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trends in plant science
perspectives
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30 Emons, A.M.C. (1989) Helicoidal microfibril
deposition in a tip-growing cell and
microtubules alignment during tip
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31 McCann, M.C. and Roberts, K. (1991)
Architecture of the primary cell wall. In
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Form (Lloyd, C.W., ed.), pp. 109–129, Academic
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33 Felle, H.H. and Hepler, P.K. (1997) The cytosolic
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ratio imaging. Plant Phys. 114, 39–45
34 De Ruijter, C.A. et al. (1998) Lipochitooligosaccharides re-initiate root hair tip
growth in Vicia sativa with high calcium and
spectrin-like antigen at the tip. Plant J. 13,
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35 Ehrhardt, D.W. et al. (1996) Calcium spiking
in plant root hairs responding to Rhizobium
nodulation signals. Cell 85, 573–681
Does a light-harvesting
protochlorophyllide a/bbinding protein complex exist?
Gregory A. Armstrong, Klaus Apel and Wolfhart Rüdiger
Recent in vitro studies have led to speculation that a novel light-harvesting
protochlorophyllide a/b-binding protein complex (LHPP) might exist in dark-grown
angiosperms. Structurally, it has been suggested that LHPP consists of a 5:1 ratio
of dark-stable ternary complexes of the light-dependent NADPH: protochlorophyllide
oxidoreductases A and B containing nonphotoactive protochlorophyllide b and
photoactive protochlorophyllide a, respectively. Functionally, LHPP has been
hypothesized to play major roles in establishing the photosynthetic apparatus, in
protecting against photo-oxidative damage during greening, and in determining
etioplast inner membrane architecture. However, the LHPP model is not compatible
with other studies of the pigments and the pigment–protein complexes of dark-grown
angiosperms. Protochlorophyllide b, which is postulated to be the major lightharvesting pigment of LHPP, has, for example, never been detected in etiolated
seedlings. This raises the question: does LHPP exist?
L
ight profoundly influences plant
development and allows photosynthesis to occur, but it also represents a
tremendous risk. Photo-oxidative damage
initiated by excited state photosensitizing
molecules, such as chlorophylls and their
biosynthetic precursors, can be lethal.
Angiosperms that germinate in darkness in
the soil enter the seedling developmental program known as skotomorphogenesis (Fig. 1).
However, such seedlings must be prepared
for a subsequent light-triggered switch to
photomorphogenesis. Upon illumination, the
leaves of etiolated angiosperms synthesize
and accumulate large quantities of chloro40
January 2000, Vol. 5, No. 1
phylls a and b. Seedlings are particularly susceptible to photo-oxidative damage during
this transition to photoautotrophy.
The presence or absence of light dramatically
influences plastid development. Dark-grown
angiosperm seedlings contain an achlorophyllous plastid type known as the etioplast, which
is transformed into a photosynthetically competent chloroplast during photomorphogenesis1.
The etioplast is defined by the presence of two
types of internal membranes, the lattice-like
prolamellar body, which is composed of interconnected tubules, and the unstacked prothylakoids. Etioplasts characteristically accumulate
the chlorophyll precursor protochlorophyllide
Anne Mie C. Emons is at the Laboratory of
Experimental Plant Morphology and Cell
Biology, Dept of Plant Sciences,
Wageningen University, Arboretumlaan 4,
6703 BD Wageningen, The Netherlands
(tel 131 317 484329;
fax 131 317 485005;
e-mail [email protected]);
Bela M. Mulder is at the Condensed Matter
Division of the FOM Institute for Atomic and
Molecular Physics, Kruislaan 407, 1098 SJ
Amsterdam, The Netherlands
(tel 131 20 6081231;
fax 131 20 6684106;
e-mail [email protected]).
(Pchlide), more specifically protochlorophyllide
a (Pchlide a)2–4. Illumination of etioplasts initiates the dispersal of the prolamellar body and
the formation of thylakoid membranes containing the pigment–protein complexes of the
photosynthetic apparatus.
In this context, recent in vitro reconstitution
experiments have been interpreted as providing
evidence for a novel light-harvesting Pchlide
a/b-binding protein complex5, termed LHPP
by analogy to the ubiquitous light-harvesting
chlorophyll a/b-binding proteins (LHCP) of
green plants. LHPP is speculated to:
• Serve as the central structural determinant
of the prolamellar body in etioplasts.
• Be essential for the establishment of the
photosynthetic apparatus.
• Confer photoprotection on greening
seedlings by dissipating excess light
energy, thereby minimizing Pchlideinduced photo-oxidative damage.
On the one hand, if they are correct, these
hypotheses would have a major impact on our
understanding of the seedling transition from
skotomorphogenesis to photomorphogenesis.
On the other hand, to date there are no in vivo
data that directly support the existence of an
LHPP complex6. Here, we critically analyse
the LHPP model in light of the current literature on the properties of etioplast membranes,
pigment–protein complexes and pigments.
Roles of the light-dependent PORA and
PORB proteins in etioplast formation
and photo-oxidative protection
The presence of the prolamellar body and the
accumulation of Pchlide a in etioplasts are
known to correlate with large quantities of the
strictly light-dependent NADPH:protochlorophyllide oxidoreductase (POR; 1.3.1.33)1,7–9.
This nuclear-encoded but plastid-localized
protein is unusual in that it mediates the only
1360 - 1385/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S1360-1385(99)01513-7
trends in plant science
Perspectives
light-requiring reaction in the chlorophyll
biosynthetic pathway, namely the reduction of
Pchlide a to chlorophyllide a (Chlide a)10,11.
Because of this light dependency, POR is not
simply an enzyme but, more accurately, a
plastid-specific photon sensor that triggers
pigment biosynthesis and membrane reorganization during the transformation of etioplasts to
chloroplasts.
Within etioplasts POR is localized almost
exclusively in the prolamellar body and is by
far the most abundant protein in this structure9,12. Dark-stable Pchlide:NADPH:POR
ternary complexes are organized in such a way
that photon absorption by the pigment leads to
its immediate reduction by NADPH to
Chlide1. The binding of Pchlide and NADPH
is apparently required for the stability and
membrane association of the POR polypeptide
in this context7,8,13,14. Pchlide a in the ternary
complexes is termed photoactive because it
can be converted to Chlide a by a single millisecond flash illumination, even at temperatures as low as 2608C (Refs 15,16). In situ
spectroscopy has been used extensively as a
tool for studying Pchlide photoreduction
because characteristic absorbance and emission maxima have been identified for the different physicochemical states of Pchlide and
Chlide. The photoactive Pchlide a in aggregated POR ternary complexes within the prolamellar body has an in situ low temperature
fluorescence emission maximum at 655 nm
(Pchlide-F655)17. Nonphotoactive Pchlide a,
a heterogeneous pigment fraction that is not
immediately reduced upon illumination, has
an emission maximum at 632 nm (PchlideF632).
Although initial studies with barley and oat
suggested that POR was encoded by a single
gene that was negatively regulated by light,
persistent reports of multiple immunoreactive
polypeptides led to the recent identification of
two differentially light-regulated genes,
PORA and PORB, in Arabidopsis and barley18,19. Both POR mRNAs are expressed in
etiolated seedlings but only PORB mRNA
continues to accumulate in light-grown plants.
The cytosolic precursor of barley PORA
(NADPH:protochlorophyllide oxidoreductase
A), but not barley PORB (NADPH:protochlorophyllide oxidoreductase B), has been
reported to be imported into plastids in a
strictly Pchlide-dependent fashion20. Pea
(Pisum sativum), in contrast with barley and
Arabidopsis, contains only a single POR gene
in spite of the presence of two distinct
polypeptides detected by an anti-POR antiserum1,21,22.
The data collected from the Arabidopsis
and barley systems have motivated recent
hypotheses that PORA and PORB might have
unique functions in etiolated seedlings and at
the onset of greening10,20. Specifically, PORA has
been proposed to play a special role in:
• Formation of POR ternary complexes containing photoactive Pchlide-F655.
• Prolamellar body assembly.
• Protection against photo-oxidative damage
caused by nonphotoactive Pchlide acting as
a photosensitizer.
Several of these hypotheses have been investigated in Arabidopsis in vivo by constitutively
overexpressing either PORA or PORB in
seedlings that contained little or no chlorophyll and that were severely depleted of endogenous POR (Refs 23,24). POR depletion can be
achieved in wild-type seedlings grown in continuous far-red light, which acts through the
phytochrome photoreceptor system to abolish
PORA and strongly down-regulate PORB
mRNA accumulation23,25. Alternatively, in the
dark-grown cop1 constitutive photomorphogenic mutant26 [also referred to as det340
(Ref. 27)], POR mRNA accumulation is drastically reduced even in the absence of light24,27.
Such POR-depleted seedlings are characterized by the complete or nearly complete
absence of photoactive Pchlide-F655 and the
prolamellar body, and by a high ratio of nonphotoactive to photoactive Pchlide a.
These overexpression studies using transgenic Arabidopsis seedlings indicate that in a
POR-depleted background either PORA or
PORB offers substantial protection against
photo-oxidative damage, and that each alone
is sufficient for the accumulation of photoactive Pchlide-F655 and the formation of the
prolamellar body membrane23,24. Therefore,
PORA and PORB appear to be qualitatively
interchangeable with respect to their functions
in etioplast formation and photoprotection.
A new model for specific functions of
PORA and PORB: a critical analysis of
the evidence for an LHPP complex
In vitro reconstitution experiments were performed recently5 with the two barley POR
enzymes19 and Zn-analogues of Pchlide b and
Pchlide a, ZnPP b and ZnPP a (Ref. 28). These
experiments led to the hypothesis of a novel
Pchlide-protein complex, termed LHPP, and a
new proposal for the in vivo functions of
PORA and PORB (Ref. 5). The heterooligomeric LHPP complex described is
thought to consist of a 5:1 ratio of the in vitrotranslated light-dependent barley PORA and
PORB proteins, which are proposed to specifically bind ZnPP b and ZnPP a, respectively.
Only the PORB-bound ZnPP a in the LHPP
complex appears to be reduced immediately
upon illumination, whereas the PORA-bound
ZnPP b is proposed to function initially as a
light-harvesting pigment. Energy transfer
from ZnPP b to ZnPP a is speculated to provide a mechanism for photoprotection during
the early stages of seedling greening.
Furthermore, LHPP is proposed to serve as the
main structural determinant of the prolamellar body membrane.
Although this is an intriguing model, a
major concern is that the broad conclusions
made about the in vitro formation of an LHPP
complex, and its possible implications in vivo,
do not reflect the experimental evidence.
First, it has been assumed, but not demonstrated, that the 5:1 ratio of ZnPP b to ZnPP a
reported for the LHPP complex in vitro can be
extrapolated to the Pchlide present in etioplast
inner membranes in vivo5. If true, dark-grown
angiosperms would contain predominantly
Pchlide b rather than Pchlide a, and the former
pigment should be detectable both in situ in
intact leaves and upon extraction with organic
solvents. Given the central role assigned to
Pchlide b as the light-harvesting pigment of
LHPP, it is therefore remarkable that no evidence for its existence in etiolated barley
seedlings has been presented. The unpublished result that Pchlide b is always present
in variable proportions relies on the statement
that extracted total Pchlide displays spectroscopic features reminiscent of both Pchlide a
and Pchlide b (Ref. 5). However, this finding
is at odds with independent studies in which
Pchlide b was not detected in any of the etiolated angiosperms that were examined29,
including barley2,4. Low temperature fluorescence measurements are routinely used to
differentiate between nonphotoactive and
photoactive Pchlide in situ1, and absorption
measurements made at visible wavelengths
can readily distinguish Pchlide a from Pchlide
b (Ref. 28). However, the room temperature
fluorescence analyses performed in conjunction with the LHPP model5 do not permit a
clear distinction between Pchlide a and
Pchlide b. When barley etioplast membranes
containing the natural endogenous mixture of
PORA and PORB were analysed, either by
solubilization and direct spectrophotometry or
by extraction with organic solvents and
HPLC, Pchlide a was readily identified, but no
traces of Pchlide b were detected4. Only
Chlide a was obtained from these prolamellar
body membranes upon irradiation. Pchlide b
added to etioplast membranes, either before or
after solubilization, proved to be stable in
darkness and convertible to Chlide b upon
irradiation4 (H. Klement and W. Rüdiger,
unpublished). Therefore, had endogenous
Pchlide b actually been present in barley etioplasts it might have been expected to be photoactive, in contrast with the prediction made
by the LHPP model5. The argument that
Pchlide b might per se be too unstable to survive extraction with organic solvents is unconvincing given the recovery of exogenous
Pchlide b or ZnPP b, together with the corresponding hydroxy compounds, after incubation
of these pigments with etioplast membranes4
(H. Klement and W. Rüdiger, unpublished).
January 2000, Vol. 5, No. 1
41
trends in plant science
Perspectives
Fig. 1. A germinating angiosperm enters one of two developmental programs, skotomorphogenesis or photomorphogenesis, depending upon whether the seedling emerges in the dark or in the
light. The presence of light not only alters seedling morphology, but also triggers the transformation
of the etioplasts of dark-grown seedlings into photosynthetically competent chloroplasts. This
transformation necessitates a major reorganization of the plastid inner membranes and the synthesis of large quantities of photosynthetic pigments, in particular chlorophylls. Light is directly
required for chlorophyll synthesis at the level of the enzymatic reduction of protochlorophyllide a
to chlorophyllide a, which is an immediate precursor of chlorophylls a and b. This light- and
NADPH-dependent reduction of a double bond can be performed by either one of two structurally
related enzymes, the NADPH:protochlorophyllide oxidoreductases A and B (PORA and PORB).
Second, in the absence of detectable
Pchlide b in etiolated angiosperms, the in vivo
significance of the high degree of substrate
specificity of barley PORA for ZnPP b and of
barley PORB for ZnPP a reported in vitro5 is
questionable. Furthermore, no such substrate
discrimination has been observed with solubilized POR from wheat prolamellar bodies28,
with highly purified POR from oat etioplasts30,
or with bacterially overexpressed pea POR
(M.P. Timko, pers. commun.).
However, let us for a moment assume that
etiolated angiosperms do indeed contain large
quantities of Pchlide b and that PORA and
PORB specifically bind Pchlide b and Pchlide
a, respectively. The LHPP model also predicts
that Pchlide b bound to PORA is nonphotoactive and that this pigment transfers its excitation energy to photoactive Pchlide a bound
42
January 2000, Vol. 5, No. 1
to PORB during the initial stages of illumination5. It is noteworthy that in vivo energy
transfer between pigment species, including
nonphotoactive and photoactive Pchlide and
different photoactive Pchlide forms, is a well
known phenomenon17,31,32 and not a novel feature of the LHPP model5. This issue aside, a
consequence of the proposed 5:1 stoichiometry of Pchlide b:NADPH:PORA to Pchlide
a:NADPH:PORB in the LHPP is that no more
than a sixth of the total Pchlide present in etiolated seedlings should be photoactive, and
hence immediately reduced to Chlide by flash
illumination. However, this prediction is in
conflict with the literature. Even if one were
to assume that standard room temperature
fluorescence analyses of pigment extracts are
somehow biased against the detection of
Pchlide b, it would nevertheless be implausible
that at least 85% of the total Pchlide
in mature etioplasts is nonphotoactive.
The ratio of nonphotoactive to photoactive
Pchlide steadily decreases as prolamellar
bodies form during the development of
etiolated seedlings22,33. In mature etioplasts of angiosperms, including barley and
Arabidopsis, most of the total Pchlide is
photoreduced to Chlide a by flash illumination15,24,25,27,34. Similarly, pigment measurements of isolated prolamellar body membranes
reveal larger amounts of photoactive than of
nonphotoactive Pchlide14,35. Therefore, the
available data on the pigments and pigment–protein complexes of etioplasts are not
consistent with the 5:1 ratio of nonphotoactive
to photoactive Pchlide mandated by the LHPP
model. Such an excess of nonphotoactive
Pchlide would indeed be more likely to cause
rather than to prevent pigment photo-oxidation in vivo, because energy transfer to photoactive Pchlide would be much faster than
NADP1 exchange after catalysis. Indeed, the
integration of most of the pigment molecules
into photoactive Pchlide:NADPH:POR
ternary complexes is itself sufficient to ensure
that only Pchlide photoreduction, and not
other undesired side reactions of the excited
state pigment, can occur at a significant rate.
What conclusions can be drawn regarding
the 5:1 stoichiometry of PORA to PORB proposed for the barley LHPP (Ref. 5)?
Throughout the above discussion the assumption has been made that the reported in vitro
ratio of ZnPP b:NADPH:PORA to ZnPP
a:NADPH:PORB is correct. However, it is
important to recognize that the stoichiometry
of the individual components of the reconstituted LHPP was not actually determined.
Rather, the 5:1 ratio refers to the relative input
quantities of the monomeric PORA and
PORB ternary complexes in sample mixtures
that subsequently appear to have formed hetero-oligomeric LHPP (Ref. 5). With respect to
the pigments, the 5:1 ratio is based on the
untested assumption that monomeric PORA
and PORB ternary complexes bind the same
number of ZnPP b and ZnPP a molecules,
respectively. Neither the stoichiometry of
PORA to PORB nor that of ZnPP b to ZnPP a
were, in fact, reported for the putative LHPP
complex isolated by gel filtration.
The 5:1 stoichiometry of PORA to PORB
hypothesized for the barley LHPP (Ref. 5) can
also be considered in light of the in vivo data
from other studies. Although a tryptic peptide
digest did reveal PORA to be the dominant
POR polypeptide of etiolated barley36, the
ratio of PORA to PORB has never been measured directly. However, the relative steadystate amounts of the PORA and PORB
mRNAs in both barley and Arabidopsis do
closely parallel the relative steady-state
amounts of two polypeptides, detected in
trends in plant science
Perspectives
western blots, that are thought to correspond to
PORA and PORB (Refs 18,19,25). In the context of LHPP, it should be noted that PORA
mRNA is much more abundant than PORB
mRNA in etiolated barley19, but not in etiolated Arabidopsis seedlings18. Furthermore,
the total quantities of either PORA or PORB
mRNA in Arabidopsis can be specifically
manipulated by their constitutive overexpression without altering the accumulation of the
endogenous POR mRNAs (Refs 23,24). Such
studies provide no evidence that alterations in
the PORA to PORB ratio alone, independent
of the total quantity of POR, dramatically
influence either POR-mediated in vivo photoprotection or the extent of the prolamellar
body membrane and the spectroscopic properties of photoactive Pchlide:NADPH:POR
complexes23,24 (F. Franck, K. Apel and G.A.
Armstrong, unpublished). The amount of photoactive Pchlide present in situ is, for example, a direct function of the total POR content.
At least in these respects, Arabidopsis PORA
and PORB appear to be functionally equivalent. Finally, pea provides an example of an
angiosperm that apparently contains only one
POR gene1,21. Therefore, the available in vivo
data from Arabidopsis and pea are not consistent with specific functions for PORA and
PORB within an LHPP complex of the type
postulated for barley.
What should be made of the assertion that
the properties of the in vitro reconstituted
LHPP complex are comparable to the physiological properties of LHPP within the prolamellar body of etioplasts5? Indeed, no such
comparison is possible because an authentic
LHPP complex has not been shown to exist
in situ in the prolamellar body, nor has such
a complex been purified from etioplasts. The
analogy that has been made between the
spectroscopic properties of the aggregated
photoactive Pchlide:NADPH:POR complexes present in the prolamellar body1,17 and
of the lipid-treated LHPP complexes produced in vitro5 is not valid because the ZnPP
a in these complexes has not been demonstrated to be photoactive. Furthermore, the
important role that the NADPH in POR
ternary complexes plays in maintaining the
structure of the prolamellar body14 has not
been addressed in the in vitro characterization of LHPP (Ref. 5).
With respect to the reported energy transfer from ZnPP b to ZnPP a within the LHPP,
470 nm light was used under the assumption
that this wavelength excites only ZnPP b and
its photoreduction product Zn pheophorbide
b, but not ZnPP a and Zn pheophorbide a
(Ref. 5). However, no excitation spectra of the
relevant POR-bound pigments were presented
to demonstrate the absolute wavelength specificity that would be required to make this conclusion. Therefore, whether in vitro energy
transfer from ZnPP b to ZnPP a occurs has yet
to be tested rigorously.
Concluding remarks
Convincing evidence for the existence in vivo
of an LHPP pigment–protein complex of the
type proposed5 requires that several features
be demonstrated, such as that:
• Etiolated angiosperms contain Pchlide b,
and, indeed, large amounts of this pigment.
• About 85% of the total Pchlide in etioplasts
is nonphotoactive.
• An enzymatically active LHPP complex
containing a 5:1 ratio of nonphotoactive
Pchlide b:NADPH:PORA to photoactive
Pchlide a:NADPH:PORB ternary complexes can be isolated from etioplast
membranes.
Such data have not been presented.
Furthermore, the in vitro characterization of
LHPP (Ref. 5) also neglected to document key
results including:
• Direct measurement of the PORA to PORB
and the ZnPP b to ZnPP a stoichiometries.
• Ability of the LHPP mixed with lipids
to reduce ZnPP a in a light-dependent
fashion.
• Selectivity of the wavelength of light chosen to demonstrate energy transfer from
ZnPP b to ZnPP a.
To date, the available data are insufficient to
demonstrate the existence of an LHPP complex in vitro or in vivo. If the proposed structure and properties of LHPP could be
confirmed it would be a truly remarkable pigment–protein complex. However, in the absence of additional experimental evidence,
one must conclude that LHPP does not exist.
Acknowledgements
We would like to thank Belá Böddi (Eötvös
University Budapest, Hungary), Fabrice
Franck (University of Liège, Belgium), and
Margareta Ryberg and Christer Sundqvist
(Göteborg University, Sweden) for their
active participation in the preparation of this
manuscript, Constantin Rebeiz (University of
Illinois at Urbana, USA) for helpful discussions, and Michael Timko (University of
Virginia, USA) for communicating unpublished results. This manuscript is dedicated to
Prof. Hubert Ziegler on the occasion of his
75th birthday.
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27 Lebedev, N. et al. (1995) Chlorophyll synthesis
in a deetiolated (det340) mutant of Arabidopsis
without NADPH-protochlorophyllide (PChlide)
oxidoreductase (POR) A and photoactive
PChlide-F655. Plant Cell 7, 2081–2090
28 Schoch, S. et al. (1995) Photoreduction of zinc
protopheophorbide b with NADPHprotochlorophyllide oxidoreductase from
etiolated wheat (Triticum aestivum L.). Eur. J.
Biochem. 229, 291–298
29 Rebeiz, C.A. et al. (1999) Chloroplast biogenesis
80. Proposal of a unified multibranched
chlorophyll a/b biosynthetic pathway.
Photosynthetica 36, 117–128
30 Klement, H. et al. (1999) Pigment-free
NADPH:protochlorophyllide oxidoreductase
from Avena sativa L.: purification and substrate
specificity. Eur. J. Biochem. 265, 862–874
31 Kahn, A. et al. (1970) Energy transfer between
protochlorophyllide molecules: evidence for
multiple chromophores in the photoactive
protochlorophyllide–protein complex in vivo and
in vitro. J. Mol. Biol. 48, 85–101
32 Sironval, C. and Brouers, M. (1970) The
reduction of protochlorophyllide into
chlorophyllide: II. The temperature dependence
of the P657–647 → P688–676 phototransformation.
Photosynthetica 4, 38–47
33 Klein, S. and Schiff, J.A. (1972) The correlated
appearance of prolamellar bodies,
photochlorophyll(ide) species, and the Shibata
shift during development of bean etioplasts in the
dark. Plant Physiol. 49, 619–626
34 Franck, F. et al. (1999) ProtochlorophyllideNADP1 and protochlorophyllide-NADPH
complexes and their regeneration after flash
illumination in leaves and etioplast membranes of
dark-grown wheat. Photosynth. Res. 59, 53–61
35 Ikeuchi, M. and Murakami, S. (1983) Separation
and characterization of prolamellar bodies and
prothylakoids from squash etioplasts. Plant Cell
Physiol. 24, 71–80
36 Benli, M. et al. (1991) Effect of light on the
NADPH-protochlorophyllide oxidoreductase of
Arabidopsis thaliana. Plant Mol. Biol. 16, 615–625
Gregory A. Armstrong* and Klaus Apel are
at the Institute for Plant Sciences, Plant
Genetics, Swiss Federal Institute of
Technology (ETH), Universitätstr. 2, CH8092 Zürich, Switzerland; Wolfhart Rüdiger
is at the Botanisches Institut der LudwigMaximilians-Universität München,
Menzingerstr. 67, D-80638 München,
Germany.
*Author for correspondence
(tel 141 1 632 3700; fax 141 1 632 1081;
e-mail [email protected]).
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January 2000, Vol. 5, No. 1
perspectives
Equisetum hyemale. Protoplasma 117,
68–81
30 Emons, A.M.C. (1989) Helicoidal microfibril
deposition in a tip-growing cell and
microtubules alignment during tip
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31 McCann, M.C. and Roberts, K. (1991)
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32 Haigler, C.H. and Brown, R.M. (1986) Transport
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33 Felle, H.H. and Hepler, P.K. (1997) The cytosolic
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hairs as revealed by Ca21-selective
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34 De Ruijter, C.A. et al. (1998) Lipochitooligosaccharides re-initiate root hair tip
growth in Vicia sativa with high calcium and
spectrin-like antigen at the tip. Plant J. 13,
341–350
35 Ehrhardt, D.W. et al. (1996) Calcium spiking
in plant root hairs responding to Rhizobium
nodulation signals. Cell 85, 573–681
Does a light-harvesting
protochlorophyllide a/bbinding protein complex exist?
Gregory A. Armstrong, Klaus Apel and Wolfhart Rüdiger
Recent in vitro studies have led to speculation that a novel light-harvesting
protochlorophyllide a/b-binding protein complex (LHPP) might exist in dark-grown
angiosperms. Structurally, it has been suggested that LHPP consists of a 5:1 ratio
of dark-stable ternary complexes of the light-dependent NADPH: protochlorophyllide
oxidoreductases A and B containing nonphotoactive protochlorophyllide b and
photoactive protochlorophyllide a, respectively. Functionally, LHPP has been
hypothesized to play major roles in establishing the photosynthetic apparatus, in
protecting against photo-oxidative damage during greening, and in determining
etioplast inner membrane architecture. However, the LHPP model is not compatible
with other studies of the pigments and the pigment–protein complexes of dark-grown
angiosperms. Protochlorophyllide b, which is postulated to be the major lightharvesting pigment of LHPP, has, for example, never been detected in etiolated
seedlings. This raises the question: does LHPP exist?
L
ight profoundly influences plant
development and allows photosynthesis to occur, but it also represents a
tremendous risk. Photo-oxidative damage
initiated by excited state photosensitizing
molecules, such as chlorophylls and their
biosynthetic precursors, can be lethal.
Angiosperms that germinate in darkness in
the soil enter the seedling developmental program known as skotomorphogenesis (Fig. 1).
However, such seedlings must be prepared
for a subsequent light-triggered switch to
photomorphogenesis. Upon illumination, the
leaves of etiolated angiosperms synthesize
and accumulate large quantities of chloro40
January 2000, Vol. 5, No. 1
phylls a and b. Seedlings are particularly susceptible to photo-oxidative damage during
this transition to photoautotrophy.
The presence or absence of light dramatically
influences plastid development. Dark-grown
angiosperm seedlings contain an achlorophyllous plastid type known as the etioplast, which
is transformed into a photosynthetically competent chloroplast during photomorphogenesis1.
The etioplast is defined by the presence of two
types of internal membranes, the lattice-like
prolamellar body, which is composed of interconnected tubules, and the unstacked prothylakoids. Etioplasts characteristically accumulate
the chlorophyll precursor protochlorophyllide
Anne Mie C. Emons is at the Laboratory of
Experimental Plant Morphology and Cell
Biology, Dept of Plant Sciences,
Wageningen University, Arboretumlaan 4,
6703 BD Wageningen, The Netherlands
(tel 131 317 484329;
fax 131 317 485005;
e-mail [email protected]);
Bela M. Mulder is at the Condensed Matter
Division of the FOM Institute for Atomic and
Molecular Physics, Kruislaan 407, 1098 SJ
Amsterdam, The Netherlands
(tel 131 20 6081231;
fax 131 20 6684106;
e-mail [email protected]).
(Pchlide), more specifically protochlorophyllide
a (Pchlide a)2–4. Illumination of etioplasts initiates the dispersal of the prolamellar body and
the formation of thylakoid membranes containing the pigment–protein complexes of the
photosynthetic apparatus.
In this context, recent in vitro reconstitution
experiments have been interpreted as providing
evidence for a novel light-harvesting Pchlide
a/b-binding protein complex5, termed LHPP
by analogy to the ubiquitous light-harvesting
chlorophyll a/b-binding proteins (LHCP) of
green plants. LHPP is speculated to:
• Serve as the central structural determinant
of the prolamellar body in etioplasts.
• Be essential for the establishment of the
photosynthetic apparatus.
• Confer photoprotection on greening
seedlings by dissipating excess light
energy, thereby minimizing Pchlideinduced photo-oxidative damage.
On the one hand, if they are correct, these
hypotheses would have a major impact on our
understanding of the seedling transition from
skotomorphogenesis to photomorphogenesis.
On the other hand, to date there are no in vivo
data that directly support the existence of an
LHPP complex6. Here, we critically analyse
the LHPP model in light of the current literature on the properties of etioplast membranes,
pigment–protein complexes and pigments.
Roles of the light-dependent PORA and
PORB proteins in etioplast formation
and photo-oxidative protection
The presence of the prolamellar body and the
accumulation of Pchlide a in etioplasts are
known to correlate with large quantities of the
strictly light-dependent NADPH:protochlorophyllide oxidoreductase (POR; 1.3.1.33)1,7–9.
This nuclear-encoded but plastid-localized
protein is unusual in that it mediates the only
1360 - 1385/00/$ – see front matter © 2000 Elsevier Science Ltd. All rights reserved. PII: S1360-1385(99)01513-7
trends in plant science
Perspectives
light-requiring reaction in the chlorophyll
biosynthetic pathway, namely the reduction of
Pchlide a to chlorophyllide a (Chlide a)10,11.
Because of this light dependency, POR is not
simply an enzyme but, more accurately, a
plastid-specific photon sensor that triggers
pigment biosynthesis and membrane reorganization during the transformation of etioplasts to
chloroplasts.
Within etioplasts POR is localized almost
exclusively in the prolamellar body and is by
far the most abundant protein in this structure9,12. Dark-stable Pchlide:NADPH:POR
ternary complexes are organized in such a way
that photon absorption by the pigment leads to
its immediate reduction by NADPH to
Chlide1. The binding of Pchlide and NADPH
is apparently required for the stability and
membrane association of the POR polypeptide
in this context7,8,13,14. Pchlide a in the ternary
complexes is termed photoactive because it
can be converted to Chlide a by a single millisecond flash illumination, even at temperatures as low as 2608C (Refs 15,16). In situ
spectroscopy has been used extensively as a
tool for studying Pchlide photoreduction
because characteristic absorbance and emission maxima have been identified for the different physicochemical states of Pchlide and
Chlide. The photoactive Pchlide a in aggregated POR ternary complexes within the prolamellar body has an in situ low temperature
fluorescence emission maximum at 655 nm
(Pchlide-F655)17. Nonphotoactive Pchlide a,
a heterogeneous pigment fraction that is not
immediately reduced upon illumination, has
an emission maximum at 632 nm (PchlideF632).
Although initial studies with barley and oat
suggested that POR was encoded by a single
gene that was negatively regulated by light,
persistent reports of multiple immunoreactive
polypeptides led to the recent identification of
two differentially light-regulated genes,
PORA and PORB, in Arabidopsis and barley18,19. Both POR mRNAs are expressed in
etiolated seedlings but only PORB mRNA
continues to accumulate in light-grown plants.
The cytosolic precursor of barley PORA
(NADPH:protochlorophyllide oxidoreductase
A), but not barley PORB (NADPH:protochlorophyllide oxidoreductase B), has been
reported to be imported into plastids in a
strictly Pchlide-dependent fashion20. Pea
(Pisum sativum), in contrast with barley and
Arabidopsis, contains only a single POR gene
in spite of the presence of two distinct
polypeptides detected by an anti-POR antiserum1,21,22.
The data collected from the Arabidopsis
and barley systems have motivated recent
hypotheses that PORA and PORB might have
unique functions in etiolated seedlings and at
the onset of greening10,20. Specifically, PORA has
been proposed to play a special role in:
• Formation of POR ternary complexes containing photoactive Pchlide-F655.
• Prolamellar body assembly.
• Protection against photo-oxidative damage
caused by nonphotoactive Pchlide acting as
a photosensitizer.
Several of these hypotheses have been investigated in Arabidopsis in vivo by constitutively
overexpressing either PORA or PORB in
seedlings that contained little or no chlorophyll and that were severely depleted of endogenous POR (Refs 23,24). POR depletion can be
achieved in wild-type seedlings grown in continuous far-red light, which acts through the
phytochrome photoreceptor system to abolish
PORA and strongly down-regulate PORB
mRNA accumulation23,25. Alternatively, in the
dark-grown cop1 constitutive photomorphogenic mutant26 [also referred to as det340
(Ref. 27)], POR mRNA accumulation is drastically reduced even in the absence of light24,27.
Such POR-depleted seedlings are characterized by the complete or nearly complete
absence of photoactive Pchlide-F655 and the
prolamellar body, and by a high ratio of nonphotoactive to photoactive Pchlide a.
These overexpression studies using transgenic Arabidopsis seedlings indicate that in a
POR-depleted background either PORA or
PORB offers substantial protection against
photo-oxidative damage, and that each alone
is sufficient for the accumulation of photoactive Pchlide-F655 and the formation of the
prolamellar body membrane23,24. Therefore,
PORA and PORB appear to be qualitatively
interchangeable with respect to their functions
in etioplast formation and photoprotection.
A new model for specific functions of
PORA and PORB: a critical analysis of
the evidence for an LHPP complex
In vitro reconstitution experiments were performed recently5 with the two barley POR
enzymes19 and Zn-analogues of Pchlide b and
Pchlide a, ZnPP b and ZnPP a (Ref. 28). These
experiments led to the hypothesis of a novel
Pchlide-protein complex, termed LHPP, and a
new proposal for the in vivo functions of
PORA and PORB (Ref. 5). The heterooligomeric LHPP complex described is
thought to consist of a 5:1 ratio of the in vitrotranslated light-dependent barley PORA and
PORB proteins, which are proposed to specifically bind ZnPP b and ZnPP a, respectively.
Only the PORB-bound ZnPP a in the LHPP
complex appears to be reduced immediately
upon illumination, whereas the PORA-bound
ZnPP b is proposed to function initially as a
light-harvesting pigment. Energy transfer
from ZnPP b to ZnPP a is speculated to provide a mechanism for photoprotection during
the early stages of seedling greening.
Furthermore, LHPP is proposed to serve as the
main structural determinant of the prolamellar body membrane.
Although this is an intriguing model, a
major concern is that the broad conclusions
made about the in vitro formation of an LHPP
complex, and its possible implications in vivo,
do not reflect the experimental evidence.
First, it has been assumed, but not demonstrated, that the 5:1 ratio of ZnPP b to ZnPP a
reported for the LHPP complex in vitro can be
extrapolated to the Pchlide present in etioplast
inner membranes in vivo5. If true, dark-grown
angiosperms would contain predominantly
Pchlide b rather than Pchlide a, and the former
pigment should be detectable both in situ in
intact leaves and upon extraction with organic
solvents. Given the central role assigned to
Pchlide b as the light-harvesting pigment of
LHPP, it is therefore remarkable that no evidence for its existence in etiolated barley
seedlings has been presented. The unpublished result that Pchlide b is always present
in variable proportions relies on the statement
that extracted total Pchlide displays spectroscopic features reminiscent of both Pchlide a
and Pchlide b (Ref. 5). However, this finding
is at odds with independent studies in which
Pchlide b was not detected in any of the etiolated angiosperms that were examined29,
including barley2,4. Low temperature fluorescence measurements are routinely used to
differentiate between nonphotoactive and
photoactive Pchlide in situ1, and absorption
measurements made at visible wavelengths
can readily distinguish Pchlide a from Pchlide
b (Ref. 28). However, the room temperature
fluorescence analyses performed in conjunction with the LHPP model5 do not permit a
clear distinction between Pchlide a and
Pchlide b. When barley etioplast membranes
containing the natural endogenous mixture of
PORA and PORB were analysed, either by
solubilization and direct spectrophotometry or
by extraction with organic solvents and
HPLC, Pchlide a was readily identified, but no
traces of Pchlide b were detected4. Only
Chlide a was obtained from these prolamellar
body membranes upon irradiation. Pchlide b
added to etioplast membranes, either before or
after solubilization, proved to be stable in
darkness and convertible to Chlide b upon
irradiation4 (H. Klement and W. Rüdiger,
unpublished). Therefore, had endogenous
Pchlide b actually been present in barley etioplasts it might have been expected to be photoactive, in contrast with the prediction made
by the LHPP model5. The argument that
Pchlide b might per se be too unstable to survive extraction with organic solvents is unconvincing given the recovery of exogenous
Pchlide b or ZnPP b, together with the corresponding hydroxy compounds, after incubation
of these pigments with etioplast membranes4
(H. Klement and W. Rüdiger, unpublished).
January 2000, Vol. 5, No. 1
41
trends in plant science
Perspectives
Fig. 1. A germinating angiosperm enters one of two developmental programs, skotomorphogenesis or photomorphogenesis, depending upon whether the seedling emerges in the dark or in the
light. The presence of light not only alters seedling morphology, but also triggers the transformation
of the etioplasts of dark-grown seedlings into photosynthetically competent chloroplasts. This
transformation necessitates a major reorganization of the plastid inner membranes and the synthesis of large quantities of photosynthetic pigments, in particular chlorophylls. Light is directly
required for chlorophyll synthesis at the level of the enzymatic reduction of protochlorophyllide a
to chlorophyllide a, which is an immediate precursor of chlorophylls a and b. This light- and
NADPH-dependent reduction of a double bond can be performed by either one of two structurally
related enzymes, the NADPH:protochlorophyllide oxidoreductases A and B (PORA and PORB).
Second, in the absence of detectable
Pchlide b in etiolated angiosperms, the in vivo
significance of the high degree of substrate
specificity of barley PORA for ZnPP b and of
barley PORB for ZnPP a reported in vitro5 is
questionable. Furthermore, no such substrate
discrimination has been observed with solubilized POR from wheat prolamellar bodies28,
with highly purified POR from oat etioplasts30,
or with bacterially overexpressed pea POR
(M.P. Timko, pers. commun.).
However, let us for a moment assume that
etiolated angiosperms do indeed contain large
quantities of Pchlide b and that PORA and
PORB specifically bind Pchlide b and Pchlide
a, respectively. The LHPP model also predicts
that Pchlide b bound to PORA is nonphotoactive and that this pigment transfers its excitation energy to photoactive Pchlide a bound
42
January 2000, Vol. 5, No. 1
to PORB during the initial stages of illumination5. It is noteworthy that in vivo energy
transfer between pigment species, including
nonphotoactive and photoactive Pchlide and
different photoactive Pchlide forms, is a well
known phenomenon17,31,32 and not a novel feature of the LHPP model5. This issue aside, a
consequence of the proposed 5:1 stoichiometry of Pchlide b:NADPH:PORA to Pchlide
a:NADPH:PORB in the LHPP is that no more
than a sixth of the total Pchlide present in etiolated seedlings should be photoactive, and
hence immediately reduced to Chlide by flash
illumination. However, this prediction is in
conflict with the literature. Even if one were
to assume that standard room temperature
fluorescence analyses of pigment extracts are
somehow biased against the detection of
Pchlide b, it would nevertheless be implausible
that at least 85% of the total Pchlide
in mature etioplasts is nonphotoactive.
The ratio of nonphotoactive to photoactive
Pchlide steadily decreases as prolamellar
bodies form during the development of
etiolated seedlings22,33. In mature etioplasts of angiosperms, including barley and
Arabidopsis, most of the total Pchlide is
photoreduced to Chlide a by flash illumination15,24,25,27,34. Similarly, pigment measurements of isolated prolamellar body membranes
reveal larger amounts of photoactive than of
nonphotoactive Pchlide14,35. Therefore, the
available data on the pigments and pigment–protein complexes of etioplasts are not
consistent with the 5:1 ratio of nonphotoactive
to photoactive Pchlide mandated by the LHPP
model. Such an excess of nonphotoactive
Pchlide would indeed be more likely to cause
rather than to prevent pigment photo-oxidation in vivo, because energy transfer to photoactive Pchlide would be much faster than
NADP1 exchange after catalysis. Indeed, the
integration of most of the pigment molecules
into photoactive Pchlide:NADPH:POR
ternary complexes is itself sufficient to ensure
that only Pchlide photoreduction, and not
other undesired side reactions of the excited
state pigment, can occur at a significant rate.
What conclusions can be drawn regarding
the 5:1 stoichiometry of PORA to PORB proposed for the barley LHPP (Ref. 5)?
Throughout the above discussion the assumption has been made that the reported in vitro
ratio of ZnPP b:NADPH:PORA to ZnPP
a:NADPH:PORB is correct. However, it is
important to recognize that the stoichiometry
of the individual components of the reconstituted LHPP was not actually determined.
Rather, the 5:1 ratio refers to the relative input
quantities of the monomeric PORA and
PORB ternary complexes in sample mixtures
that subsequently appear to have formed hetero-oligomeric LHPP (Ref. 5). With respect to
the pigments, the 5:1 ratio is based on the
untested assumption that monomeric PORA
and PORB ternary complexes bind the same
number of ZnPP b and ZnPP a molecules,
respectively. Neither the stoichiometry of
PORA to PORB nor that of ZnPP b to ZnPP a
were, in fact, reported for the putative LHPP
complex isolated by gel filtration.
The 5:1 stoichiometry of PORA to PORB
hypothesized for the barley LHPP (Ref. 5) can
also be considered in light of the in vivo data
from other studies. Although a tryptic peptide
digest did reveal PORA to be the dominant
POR polypeptide of etiolated barley36, the
ratio of PORA to PORB has never been measured directly. However, the relative steadystate amounts of the PORA and PORB
mRNAs in both barley and Arabidopsis do
closely parallel the relative steady-state
amounts of two polypeptides, detected in
trends in plant science
Perspectives
western blots, that are thought to correspond to
PORA and PORB (Refs 18,19,25). In the context of LHPP, it should be noted that PORA
mRNA is much more abundant than PORB
mRNA in etiolated barley19, but not in etiolated Arabidopsis seedlings18. Furthermore,
the total quantities of either PORA or PORB
mRNA in Arabidopsis can be specifically
manipulated by their constitutive overexpression without altering the accumulation of the
endogenous POR mRNAs (Refs 23,24). Such
studies provide no evidence that alterations in
the PORA to PORB ratio alone, independent
of the total quantity of POR, dramatically
influence either POR-mediated in vivo photoprotection or the extent of the prolamellar
body membrane and the spectroscopic properties of photoactive Pchlide:NADPH:POR
complexes23,24 (F. Franck, K. Apel and G.A.
Armstrong, unpublished). The amount of photoactive Pchlide present in situ is, for example, a direct function of the total POR content.
At least in these respects, Arabidopsis PORA
and PORB appear to be functionally equivalent. Finally, pea provides an example of an
angiosperm that apparently contains only one
POR gene1,21. Therefore, the available in vivo
data from Arabidopsis and pea are not consistent with specific functions for PORA and
PORB within an LHPP complex of the type
postulated for barley.
What should be made of the assertion that
the properties of the in vitro reconstituted
LHPP complex are comparable to the physiological properties of LHPP within the prolamellar body of etioplasts5? Indeed, no such
comparison is possible because an authentic
LHPP complex has not been shown to exist
in situ in the prolamellar body, nor has such
a complex been purified from etioplasts. The
analogy that has been made between the
spectroscopic properties of the aggregated
photoactive Pchlide:NADPH:POR complexes present in the prolamellar body1,17 and
of the lipid-treated LHPP complexes produced in vitro5 is not valid because the ZnPP
a in these complexes has not been demonstrated to be photoactive. Furthermore, the
important role that the NADPH in POR
ternary complexes plays in maintaining the
structure of the prolamellar body14 has not
been addressed in the in vitro characterization of LHPP (Ref. 5).
With respect to the reported energy transfer from ZnPP b to ZnPP a within the LHPP,
470 nm light was used under the assumption
that this wavelength excites only ZnPP b and
its photoreduction product Zn pheophorbide
b, but not ZnPP a and Zn pheophorbide a
(Ref. 5). However, no excitation spectra of the
relevant POR-bound pigments were presented
to demonstrate the absolute wavelength specificity that would be required to make this conclusion. Therefore, whether in vitro energy
transfer from ZnPP b to ZnPP a occurs has yet
to be tested rigorously.
Concluding remarks
Convincing evidence for the existence in vivo
of an LHPP pigment–protein complex of the
type proposed5 requires that several features
be demonstrated, such as that:
• Etiolated angiosperms contain Pchlide b,
and, indeed, large amounts of this pigment.
• About 85% of the total Pchlide in etioplasts
is nonphotoactive.
• An enzymatically active LHPP complex
containing a 5:1 ratio of nonphotoactive
Pchlide b:NADPH:PORA to photoactive
Pchlide a:NADPH:PORB ternary complexes can be isolated from etioplast
membranes.
Such data have not been presented.
Furthermore, the in vitro characterization of
LHPP (Ref. 5) also neglected to document key
results including:
• Direct measurement of the PORA to PORB
and the ZnPP b to ZnPP a stoichiometries.
• Ability of the LHPP mixed with lipids
to reduce ZnPP a in a light-dependent
fashion.
• Selectivity of the wavelength of light chosen to demonstrate energy transfer from
ZnPP b to ZnPP a.
To date, the available data are insufficient to
demonstrate the existence of an LHPP complex in vitro or in vivo. If the proposed structure and properties of LHPP could be
confirmed it would be a truly remarkable pigment–protein complex. However, in the absence of additional experimental evidence,
one must conclude that LHPP does not exist.
Acknowledgements
We would like to thank Belá Böddi (Eötvös
University Budapest, Hungary), Fabrice
Franck (University of Liège, Belgium), and
Margareta Ryberg and Christer Sundqvist
(Göteborg University, Sweden) for their
active participation in the preparation of this
manuscript, Constantin Rebeiz (University of
Illinois at Urbana, USA) for helpful discussions, and Michael Timko (University of
Virginia, USA) for communicating unpublished results. This manuscript is dedicated to
Prof. Hubert Ziegler on the occasion of his
75th birthday.
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Gregory A. Armstrong* and Klaus Apel are
at the Institute for Plant Sciences, Plant
Genetics, Swiss Federal Institute of
Technology (ETH), Universitätstr. 2, CH8092 Zürich, Switzerland; Wolfhart Rüdiger
is at the Botanisches Institut der LudwigMaximilians-Universität München,
Menzingerstr. 67, D-80638 München,
Germany.
*Author for correspondence
(tel 141 1 632 3700; fax 141 1 632 1081;
e-mail [email protected]).
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