Update TRENDS in Plant Science Vol.10 No.7 July 2005 309

that tumour cells generate a differential biophotonsignature [7]. Bennett et al. [5] demonstrate that rapidbiophoton generation (BG) is directly related to theactivation of gene-for-gene mediated HRs, not only inArabidopsis but also in tomato and French bean. Theelegant non-destructive analysis facilitated investigationof the source of BG using various inhibitors. Intriguingly,BG was not suppressed by inhibiting NADPH oxidaseactivity using diphenyliodonium chloride but was com-promised by inhibiting NO generation and by applying theCa2C channel blocker LaCl3. In the AvrRpm1–RPM1interaction (studied in detail in [5]), the signallingsequence of Avr protein recognition and Ca2C influxleading to NO synthesis would appear to be required forthe HR, rather than for Ca2C influx leading to NADPHoxidase activation and H2O2 accumulation. Perhapsincorrectly, detection of H2O2 by histochemical staininghas often been used as an HR marker [4,8,9]. In afascinating image, similar to Figure 1, Bennett et al. [5]show, through quantitative analysis, the differentialtiming of BG that occurs at different positions within asingle infiltration site. As the authors point out, thespatial analysis provides a glimpse of further detailedobservations that should be possible when the technique isrefined to examine BG from individual cells, challengednot only by bacteria but also by fungi.

Photons on the horizon

The origin of biophotons remains puzzling. The most likelysource of the rapid burst is via the products of lipidperoxidation that are generated because of membranedamage at the onset of the HR. However, other sources arepossible, such as the direct interaction between NO andozone, and other free-radical-mediated photon emissions[10]. Are biophotons signalling molecules? Is the delayedand weaker burst detected during the compatible reactionwith DC3000 caused by the same biochemical reaction?Whatever the answers to such questions, I predict that the

Corresponding author: Beale, S.I. (sib@brown.edu).Available online 13 June 2005

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pioneering study by Bennett et al. [5] will lead towidespread use of the technique as an invaluable tool inthe dissection of the role of programmed cell death indisease resistance. The value of the approach has alreadybeen demonstrated in studies of proteins interacting withthe product of the RPM1 resistance gene [11].

References

1 Axtell, M.J. et al. (2003) Genetic and molecular evidence that thePseudomonas syringae type III effector protein AvrRpt2 is a cysteineprotease. Mol. Microbiol. 49, 1537–1546

2 Mindrinos, M. et al. (1994) The A. thaliana disease resistance geneRPS2 encodes a protein containing a nucleotide-binding site andleucine-rich repeats. Cell 78, 1089–1099

3 Chisholm, S.T. et al. (2005) Molecular characterization of proteolyticcleavage sites of the Pseudomonas syringae effector AvrRpt2. Proc.Natl. Acad. Sci. U. S. A. 102, 2087–2092

4 Grant, M. et al. (2000) The RPM1 plant disease resistance genefacilitates a rapid and sustained increase in cytosolic calcium that isnecessary for the oxidative burst and hypersensitive cell death. PlantJ. 23, 441–450

5 Bennett, M. etal. (2005) Biophoton imaging: anondestructive method forassaying R gene responses. Mol. Plant–Microbe Interact. 18, 95–102

6 Gassmann, W. et al. (1999) The Arabidopsis RPS4 bacterial-resistancegene is a member of the TIR–NBS–LRR family of disease-resistancegenes. Plant J. 20, 265–277

7 Kalikin, L.M. et al. (2003) In vivo visualization of metastatic prostatecancer and quantitation of disease progression in immunocompro-mised mice. Cancer Biol. Ther. 2, 656–660

8 Torres, M.A. et al. (2002) Arabidopsis gp91phox homologues AtrbohDand AtrbohF are required for accumulation of reactive oxygenintermediates in the plant defense response. Proc. Natl. Acad. Sci.U. S. A. 99, 517–522

9 Bestwick, C.S. et al. (1998) Localized changes in peroxidase activityaccompany hydrogen peroxide generation during the developmentof a nonhost hypersensitive reaction in lettuce. Plant Physiol. 118,1067–1078

10 Slawinski, J. (2003) Biophotons from stressed and dying organisms:toxicological aspects. Indian J. Exp. Biol. 41, 483–493

11 Al-Daoude, A. et al. (2005) RIN13 is a positive regulator of the plantdisease resistance protein RPM1. Plant Cell 17, 1016–1028

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doi:10.1016/j.tplants.2005.05.007

Green genes gleaned

Samuel I. Beale

Division of Biology and Medicine, Brown University, Providence, RI 02912, USA

A recent paper by Ayumi Tanaka and colleagues

identifying an Arabidopsis thaliana gene for 3,8-divinyl

(proto)chlorophyllide 8-vinyl reductase brings a satisfy-

ing conclusion to the hunt for genes encoding enzymes

for the steps in the chlorophyll biosynthetic pathway.

Now, at least in angiosperm plants represented by

Arabidopsis, genes for all 15 steps in the pathway from

glutamyl-tRNA to chlorophylls a and b have been

identified.

Monovinyl and divinyl chlorophylls

The chlorophyll a and b molecules in all plants and algaecontain a vinyl group at position 3 and an ethyl group atposition 8 in the tetrapyrrole macrocycle (Figure 1). Theethyl group is formed from a vinyl group that is present atthe corresponding position in chlorophyll precursors. A

9

1 2 3

56

8

1112

O

O OCH3

CO C

HH

HO

H

N

N

N

NMg

O

O OCH3

CO C

HH

HO

H

N

N

N

NMg

O

O OCH3

CO C

HH

O

H H

H

N

N

N

NMg

O OHC

H2N OC

O tRNAGlu

H

O OHC

H2N OC

H

H

δ-Aminolevulinic acid

NH2

O OHC

O

NNH2

O OHC

O

HO

C

H

Porphobilinogen

NH

NH

HN

HN

O OH

C

O

OH

C

OHO

CO

OH

C

O

OH

C

O

HO

C

HOH2C

O

HOC

O

OH

C

NH

NH

HN

HN

O OHC

O OHC

O

OH

C

OHO

CO

OH

C

O

OH

CO

HO

C

O

HO

C

NH

NH

HN

HN

O OH

C

O OH

C

O

OH

C

OHO

C

NH

NH

HN

HN

O OH

C

O OH

C

NH

N

N

HN

O OH

C

O OH

C

O OH

C

O OH

C

N

N

N

NMg

O OCH3

C

O OH

C

N

N

N

NMg

O

O OCH3

CH

O

HO

C

N

N

N

NMg

12

CHO

13

14 15

13

10

7

4

O

O OCH3

CH

O

HO

C

N

N

N

NMg

8

3

O

O OCH3

CO C

HH

O

H H

H

N

N

N

NMg

CHO

14

O

O OCH3

CO C

HH

HO

H

N

N

N

NMg

L-glutamic acid1-semialdehyde

Coproporphyrinogen III Uroporphyrinogen III Hydroxymethylbilane

Protoporphyrinogen IX Protoporphyrin IX Mg-protoporphyrin IX

Protochlorophyllide Divinyl protochlorophyllide Mg-protoporphyrin IXmonomethyl ester

Chlorophyllide a Divinyl chlorophyllide a

Chlorophyll a Chlorophyllide b Chlorophyll b

TRENDS in Plant Science

L-glutamyl-tRNA

Figure 1. The chlorophyll biosynthetic pathway in angiosperms. Numbered arrows refer to the enzymes listed in Table 1. Reactions 12 and 13 can occur in either order,

depending on the availability of substrates. Reaction 14 can use either of the two substrates indicated. The position numbers of the two vinyl groups are indicated

for 3,8-divinyl protochlorophyllide.

Update TRENDS in Plant Science Vol.10 No.7 July 2005310

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Update TRENDS in Plant Science Vol.10 No.7 July 2005 311

mutant strain of maize has been described that containsabnormal chlorophyll a and b molecules that have vinylgroups at positions 3 and 8 [1]. The mutant plants arephotosensitive but are able to photosynthesize, albeitpoorly. More recently, the marine prochlorophyte Pro-chlorococcus marinus was found naturally to contain onlydivinyl chlorophylls a and b [2].

Divinyl reductase cloned

Ayumi Tanaka and colleagues [3] isolated a mutant strain ofArabidopsis thaliana that accumulates divinyl chloro-phylls. By map-based cloning, the gene responsible for themutant phenotype was identified. The gene, At5G18660, isable to complement the mutant plants. The predicted geneproduct resembles isoflavone reductase. Expression of thegene in Escherichia coli yields a protein that catalyzesNADPH-dependent conversion of 3,8-divinylchlorophyll ato (3-monovinyl)chlorophyll a, thus establishing the geneproduct as a 3,8-divinyl(proto) chlorophyllide 8-vinylreductase (DVR) enzyme. A homolog of At5G18660 hasbeen found in the genome of the cyanobacterium Synecho-coccus sp. WH8102 (which contains normal chlorophyll a)but not in that of the divinyl chlorophyll-containingP. marinus. With the inclusion of the gene for DVR, all thegenes for the chlorophyll biosynthetic steps in angiospermplants have now been identified (Table 1).

Table 1. Genes encoding the enzymes in the chlorophyll

biosynthetic pathway in angiosperms

Stepa Enzyme name Gene

name(s)b

1 Glutamyl-tRNA reductase HEMA1

HEMA2

HEMA3

2 Glutamate 1-semialdehyde aminotransferase

(Glutamate 1-semialdehyde aminomutase)

GSA1

(HEML1)

GSA2

(HEML2)

3 Porphobilinogen synthase

(5-Aminolevulinate dehydratase)

HEMB1

HEMB2

4 Hydroxymethylbilane synthase

(Porphobilinogen deaminase)

HEMC

5 Uroporphyrinogen III synthase

(Uroporphyrinogen III co-synthase)

HEMD

6 Uroporphyrinogen decarboxylase HEME1

HEME2

7 Coproporphyrinogen oxidative decarboxylase HEMF1

HEMF2

8 Protoporphyrinogen oxidase HEMG1

HEMG2

9 Mg chelatase D subunit CHLD

Mg chelatase H subunit CHLH

Mg chelatase I subunit CHLI1

CHLI2

10 Mg-protoporphyrin IX methyltransferase CHLM

11 Mg-protoporphyrinogen IX monomethylester

cyclase

CRD1

(ACSF )

12 Divinyl reductase DVR

13 NADPH:protochlorophyllide oxidoreductase PORA

PORB

PORC

14 Chlorophyll synthase CHLG

15 Chlorophyllide a oxygenase CAO (CHL)aThe step numbers correspond to the numbers in Figure 1.bThe gene names are those given for Arabidopsis thaliana. Alternative names for

enzymes and genes are indicated by parentheses. Multiple genes for a given

enzyme are indicated by numerical suffixes, except for the POR genes (step 13) for

which letter suffixes are used.

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Mendel and chlorophyll synthesis

In a sense, the search for genes involved in chlorophyllsynthesis can be said to have begun with Gregor Mendel,who noted the inheritance of differences in the tendency ofpea cotyledons to retain or lose their green color as theymatured (cited in [4]). As it turns out, the gene responsiblefor this trait encodes an enzyme that is involved in thedegradation, rather than in the formation, of chlorophyll[4]. Nevertheless, Mendel’s observations can be consideredto be the first systematic attempt to study the genetics ofchlorophyll.

Relationship of chlorophyll to heme

An important early contribution to the genetics ofchlorophyll synthesis was the discovery by Sam Granickthat mutant strains of the alga Chlorella vulgaris thatwere unable to synthesize chlorophyll accumulatedporphyrins that were known to be related to precursorsof heme [5]. These results established that heme andchlorophyll share common biosynthetic steps andsuggested that the enzymes that catalyze these steps,and the genes that encode them, are also shared.

The genetics of heme biosynthesis, and therefore theportion of the chlorophyll pathway that is shared withthat for heme, was advanced significantly by Sasarmanand co-workers, who identified a series of genes in E. coliwhose mutation resulted in respiratory deficiency owingto the inability to synthesize heme [6]. These results, andsimilar results obtained with yeast (e.g. [7]), led to theidentification of genes for each of the enzymes in thepathway from the universal tetrapyrrole precursor,5-aminolevulinate, to the last common precursor of hemeand chlorophyll, protoporphyrin IX, as well as that for theenzyme that inserts iron into protoporphyrin IX to formheme. Later genetic results confirmed enzyme-basedconclusions that E. coli, as well as most other bacteriaand all plants, synthesize 5-aminolevulinate from gluta-mate by a different pathway, catalyzed by differentenzymes than those of yeast and animals, which form5-aminolevulinate from succinate and glycine [8].

Mutant strains of E. coli and yeast with defects inspecific steps of tetrapyrrole synthesis have been aninvaluable resource for obtaining genes for the corre-sponding steps from other species by complementationcloning. One important motivation for pursuing studies onthe genetics of heme synthesis was to understand themolecular bases for a group of inherited metabolicdiseases of heme metabolism in humans, collectivelyknown as porphyrias.

Chlorophyll synthesis branch

A major advance toward understanding the moleculargenetics of chlorophyll synthesis was the identification of alarge ‘photosynthetic gene cluster’ in the photosyntheticbacterium Rhodobacter capsulatus. This cluster containsmost or all the genes for the structural components ofthe photosynthetic apparatus as well as those that encodeenzymes for all the biosynthetic steps between proto-porphyrin IX and bacteriochlorophyll [9]. By systematicinactivation of these genes and examination of the mutantphenotypes, most of the biosynthetic genes were matched

Update TRENDS in Plant Science Vol.10 No.7 July 2005312

with specific enzymes [10]. Later, homologs of many ofthese genes were found in plants [11].

Significance of DVR

In addition to the general satisfaction provided by Tanakaand colleagues completing the list of genes for chlorophyllsynthesis, what have we learned? First, mutation ofAt5G18660 in Arabidopsis results in the completeabolition of monovinyl chlorophylls, which implies thatthis species contains only one functional DVR. Thisconclusion conflicts with earlier hypotheses that invokemultiple DVR enzymes to account for the different relativecellular abundances of monovinyl and divinyl protochloro-phyllides and chlorophyllides in rapidly greening tissuesof different plant species [12]. Second, some organismscontain monovinyl chlorophylls and thus presumablyhave DVR enzymes even though they apparently lackhomologs to At5G18660. For example, R. capsulatus doesnot contain an At5G18660 homolog but it does contain adifferent gene that might code for a DVR [10]. Thisindicates that different species can use divergent DVRenzymes to catalyze the vinyl reduction reaction.

What else is there to discover?

In addition to the likely existenceofalternate DVR-encodinggenes mentioned above, there are other loose ends in thegenetics of chlorophyll synthesis, particularly for non-angiosperm organisms. First, several chlorophyll syn-thetic steps in plants and algae (Figure 1, steps 7, 8 and 11)require O2 but anaerobic photosynthetic bacteria synthe-size (bacterio)chlorophylls in the absence of O2. In somebut not in all cases, alternative enzymes for the anaerobicreactions, encoded by different genes, have been identi-fied. Second, although angiosperms require light forchlorophyll synthesis, most other plants, as well as mostalgae and all photosynthetic bacteria (including cyano-bacteria), do not require light because they contain alter-native enzymes to circumvent the step catalyzed bylight-dependent NADPH:protochlorophyllide oxidoreduc-tase (Figure 1, step 13). In eukaryotic species, the genesfor the light-independent reduction enzyme are containedin the plastid genome, whereas the genes for all otherenzymes of chlorophyll synthesis are contained in thenucleus. However, in the alga Chlamydomonas reinhardtii,several nuclear genes are required for light-independentchlorophyll synthesis [13]. The functions of these genesremain to be determined. Finally, several other genes andgene products in plants and algae have recently been

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described that, although not strictly required for chloro-phyll synthesis, greatly affect the rate of chlorophyllformation and the regulation of this process [14,15]. It islikely that further studies of chlorophyll synthesis willyield additional genes that have an important bearing onthis process; these genes will need to be incorporated intothe conceptual framework to achieve a better under-standing of this biosynthetic activity that is fundamentalto all photosynthetic organisms.

AcknowledgementsI thank Luiza A. Nogaj for helpful comments on the manuscript.

References

1 Bazzaz, M.B. et al. (1982) 4-Vinyl-4-desethyl chlorophyll a: character-ization of a new naturally occurring chlorophyll using fast atombombardment, field desorption and “in beam” electron impact massspectroscopy. Tetrahedron Lett. 23, 1211–1214

2 Goericke, R. and Repeta, D.J. (1992) The pigments of Prochlorococcusmarinus: the presence of divinylchlorophyll a and b in a marineprocaryote. Limnol. Oceanogr. 37, 425–433

3 Nagata, N. et al. (2004) Identification of a vinyl reductase gene forchlorophyll synthesis in Arabidopsis thaliana and implications for theevolution of Prochlorococcus species. Plant Cell 17, 233–240

4 Tomas, H. et al. (1996) Gregor Mendel’s green and yellow pea seeds.Bot. Acta 109, 3–4

5 Granick, S. (1950) The structural and functional relationshipsbetween heme and chlorophyll. Harvey Lect. 44, 220–245

6 Sasarman, A. et al. (1968) Hemin-deficient mutants of Escherichia coliK-12. J. Bacteriol. 96, 570–572

7 Amillet, J-M. and Labbe-Bois, R. (1995) Isolation of the gene HEM4encoding uroporphyrinogen III synthase in Saccharomyces cerevisiae.Yeast 11, 419–424

8 Li, J-M. et al. (1989) Cloning and structure of the hemA gene ofEscherichia coli K-12. Gene 82, 209–217

9 Zsebo, K.M. and Hearst, J.E. (1984) Genetic-physical mapping of aphotosynthetic gene cluster from R. capsulata. Cell 37, 937–947

10 Bollivar, D.W. et al. (1994) Directed mutational analysis of bacterio-chlorophyll a biosynthesis in Rhodobacter capsulatus. J. Mol. Biol.237, 622–640

11 Jensen, P.E. et al. (1996) Structural genes for Mg-chelatase subunits ofbarley: Xantha-f, -g and -h. Mol. Gen. Genet. 250, 383–394

12 Parham, R. and Rebeiz, C.A. (1992) Chloroplast biogenesis: [4-vinyl]chlorophyllide a reductase is a divinyl chlorophyllide a-specific,NADPH-dependent enzyme. Biochemistry 31, 8460–8464

13 Ford, C. and Wang, W-y. (1980) Temperature-sensitive yellow mutantsof Chlamydomonas reinhardtii. Mol. Gen. Genet. 180, 5–10

14 Larkin, R.M. et al. (2003) GUN4, a regulator of chlorophyll synthesisand intracellular signaling. Science 299, 902–906

15 Meskauskiene, R. et al. (2001) FLU: a negative regulator ofchlorophyll biosynthesis in Arabidopsis thaliana. Proc. Natl. Acad.Sci. U. S. A. 98, 12826–12831

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doi:10.1016/j.tplants.2005.05.005