Light-harvesting Chlorophyll Binding Research Articles

English

A glasshouse experiment was conducted to compare the performance of a stay-green QTL introgression line (RSG 03123) and its parental lines (B35, a stay-green and R16, a high yielding cultivar with rapid senescence) after flowering, when water-limited (WL) or well-watered (WW). Flowering time and plant height were similar in RSG 03123 and the recurrent parent, R16. Under WW conditions, retention of green leaf area (GLA) and chlorophyll was similar in RSG 03123 and R16, whereas under WL conditions RSG 03123 retained significantly (P<0.05) more GLA. The rate of senescence was also lower in RSG 03123 as compared to R16 under WL conditions. B35 displayed a delayed onset of senescence under both WW and WL conditions while both R16 and RSG 01323 displayed an earlier onset of senescence under WL conditions. Photochemical efficiency of photosystem II (ΦPSII), gas exchange characteristics, and total soluble carbohydrates contents, in both leaf and stem, were higher in RSG 03123 than in R16 under WL conditions. These parameters were enhanced in RSG 03123 due to higher retention of chloroplast proteins, such as light-harvesting chlorophyll binding protein complex of PSII (LHCPII), oxygen-evolving complex 33 kDa (OEC33), phosphoenolpyruvate carboxylase (PEPC) and the large subunit of ribulose bisphosphate carboxylase-oxygenase (Rubisco). The stay-green characteristics of RSG 03123 were intermediate between R16 and B35; nonetheless, the results show that stay-green QTL are functional during senescence and improve tolerance to water limitation after flowering. Key words: Stay-green QTL, introgression, drought, photosynthesis.

Domain Organization in the 54-kDa Subunit of the Chloroplast Signal Recognition Particle

Chloroplast signal recognition particle (cpSRP) is a heterodimer composed of an evolutionarily conserved 54-kDa GTPase (cpSRP54) and a unique 43-kDa subunit (cpSRP43) responsible for delivering light-harvesting chlorophyll binding protein to the thylakoid membrane. While a nearly complete three-dimensional structure of cpSRP43 has been determined, no high-resolution structure is yet available for cpSRP54. In this study, we developed and examined an in silico three-dimensional model of the structure of cpSRP54 by homology modeling using cytosolic homologs. Model selection was guided by single-molecule Förster resonance energy transfer experiments, which revealed the presence of at least two distinct conformations. Small angle x-ray scattering showed that the linking region among the GTPase (G-domain) and methionine-rich (M-domain) domains, an M-domain loop, and the cpSRP43 binding C-terminal extension of cpSRP54 are predominantly disordered. Interestingly, the linker and loop segments were observed to play an important role in organizing the domain arrangement of cpSRP54. Further, deletion of the finger loop abolished loading of the cpSRP cargo, light-harvesting chlorophyll binding protein. These data highlight important structural dynamics relevant to cpSRP54’s role in the post- and cotranslational signaling processes.

GTP Hydrolysis is Not Required for Post‐translational Protein Insertion by a Chloroplast Signal Recognition Particle

Light harvesting chlorophyll binding proteins (LHCPs) are a family of nuclear encoded chloroplast thylakoid proteins that are synthesized in the cytosol, imported into the chloroplast stroma, and subsequently directed to the thylakoid membrane. LHCP targeting/insertion into thylakoid membranes requires GTP, a thylakoid protein insertase, and two prokaryote derived GTPases, chloroplast signal recognition particle (cpSRP) and its membrane receptor (cpFtsY). cpSRP binds LHCP in the stroma to form a soluble targeting complex that is directed to thylakoids owing to affinity of cpSRP for cpFtsY, an interaction requiring GTP. LHCP insertion requires Albino‐3 (Alb3), which stimulates GTP hydrolysis by cpSRP/cpFtsY and releases cpSRP from cpFtsY. The role of GTP in LHCP insertion is less clear. Using assays that reconstitute LHCP integration into isolated thylakoids, we have demonstrated that GTP hydrolysis is not required for LHCP insertion. GMP‐PNP, a non‐hydrolysable analogue of GTP, is sufficient to support LHCP integration. Surprisingly, LHCP integration supported by GMP‐PNP, and not GTP, is sensitive to elevated concentrations of free cpSRP/cpFtsY. Our data supports a mechanism in which guanine nucleotide binding by cpSRP/cpFtsY is required for LHCP release from cpSRP whereas GTP hydrolysis serves to recycle cpSRP from cpFtsY for subsequent rounds of targeting. It is hypothesized that guanine nucleotide binding will only serve to release LHCP from cpSRP in the presence of an available Alb3 insertase to prevent unproductive targeting.

Discovery of a Chlorophyll Binding Protein Complex Involved in the Early Steps of Photosystem II Assembly in Synechocystis

Efficient assembly and repair of the oxygen-evolving photosystem II (PSII) complex is vital for maintaining photosynthetic activity in plants, algae, and cyanobacteria. How chlorophyll is delivered to PSII during assembly and how vulnerable assembly complexes are protected from photodamage are unknown. Here, we identify a chlorophyll and β-carotene binding protein complex in the cyanobacterium Synechocystis PCC 6803 important for formation of the D1/D2 reaction center assembly complex. It is composed of putative short-chain dehydrogenase/reductase Ycf39, encoded by the slr0399 gene, and two members of the high-light-inducible protein (Hlip) family, HliC and HliD, which are small membrane proteins related to the light-harvesting chlorophyll binding complexes found in plants. Perturbed chlorophyll recycling in a Ycf39-null mutant and copurification of chlorophyll synthase and unassembled D1 with the Ycf39-Hlip complex indicate a role in the delivery of chlorophyll to newly synthesized D1. Sequence similarities suggest the presence of a related complex in chloroplasts.

Three-Dimensional Structure of the 54-Kda Subunit of the Chloroplast Signal Recognition Particle using Molecular Modeling

The chloroplast signal recognition particle is a heterodimeric complex of the 54kDa cytosolic signal recognition particle homologue (cpSRP54), and a novel 43kDa subunit (cpSRP43). While a nearly complete three-dimensional structure of cpSRP43 has been obtained, no structure is yet available for cpSRP54. The three-dimensional structure for cpSRP54 could provide valuable information for the rationalization of the extensive information already available regarding its function, and in the understanding of the as yet undetermined mechanism of light harvesting chlorophyll binding protein's (LHCP) insertion into the thylakoid membrane. In this study, we developed an in silico, three-dimensional structural model of cpsRP54 using a combination of homology modeling, de novo structure prediction and molecular dynamics simulation. The resulting structure is consistent with the known properties of the protein and sheds new light on some of the mechanistic details of its functioning.

C-Terminal M4 Peptide Fragment of Albino 3 Interaction with cpSRP-43

The protein complexes responsible for insertion of light harvesting chlorophyll binding protein into the thylakoid membrane of the chloroplast are the concern of this investigation. Critical for this insertion, the membrane bound protein Albino 3 has been shown to bind a cpSRP-FTSY-LHCP complex. The following study implicates the stromal, c-terminus of Albino 3 as an imperative binding interaction in this process. Isothermal titration calorimetry and 1H-15N HSQC were used to understand the binding of a 16 residue segment from this region of Albino 3 known as M4. The data show that this segment binds with 14uM affinity and that electrostatic interactions with E327 and E356 on the 43kDa subunit of cpSRP are essential for this event. Importantly, significant binding is not observed when two domains of cpSRP-43, CD2 and CD3, are separated. Far UV and intrinsic fluorescence experiments also show that, upon M4 binding, moderate conformational changes in CD2-CD3 occur.

Investigation of the Structural Stability of cpSRP43 Chromodomain2 by Hydrogen-Deuterium Exchange

The essential pathway by which light-harvesting chlorophyll binding proteins are inserted into the thylakoid of the chloroplast is mediated by a unique signal recognition particle (cpSRP). Its novel 43kDa subunit (cpSRP43) contains three chromodomains (CDs). CD2 has been shown to be significant in various aspects of the cpSRP-mediated pathway. While structural information about CD2 is known, this study aims to gain a more comprehensive understanding of the structural stability through analysis of hydrogen-deuterium exchange. 15N-labeled CD2 samples will be produced from over-expression of CD2 in E. coli cultured in labeled media. Subsequently, the readily-occurring exchange between amide hydrogen and solvent deuterium will be monitored by NMR spectroscopy. The kinetics of the exchange can provide useful information about the free energy of exchange and thus the stability.

The SCO2 protein disulphide isomerase is required for thylakoid biogenesis and interacts with LCHB1 chlorophyll a/b binding proteins which affects chlorophyll biosynthesis in Arabidopsis seedlings

The process of chloroplast biogenesis requires a multitude of pathways and processes to establish chloroplast function. In cotyledons of seedlings, chloroplasts develop either directly from proplastids (also named eoplasts) or, if germinated in the dark, via etioplasts, whereas in leaves chloroplasts derive from proplastids in the apical meristem and are then multiplied by division. The snowy cotyledon 2, sco2, mutations specifically disrupt chloroplast biogenesis in cotyledons. SCO2 encodes a chloroplast-localized protein disulphide isomerase, hypothesized to be involved in protein folding. Analysis of co-expressed genes with SCO2 revealed that genes with similar expression patterns encode chloroplast proteins involved in protein translation and in chlorophyll biosynthesis. Indeed, sco2-1 accumulates increased levels of the chlorophyll precursor, protochlorophyllide, in both dark grown cotyledons and leaves. Yeast two-hybrid analyses demonstrated that SCO2 directly interacts with the chlorophyll-binding LHCB1 proteins, being confirmed in planta using bimolecular fluorescence complementation (BIFC). Furthermore, ultrastructural analysis of sco2-1 chloroplasts revealed that formation and movement of transport vesicles from the inner envelope to the thylakoids is perturbed. SCO2 does not interact with the signal recognition particle proteins SRP54 and FtsY, which were shown to be involved in targeting of LHCB1 to the thylakoids. We hypothesize that SCO2 provides an alternative targeting pathway for light-harvesting chlorophyll binding (LHCB) proteins to the thylakoids via transport vesicles predominantly in cotyledons, with the signal recognition particle (SRP) pathway predominant in rosette leaves. Therefore, we propose that SCO2 is involved in the integration of LHCB1 proteins into the thylakoids that feeds back on the regulation of the tetrapyrrole biosynthetic pathway and nuclear gene expression.

LIL3, a light-harvesting-like protein, plays an essential role in chlorophyll and tocopherol biosynthesis

The light-harvesting chlorophyll-binding (LHC) proteins are major constituents of eukaryotic photosynthetic machinery. In plants, six different groups of proteins, LHC-like proteins, share a conserved motif with LHC. Although the evolution of LHC and LHC-like proteins is proposed to be a key for the diversification of modern photosynthetic eukaryotes, our knowledge of the evolution and functions of LHC-like proteins is still limited. In this study, we aimed to understand specifically the function of one type of LHC-like proteins, LIL3 proteins, by analyzing Arabidopsis mutants lacking them. The Arabidopsis genome contains two gene copies for LIL3, LIL3:1 and LIL3:2. In the lil3:1/lil3:2 double mutant, the majority of chlorophyll molecules are conjugated with an unsaturated geranylgeraniol side chain. This mutant is also deficient in α-tocopherol. These results indicate that reduction of both the geranylgeraniol side chain of chlorophyll and geranylgeranyl pyrophosphate, which is also an essential intermediate of tocopherol biosynthesis, is compromised in the lil3 mutants. We found that the content of geranylgeranyl reductase responsible for these reactions was severely reduced in the lil3 double mutant, whereas the mRNA level for this enzyme was not significantly changed. We demonstrated an interaction of geranylgeranyl reductase with both LIL3 isoforms by using a split ubiquitin assay, bimolecular fluorescence complementation, and combined blue-native and SDS polyacrylamide gel electrophoresis. We propose that LIL3 is functionally involved in chlorophyll and tocopherol biosynthesis by stabilizing geranylgeranyl reductase.

The senescence-induced staygreen protein regulates chlorophyll degradation.

Loss of green color in leaves results from chlorophyll (Chl) degradation in chloroplasts, but little is known about how Chl catabolism is regulated throughout leaf development. Using the staygreen (sgr) mutant in rice (Oryza sativa), which maintains greenness during leaf senescence, we identified Sgr, a senescence-associated gene encoding a novel chloroplast protein. Transgenic rice overexpressing Sgr produces yellowish-brown leaves, and Arabidopsis thaliana pheophorbide a oxygenase-impaired mutants exhibiting a stay-green phenotype during dark-induced senescence have reduced expression of Sgr homologs, indicating that Sgr regulates Chl degradation at the transcriptional level. We show that the leaf stay-greenness of the sgr mutant is associated with a failure in the destabilization of the light-harvesting chlorophyll binding protein (LHCP) complexes of the thylakoid membranes, which is a prerequisite event for the degradation of Chls and LHCPs during senescence. Transient overexpression of Sgr in Nicotiana benthamiana and an in vivo pull-down assay show that Sgr interacts with LHCPII, indicating that the Sgr-LHCPII complexes are formed in the thylakoid membranes. Thus, we propose that in senescing leaves, Sgr regulates Chl degradation by inducing LHCPII disassembly through direct interaction, leading to the degradation of Chls and Chl-free LHCPII by catabolic enzymes and proteases, respectively.

Canonical Signal Recognition Particle Components Can Be Bypassed for Posttranslational Protein Targeting in Chloroplasts

The chloroplast signal recognition particle (cpSRP) and its receptor (cpFtsY) target proteins both cotranslationally and posttranslationally to the thylakoids. This dual function enables cpSRP to utilize its posttranslational activities for targeting a family of nucleus-encoded light-harvesting chlorophyll binding proteins (LHCPs), the most abundant membrane proteins in plants. Previous in vitro experiments indicated an absolute requirement for all cpSRP pathway soluble components. In agreement, a cpFtsY mutant in Arabidopsis thaliana exhibits a severe chlorotic phenotype resulting from a massive loss of LHCPs. Surprisingly, a double mutant, cpftsy cpsrp54, recovers to a great extent from the chlorotic cpftsy phenotype. This establishes that in plants, a new alternative pathway exists that can bypass cpSRP posttranslational targeting activities. Using a mutant form of cpSRP43 that is unable to assemble with cpSRP54, we complemented the cpSRP43-deficient mutant and found that this subunit is required for the alternative pathway. Along with the ability of cpSRP43 alone to bind the ALBINO3 translocase required for LHCP integration, our results indicate that cpSRP43 has developed features to function independently of cpSRP54/cpFtsY in targeting LHCPs to the thylakoid membranes.

Decreased and increased expression of the subunit CHL I diminishes Mg chelatase activity and reduces chlorophyll synthesis in transgenic tobacco plants.

The chelation of Fe2+ and Mg2+ ions forms protoheme IX and Mg-protoporphyrin IX, respectively, and the latter is an intermediate in chlorophyll synthesis. Active magnesium protoporphyrin IX chelatase (Mg-chelatase) is an enzyme complex consisting of three different subunits. To investigate the function of the CHL I subunit of Mg-chelatase and the effects of modified Mg-chelatase activity on the tetrapyrrole biosynthetic pathway, we characterized N. tabacum transformants carrying gene constructs with the Chl I cDNA sequence in antisense and sense orientation under the control of the CaMV 35S promoter. Both elevated and diminished levels of Chl I mRNA and Chl I protein led to reduced Mg-chelatase activities, reflecting a perturbation of the assembly of the enzyme complex. The transformed plants did not accumulate the substrate of Mg-chelatase, protoporphyrin IX, but the leaves contained less chlorophyll and possessed increased chlorophyll a/b ratios, as well as a deficiency of light-harvesting chlorophyll binding proteins of photosystems I and II. The expression and activity of several tetrapyrrolic enzymes were reduced in parallel to lower the Mg-chelatase activity. Consistent with the lower chlorophyll contents, the rate-limiting synthesis of 5-aminolevulinate was also decreased in the transgenic lines analyzed. The consequence of reduced Mg-chelatase on early and late steps of chlorophyll synthesis, and on the organization of light harvesting complexes is discussed.

Purification and Properties of a Novel Chloroplast Stromal Peptidase: PROCESSING OF POLYPHENOL OXIDASE AND OTHER IMPORTED PRECURSORS

Polyphenol oxidases (PPOs) are nuclear-encoded chloroplast proteins that are targeted to the thylakoid lumen by a bipartite presequence. The N-terminal part of this sequence is removed by a stromal processing peptidase (SPP), and the resulting intermediate is translocated across the thylakoid and processed to the mature protein. A 4800-fold-purified SPP processed a PPO precursor (pPPO) at a site identical to that occurring in organelle. The in vitro product of SPP action on pPPO was further processed and translocated by thylakoids. This SPP processed other precursors but was inactive toward those of light-harvesting chlorophyll binding proteins. The enzyme appeared to be a metalloendopeptidase, like previously reported SPPs. However, it differed in substrate specificity, apparent size, and, most significantly, cleavage site of pPPO. Whereas the processing sites of lumen proteins determined so far were relatively distant from the hydrophobic core of the thylakoid targeting domain, pPPO was cleaved immediately before this domain. Cleavage removed the twin arginine motif characteristic of thylakoid targeting domains of lumen proteins, which are translocated by the DeltapH-dependent pathway. The possible significance of these observations to PPO translocation mechanism is discussed. It is suggested that several SPPs may exist in chloroplasts with preferences for different subsets of precursors.

Identification of two structurally related proteins involved in proteolytic processing of precursors targeted to the chloroplast.

Two proteins of 145 and 143 kDa were identified in pea which co-purify with a chloroplast processing activity that cleaves the precursor for the major light-harvesting chlorophyll binding protein (preLHCP). Antiserum generated against the 145/143 kDa doublet recognizes only these two polypeptides in a chloroplast soluble extract. In immunodepletion experiments the antiserum removed the doublet, and there was a concomitant loss of cleavage of preLHCP as well as of precursors for the small subunit of Rubisco and the acyl carrier protein. The 145 and 143 kDa proteins co-eluted in parallel with the peak of processing activity during all fractionation procedures, but they were not detectable as a homo- or heterodimeric complex. The 145 and 143 kDa proteins were used separately to affinity purify immunoglobulins; each preparation recognized both polypeptides, indicating that they are antigenically related. Wheat chloroplasts contain a soluble species similar in size to the 145/143 kDa doublet.

Transiently expressed early light-inducible thylakoid proteins share transmembrane domains with light-harvesting chlorophyll binding proteins

The early light-inducible proteins (ELIPs) of barley chloroplasts are encoded in two multigene families yielding end products of different molecular mass. Sequencing of complete cDNA clones showed that the low and high molecular mass proteins differ by the presence or absence of a 65 amino acid peptide in the amino-terminal part of the mature proteins. Two domains of the ELIPs reveal striking similarity in amino acid sequence with two transmembrane domains of all known light-harvesting chlorophyll a/b-binding proteins from photosystem I and II and may be of importance in anchoring the polypeptides in the membrane. The cDNA sequences of two low molecular mass ELIPs differ by an insert of 5 codons in the putative transit peptide. By in vitro transcription and translation of the cloned DNA and subsequent transport of the products into chloroplasts it could be established that the precursors are processed into products of identical apparent molecular mass. In vitro translated ELIPs were incorporated into thylakoid membranes both as precursors and mature polypeptides. It is suggested that ELIPs are pigment-free substitutes for light-harvesting polypeptides in the assembly of photosynthetic units during early development of thylakoids.

Nucleotide sequence of cDNA encoding the small subunit of ribulose-1,5-bisphosphate carboxylase from maize.

We have cloned a full length cDNA for the small subunit of ribulose-1,5-bisphosphate carboxylase from C4 monocot maize, determined the complete nucleotide sequence of this cDNA and deduced its amino acid sequence. The cDNA insert included 513 bp of the coding region, and 65 and 252 nucleotides of the 5' and 3' untranslated regions, respectively. The transit and mature peptides have, respectively, 47 and 123 amino acids. Comparison with the small subunit genes from other plants revealed that the maize small subunit is similar to the wheat one, there being 73% homology between the transit peptides and 64% between the mature proteins. This indicates that there is no noteworthy difference between the C3 and C4 small subunit structures. Extreme codon bias was observed for this gene, and similar codon preferences are observed for other proteins highly expressed in maize leaf, light harvesting chlorophyll binding protein and phosphoenolpyruvate carboxylase. The results indicate that preferential codon usage for highly expressed genes occurs in maize leaf.