacteriochlorophylla from water inRhodobacter sphaeroidescells adaptingfrom respiratory to photosynthetic con-ditions: evidence for an anaerobic path-way for the formation ofisocyclicring E.FEBS Lett.371:21–24

85. Reinbothe S, Reinbothe C. 1996. Regu-lation of chlorophyll biosynthesis in an-giosperms.Plant Physiol.111:1–7

86. Reinbothe S, Reinbothe C, Apel K, Lebe-dev N. 1996. Evolution of chlorophyllbiosynthesis—the challenge to survivephotooxidation.Cell 86:703–5

87. Reinbothe S, Reinbothe C, Runge S, ApelK. 1995. Enzymatic product formationimpairs both the chloroplast receptor-binding function as well as transloca-tion competence of the NADPH:proto-chlorophyllide oxidoreductase, a nuclearencoded plastid precursor protein.J. CellBiol. 129:299–308

88. Reinbothe S, Runge S, Reinbothe C,van Cleve B, Apel K. 1995. Substrate-dependent transport of the NADPH:pro-tochlorophyllide oxidoreductase into iso-lated plastids.Plant Cell7:161–72

89. Reinbothe S, Reinbothe C, Holtorf H,Apel K. 1995. Two NADPH:proto-chlorophyllide oxidoreductases in bar-ley: evidence for the selective disappear-ance of PORA during the light-inducedgreening of etiolated seedlings.Plant Cell7:1933–40

90. Roitgrund C, Mets LJ. 1990. Localizationof two novel chloroplast genome func-

tions: trans-splicing of RNA and pro-tochlorophyllide reduction.Curr. Genet.17:147–53

91. Reith M, Munholland J. 1993. A high-resolution gene map of the chloroplastgenome of the red algaProphyra pur-purea. Plant Cell5:465–75

92. Richard M, Tremblay C, Bellemare G.1994. Chloroplastic genomes ofGinkgobiloba and Chlamydomonas moewusiicontain achlB gene encoding one sub-unit of a light-independent protochloro-phyllide reductase.Curr. Genet.26:159–65

93. Runge S, van Cleve B, Lebedev N,Armstrong G, Apel K. 1995. Isolationand classification of chlorophyll-deficientxanthamutants ofArabidopsis thaliana.Planta197:490–500

94. Runge S, Sperling U, Frick G, ApelK, Armstrong GA. 1996. Distinct rolesfor light-dependent NADPH-protoch-lorophyllide oxidoreductases (POR) Aand B during greening in higher plants.Plant J.9:513–23

95. Sager R. 1955. Inheritance in the greenalgaChlamydomonas reinhardtii. Genet-ics40:476–89

96. Sager R. 1959. The architecture of thechloroplast in relation to its photosyn-thetic activities.Brookhaven Symp. Biol.11:101–17

97. Sager R. 1961. Photosynthetic pigmentsin mutant strains ofChlamydomonasreinhardtii. Carnegie Inst. Wash. Yearb.60:374–76

98. Schneegurt MA, Beale IS. 1992. Ori-gin of the chlorophyll b formyl oxy-gen in Chlorella vulgaris. Biochemistry31:11677–83

99. Schulz R, Steinmüller K, Klaas M, For-reiter C, Rasmussen S, et al. 1989. Nu-cleotide sequence of a cDNA codingfor the NADPH- protochlorophyllide ox-idoreductase (PCR) of barley (Hordeumvulgare L.) and its expression inEs-cherichia coli.Mol. Gen. Genet.217:355–61

100. Scolnik P, Bartley GE. 1996. A tableof some cloned plant genes involved inisoprenoid biosynthesis.Plant Mol. Biol.Rep.14:305–19

101. Simpson D, Machold O, Hoyer-HansenG, von Wettstein D. 1985.Chlorina mu-tants of barley (Hordeum vulgareL.)Carlsberg Res. Commun.50:223–38

102. Smith C, Suzuki JY, Bauer CE. 1996.Cloning and characterization of thechlorophyll biosynthesis genechlM fromSynechocystisPCC 6803 by complemen-tation of a bacteriochlorophyll biosyn-

Ann

u. R

ev. G

enet

. 199

7.31

:61-

89. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

Acc

ess

prov

ided

by

AL

I: A

cade

mic

Lib

rari

es o

f In

dian

a on

08/

18/1

5. F

or p

erso

nal u

se o

nly.

P1: ARS



thesis mutant ofRhodobacter capsulatus.Plant Mol. Biol.30:1307–14

103. Smith KM. 1991. The structure andbiosynthesis of bacteriochlorophylls. InBiosynthesis of Tetrapyrroles,ed. PMJordan, 19:237–56. Amsterdam: NewCompr. Biochem.

104. Spano AJ, He Z, Timko MP. 1992.NADPH:protochlorophyllide oxidore-ductases in white pine (Pinus strobus)and loblolly pine (P. taeda.) Evidence forlight and developmental regulation andconservation in gene organization andprotein structure between angiospermsand gymnosperms.Mol. Gen. Genet.236:86–95

105. Spano, AJ, He Z, Michel H, Hunt DF,Timko MP. 1992. Molecular cloning, nu-clear structure, and developmental ex-pression of NADPH:protochlorophyllideoxidoreductase in pea (Pisum sativumL.)Plant Mol. Biol. 18:967–72

106. Santel HJ, Apel K. 1981. The pro-tochlorophyllide holochrome of barley.The effect of light on the NADPH-protochlorophyllide oxidoreductase.Eur.J. Biochem120:95–103

107. Shinozaki K, Ohme M, Tanaka M, Waka-sugi T, Hayashida N, et al. 1986. The com-plete nucleotide sequence of the tobaccochloroplast genome: its gene organiza-tion and expression.EMBO J.5:2043–49

108. Suzuki JY, Bauer CE. 1992. Light-independent chlorophyll biosynthesis:involvement of the chloroplast genechlL( frxC). Plant Cell4:929–40

109. Suzuki JY, Bauer CE. 1995. A prokary-otic origin for light-dependent chloro-phyll biosynthesis of plants.Proc. Natl.Acad. Sci. USA92:3749–53

110. Suzuki JY, Bauer CE. 1995. Alteredmonovinyl and divinyl protochlorophyl-lide pools inbchJmutants ofRhodobactercapsulatus. J. Biol. Chem.270:3732–40

111. Tam L-W, Lefebvre PA. 1993. Cloning offlagellar genes inChlamydomonas rein-hardtii by DNA insertional mutagenesis.Genetics135:375–84

112. Taylor DP, Cohen SN, Clark WG, MarrsBL. 1983. Alignment of the genetic andrestriction maps of the photosynthetic re-gion of theRhodopseudomonas capsulatachromosome by a conjugation-mediatedmarker rescue technique.J. Bacteriol.154:580–90

113. Ulrike O, Bauer CE, Rüdiger W. 1997.Characterization of chlorophylla andbacteriochlorophylla synthetases by het-erologous expression inE. coli. J. Biol.Chem.272:9671–76

114. von Wettstein D, Gough S, Kannan-

gara CG. 1995. Chlorophyll biosynthesis.Plant Cell7:1039–57

115. Wakao N, Yokoi N, Isoyama N, Hi-raishi A, Shimada K, et al. 1996. Dis-covery of natural photosynthesis usingZn-containing bacteriochlorophyll in anaerobic bacteriumAcidiphilium rubrum.Plant Cell Physiol.37:899–93

116. Wakasugi T, Hirose T, Horihata M,Tsudzuki T, Koessel H, Sugiura M. 1996.Creation of a novel protein-coding regionat the RNA level in black pine chloro-plasts: The pattern of RNA editing in thegymnosperm chloroplast is different fromthat in angiosperms.Proc. Natl. Acad. Sci.USA93:8766–70

117. Wang W-Y, Wang WL, Boynton JE, Gill-ham NW. 1974. Genetic control of chloro-phyll biosynthesis inChlamydomonas. J.Cell Biol. 63:806–23

118. Walker CJ, Weinstein JD. 1994. Themagnesium-insertion step of chlorophyllbiosynthesis is a two-stage reaction.Biochem. J.299:277–84

119. Walker CJ, Mansfield KE, Smith KM,Castelfranco PA. 1989. Incorporation ofatmospheric oxygen into the carbonylfunctionality of the protochlorophyllideisocyclic ring.Biochem. J.257:599–602

120. Wellington CL, Beatty JT. 1989. Pro-moter mapping and nucleotide sequenceof the bchCbacteriochlorophyll biosyn-thesis gene fromRhodobacter capsulatus.Gene83:251–61

121. Wilks HM, Timko MP. 1995. A light-dependent complementation system foranalysis of NADPH:protochlorophyllideoxidoreductase: identification of mutage-nesis of two conserved residues that areessential for enzyme activity.Proc. Natl.Acad. Sci. USA92:724–28

122. Wolfe KH, Morden CW, Palmer JD. 1992.Function and evolution of a minimal plas-tid genome from a nonphotosynthetic par-asitic plant.Proc. Natl. Acad. Sci. USA89:10648–52

123. Yang ZY, Bauer CE. 1990.Rhodobac-ter capsulatusgenes involved in earlysteps of the bacteriochlorophyll biosyn-thetic pathway.J. Bacteriol. 172:5001–10

124. Yen H-C, Marrs BL. 1976. Map of genesfor carotenoid and bacteriochlorophyllbiosynthesis inRhodopseudomonas cap-sulata. J. Bacteriol.126:619–29

125. Yildiz FH, Gest H, Bauer CE. 1992. Con-servation of the photosynthesis gene clus-ter inRhodospirillum centenum. Mol. Mi-crobiol. 6:2683–91

126. Yoshida K, Chen RM, Tanaka A, Ter-amoto H, Tanaka R, et al. 1995. Cor-

Ann

u. R

ev. G

enet

. 199

7.31

:61-

89. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

Acc

ess

prov

ided

by

AL

I: A

cade

mic

Lib

rari

es o

f In

dian

a on

08/

18/1

5. F

or p

erso

nal u

se o

nly.

P1: ARS



related changes in the activity, amountof protein, and abundance of transcriptof NADPH:protochlorophyllide oxidore-ductase and chlorophyll accumulationduring greening of cucumber cotyledons.Plant Physiol.109:231–38

127. Youvan DC, Bylina EJ, Alberti M, Be-gusch H, Hearst JE. 1984. Nucleotide

and deduced polypeptide sequences of thephotosynthetic reaction center, B870 an-tenna, and flanking polypeptides fromR.capsulata. Cell 37:949–57

128. Zsebo KM, Hearst JE. 1984. Geneticphysical mapping of a photosyntheticgene cluster fromR. capsulata. Cell37:937–47

Ann

u. R

ev. G

enet

. 199

7.31

:61-

89. D

ownl

oade

d fr

om w

ww

.ann

ualr

evie

ws.

org

Acc

ess

prov

ided

by

AL

I: A

cade

mic

Lib

rari

es o

f In

dian

a on

08/

18/1

5. F

or p

erso

nal u

se o

nly.