Light-harvesting Chlorophyll A/b-binding Protein Of Photosystem II Research Articles

Induction of acclimative proteolysis of the light-harvesting chlorophyll a/b protein of photosystem II in response to elevated light intensities

Most plants have the ability to respond to fluctuations in light to minimize damage to the photosynthetic apparatus. A proteolytic activity has been discovered that is involved in the degradation of the major light-harvesting chlorophyll a/b-binding protein of photosystem II (LHCII) when the antenna size of photosystem II is reduced upon acclimation of plants from low to high light intensities. This ATP-dependent proteolytic activity is of the serine or cysteine type and is associated with the outer membrane surface of the stroma-exposed thylakoid regions. The identity of the protease is not known, but it does not correspond to the recently identified chloroplast ATP-dependent proteases Clp and FtsH, which are homologs to bacterial enzymes. The acclimative response shows a delay of 2 d after transfer of the leaves to high light. This lag period was shown to be attributed to expression or activation of the responsible protease. Furthermore, the LHCII degradation was found to be regulated at the substrate level. The degradation process involves lateral migration of LHCII from the appressed to the nonappressed thylakoid regions, which is the location for the responsible protease. Phosphorylated LHCII was found to be a poor substrate for degradation in comparison with the unphosphorylated form of the protein. The relationship between LHCII degradation and other regulatory proteolytic processes in the thylakoid membrane, such as D1-protein degradation, is discussed.

A chloroplast processing enzyme functions as the general stromal processing peptidase.

A highly specific stromal processing activity is thought to cleave a large diversity of precursors targeted to the chloroplast, removing an N-terminal transit peptide. The identity of this key component of the import machinery has not been unequivocally established. We have previously characterized a chloroplast processing enzyme (CPE) that cleaves the precursor of the light-harvesting chlorophyll a/b binding protein of photosystem II (LHCPII). Here we report the overexpression of active CPE in Escherichia coli. Examination of the recombinant enzyme in vitro revealed that it cleaves not only preLHCPII, but also the precursors for an array of proteins essential for different reactions and destined for different compartments of the organelle. CPE also processes its own precursor in trans. Neither the recombinant CPE nor the native CPE of chloroplasts process a preLHCPII mutant with an altered cleavage site demonstrating that both forms of the enzyme are sensitive to the same structural modification of the substrate. The transit peptide of the precursor of ferredoxin is released by a single cleavage event and found intact after processing by recombinant CPE and a chloroplast extract as well. These results provide the first direct demonstration that CPE is the general stromal processing peptidase that acts as an endopeptidase. Significantly, recombinant CPE cleaves in the absence of other chloroplast proteins, and this activity depends on metal cations, such as zinc.

A Soluble Chloroplast Protease Processes the Euglena Polyprotein Precursor to the Light Harvesting Chlorophyll a/b Binding Protein of Photosystem II

The Euglena light harvesting chlorophyll a/b binding protein of photosystem II (LHCPII) is synthesized as a polyprotein precursors composed of 8 LHCPIIs covalently joined by a decapeptide. A soluble chloroplast protease releases LHCPII from the polyprotein. The polyprotein processing peptidase has a pH optima between 8.0 and 9.0. It is inhibited by Zn, Cu, phenylmethylsulfonyl fluoride and E64 suggesting it is a novel thiol protease.

Reconstitution and pigment-binding properties of recombinant CP29.

The minor light-harvesting chlorophyll-a/b-binding protein CP29 (Lhcb4), overexpressed in Escherichia coli, has been reconstituted in vitro with pigments. The recombinant pigment-protein complexes show biochemical and spectral properties identical to the native CP29 purified from maize thylakoids. The xanthophyll lutein is the only carotenoid necessary for reconstitution, a finding consistent with the structural role of two lutein molecules/polypeptide suggested by the crystallographic data for the homologous protein light-harvesting chlorophyll-a/b-binding protein of photosystem II (LHCII). The CP29 protein scaffold can accommodate different chromophores. This conclusion was deduced by the observation that the pigment composition of the reconstituted protein depends on the pigments present in the reconstitution mixture. Thus, in addition to a recombinant CP29 identical to the native one, two additional forms of the complex could be obtained by increasing chlorophyll b content. This finding is typical of CP29 because the major LHCII complex shows an absolute selectivity for chromophore binding [Plumley, F. G. & Schmidt, G. W. (1987) Proc. Natl Acad. Sci. USA 84, 146-150; Paulsen, H., Rümler, U. & Rüdiger, W. (1990) Planta (Heidelb.) 181, 204-211], and it is consistent with the higher stability of CP29 during greening and in chlorophyll b mutants compared with LHCII.

Characterization of the unique intron-exon junctions of Euglena gene(s) encoding the polyprotein precursor to the light-harvesting chlorophyll a/b binding protein of photosystem II.

The precursor to the Euglena light harvesting chlorophyll a/b binding protein of photosystem II (LHCPII) is a polyprotein containing multiple copies of LHCPII covalently joined by a decapeptide linker. cDNA and genomic clones encoding the 5' and 3' end of a 6.6 kb LHCPII mRNA were sequenced. A 3.1 kb genomic region encoding 1.05 kb of the 5' end of LHCPII mRNA contains 4 introns. A 7.6 kb genomic region encoding 3.3 kb of the 3' end of LHCPII mRNA contains 10 introns. The 5' and 3' ends of the 14 identified Euglena introns lacked the conserved dinucleotides (5'-GT and AG-3') found at the termini of virtually every characterized nuclear pre-mRNA intron. A common consensus splice site selection sequence could not be identified. The Euglena introns do not have the structural characteristics of group I and group II introns. The only structural feature common to all Euglena introns was the ability of short stretches of nucleotides at the 5' and 3' ends of the introns to base pair, forming a stable stem-loop with the 5' and 3' splice site juxtaposed for splicing but displaced by 2 nucleotides. The 26 nucleotide sequence at the 5' end of LHCPII mRNA is absent from the genomic sequence and identical to the 5' end of one of the small Euglena SL-RNAs indicating that it is post-transcriptionally added by trans-splicing.

Photo and Nutritional Regulation of the Light-Harvesting Chlorophyll a/b-Binding Protein of Photosystem II mRNA Levels in Euglena

In Euglena gracillis var bacillaris, light exposure increases the level of mRNA encoding the light-harvesting chlorophyll a/b-binding protein of photosystem II (LHCPII) approximately twofold. LHCPII mRNA levels increased in the dark upon either malate or ethanol addition. LHCPII mRNA is present but LHCPII is not synthesized in the bleached mutants W(3)BUL and W(10)BSmL, which lack protochlorophyll(ide) and most if not all of the chloroplast genome. Light exposure increased LHCPII mRNA levels in W(3)BUL but not in W(10)BSmL. Carbon availability and light acting through a nonchloroplast photoreceptor appear to regulate LHCPII mRNA levels. A chloroplast photoreceptor and/or a product produced by the chloroplast appear to regulate LHCPII mRNA translation.

Characterization of a Euglena gene encoding a polyprotein precursor to the light-harvesting chlorophyll a/b-binding protein of photosystem II

A 7.4 kb segment of a Euglena nuclear gene encoding a portion of the polyprotein precursor to the light-harvesting chlorophyll a/b-binding protein of photosystem II (LHCPII) has been isolated and sequenced. Nine exons can be assembled into a continuous open reading frame encoding 113 amino acids of the C-terminus of an LHCPII, followed by 4 complete LHCPIIs. The intron-exon junctions required to maintain maximum amino acid sequence homology between the LHCPIIs encoded by the Euglena genomic clone and Arabidopsis LHCPII do not conform to the consensus splice site sequences present in most eukaryotic organisms. Three types of LHCPIIs are contained within the polyprotein precursor. There is greater than 90% sequence identity at both the protein and nucleic acid level within a type while between types, there is less than 65% sequence identity. All Euglena LHCPIIs contain three hydrophobic membrane-spanning domains in the same positions as found in other LHCPIIs. Hybridization of genomic Southern blots with a probe encoding LHCPII suggests that the Euglena genome encodes a large number (30-50) of LHCPIIs. A probe derived from the 3' end of the sequenced genomic clone hybridizes with equal intensity to 2-3 genomic fragments on Southern blots. The probe encoding LHCPII hybridizes to RNAs of 9.5 and 6.6 kb on northern blots of total RNA while the 3'-end probe hybridizes only to the 6.6 kb RNA. The 6.6 kb LHCPII band detected on northern blots appears to be composed of transcription products derived from 2-3 LHCPII genes. Euglena appears to be unique in that a large number of LHCPIIs are encoded by a small number (3-5) of polyprotein genes.

Expression of a monocot LHCP promoter in transgenic rice.

The beta-glucuronidase (GUS) gene linked to the promoter of rice light harvesting chlorophyll a/b-binding protein of photosystem II (LHCPII) was introduced into rice by electroporation. Expression of the GUS gene with the LHCP promoter in transformed protoplasts and callus was much lower than with the cauliflower mosaic virus (CaMV) 35S promoter. In green organs, however, LHCP-GUS activity with the LHCP promoter was 10-fold higher than that with the CaMV 35S promoter. Expression of the LHCP-GUS gene was detected in leaves, stems and floral organs but not in roots. Histochemical analysis of leaves showed that the GUS gene was expressed specifically in green tissues. In selfed seeds of the transformants, GUS gene expression was detectable only in the germinated embryos after greening. Northern blot analysis revealed that transcription of the GUS gene with the LHCP promoter was not detectable in dark-grown seedlings of transformants but was induced by exposure to light. These results show that regulation of the introduced chimeric gene in transgenic plants was characteristic of the LHCP gene itself.

Classification and Characterization of cDNA That Encodes the Light-Harvesting Chlorophyll a/b Binding Protein of Photosystem II from Rice

Thirteen cDNA clones for the light-harvesting chlorophyll a/b binding protein of photosystem II (LHCPII) from rice were constructed; they were grouped into two classes that were based on restriction enzyme maps. The nucleotide sequences from two clones that belonged to different classes were determined and compared. The clones contained a 798-bp and 792-bp open reading frame capable of coding for polypeptides with 265 and 263 amino acids, respectively (...)

The gene for the 10 kDa phosphoprotein of photosystem II is located in chloroplast DNA

Nucleotide sequencing of a region of wheat chloroplast DNA between the genes for the 47 kDa chlorophyll a-binding protein of photosystem II ( psbB) and cytochrome b-563 ( petB) has revealed an open reading frame of 73 codons. This open reading frame has been identified as the gene ( psbH) for the 10 kDa phosphoprotein of photosystem II by comparison with the published N-terminal amino acid sequence and amino acid composition of the purified spinach protein. The predicted sequence of the protein shows some homology with the N-terminal region of the light-harvesting chlorophyll a/ b-binding protein of photosystem II (LHCII).

Differential expression of six light-harvesting chlorophyll a/b binding protein genes in maize leaf cell types.

Bundle sheath chloroplasts of maize leaves contain about one-fourth as much light-harvesting chlorophyll a/b binding protein of photosystem II (LHCP-II) as do mesophyll chloroplasts. We have determined that this difference is, in part, the result of differential expression of different LHCP-II genes. We have prepared and partially characterized cDNA clones specific for six LHCP-II genes of maize. Transcripts of these six LHCP-II genes are present at vastly different levels and account for about 95% of total LHCP-II mRNAs in bundle sheath and mesophyll cells of illuminated dark-grown maize leaves. Three genes are preferentially expressed in mesophyll cells, and their mRNAs constitute about 54% of the total LHCP-II transcripts in greening (24 hr) maize leaves. Two genes are expressed equally in bundle sheath and mesophyll cells. Most interestingly, the RNA of one gene that contributes about 8% of the total LHCP-II transcripts in leaves greening for 24 hr is present at a much higher level in bundle sheath than in mesophyll cells. Moreover, immunoblot analysis of maize thylakoids reveals at least five sizes of LHCP-II; these also differ from one another in their relative abundance in bundle sheath and mesophyll cells of developing maize leaves.