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First published online March 26, 2004; 10.1104/pp.103.033613 Plant Physiology 134:1355-1365 (2004) © 2004 American Society of Plant Biologists A Novel Role of Water-Soluble Chlorophyll Proteins in the Transitory Storage of Chorophyllide1,2Lehrstuhl für Pflanzenphysiologie, Universität Bayreuth, D–95447 Bayreuth, Germany (C.R.); Department of Biomolecular Science, Toho University, 2–2–1 Miyama, Funabashi, Chiba 274–8510, Japan (H.S.); Université Joseph Fourier et Centre National de la Recherche Scientifique, Unité Mixte de Recherche 5575, F–38041 Grenoble cedex 9, France (J.-P.A., S.R.); and Ruhr-Universität Bochum, Lehrstuhl für Pflanzenphysiologie, D–44801 Bochum, Germany (S.R.)
All chlorophyll (Chl)-binding proteins involved in photosynthesis of higher plants are hydrophobic membrane proteins integrated into the thylakoids. However, a different category of Chl-binding proteins, the so-called water-soluble Chl proteins (WSCPs), was found in members of the Brassicaceae, Polygonaceae, Chenopodiaceae, and Amaranthaceae families. WSCPs from different plant species bind Chl a and Chl b in different ratios. Some members of the WSCP family are induced after drought and heat stress as well as leaf detachment. It has been proposed that this group of proteins might have a physiological function in the Chl degradation pathway. We demonstrate here that a protein that shared sequence homology to WSCPs accumulated in etiolated barley (Hordeum vulgare) seedlings exposed to light for 2 h. The novel 22-kD protein was attached to the outer envelope of barley etiochloroplasts, and import of the 27-kD precursor was light dependent and induced after feeding the isolated plastids the tetrapyrrole precursor 5-aminolevulinic acid. HPLC analyses and spectroscopic pigment measurements of acetone-extracted pigments showed that the 22-kD protein is complexed with chlorophyllide. We propose a novel role of WSCPs as pigment carriers operating during light-induced chloroplast development.
In light-grown seedlings and mature green plants, chlorophyll (Chl) a and Chl b as well as carotenoids are bound to various proteins within PSI and PSII (Ort and Yocum, 1996  ). These proteins differ in their Chl-binding properties: one group binds only Chl a, whereas the other can bind both Chl a and Chl b (for reviews, see von Wettstein et al., 1995; Green and Durnford, 1996). The first group is represented by the plastid-encoded pigment-binding proteins of the actual reaction centers (PSI-A/B and D1/D2 in PSII) and the photosynthetic cores of PSII (CP43 and CP47).
The second group comprises the light-harvesting Chl a/b-binding proteins LHCI and LHCII of PSI and PSII, respectively (Dreyfuss and Thornber, 1994
In dark-grown (etiolated) seedlings, protochlorophyllide (Pchlide) accumulates instead of Chl. This compound is the immediate precursor of chlorophyllide (Chlide) and differs from it by a double bond in ring D of the tetrapyrrole ring system (for details, see von Wettstein et al., 1995 In this study, a biochemical approach was taken to identify putative Chlide carriers. We discovered a novel pigment-binding protein, which transiently accumulated as a soluble protein in the stroma of barley (Hordeum vulgare) etiochloroplasts, present in dark-grown plants that had been exposed to light for 2 h. The pigment-binding properties, chloroplast import characteristics, and the temporal expression pattern of this protein were compatible with its role as a Chlide carrier. This protein, named FCBP (free Chl[ide]-binding protein), was found to be a novel member of the family of water-soluble Chl proteins (WSCPs) of the Brassicaceae family, which thus far have only been implicated in pigment binding during photosynthetic acclimation and leaf senescence. Our results show for the first time, to our knowledge, that WSCPs, including FCBP, also operate in the shuttling of Chl intermediates during seedling de-etiolation.
Previous work had shown that etiochloroplasts isolated from etiolated barley plants, which had been exposed to light for a few hours, are experimentally depleted of endogenous PORA and PORB (Reinbothe et al., 1996 ). Due to their light-induced destabilization (C. Reinbothe et al., 1995; S. Reinbothe et al., 1995b, 1995c), PORA and PORB are degraded during plastid isolation in the light (Reinbothe et al., 1996). However, etiochloroplasts contain substantial amounts of the cytosolic PORA precursor (pPORA) at their outer envelope (Reinbothe et al., 1996). Because of the light-induced decline in the level of Pchlide, which is likely to be synthesized in the plastid envelope (Pineau et al., 1986; Joyard et al., 1990), import of the pPORA was blocked (Reinbothe et al., 1996). Import could be restored upon feeding isolated etiochloroplasts the Pchlide precursor 5-aminolevulinic acid (5-ALA; Reinbothe et al., 1996). Then, the envelope-bound precursor was chased into the plastids and processed to mature size (Reinbothe et al., 1996). We exploited these previous observations in all subsequent experiments. Briefly, 5-d-old dark-grown barley seedlings were exposed to white light for 2 h, and plastids were isolated by Percoll density gradient centrifugation and purified. The whole plastid isolation and purification procedure was performed in white light and led to etiochloroplasts that were devoid of endogenous PORA and PORB (Fig. 1 , Etiochloroplasts, lane 0). These PORA/B-depleted plastids were subsequently incubated either in darkness or white light with 5-ALA in the presence of 5 mM Mg-ATP (see "Materials and Methods").
Stromal proteins were recovered from lysed etiochloroplasts after various stages of import and subjected to immunoprecipitation (Wiedmann et al., 1987 ), using an antiserum against pPORA (Schulz et al., 1989). We assumed that nascent PORA-Pchlide-NADPH complexes formed upon import may reduce their Pchlide to Chlide and in turn would be degraded if the samples were illuminated. As shown previously, there is a light-induced, ATP- and metal ion-dependent, multicomponent stromal protease that degrades freshly formed POR-product complexes (C. Reinbothe et al., 1995; S. Reinbothe et al., 1995a). This so-called POR-degrading protease is unable to attack POR-Pchlide-NADPH complexes in darkness (C. Reinbothe et al., 1995; S. Reinbothe et al., 1995a). Figure 1 shows the results of the immunoprecipitations. They unveiled the transient accumulation of mature PORA in the stroma of the 5-ALA-fed, dark-incubated etiochloroplasts and its subsequent targeting to the thylakoids (Fig. 1A). By contrast, no mature PORA could be seen in the 5-ALA-fed, illuminated samples (Fig. 1B). This finding supports our assumption that the imported and processed pPORA was degraded. We reasoned that Chlide (a possible phototoxin) released during the degradation of imported PORA would not exist free in the stroma but would be bound by other carrier proteins, such as members of the LHC superfamily (see introduction).
To detect such putative Chlide-binding proteins, immunoprecipitations were performed using a polyclonal antiserum against LHCII (Apel and Kloppstech, 1980
Figure 2A
shows that a 22-kD protein could be immunoprecipitated from the stroma of 5-ALA-fed, illuminated etiochloroplasts. In addition to the main, prominent band, a slightly smaller band and a slightly larger band were observed (Fig. 2A). All three transiently accumulated as soluble proteins in the stroma. Their banding pattern was reminiscent of that observed for LHCII in the thylakoids (Apel and Kloppstech, 1980
The appearance of the group of 22-kD proteins, which we collectively named FCBP, was investigated further. When total etiochloroplast proteins were probed with the anti-LHCII antiserum, a larger, 27-kD protein was observed in addition to the main 22-kD protein (Fig. 3A , lane 1). Pretreatment of isolated etiochloroplasts with thermolysin prior to protein extraction led to the disappearance of the 27-kD band (Fig. 3A, lane 2). A protein of similar electrophoretic mobility could be detected when isolated mixed envelopes, which had been prepared from purified barley etiochloroplasts, were probed with the anti-LHCII antiserum (Fig. 3B, lane 3). This protein disappeared upon thermolysin treatment prior to plastid fractionation (Fig. 3B, lane 4). The inner plastid envelope membrane protein Tic110 remained protease resistant, however (Fig. 3D, lanes 5 and 6). These results demonstrated that the isolated etiochloroplasts were largely intact. Upon feeding such etiochloroplasts 5-ALA in white light, the level of 27-kD protein dropped, and increasing amounts of 22-kD FCBP were detectable in the total etiochloroplast samples (Fig. 3C, Total, lanes 1–3). Fractionation experiments with etiochloroplasts, which had been treated with thermolysin after import, and subsequent immunoprecipitations with the anti-LHCII antiserum revealed that the 27-kD protein was imported and processed to mature size. The resulting 22-kD protein accumulated transiently in the stroma and subsequently was targeted to the inner membranes of chloroplasts (Fig. 3, Stroma versus Membranes).
We next analyzed whether the freshly imported, processed FCBP may be complexed with Chlide. When we assayed immunocomplexes of stromal protein recovered after different stages of import with the anti-LHCII antiserum, unexpectedly no Chlide fluorescence could be seen using low temperature spectroscopy at 77 K (data not shown). Room temperature analyses of the same samples showed that there was acetone-extractable pigment in the illuminated etiochloroplasts, however (Fig. 4B ). Its emission maximum at 652 nm was consistent with that of Chlide b. Additional fluorescence emission and excitation analyses at different excitation and emission wavelengths proved the identity of FCBP-extracted pigment as Chlide b (data not shown). When immunocomplexes were recovered from stromal extracts of 5-ALA-fed but dark-incubated etiochloroplasts using the PORA antiserum, Pchlide was the only acetone-extractable pigment (Fig. 4A). Because Pchlide a and Pchlide b display very similar fluorescence spectra at the chosen excitation wavelength of 440 nm, no definite answer on the identity of the detected pigment could be given at this stage. When import reactions were performed in the presence of 3H-5-ALA, 3H-Chlide b and 3H-Pchlide were found (Fig. 4, C and D). This result showed that de novo synthesized pigments bound to the FCBP and PORA, respectively.
Fluorescence spectroscopy of acetone-extracted pigments does not allow distinguishing of whether porphyrins and chlorins, such as Pchlide and Chlide, may be present in their esterified or nonesterified forms. As shown previously, the phytol chain has no influence on the spectral properties of the dissolved compounds (for example, see Helfrich et al., 1994 ). We consequently sought for alternative methods permitting the identification of FCBP-extracted and PORA-extracted pigments.
In the first experiment, differential solvent extractability of FCBP-bound and PORA-bound pigments with mixtures of acetone and hexane (see "Materials and Methods") was tested. This method had previously been introduced by Helfrich et al. (1994)
As a second approach, we analyzed acetone-extracted pigments by HPLC. As shown previously, Pchlide a and Pchlide b can well be resolved on C18 reverse phase columns (Scheumann et al., 1999 ; C. Reinbothe et al., 2003), whereas Chlide a and Chlide b as well as their esterified products are separable on C30 reversed phase materials (S. Reinbothe et al., 2003). During HPLC, pigment detection and identification was made by absorbance measurements, using a photodiode array detector (see "Materials and Methods"). For comparison, synthetic standards were prepared. These have been characterized (C. Reinbothe, S. Pollmann, C. Desvignes, M. Weigele, E. Beck, and S. Reinbothe, unpublished data).
Figure 6A
shows a representative HPLC chromatogram of PORA-extracted pigments. The inset depicts an absorbance spectrum of resolved pigment. Both the retention time of 12.5 min and the absorbance profile (Fig. 6A) are consistent with values previously reported for Pchlide b (Scheumann et al., 1999
Coinjection experiments were performed in order to prove the identity of PORA-extracted Pchlide b. Ten-fold molar excess of synthetic Pchlide b was added to the sample prior to separation. Figure 6B shows an absorption spectrum of synthetic Pchlide b, which was identical to that reported before for pigments extracted from the PORA (Fig. 6A, inset). The retention time of the synthetic standard was also indistinguishable from that of the PORA-extracted pigment, strongly suggesting that both compounds were identical.
Figure 6C shows a representative HPLC chromatogram of FCBP-extracted pigments after separation on a C30 column. The inset highlights the absorption properties of resolved pigment. The shape of the curve and the main absorption maxima were consistent with those reported for Chl(ide) b (e.g. Ito et al., 1996 We next purified and sequenced the 22-kD FCBP. Import reactions were scaled up 103-fold and immunoprecipitates recovered with the anti-LHCII antiserum separated electrophoretically. Then, the 22-kD band was eluted, treated with either Staphylococcus aureus protease V8 or endoproteinase Lys C, and the mixtures subjected to HPLC. Several peptides were obtained that were subsequently sequenced.
Figure 7A
shows a sequence of 29 amino acids that could be constructed from three overlapping peptides. In this sequence, three positions could not be determined with certainty (Fig. 7A, dots). In addition, a fourth short peptide sequence was obtained. Upon aligning the resulting overall sequence with protein sequences deduced from the data banks, a relationship of the 22-kD protein to a group of previously identified seed storage proteins possessing the so-called Kunitz trypsin proteinase inhibitor motif became apparent (Fig. 7A). Interestingly, this Kunitz motif was also found in a distantly related member of the Chl-binding protein family, the so-called WSCPs of the Brassicaceae family, including Lepidium virginicum (Murata and Ishikawa, 1981
All WSCPs characterized thus far are able to bind Chl a and Chl b (for summary, see Satoh et al., 1998 ). Despite their similarity in terms of hydrophilicity, molecular mass, and subunit composition, two different subgroups of WSCPs have been discerned (Satoh et al., 1998). Members of the first group, comprising WSCPs from cauliflower, wild mustard, and brussels sprouts, show a Chl a content that is approximately 6- to 10-fold higher than that of Chl b (Murata and Murata, 1971; Murata et al., 1971; Kamimura et al., 1997; Satoh et al., 1998). Members of the second group are characterized by approximately equimolar contents of Chl a and Chl b (Murata and Ishikawa, 1981; Itoh et al., 1982). The obtained partial sequence information shown in Figure 7A suggested that the barley FCBP may belong to group II WSCPs. We therefore reprobed western blots containing total etiochloroplast samples with a heterologous antiserum against the WSCP of L. virginicum. Figure 7B demonstrates that the antiserum cross-reacted with the previously identified 22-kD and 27-kD proteins (Fig. 7B, lane 1). Pretreatment of isolated etiochloroplasts with thermolysin degraded the 27-kD protein but did not affect the level of the 22-kD protein (Fig. 7B, lane 2). A similar protease-sensitive 27-kD band was detectable in mixed outer and inner plastid envelope membranes (Fig. 7B, lanes 3 and 4). Upon feeding isolated intact etiochloroplasts 5-ALA in white light, the 27-kD protein was imported and processed, as judged from the increased amounts of 22-kD protein on the blots (Fig. 7C, L). In dark controls, no import occurred (Fig. 7C, D).
In this study, a novel water-soluble pigment-binding protein was discovered in barley etiochloroplasts. This protein is related to a family of WSCPs studied in the Brassicaceae family. In contrast to previously described pigment-binding proteins, such as LHCII, its minor species CP29, CP26, CP24, and CP14, as well as ELIPs and PsbS, the barley WSCP, which we henceforth refer to as hvWSCP, was found to contain Chl(ide) (Fig. 4). The aqueous solubulity of this pigment combined with an HPLC retention time identical to that of Chlide b (Fig. 6) indicates that the pigment bound to hvWSCP is nonesterified. Moreover, hvWSCP precursor molecules (hvpWSCP) accumulated at the outer envelope of developing barley etiochloroplasts in vivo and were imported into the plastids only under conditions where Chlide b was produced (Figs. 1, 3, and 7).
The time course experiments shown in Figures 1 and 3 suggest that Chlide b may originate during the degradation of imported pPORA. Previous in vitro reconstitution experiments showing high affinity binding of Pchlide b to PORA and its conversion to Chlide b (C. Reinbothe et al., 1999
hvWSCP is remarkable in at least three more aspects. First, it could not be detected by in situ fluorescence measurements at –196°C (77 K). This finding suggests that Chlide b may be present in a nonphotoexcitable form if bound to hvWSCP or that it may dissipate its excitation energy in a radiation-less manner, for example, by interacting with carotenoids present in etioplasts. Interestingly, recombinant WSCP of cauliflower has recently been demonstrated to lack carotenoids but to quench triplet excited Chl and singlet oxygen in an as yet unknown manner (Schmidt et al., 2003
The second remarkable point is the apparent absence of structural similarity between hvWSCP and related WSCPs to other known Chl-binding proteins. The partial amino acid sequence obtained for hvWSCP indicated a relationship to the Kunitz trypsin inhibitor motif present in certain seed storage proteins. But it also unveiled that the Kunitz motif is a signature of WSCPs. As shown in Figure 7A, the Kunitz trypsin inhibitor motif is located adjacent to the (F/Y)DPLGL motif found in the hinge region between helix 2 and helix 3 of LHCII (Kühlbrandt et al., 1994
The function of the Kunitz trypsin inhibitor motif is not yet understood. A role in inhibiting stromal proteases is conceivable but has not been demonstrated. This lack of information may be due to some specific cofactor requirements not met in previous in vitro tests using trypsin (Kamimura et al., 1997
As a third point, it is noteworthy that WSCPs thus far are believed to lack typical chloroplast transit peptides (Satoh et al., 1998
Satoh et al. (1998)
Plastid Isolation
Seeds of barley (Hordeum vulgare) L. cv Carina were grown on moist vermiculite for 5 d in the dark and exposed to white light (30 W m–2, provided by fluorescent bulbs) for 2 h (Reinbothe et al., 1996
Fifty-microliter import assays consisted of 25 µL of a doubly concentrated import buffer lacking ATP (see above), 2 µL of Mg-ATP (5 mM final concentration), and 10 µL of the isolated, resuspended plastids. The import reaction was initiated by adding 2.5 µL of 5-ALA (0.5 mM final concentration; S. Reinbothe et al., 1995c
Stromal polypeptides were subjected to gel filtration on Sephadex G15 (S. Reinbothe et al., 1995c
Total protein was extracted from nonfractionated etiochloroplasts, which had been recovered from the import mixtures by centrifugation, as described previously (S. Reinbothe et al., 1995c
Pigments were extracted from stromal fractions of lysed barley etiochloroplasts with 100% acetone containing 0.1% (v/v) diethyl pyrocarbonate. Separation by HPLC was performed on a C18 reverse phase silica gel column (250 x 4.6 mm, Nucleosil ODS 5 µm; Macherey-Nagel, Duren, Germany) as described by Scheumann et al. (1999)
For separation of Chlides a and b and their esterified products, a C30 reverse phase column (250 x 4.6 mm, 5 µm; YMC, Wilmington, NC; Fraser et al., 2000
Differential pigment extraction with acetone and hexane was performed according to Helfrich et al. (1994)
Fluorescence spectroscopy of acetone-extracted pigments was performed in a Perkin Elmer LS50B spectrometer (Foster City, CA) at room temperature using the indicated excitation and emission wavelengths (Reinbothe et al., 1999
This work was inaugurated in the Department of Prof. Dr. E. W. Weiler at the Institute for Plant Physiology, Ruhr-Universität Bochum, Bochum, Germany. We thank E.W. for his stimulating interest and continuous support of the work. We thank Dr. S. Pollmann, Bochum, for expert help with HPLC. Critical reading of the manuscript by Dr. J. Gray, University of Toledo, is gratefully acknowledged. Received September 19, 2003; returned for revision December 3, 2003; accepted December 3, 2003.
1 This work was supported by the Deutsche Forschungsgemeinschaft, Bonn, Germany (grant no. RE1465/1–1,1–2 to C.R.). 
2 This article is dedicated to Régis Mache on the occasion of his 70th birthday. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.033613. * Corresponding author; e-mail steffen.reinbothe{at}ujf-grenoble.fr; fax 0049–921–75–77–442.
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-helices (Kühlbrandt et al., 1994
