The Plant Cell, Vol. 8, 763-769, May 1996 O 1996 American Society of Plant Physiologists

REVIEW ARTICLE

PORA and PORB, Two Light-Dependent Protochlorophyllide-Reducing Enzymes of Angiosperm Chlorophyll Biosynthesis

Steffen Reinbothe,ai’ Christiane Reinbothe,a Nikolai Lebedev,b and Klaus Apela a lnstitute for Plant Sciences, Department of Genetics, Swiss Federal lnstitute of Technology Zurich (ETH), ETH-Zentrum, Universitatsstrasse 2, CH-8092 Zurich, Switzerland

A.N. Bakh lnstitute of Biochemistry, Russian Academy of Sciences, Leninsky pr 33, Moscow 117071, Russia

INTRODUCTION

Chloroplasts are the most abundant organelles in a plant cell. Besides their familiar roles in photosynthesis, chloroplasts also perform important functions during numerous other metabolic processes, such as nitrogen assimilation, amino acid biosynthesis, and tetrapyrrole production. ln particular, a major route of chloroplast anabolism is devoted to the syn- thesis of chlorophyll (Chl; von Wettstein et al., 1995). As the “pigment of life,” Chl plays a fundamental role in the energy absorption and transduction activities of all photosynthetic organisms (Bogorad, 1967; von Wettstein et al., 1971).

In angiosperms, Chl synthesis is dependent on light (Granick, 1950). NADPH:protochlorophyllide oxidoreductase (POR; EC 1.3.1.33) catalyzes the only known light-requiring step of tetrapyrrole biosynthesis, the reduction of proto- chlorophyllide (Pchlide) to chlorophyllide (Chlide) (Griffiths, 1978; Apel et al., 1980). Both the POR(A) enzyme and its substrate, Pchlide, accumulate to high levels in dark-grown plants. Together with NADPH, they form a ternary complex that instantaneously converts Pchlide to Chlide when illuminated. The operation of PORA hence helps prevent photodynamic damage by large amounts of not immediately photoconvert- ible Pchlide.

The function of PORA is confined to the very early stages of transition from etiolated to light growth (Apel, 1981; Batschauer and Apel, 1984; Mosinger et al., 1985; Forreiter et al., 1990). The amounts of both PORA protein and porA mRNA decrease drastically soon after the beginning of illumi- nation (Mapleston and Griffiths, 1980; Santel and Apel, 1981; Forreiter et al., 1990), due in part to rapid proteolytic turnover of the enzyme protein (Kay and Griffiths, 1983; Hauser et al., 1984).

To perform the reduction of Pchlide to Chlide during the fi- nal stages of the light-induced greening and to sustain Chl

To whom correspondence should be addressed

synthesis in mature leaves, a second Pchlide-reducing en- zyme has to operate in angiosperms. This enzyme, termed PORB, is active under all tested conditions and drives Chlide synthesis particularly in green plants (Holtorf et al., 1995). This review summarizes our current knowledge of the different POR enzymes in angiosperms, their differential expression in response to light, and their putative roles in Chl biosynthe- sis and chloroplast development.

PORA-THE SHORT LlFETlME OF AN EXTRAORDINARY ENZYME

PORA-A “Suicidal” Enzyme?

In higher plants, the synthesis and assembly of Chl (used to refer collectively to Chls a and b) into the two photosyntheti- cally active thylakoid membrane complexes are regulated by light (Bogorad, 1967; von Wettstein et al., 1971, 1995). Chl is controlled at multiple levels in both the nucleocytoplasmic and plastid compartments and includes processes such as tran- scription, post-transcriptional RNA processing and modification, and post-translational protein import and protein stabilization (van Grinsven and Kool, 1988; Thompson and White, 1991). The establishment of the photosynthetic apparatus during the transition from dark (etiolated) to light growth leads to the visi- ble greening of the plant (Thorne, 1971). During this process, etioplasts differentiate into chloroplasts (Virgin et al., 1963; Henningsen, 1970; Kirk and Tilney-Basset, 1978). Simultane- ously, leaf development proceeds (Mullet, 1988).

When angiosperm seedlings are grown in darkness, plastid development is arrested at an early stage, giving rise to the etioplast (Virgin et al., 1963). Within this organelle, the prolamel- lar body forms a paracrystalline structure (Virgin et al., 1963; Henningsen, 1970) composed of lipids and proteins (HByer- Hansen and Simpson, 1977). Among these macromolecules,

764 The Plant Cell

the one protein that predominares is PORA (Ikeuchi and Murakami, 1983; Dehesh and Ryberg, 1985). Together with its two substrates, Pchlide and NADPH, PORA forms a ter- nary complex (Griffiths, 1978; Apel et al., 1980). When illuminated, PORA photoreduces its Pchlide to Chlide (Griffiths, 1978; Apel et al., 1980; Mapleston and Griffiths, 1980; Santel and Apel, 1981), and simultaneously, the prolamellar body be- gins to disintegrate (Virgin et al., 1963; Henningsen, 1970). During catalysis, PORA is inactivated and subsequently degraded (Santel and Apel, 1981; Kay and Griffiths, 1983; Hauser et al., 1984; Forreiter et al., 1990).

Attracted by the extraordinary behavior of PORA- its require- ment for but also its sensitivity to light-scientists have made great effort to purify this protein and to characterize its cata- lytic properties (summarized in Schulz and Senger, 1993). These studies were reinforced by the recent cloning of POR cDNAs from both angiosperms, such as barley (Schulz et al., 1989; Holtorf et al., 1995), wheat (Teakle and Griffiths, 1993), oat (Darrah et al., 1990), pea (Spano et al., 1992a), and Arabidopsis (Benli et al., 1991; Armstrong et al., 1995), and gymnosperms, such as pine (Spano et al., 1992b; Forreiter and Apel, 1993), as well as from cyanobacteria, such as Syn- echocysfis sp strain PCC 6803 (Suzuki and Bauer, 1995).

The reaction catalyzed by POR is a ffans-reduction of the double bond in ring D of the tetrapyrrole ring system (Begley and Young, 1989), as shown in Figure 1. This reaction requires NADPH as cosubstrate as well as light (Griffiths, 1978; Apel et al., 1980; Santel and Apel, 1981). During catalysis, POR first binds NADPH, then Pchlide. A ternary POR-Pchlide-NADPH complex is formed. This complex is spectroscopically detect- able (Figure 2, P ~ h l i d e ~ ~ ~ - ~ ~ ~ nm) in prolamellar bodies of wheat and barley etioplasts (Griffiths, 1978; Oliver and Griffiths, 1982) and in greening barley leaves (Franck and Strzalka, 1992), and can also be reconstituted in vitro either with detergent-solubilized enzymes, such as those from wheat or barley etioplasts (Griffiths, 1978; Apel et al., 1980; Santel and Apel, 1981; Oliver and Griffiths, 1982; Schoch et al., 1995), or with the cDNA-encoded, in vitro-synthesized PORA precur-

NADPH, Light

POR

PrOlochiomphyllide Chloiophyliide

Figure 1. The Reaction Catalyzed by POR

POR catalyzes the only known light-requiring step of tetrapyrrole bio- synthesis, the NADPH-dependent trans-reduction of the double bond in ring D of Pchlide to Chlide.

POR : NADPH : Pchlide Pchlide (650-657)

i 6 2 8 - 6 3 2 ) Y

POR : NADPH POR : NADP' : Chlide (678-690)

POR : NADPH : Chlide NADP+ (684-696)

Chlide (672-680)

I b, Degradation

Figure 2. Steps in the Reaction Mechanism Catalyzed by POR.

During POR-driven Pchlide reduction to Chlide, several intermediate steps can be distinguished spectroscopically. Although the PORA en- zyme appears to be used repeatedly for catalysis in etiolated plants at the very beginning of illumination (route a), a light-induced protease degrades freshly formed PORA-NADPH-Chlide complexes in etio- chloroplasts and also in chloroplasts (route b).

sor protein of barley (Holtorf et al., 1995; Reinbothe et al., 1995a, 1995b).

Based on sequence comparison with other proteins found in the data banks, it has been proposed (Wilks and Timko, 1995) that NADPH binds to a region of the POR polypeptide that is similar to that of short-chain alcohol dehydrogenases (Baker, 1994). Active site residues in the pea enzyme, such as Tyr- 225 and Lys-279, are involved in NADPH binding (Wilks and Timko, 1995), whereas two or even three of the evolutionarily conserved cysteine residues have been implicated as par- ticipating in Pchlide binding and/or catalysis (Oliver and Griffiths, 1981; Dehesh et al., 1986; Teakle and Griffiths, 1993). Dueto the presence of the tetrapyrrole ring system, the ternary POR-Pchlide-NADPH complex is able to absorb light, in par- ticular blue light, and thus is photoactive (summarized in Ryberg and Sundqvist, 1991).

Light absorption sets in motion a series of spectral changes that reflect individual steps in enzyme catalysis, as shown in Figure 2 (Oliver and Griffiths, 1981; Franck and Strzalka, 1992). In contrast to NADP+, which is rapidly displaced by fresh NADPH, Chlide appears to remain bound to the POR poly- peptide in vivo. Its release is a relatively slow process, as seen by the Shibata shift (Shibata, 1957), that is, a spectral change

gested that after the reiease of Chlide, a new molecule of Pchlide binds to the POR-NADPH complex to re-form the pho- toactive ternary complex (Figure 2, route a; Oliver and Griffiths, 1982). By proceeding repeatedly through this cycle of substrate binding, product formation, and product release, POR has been proposed to be used several times for catalysis (Oliver and Griffiths, 1982), as expected for an ordinary enzyme.

Recent results obtained for the cDNA-encoded PORA pro- tein of barley do not appear to support this explanation.

Of chlide684-696 nm to Chlides72-680 nm (Figure 2). It was SUg-

Spotlight on POR Function 765

Chlorophyllide is not easily dissociated from the enzyme (Reinbothe et al., 1995b). Rather, the pigment remains tightly bound to PORA and can be released only by denaturation, such as heating or treatment with SDS (Reinbothe et al., 1995b), suggesting that each PORA molecule may be used for catalysis only once. Hence, PORA might be regarded as a suicida1 enzyme.

In contrast to this view, Nielsen (1974, 1975) has provided evidence that Pchlide may replace Chlide from the substrate

ments (Apel and Kloppstech, 1980; Bennett, 1981; Kim et al., 1994). It might also be that such Chlide-containing peptides of PORA can regulate the whole assembly pathway of the pho- tosynthetically active thylakoid membrane complexes. In particular, synthesis of the plastid-encoded Chl binding pro- teins has been suggested to be regulated cotranslationally (for a critical discussion, see Kim et al., 1994).

",,,) exists, Chlide does not leave the enzyme, but it can do so if free Pchlide is produced by feeding the pig- ment precursor 5-aminolevulinic acid. Similarly, excess Pchlide that accumulates in the dark in the figrina 034 mutant of bar- ley can be successively converted to Chlide by short light flashes'at 6 to 10°C (Nielsen, 1974). Because there is a pool of photoinactive Pchlide also in wild-type etioplasts that can replace Chlide from the PORA enzyme, one may conclude that PORA, at least during the transition from dark to light growth, can be used severa1 times for subsequent rounds of catalysis.

Proteolytic Degradation of PORA

Pioneering studies with POR(A) have suggested that photocon- version of Pchlide and transformation of the prolamellar body may represent a single photodependent process (Kahn, 1968). Re-formation of POR(A)-Pchlide-NADPH complexes, which would stabilize the prolamellar body, thus does not seem to occur in vivo. Instead, proteolytic degradation of the POR(A)- Chlide complex(Figure2, route b) maycontribute to the disin- tegration of the prolamellar body. In line with this possibility, PORA-Chlide complexes were found to be highly suscepti- ble to degradation by plastid proteases in vitro and in organello, whereas PORA-substrate complexes, such as PORA-Pchlide or PORA-Pchlide-NADPH, were protease resistant (Reinbothe et al., 1995; Reinbothe et al., 1995~).

Although these findings may imply an involvement of pro- teolysis in the breakdown of the prolamellar body, there is one important argument against this possibility. The protease ac- tivity that degrades POR is not active in etioplasts (Reinbothe et al., 1995; Reinbothe et al., 199%). As a nuclear gene prod- uct, it appears in the light (Reinbothe et al., 1995; Reinbothe et al., 1995c) and reaches its final activity only when the disin- tegration of the prolamellar body has been completed (Virgin et al., 1963; Henningsen, 1970). Despite this fact, it has been speculated that the proteolytic degradation of PORA can have an important physiological meaning in vivo (Kay and Griffiths, 1983; Hauser et al., 1984). Proteolytic fragments of PORA, such as those generated in vitro (Reinbothe et al., 1995), can serve as vehicles for the transfer of freshly formed Chlide to the de- veloging thylakoids. 60th nuclear- and plastid-encoded Chl binding proteins are stable only in the presence of their pig-

To accomplish its role in the buildup of the photosynthetic ap- paratus during the very early stages of transition from etiolated to light growth, PORA would have to interact with Pchlide and photoreduce the pigment within the prolamellar body close to the developing thylakoids. Interestingly, plastids solve this problem by coupling the import of the cytosolic precursor of PORA (pPORA) to the supply of Pchlide (Reinbothe et al., 1995a, 1995b).

In vitro import studies performed with the radioactively la- beled PORA precursor polypeptide of barley synthesized by coupled transcriptionhranslation of a porkspecific cDNA (Schulz et al., 1989) and isolated plastids show that etioplasts efficiently sequester pPORA (Figure 3). Pchlide inside the etioplasts is required for the import of the pPORA and its processing to mature size. During the step of precursor translocation, pPORA binds Pchlide; then the transit peptide is removed, followed by targeting of the ternary PORA-Pchlide-NADPH complex to the prolamellar body (Reinbothe et al., 1995a).

Soon after the beginning of illumination, the leve1 of Pchlide declines drastically (Sundqvist, 1974; Mapleston and Griffiths, 1980), and the developing etiochloroplasts lose their capabil- ity to sequester pPORA(Reinbothe et al., 1995a, 1995~). Due to the depletion in the pool of translocation-active Pchlide, chlo- roplasts can no longer import pPORA (Figure 3). However, they still can import other precursor proteins, such as the precursor of the small subunit of ribulose-l,5-bisphosphate carboxy- lase/oxygenase and a dihydrofolate reductase reporter protein of mouse fused to the transit peptide of plastocyanin of Silene prafensis (Reinbothe et al., 1995a). When such chloroplasts are supplied with Pchlide, for example, by incubating the or- ganelles with 5-aminolevulinic acid, a precursor of Pchlide, their import capacity for pPORA can be restored (Figure 3). Similarly, mutants of barley that are defective in the regulation of chlorophyll biosynthesis, such as the figrina mutants (sum- marized in Henningsen et al., 1993), import pPORA under conditions that lead to excess plastidic Pchlide accumulation (Reinbothe et al., 1995~).

In wild-type barley plants grown in dark and light cycles, the transport competence of the chloroplasts for pPORA is at a minimum during the last hours of the day, when Pchlide lev- els are low, but is considerable at the end of the night, when Pchlide has accumulated substantially (Reinbothe et al., 1995a). Hence, one can conclude that the Pchlide-dependent plastid import pathway of the pPORA still remains operative

766 The Plant Cell

EPb e d

ECPa b e d

CPa b e d

- IpPOR

POR

Figure 3. The Substrate Dependency of pPORA's Plastid Import.

Barley etioplasts (EP), etiochloroplasts (ECP), and chloroplasts (CP)were isolated from etiolated seedlings, dark-grown seedlings that hadbeen exposed to light for 30 min, and light-grown seedlings, respec-tively. Each of the various plastid samples was then divided into twoequal parts. One half was incubated with 5-aminolevulinic acid (0.5mM final concentration); the other half was incubated with phosphatebuffer alone instead of 5-aminolevulinic acid. The different plastidswere then incubated with the radioactively labeled pPORA of barleysynthesized by coupled in vitro transcription/translation of the porA-specific cDNA clone A7 (Schulz et al., 1989) in the dark for 15 min.35S-methionine-labeled pPORA (pPOR) molecules that had not beensequestered by the plastids and thus remained in the supernatant af-ter centrifugation of the assays were precipitated with trichloroaceticacid, separated electrophoretically, and detected by autoradiography(top). Similarly, the mature radiolabeled PORA (POR) was recoveredfrom thermolysin-treated, intact plastids by sonication and precipita-tion with trichloroacetic acid and was run in a separate denaturingpolyacrylamide gradient and detected by autoradiography (bottom).Shown are the levels of the 44-kD pPORA and 36-kD PORA, eachbefore (lanes a and c) and after (lanes b and d) incubation with plastidscontaining (lanes a and b each) or lacking (lanes c and d each) theexogenous 5-aminolevulinic acid-derived Pchlide. Note that etioplastsand etiochloroplasts, due to their high endogenous level of Pchlide,sequester pPORA in the absence of additional Pchlide produced by5-aminolevulinic acid feeding. By contrast, chloroplasts, which lacktranslocation-active Pchlide, can import pPORA only when their en-dogenous level of Pchlide is raised by 5-aminolevulinic acid feeding.

in chloroplasts. This conclusion is valid not only for barley butalso for chloroplasts from pea, wheat, and Arabidopsis (C.Reinbothe, S. Reinbothe, and K. Apel, unpublished data).

Further evidence for operation of the Pchlide-dependentplastid import pathway of the pPORA in vivo is provided byprotein gel blot analyses that demonstrate the presence of a44-kD protein among plastid proteins of barley seedlings thathad been exposed to light for 8 hr (S. Reinbothe, C. Reinbothe,D. Neumann, and K. Apel, submitted manuscript). This 44-kDprotein is likely to represent pPORA that, due to the depletionin the pool of translocation-active Pchlide within the etio-chloroplasts, accumulates at the outer plastid envelope. WhenPchlide formation is induced by feeding the plastids 5-amino-levulinic acid, pPORA is chased into the organelles, processedto mature size, and targeted to the thylakoids.

Remarkably, the PORA also can be released from the plastidenvelope if Pchlide is supplied from the outside of the etio-chloroplasts. However, this substrate-induced release ofpPORA was not observed in the presence of a cytosolic 70-kD heat shock cognate protein (Hsc70) that is contained inwheat germ extracts added to the incubation mixtures. By anal-ogy with other members of the chaperonin family, the cytosolicHsc70 appears to stabilize a transport-competent conforma-tion of the envelope-bound pPORA.

When enzymatic Chlide formation was induced by incubat-ing the in vitro-synthesized pPORA with Pchlide and NADPHin the light before import, the ATP-dependent binding of theprecursor protein to the chloroplasts was abolished (Reinbotheetal., 1995b). In vitro processing experiments with leader pep-tidase preparations further demonstrated that Chlide causesa conformational change through which the transit peptide ofthe pPORA is rendered inaccessible to interact with the plastidenvelope (Reinbothe et al., 1995b). Hence, Hsc70 does notseem able to denature the tightly folded pPORA-Chlide com-plex in vitro. Unfolding of pPORA is likely to occur not beforebut rather during its translocation (Reinbothe et al., 1995b).

The actual driving forces for translocation appear to be ATPand the interaction between intraplastidic Pchlide and pPORA.We assume that there are three binding sites for Pchlide inpPORA: one in the transit peptide and two in the mature partof the protein. Alternative binding of Pchlide to these three pig-ment binding sites appears to determine the direction in whichthe unfolded precursor will move.

The Pchlide-dependent import of the precursor into theplastids appears to be devoted to the specific task of supply-ing the prolamellar body with both PORA and Pchlide. Inaddition, the operation of this import pathway has been pro-posed to be part of a protective mechanism through whichplants lower the risk that excited Pchlide molecules will causephotooxidation of the plastid compartment during the very earlystages of transition from etiolated to light growth (Reinbotheet al., 1995a, 1995b). By contrast, the physiological significanceof the exogenous Pchlide-induced release of the envelope-bound pPORA to the cytosol is as yet undetermined. It isconceivable, however, that it might be part of a regulatory mech-anism through which nuclear gene expression can becontrolled by tetrapyrrole signals that are liberated from theplastid.

Light-Depressed porA Gene Transcription

The rapid degradation of PORA-Chlide complexes and theimpairment of pPORA's plastid import appear to be only twoof three mechanisms through which plants confine the func-tion of PORA to the very early stages of the light-inducedgreening. The third level of control is that of porA gene tran-scription. The port gene is active only in etiolated seedlings,but its transcription declines soon after the beginning of illu-mination (Apel, 1981; Batschauer and Apel, 1984; Mosinger

Spotlight on POR Function 767

et al., 1985; Forreiter et al., 1990). Phytochrome, presumably the phytochrome A holocomplex, is responsible for porA gene inactivation (Batschauer and Apel, 1984; Mosinger et al., 1985). Conditions that favor the formation of the active phytochrome holocomplex, such as far-red light treatment, lead to a drastic reduction in porA gene transcription and porA transcript abun- dance (Batschauer and Apel, 1984; Mosinger et al., 1985).

In constitutive morphogenetic mutants of Arabidopsis, in which certain steps in the signal transduction pathway of phytochrome action are already active in the dark, such as in the deetiolated 1 (detl; Chory et al., 1989), constitutive pho- tomorphogenetic 9 (cop9; Wei and Deng, 1992), or det340 (Lebedev et al., 1995) mutants, noporA transcript was detected (Lebedev et al., 1995). Prolamellar bodies comparable to those in etiolated wild-type seedlings are lacking (Chory et al., 1989; Lebedev et al., 1995) or structurally impaired (Wei and Deng, 1992), PORA is absent, and normal greening, despite the pres- ente of the nuclear- and plastid-encoded light-harvesting Chl binding protein transcripts (Chory et al., 1989; Wei and Deng, 1992), is delayed (Lebedev et al., 1995). To pose theporA gene under the control of phytochrome thus appears to have a strong impact on the control of plastid development both in the dark and in the light.

PORB-THE "TRUE Pchlide-REDUCING ENZYME

Even though PORA's role in etioplast-to-chloroplast differenti- ation appears to be multivalent, its function during Chl synthesis in green plants is limited. Dueto the cumulative effect of light on the expression of the porA gene, on the plastid import of the cytosolic precursor protein, and on the stability of the im- ported enzyme, PORA disappears from illuminated seedlings long before Chl accumulation has reached its maximum rate. Thus, another Pchlide-reducing enzyme must exist that drives Chl synthesis in the absence of PORA. Particularly in green plants, there is a continuous need to replace those pigment molecules that become damaged during light trapping and energy transduction (see, e.g., De Las Rivas et al., 1993).

The recently discovered PORB (Holtorf et al., 1995) in fact is a likely candidate for a Pchlide-reducing enzyme that is oper- ative in green plants. PORB is closely related to PORA in its predicted amino acid sequence as well as in its catalytic prop- erties as a light- and NADPH-dependent enzyme (Holtorf et al., 1995). Thepor6 gene is active both in the dark and in the light and does not respond to phytochrome (Holtorf et al., 1995). Both por6 mRNA and PORB protein accumulate in etiolated, illuminated, and light-adapted plants (Holtorf et al., 1995). Although the plastid import pathway of PORB does not re- quire Pchlide, PORB is still able to interact with the pigment (Reinbothe et al., 1995~). PORB imported into chloroplasts in vitro is stable in the dark, demonstrating its binding to Pchlide and NADPH, but its leve1 declines within a few minutes of illu- mination (Reinbothe et al., 1995~).

Light-driven enzymatic product formation destines PORB for degradation by plastid proteases. In vivo, pronounced fluc- tuations in PORB protein levels must thus be expected to occur in seedlings grown under alternating dark and light cycles, that is, under conditions reflecting those in nature. However, such oscillations have not been observed in previous experiments (Holtorf et al., 1995). Oscillations in porB mRNA concentra- tion with a maximum during the day and a minimum during the night appear to compensate for the preferential loss of PORB in the light (Holtorf et al., 1995). Changes in the activity of the POR-degrading protease seem also to modulate the ac- tua1 PORB enzyme concentration. Dueto its ATP requirement and pH optimum at pH 6.5 (Reinbothe et al., 1995), the POR- degrading protease is expected to be most active in the early hours of the day (before maximal levels of porB mRNA drive full PORB protein synthesis) but apparently would be inactive throughout the early night.

CONCLUSIONS AND PERSPECTIVES

The results reviewed in this article show that Chl synthesis in angiosperms is controlled by two different light-dependent POR enzymes. Both PORA and PORB are light- and NADPH- dependent Pchlide-reducing enzymes. As nuclear gene products, the cytosolic precursors of PORA and PORB are transported post-translationally into the plastids. Although the translocation of pPORA requires Pchlide, that of pPORB does not. Etioplasts containing translocation-active Pchlide can im- port pPORA, but chloroplasts cannot. However, both plastid types can sequester equally well pPORB and other cytosolic precursor proteins.

Based on sequence comparison, we postulate that the differ- ence in the plastid import pathways of pPORA and pPOR6 could be due to the existence of a Pchlide binding site in pPORA's transit peptide. This pigment binding domain could interact with Pchlide that is formed in the plastid envelope (Pineau et al., 1986, 1993). Pchlide binding to pPORA's transit peptide would thus induce the translocation step. However, dueto the postulated lack of such a pigment binding domain in its transit peptide, pPOR6 would be imported into the plastids in a Pchlide-independent manner.

In addition to these differences in protein translocation across the plastid envelope, PORA and PORB are also distinct in their expression patterns. Although theporA gene is active only tran- siently in etiolated seedlings at the beginning of illumination, the por6 gene is operative also in green plants. This implies that the porA and por6 genes may contain different cis- regulatory elements in their promoters.

Studies with transgenic plants to investigate the actual func- tions of PORA and PORB are very appealing. In particular, mutants expressing virtually no porA mRNA, such as porA gene-deficient mutants or mutants that mimic the presence of the functional phytochrome holocomplex, could be used as

768 The Plant Cell

a background for functional complementation and to study the role of the pPORA for Chl biosynthesis and chloroplast devel-

investigate the effects of engineered mutations on PORA pro- tein function.

Darrah, EM., Kay, S.A., Teakle, J.R., and Griffiths, W.T. (1990). Clon- ing and sequencing of protochlorophyllide reductase. Biochem. J.

Dehesh, K., and Ryberg, M. (1985). The NADPH-protochlorophyllide oxidoreductase is the major protein constituent of prolamellar bod- ies in wheat (7iiticum aestivum L.). Planta 164, 396-399.

Dehesh, K., Klaas, M., Hauser, I., and Apel, K. (1986). Light-

opment. In particular, this approach could be exploited to 265, 789-798.

ACKNOWLEDGMENTS

We are grateful to members of the group, in particular to Dieter Rubli and Barbara van Cleve, for their help in preparing the figures.

Received November 10, 1995; accepted March 11, 1996

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DOI 10.1105/tpc.8.5.763 1996;8;763-769Plant Cell

S. Reinbothe, C. Reinbothe, N. Lebedev and K. ApelChlorophyll Biosynthesis.

PORA and PORB, Two Light-Dependent Protochlorophyllide-Reducing Enzymes of Angiosperm

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