PORA and PORB, Two Light-Dependent ... - The Plant Cell

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 ternary
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 begins
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 requirement
for but also its sensitivity to light-scientists have made
great effort to purify this protein and to characterize its catalytic
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 Synechocysfis
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 detectable
(Figure 2, P~hlide~~~-~~~
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
Chlide
(672-680)
POR : NADP' : Chlide
(678-690)
POR : NADPH : Chlide NADP+
(684-696)
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 participating
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 particular
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 polypeptide
in vivo. Its release is a relatively slow process, as seen
by the Shibata shift (Shibata, 1957), that is, a spectral change
Of chlide684-696 nm to Chlides72-680 nm (Figure 2). It was SUggested
that after the reiease of Chlide, a new molecule of
Pchlide binds to the POR-NADPH complex to re-form the photoactive
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 protein
of barley do not appear to support this explanation.

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
",) 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-
Spotlight on POR Function 765
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).
To accomplish its role in the buildup of the photosynthetic apparatus
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 labeled
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 capability
to sequester pPORA(Reinbothe et al., 1995a, 1995~). Due
to the depletion in the pool of translocation-active Pchlide, chloroplasts
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 carboxylase/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 organelles
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 (summarized
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 levels
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
pPOR
POR
EP
bed
ECP
a b e d
CP
abed
- I
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 had
been exposed to light for 30 min, and light-grown seedlings, respectively.
Each of the various plastid samples was then divided into two
equal parts. One half was incubated with 5-aminolevulinic acid (0.5
mM final concentration); the other half was incubated with phosphate
buffer alone instead of 5-aminolevulinic acid. The different plastids
were then incubated with the radioactively labeled pPORA of barley
synthesized by coupled in vitro transcription/translation of the porAspecific
cDNA clone A7 (Schulz et al., 1989) in the dark for 15 min.
35 S-methionine-labeled pPORA (pPOR) molecules that had not been
sequestered by the plastids and thus remained in the supernatant after
centrifugation of the assays were precipitated with trichloroacetic
acid, separated electrophoretically, and detected by autoradiography
(top). Similarly, the mature radiolabeled PORA (POR) was recovered
from thermolysin-treated, intact plastids by sonication and precipitation
with trichloroacetic acid and was run in a separate denaturing
polyacrylamide gradient and detected by autoradiography (bottom).
Shown are the levels of the 44-kD pPORA and 36-kD PORA, each
before (lanes a and c) and after (lanes b and d) incubation with plastids
containing (lanes a and b each) or lacking (lanes c and d each) the
exogenous 5-aminolevulinic acid-derived Pchlide. Note that etioplasts
and etiochloroplasts, due to their high endogenous level of Pchlide,
sequester pPORA in the absence of additional Pchlide produced by
5-aminolevulinic acid feeding. By contrast, chloroplasts, which lack
translocation-active Pchlide, can import pPORA only when their endogenous
level of Pchlide is raised by 5-aminolevulinic acid feeding.
in chloroplasts. This conclusion is valid not only for barley but
also for chloroplasts from pea, wheat, and Arabidopsis (C.
Reinbothe, S. Reinbothe, and K. Apel, unpublished data).
Further evidence for operation of the Pchlide-dependent
plastid import pathway of the pPORA in vivo is provided by
protein gel blot analyses that demonstrate the presence of a
44-kD protein among plastid proteins of barley seedlings that
had been exposed to light for 8 hr (S. Reinbothe, C. Reinbothe,
D. Neumann, and K. Apel, submitted manuscript). This 44-kD
protein is likely to represent pPORA that, due to the depletion
in the pool of translocation-active Pchlide within the etiochloroplasts,
accumulates at the outer plastid envelope. When
Pchlide formation is induced by feeding the plastids 5-aminolevulinic
acid, pPORA is chased into the organelles, processed
to mature size, and targeted to the thylakoids.
Remarkably, the PORA also can be released from the plastid
envelope if Pchlide is supplied from the outside of the etiochloroplasts.
However, this substrate-induced release of
pPORA was not observed in the presence of a cytosolic 70kD
heat shock cognate protein (Hsc70) that is contained in
wheat germ extracts added to the incubation mixtures. By analogy
with other members of the chaperonin family, the cytosolic
Hsc70 appears to stabilize a transport-competent conformation
of the envelope-bound pPORA.
When enzymatic Chlide formation was induced by incubating
the in vitro-synthesized pPORA with Pchlide and NADPH
in the light before import, the ATP-dependent binding of the
precursor protein to the chloroplasts was abolished (Reinbothe
etal., 1995b). In vitro processing experiments with leader peptidase
preparations further demonstrated that Chlide causes
a conformational change through which the transit peptide of
the pPORA is rendered inaccessible to interact with the plastid
envelope (Reinbothe et al., 1995b). Hence, Hsc70 does not
seem able to denature the tightly folded pPORA-Chlide complex
in vitro. Unfolding of pPORA is likely to occur not before
but rather during its translocation (Reinbothe et al., 1995b).
The actual driving forces for translocation appear to be ATP
and the interaction between intraplastidic Pchlide and pPORA.
We assume that there are three binding sites for Pchlide in
pPORA: one in the transit peptide and two in the mature part
of the protein. Alternative binding of Pchlide to these three pigment
binding sites appears to determine the direction in which
the unfolded precursor will move.
The Pchlide-dependent import of the precursor into the
plastids appears to be devoted to the specific task of supplying
the prolamellar body with both PORA and Pchlide. In
addition, the operation of this import pathway has been proposed
to be part of a protective mechanism through which
plants lower the risk that excited Pchlide molecules will cause
photooxidation of the plastid compartment during the very early
stages of transition from etiolated to light growth (Reinbothe
et al., 1995a, 1995b). By contrast, the physiological significance
of the exogenous Pchlide-induced release of the envelopebound
pPORA to the cytosol is as yet undetermined. It is
conceivable, however, that it might be part of a regulatory mechanism
through which nuclear gene expression can be
controlled by tetrapyrrole signals that are liberated from the
plastid.
Light-Depressed porA Gene Transcription
The rapid degradation of PORA-Chlide complexes and the
impairment of pPORA's plastid import appear to be only two
of three mechanisms through which plants confine the function
of PORA to the very early stages of the light-induced
greening. The third level of control is that of porA gene transcription.
The port gene is active only in etiolated seedlings,
but its transcription declines soon after the beginning of illumination
(Apel, 1981; Batschauer and Apel, 1984; Mosinger

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 differentiation
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 imported
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 operative
in green plants. PORB is closely related to PORA in its
predicted amino acid sequence as well as in its catalytic properties
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 require
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 illumination
(Reinbothe et al., 1995~).
Spotlight on POR Function 767
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 development.
In particular, this approach could be exploited to
investigate the effects of engineered mutations on PORA protein
function.
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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PORA and PORB, Two Light-Dependent Protochlorophyllide-Reducing Enzymes of Angiosperm
Chlorophyll Biosynthesis.
S. Reinbothe, C. Reinbothe, N. Lebedev and K. Apel
Plant Cell 1996;8;763-769
DOI 10.1105/tpc.8.5.763
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