Light-harvesting Chlorophyll A/b Protein Research Articles

Normalization using ploidy and genomic DNA copy number allows absolute quantification of transcripts, proteins and metabolites in cells

BackgroundQuantification of transcripts, proteins, or metabolites is straightforward when the factor used to normalize these values remains constant between samples. However, normalization factors often vary among samples and thus must be developed for each new analytical method.ResultsWe demonstrate quantification of transcript and protein levels in Arabidopsis based on genomic DNA copy number. We extracted total nucleic acid from 3-week-old rosette leaves of wild-type Arabidopsis and the pale-green/dwarf mutant, abc4, and quantified the number of transcripts by quantitative reverse-transcription PCR using genomic DNA copy number and ploidy (as determined by cytometry) for normalization. Our data indicated that normalization using genes commonly employed as references resulted in inaccuracies in transcript levels of the genes RBC-L and RBC-S (encoding the large and small subunits, respectively, of ribulose 1,5-bisphosphate carboxylase/oxygenase) in wild type and mutant. Normalization using genomic DNA copy number and ploidy, however, appropriately showed that the RBC-L and RBC-S transcript levels per cell in the mutant were significantly lower than that in wild type. Furthermore, quantification revealed that a cell of a 3-week-old wild-type Arabidopsis rosette leaf had an average of 7.5 × 103 transcripts of RBC-L, 9.9 × 103 transcripts of RBC-S, and 1.4 × 106 18S rRNA. We similarly analyzed the accumulation of RBC-L and LHCP (light-harvesting chlorophyll a/b protein) in wild type and mutant based on ploidy and genomic DNA copy number that was determined by direct quantitative PCR analysis of extracts using a DNA polymerase tolerant to a wide range of common PCR inhibitors. Furthermore, we estimated the number of RBC-L molecules (2.63 × 108) and chlorophyll molecules (1.85 × 109) in each cell in 3-week-old wild-type rosette leaves; these values had relatively low coefficients of variation, underscoring the reliability of our method.ConclusionGenomic DNA copy number and ploidy are useful as general normalization factors, providing an easy method for determining the number of transcripts, proteins, and metabolites in a cell.

A chloroplast homologue of the signal recognition particle subunit SRP54 is involved in the posttranslational integration of a protein into thylakoid membranes.

The mechanisms involved in the integration of proteins into the thylakoid membrane are largely unknown. However, many of the steps of this process for the light-harvesting chlorophyll a/b protein (LHCP) have been described and reconstituted in vitro. LHCP is synthesized as a precursor in the cytosol and posttranslationally imported into chloroplasts. Upon translocation across the envelope membranes, the N-terminal transit peptide is cleaved, and the apoprotein is assembled into a soluble "transit complex" and then integrated into the thylakoid membrane via three transmembrane helices. Here we show that 54CP, a chloroplast homologue of the 54-kDa subunit of the mammalian signal recognition particle (SRP54), is essential for transit complex formation, is present in the complex, and is required for LHCP integration into the thylakoid membrane. Our data indicate that 54CP functions posttranslationally as a molecular chaperone and potentially pilots LHCP to the thylakoids. These results demonstrate that one of several pathways for protein routing to the thylakoids is homologous to the SRP pathway and point to a common evolutionary origin for the protein transport systems of the endoplasmic reticulum and the thylakoid membrane.

Stromal factor plays an essential role in protein integration into thylakoids that cannot be replaced by unfolding or by heat shock protein Hsp70.

The light-harvesting chlorophyll a/b protein (LHCP) is an integral thylakoid membrane protein. It is made in the cytosol as a precursor (pLHCP), imported into chloroplasts, and subsequently integrated into thylakoids. Integration of pLHCP into thylakoids requires a stromal protein factor that functions in part to maintain the solubility and integration competence of pLHCP. Recently, it was reported that unfolded pLHCP was sufficient for integration and that the stromal factor, identified as the plastid Hsp70, was required only to prevent pLHCP refolding [Yalovsky, S., Paulsen, H., Michaeli, D., Chitnis, P. R. & Nechushtai, R. (1992) Proc. Natl. Acad. Sci. USA 89, 5616-5619]. Our studies, using more rigorous criteria for integration, show that unfolded pLHCP is not sufficient; stromal factor is an absolute requirement for integration. Furthermore, experiments with purified Hsp70 as well as Hsp70-depleted stromal extract demonstrate that Hsp70 is not the stromal factor. These results plus the finding that pLHCP diluted out of urea is relatively stable as a substrate for integration point to an additional role for the stromal factor in targeting and/or membrane translocation.

Pith cells of poplar contain photosynthetically active chloroplasts

Pith cells of young poplar (Populus x canadensis Moench) twigs were found to contain chlorophylls a and b. In addition, it was shown that pith cells also have a considerable amount of light-harvesting chlorophyll a/b protein (LHCP), which was identified by Western blotting and localized by immunogold labelling of ultrathin sections. The data strongly indicate that these cells, though they are completely covered by wood and bark and thus are accessible only to very low amounts of light, possess a functionally active photosynthetic apparatus. Evidence for this was found by feeding isolated longitudinal sections of pith with radioactively labelled carbon dioxide and exposing them to light. After incubation, reduced carbohydrates could be detected by thinlayer chromatography, indicating that photosynthesis occurs.

Protein-specific energy requirements for protein transport across or into thylakoid membranes. Two lumenal proteins are transported in the absence of ATP.

Cytosolically synthesized thylakoid proteins must be translocated across the chloroplast envelope membranes, traverse the stroma, and then be translocated into or across the thylakoid membrane. Protein transport across the envelope requires ATP hydrolysis but not electrical or proton gradients. The energy requirements for the thylakoid translocation step were studied here for the light-harvesting chlorophyll a/b protein (LHCP), an integral membrane protein, and for several thylakoid lumen-resident proteins: plastocyanin and OE33, OE23, and OE17 (the 33-, 23-, and 17-kDa subunits of the oxygen-evolving complex, respectively). Dissipation of the thylakoid protonmotive force during an in organello protein import assay partially inhibited the thylakoid localization of LHCP and OE33, totally inhibited localization of OE23 and OE17, and had no effect on localization of plastocyanin. We used reconstitution assays for LHCP insertion and for OE23 and OE17 transport into isolated thylakoids to investigate the energy requirements in detail. The results indicated that LHCP insertion absolutely requires ATP hydrolysis and is enhanced by a transthylakoid delta pH and that transport of OE23 and OE17 is absolutely dependent upon a delta pH. Surprisingly, OE23 and OE17 transport occurred maximally in the complete absence of ATP. These results establish the thylakoid membrane as the only membrane system in which a delta pH can provide all of the energy required to translocate proteins across the bilayer. They also demonstrate that the energy requirements for integration into or translocation across the thylakoid membranes are protein-specific.

Circadian oscillations of nuclear-encoded chloroplast proteins in pea (Pisum sativum)

The diurnal and circadian expression of light-inducible chloroplast proteins, i.e. light-harvesting chlorophyll a/b protein (LHCP), early light-inducible protein (ELIP) and Fe-S Rieske, has been studied in young pea plantlets at the level of mRNA integrated into polysomal complexes and at the level of proteins. Under light-dark as well as constant light conditions the levels of the three nuclear-encoded chloroplast proteins oscillate while the investigated plastid-encoded proteins, large subunit of ribulose-1,5-bisphosphate carboxylase (LSU), reaction center protein D1 and cytochrome f, do not show oscillations at the protein level. The levels of the nuclear-encoded polysome-bound mRNAs fluctuate in parallel with the changes in the levels of poly(A) RNA which were described previously. Under constant light conditions the oscillation at the level of polysomal bound mRNA is readily dampened while the steady-state levels of the investigated nuclear-encoded proteins still fluctuate. We conclude that the extent of expression of the genes for the nuclear-encoded chloroplast proteins studied is controlled by a circadian ocillator primarily, but not exclusively, at the level of transcription.

An amino-proximal hydrophobic domain in the major light-harvesting chlorophyll a/b-protein is essential for membrane integration and protein stability

The major light-harvesting chlorophyll a/b-protein (LHCP) of higher plant chloroplasts is a nuclearencoded, integral thylakoid membrane protein that binds photosynthetic pigments and occurs in situ in an oligomeric form. We have previously examined structural and functional domains of the mature apoprotein by use of mutant LHCPs and in vitro assays for uptake and insertion. Results presented here demonstrate the effects of several mutations in the amino terminal domain of the mature apoprotein. Deletion of amino acid residues 12-58 greatly affected import into chloroplasts, while deletion or alteration of the hydrophobic region E(65)VIHARWAM(73) led to rapid degradation of the mutant LHCP. We suggest that this amino-proximal region is essential for the stability of the LHCP and its ability to integrate into the thylakoid membranes. A structural/functional relationship of this region to a previously examined hydrophobic carboxy-proximal domain [Kohorn and Tobin (1989), The Plant Cell 1, 159-166] is proposed.

A stromal protein factor maintains the solubility and insertion competence of an imported thylakoid membrane protein.

The light-harvesting chlorophyll a/b protein (LHCP) is an approximately 25,000-D thylakoid membrane protein. LHCP is synthesized in the cytosol as a precursor and must translocate across the chloroplast envelope before becoming integrally associated with the thylakoid bilayer. Previous studies demonstrated that imported LHCP traverses the chloroplast stroma as a soluble intermediate before thylakoid insertion. Here, examination of this intermediate revealed that it is a stable, discrete approximately 120,000-D species and thus either an LHCP oligomer or a complex with another component. In vitro-synthesized LHCP can be converted to a similar form by incubation with a stromal extract. The stromal component responsible for this conversion is proteinaceous as evidenced by its inactivation by heat, protease, and NEM. Furthermore, the conversion activity coelutes from a gel filtration column with a stromal protein factor(s) previously shown to be necessary for LHCP integration into isolated thylakoids. Conversion of LHCP to the 120-kD form prevents aggregation and maintains its competence for thylakoid insertion. However, conversion to this form is apparently not sufficient for membrane insertion because the isolated 120-kD LHCP still requires stroma to complete the integration process. This suggests a need for at least one more stroma-mediated reaction. Our results explain how a hydrophobic thylakoid protein remains soluble as it traverses the aqueous stroma. Moreover, they describe in part the function of the stromal requirement for insertion into the thylakoid membrane.

The greening process in cress seedlings. II. Complexing agents and 5‐aminolevulinate inhibit accumulation of cab‐mRNA coding for the light‐harvesting chlorophyll a/b protein

Dark‐green cress seedlings were treated with 5‐aminolevulinate (ALA) or the metal complexing agents 2,2′‐bipyridyl and 8‐hydroxyquinoline. The subsequent induction of cab‐mRNA coding for the light‐harvesting chlorophyll a/b protein (LHCP) by white or far‐red light was investigated. Pretreatment with ALA 48 h after sowing yielded increased levels, the same pretreatment 72 or 96 h after sowing decreased levels of cab‐mRNA. The strongest inhibition of light induction of cab‐mRNA was found by pretreatment with 2,2′‐bipyridyl. Less inhibition was obtained by pretreatment with 8‐hydroxyquinoline. Steady‐state levels of transcripts which are not light‐regulated (actin, psbA) were only slightly decreased by pretreatment with ALA or the metal chelators, whereas no decrease was found for mRNA coding for NADPH: protochlorophyllide oxidoreductase. Run‐off transcription with isolated nuclei showed that the transcription rate was reduced by pretreatment of intact plants with the metal chelator 2,2′‐bipyridyl. The results are discussed in the context of current proposals for the role of transition metals. ALA, and chlorophyll precursors in light‐induction of gene expression.

Early events in the import/assembly pathway of an integral thylakoid protein

The light-harvesting chlorophyll a/b protein (LHCP) is nuclear-encoded and must traverse the chloroplast envelope before becoming integrally assembled into thylakoid membranes. Previous studies implicated a soluble stromal form of LHCP in the assembly pathway, but relied upon assays in which the thylakoid insertion step was intentionally impaired [Cline, K., Fulsom, D. R. and Viitanen, P. V. (1989) J. Biol. Chem. 264, 14225-14232]. Here we have developed a rapid-stopping procedure, based upon the use of HgCl2, to analyze early events of the uninhibited assembly process. With this approach, we have found that proper assembly of LHCP into thylakoids lags considerably behind trans-envelope translocation. During the first few minutes of import, two distinct populations of mature-size LHCP accumulate within the chloroplast. One is the aforementioned soluble stromal intermediate, while the other is a partially (or improperly) assembled thylakoid species. Consistent with precursor/product relationships, both species reach peak levels at a time when virtually none of the imported molecules are correctly assembled. These results confirm and extend our previous interpretation, that upon import, preLHCP is rapidly processed to its mature form, giving rise to a soluble stromal intermediate. They further suggest that the stromal intermediate initially inserts into the thylakoid bilayer in a partially assembled form, which eventually becomes properly assembled into the light-harvesting complex.

PHYTOCHROME‐REGULATED EXPRESSION OF GENES ENCODING LIGHT‐HARVESTING CHLOROPHYLL a/b‐PROTEIN IN TWO LONG HYPOCOTYL MUTANTS and WILD TYPE PLANTS OF Arabidopsis thaliana*

The cab genes which encode the light-harvesting chlorophyll a/b-protein (LHCP) are expressed normally with respect to phytochrome regulation in the hy-3 and hy-5 long hypocotyl mutants of Arabidopsis thaliana. In etiolated seedlings of these mutants as well as of the wild type, 1 min of red light elevates cab mRNA levels substantially within 2 h; this increase is reversed if far-red light is given immediately after the red light treatment. We conclude that the genetic defects in these mutants do not affect steps in the signal transduction pathway leading to the regulated expression of cab genes. Additionally, the mRNA from one of the three known A. thaliana cab genes, AB140, is similar in quantity to the mRNAs from the other two, AB165 and AB180, in dark-grown seedlings of hy-3 and hy-5 as well as the parent A. thaliana (Landsberg) after a brief red light treatment. This aspect of cab gene expression differs from the strain Columbia of A. thaliana in which AB140 mRNA is the predominant message. In mature white light-grown plants of the strain Columbia, AB140 as well as a combination of AB165 and AB180 mRNAs are expressed at high levels, suggesting that AB165 and/or AB180 may be developmentally regulated.

Benzyladenine induces the appearance of LHCP-mRNA and of the relevant protein in dark-grown excised watermelon cotyledons

Cotyledons were excised from imbibed watermelon seeds, grown for 4 days in darkness on water or 10 microM benzyladenine (BA) and then tested for the presence of the light-harvesting chlorophyll a/b protein (LHCP) and its mRNA. LHCP was assayed immunologically by western blotting of SDS gels: the protein was present in plastids, but it was not recovered with the thylakoid fraction. Antibodies directed against LHCP precipitated a 32 kDa polypeptide from translation products of poly(A) RNA of cotyledons only if these had been grown on BA. Taken together the data suggest that in absence of light cytokinins are necessary for the maintenance of a detectable level of LHCP-mRNA as well as for synthesis of the protein.

An Imported Thylakoid Protein Accumulates in the Stroma When Insertion into Thylakoids is Inhibited

The light-harvesting chlorophyll a/b protein (LHCP) is synthesized in the cytosol as a precursor (pLHCP) that is imported into chloroplasts and assembled into thylakoid membranes. Under appropriate conditions, either pLHCP or LHCP will integrate into isolated thylakoids. We have identified two situations that inhibit integration in this assay. Ionophores and uncouplers inhibited integration up to 70%. Carboxyl-terminal truncations of pLHCP also interfered with integration. A 22-residue truncation reduced integration to about 25% of control, whereas a 93 residue truncation completely abolished it. When pLHCP was imported into chloroplasts in the presence of uncouplers or when truncated forms of pLHCP were used, significant amounts of the imported proteins failed to insert into thylakoids and instead accumulated in the aqueous stroma. Accumulation of stromal LHCP occurred at uncoupler concentrations required to dissipate the trans-thylakoid proton electrochemical gradient and was enhanced at reduced levels of ATP. The latter effect may be a secondary consequence of a reduction in ATP-dependent degradation within the stroma. These results indicate that the stroma is an intermediate location in the LHCP assembly pathway and provide the first evidence for a soluble intermediate during biogenesis of a chloroplast membrane protein.

Expression of Light-Harvesting Chlorophyll a/b-Protein Genes Is Phytochrome-Regulated in Etiolated Arabidopsis thaliana Seedlings

Phytochrome action results in a large and rapid increase in the light-harvesting chlorophyll a/b-protein (LHCP) mRNA level in etiolated seedlings of Arabidopsis thaliana: the RNA increase is detectable within 1 hour after 1 minute red illumination, reaches a maximum 30-fold higher than the dark level at ca. 2 hours, and decays back to dark levels by about 8 hours after the brief red illumination. S1 nuclease analysis distinguishes two kinds of mRNAs transcribed from the three members of the LHCP gene family previously characterized for Arabidopsis (LS Leutwiler, EM Meyerowitz, EM Tobin, 1986 Nucleic Acids Res 14: 4051-4064). One of these arises from the AB140 gene, while the other represents the product(s) of the AB165 and/or AB180 gene(s) (AB165/AB180 mRNA). In mature, white light-grown plants, the two kinds of mRNAs are present in nearly equal amounts. In contrast, in etiolated seedlings, 1 minute red light causes a sixfold greater increase in the level of AB140 mRNA than in the level of AB165/AB180 mRNA, although both levels are regulated by phytochrome action. The kinetics of the responses to 1 minute red light are similar for both kinds of transcripts. Additional evidence suggests that this differential expression is developmentally regulated. Because the AB140 gene offers an attractive target for further analysis of phytochrome-regulated gene expression in Arabidopsis, we have further characterized this gene by mapping its 5' and 3' transcript termini.

The chloroplast affects the transcription of a nuclear gene family

Carotenoid deficient, maize seedlings ordinarily incur extensive photooxidative damage to their chloroplasts. This damage can be minimized by growing seedlings in very dim light. Cytosolic mRNA encoding a prevalent chloroplast protein, the light-harvesting chlorophyll a/b protein (LHCP), accumulates under low-intensity light, but rapidly disappears when plants are exposed to higher intensity light. Transcription studies were performed in vitro on nuclei isolated from carotenoid deficient seedlings grown first in low-intensity light and then transferred to higher intensity light. The rate of LHCP gene transcription was rapidly reduced in high intensity light. We propose that a signal of chloroplast origin is a necessary component of optimal transcription of LHCP genes and other nuclear genes encoding chloroplast proteins. Our data indicate that the signal originates at an early stage of chloroplast development, is continuously required, for LHCP gene transcription and has a short half-life. Photooxidative damage to the chloroplast destroys its capacity for further synthesis of the signal. A significant diurnal fluctuation in LHCP transcriptional activity was also observed.

Light-Harvesting Chlorophyll a/b Protein

The apoprotein of the light-harvesting chlorophyll a/b protein (LHCP) is a major integral thylakoid membrane protein that is normally complexed with chlorophyll and xanthophylls and serves as the antenna complex of photosystem II. LHCP is encoded in the nucleus and synthesized in the cytosol as a higher molecular weight precursor that is subsequently imported into chloroplasts and assembled into thylakoids. In a previous study it was established that the LHCP precursor can integrate into isolated thylakoid membranes. The present study demonstrates that under conditions designed to preserve thylakoid structure, the inserted LHCP precursor is processed to mature size, assembled into the LHC II chlorophyll-protein complex, and localized to the appressed thylakoid membranes. Under these conditions, light can partially replace exogenous ATP in the membrane integration process.

Comparison of the effects of exogenous native phytochrome and in-vivo irradiation on in-vitro transcription in isolated nuclei from barley (Hordeum vulgare)

In barley seedlings the transcription of genes coding for the light-harvesting chlorophyll a/b protein (LHCP) is stimulated and the transcription of genes coding for the NADPH-protochlorophyllide oxidoreductase (reductase) is repressed by light working via the phytochrome system. This phytochrome-mediated control of gene expression has been studied by monitoring in-vitro transcription in isolated nuclei. Two different experimental approaches have been used to elucidate the function of phytochrome (Pfr) during the transduction of the light signal. Concentrations of phytochrome were varied experimentally either by illuminating intact plants or macerated plant material prior to the isolation of nuclei or by adding purified phytochrome (Pfr) in its native 124-kDa form to the isolated nuclei. Our results indicate that there are at least two different steps involved in the phytochrome control of specific gene expression. (i) There is a rapid and transient change in the transcription rate which is saturated by very low levels of Pfr. (ii) There is a change in the duration and the maximum range of the transient change; this step requires relatively high Pfr concentrations and thus reacts very sensitively and rapidly to changes in Pfr levels as induced by secondary irradiations. This second step, but not the first one, could be triggered by the addition of purified oat phytochrome to a reconstituted nuclear system. This effect of purified phytochrome could only be shown if nuclei isolated from red-light (R)-irradiated seedlings were used. It was thus possible to simulate the effect of an in-vivo-applied second R pulse by the addition of Pfr to nuclei isolated from R-preirradiated plants.

Chloroplast photooxidation affects the accumulation of cytosolic mRNAs encoding chloroplast proteins in maize

Maize (Zea mays L.) seedlings were grown in the presence or absence of an herbicide, norflurazon (4-chloro-5-(methylamino)-2-(α,α,α-trifluoro-m-tolyl)-pyridazinone), which prevents the accumulation of colored carotenoids. In the absence of carotenoids, plants grown in high light incur extensive photooxidative damage to their plastids, but relatively little damage elsewhere. Growth in very low light minimizes chlorophyll photooxidation and allows chloroplast development to proceed. We have previously reported that mRNA encoding light-harvesting chlorophyll a/b protein (LHCP) fails to accumulate in high-light-grown carotenoid-deficient seedlings, but accumulates normally in carotenoid-deficient seedlings grown in low light. Here we extend these results by examining the levels of translatable mRNAs encoding seven additional nuclear-encoded chloroplast proteins. When norflurazon-treated seedlings were grown in low light for 8 d and then transferred to high light for 24 h, three cytosolic mRNAs (plastocyanin, Rieske Fe-S protein, and the 33-kdalton (kDa) subunit of the photosystem II O2-evolving complex) decreased to less than 1% the amount found in untreated seedlings. Two other mRNAs (NADP malic enzyme, EC 1.1.1.40, and the 23-kDa subunit of the photosystem II O2-evolving complex) decreased significantly but not to levels as low as the first three. Levels of translatable mRNA for two other chloroplast proteins (pyruvate orthophosphate dikinase, EC 2.7.9.1, and ferredoxin NADP oxidoreductase, EC 1.18.1.2) were not reduced in nonflurazon-treated seedlings after 24 h in high light, but did not show the normal light-induced increase found in untreated plants. Photooxidative damage in the chloroplast thus affects the accumulation of a number of cytosolic mRNAs encoding proteins destined for the chloroplast.

Tissue-specific and light-dependent changes of chromatin organization in barley (Hordeum vulgare).

The DNase I sensitivity of the nuclear genes encoding the NADPH-protochlorophyllide oxidoreductase, the light-harvesting chlorophyll a/b protein (LHCP), the hordeins and a 15-kDa protein of unknown function was assayed in chromatin of etiolated and green leaves and endosperm tissue of barley (Hordeum vulgare L.). A tissue-specific differentiation of chromatin structure was found for the LHCP, hordein and 15-kDa protein genes. The genes for the LHCP and the 15-kDa protein, which are expressed in leaf tissue, display DNase I sensitivity in leaves but not in endosperm. Hordein genes which are expressed solely in endosperm, were insensitive to low levels of digestion with DNase I in leaves but sensitive in endosperm. The effect of light on chromatin structure was determined by comparing leaves of etiolated plants and plants which had been grown under a day/night cycle. Only in the case of the 15-kDa protein is there a remarkable change from a DNAse-I-sensitive configuration in etiolated leaves to a more resistant one in leaves from illuminated plants. The gene for the NADPH-protochlorophyllide oxidoreductase was found to be equally sensitive to DNase I in leaves and endosperm.

Modification of the Photosystem II light-harvesting chlorophyll [formula omitted] protein complex in maize during chill-induced photoinhibition

The changes in the functional and molecular organization of the light-harvesting chlorophyll ab protein complex (LHC II), associated with Photosystem II (PS II), are examined when maize leaves are exposed to high light at 5°C for 6 h. Chlorophyll-fluorescence kinetic analyses of thylakoids isolated from mesophyll cells of stressed and control leaves demonstrate that the stress produces a reduction in energy transfer from LHC II to PS II. The polypeptide complements of thylakoids isolated from stressed leaves showed the accumulation of a 31 kDa polypeptide; no other changes in thylakoid polypeptides were observed between control and stress leaves. The 31 kDa polypeptide was a component of purified LHC II from stressed leaves and was immunologically related to the LHC II polypeptides. Differences in fluorescence emission spectra at 77 K of purified LHC II from control and stressed leaves suggested that the stress induces a perturbation of the environment of the chlorophyll species. The appearance of the 31 kDa polypeptide correlated with a modification of LHC II and its functional association with PS II. It is suggested that this polypeptide is a precursor of LHC II polypeptides and is inserted into the membrane prior to processing.