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 morphogenesis: a dry-cleaving and freeze-substitution study. Can. J. Bot. 67, 2401–2408
31 McCann, M.C. and Roberts, K. (1991) Architecture of the primary cell wall. In The Cytoskeletal Basis of Plant Growth and Form (Lloyd, C.W., ed.), pp. 109–129, Academic Press
32 Haigler, C.H. and Brown, R.M. (1986) Transport of rosettes from the Golgi apparatus to plasma membrane in isolated mesophyll cells of
Zinnia elegans during differentiation to tracheary elements in suspension culture. Protoplasma 134, 111–120
33 Felle, H.H. and Hepler, P.K. (1997) The cytosolic Ca21concentration gradient of Sinapis alba root hairs as revealed by Ca21-selective
microelectrode tests and fura-dextran ratio imaging. Plant Phys. 114, 39–45 34 De Ruijter, C.A. et al. (1998)
Lipochito-oligosaccharides 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
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]).
L
ight profoundly influences plantdevelopment and allows photosyn-thesis 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 pro-gram 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
chloro-phylls a and b. Seedlings are particularly sus-ceptible 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 achlorophyl-lous plastid type known as the etioplast, which is transformed into a photosynthetically com-petent 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 inter-connected tubules, and the unstacked prothyl-akoids. Etioplasts characteristically accumulate the chlorophyll precursor protochlorophyllide
(Pchlide), more specifically protochlorophyllide
a (Pchlide a)2–4. Illumination of etioplasts
initi-ates the dispersal of the prolamellar body and the formation of thylakoid membranes con-taining 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 Pchlide-induced 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 litera-ture 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:protochloro-phyllide oxidoreductase (POR; 1.3.1.33)1,7–9.
This nuclear-encoded but plastid-localized protein is unusual in that it mediates the only
Does a light-harvesting
protochlorophyllide
a/b-binding protein complex exist?
Gregory A. Armstrong, Klaus Apel and Wolfhart Rüdiger
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 reorganiz-ation during the transformreorganiz-ation 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
struc-ture9,12. 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 milli-second flash illumination, even at tempera-tures 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 emis-sion maxima have been identified for the dif-ferent physicochemical states of Pchlide and Chlide. The photoactive Pchlide a in aggre-gated POR ternary complexes within the pro-lamellar 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 (Pchlide-F632).
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
bar-ley18,19. 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:proto-chlorophyllide 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 POR anti-serum1,21,22.
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
con-taining 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 inves-tigated in Arabidopsis in vivo by constitutively overexpressing either PORA or PORB in seedlings that contained little or no chloro-phyll and that were severely depleted of endogen-ous POR (Refs 23,24). POR depletion can be achieved in wild-type seedlings grown in con-tinuous 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 photomor-phogenic mutant26 [also referred to as det340
(Ref. 27)], POR mRNA accumulation is dras-tically reduced even in the absence of light24,27.
Such POR-depleted seedlings are character-ized by the complete or nearly complete absence of photoactive Pchlide-F655 and the prolamellar body, and by a high ratio of non-photoactive to non-photoactive Pchlide a.
These overexpression studies using trans-genic 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 photoac-tive 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
per-formed recently5with 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 hetero-oligomeric LHPP complex described is thought to consist of a 5:1 ratio of the in vitro-translated light-dependent barley PORA and PORB proteins, which are proposed to specifi-cally 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 pro-vide a mechanism for photoprotection during the early stages of seedling greening. Furthermore, LHPP is proposed to serve as the
main structural determinant of the prolamel-lar 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 demon-strated, 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 evi-dence for its existence in etiolated barley seedlings has been presented. The unpub-lished result that Pchlide b is always present in variable proportions relies on the statement that extracted total Pchlide displays spectro-scopic 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
etio-lated angiosperms that were examined29,
including barley2,4. Low temperature
fluor-escence 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
conjunc-tion with the LHPP model5do 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 etio-plasts it might have been expected to be pho-toactive, in contrast with the prediction made
by the LHPP model5. The argument that
Pchlide b might per se be too unstable to sur-vive extraction with organic solvents is uncon-vincing given the recovery of exogenous Pchlide b or ZnPP b, together with the corre-sponding hydroxy compounds, after incubation of these pigments with etioplast membranes4
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 vitro5is
questionable. Furthermore, no such substrate discrimination has been observed with solu-bilized 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 nonphoto-active and that this pigment transfers its exci-tation energy to photoactive Pchlide a bound
to PORB during the initial stages of illumi-nation5. 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 phenomenon17,31,32and not a novel
fea-ture of the LHPP model5. This issue aside, a
consequence of the proposed 5:1 stoichio-metry 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 etio-lated 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
etio-plasts of angiosperms, including barley and
Arabidopsis, most of the total Pchlide is
photoreduced to Chlide a by flash illumi-nation15,24,25,27,34. Similarly, pigment
measure-ments of isolated prolamellar body membranes reveal larger amounts of photoactive than of nonphotoactive Pchlide14,35. Therefore, the
available data on the pigments and pig-ment–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-oxida-tion in vivo, because energy transfer to pho-toactive Pchlide would be much faster than NADP1exchange 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 pro-posed for the barley LHPP (Ref. 5)? Throughout the above discussion the assump-tion 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 reconsti-tuted 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 het-ero-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 meas-ured directly. However, the relative steady-state 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
Fig. 1. A germinating angiosperm enters one of two developmental programs,
western blots, that are thought to correspond to PORA and PORB (Refs 18,19,25). In the con-text of LHPP, it should be noted that PORA mRNA is much more abundant than PORB mRNA in etiolated barley19, but not in
etio-lated Arabidopsis seedlings18. Furthermore,
the total quantities of either PORA or PORB mRNA in Arabidopsis can be specifically manipulated by their constitutive overexpres-sion 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 photo-protection or the extent of the prolamellar body membrane and the spectroscopic prop-erties of photoactive Pchlide:NADPH:POR complexes23,24(F. Franck, K. Apel and G.A.
Armstrong, unpublished). The amount of pho-toactive Pchlide present in situ is, for exam-ple, a direct function of the total POR content. At least in these respects, Arabidopsis PORA and PORB appear to be functionally equiva-lent. 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 consis-tent 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 physio-logical properties of LHPP within the pro-lamellar 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 com-plexes present in the prolamellar body1,17 and
of the lipid-treated LHPP complexes pro-duced in vitro5is not valid because the ZnPP
a in these complexes has not been
demon-strated to be photoactive. Furthermore, the important role that the NADPH in POR ternary complexes plays in maintaining the structure of the prolamellar body14has not
been addressed in the in vitro characteriz-ation of LHPP (Ref. 5).
With respect to the reported energy trans-fer 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 speci-ficity that would be required to make this con-clusion. 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 proposed5requires 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 com-plexes 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 cho-sen to demonstrate energy transfer from ZnPP b to ZnPP a.
To date, the available data are insufficient to demonstrate the existence of an LHPP com-plex in vitro or in vivo. If the proposed struc-ture and properties of LHPP could be confirmed it would be a truly remarkable pig-ment–protein complex. However, in the ab-sence 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 discus-sions, and Michael Timko (University of Virginia, USA) for communicating unpub-lished 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, CH-8092 Zürich, Switzerland; Wolfhart Rüdiger is at the Botanisches Institut der Ludwig-Maximilians-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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