The role of chloroplast SRP54 domains and its C-terminal tail region in post- and cotranslational protein transport in vivo.

In the chloroplast, the 54 kDa subunit of the signal recognition particle (cpSRP54) is involved in the posttranslational transport of the light-harvesting chlorophyll a/b-binding proteins (LHCPs) and the cotranslational transport of plastid-encoded subunits of the photosynthetic complexes to the thylakoid membrane. It forms a high-affinity complex with plastid-specific cpSRP43 for posttranslational transport, while a ribosome-associated pool coordinates its cotranslational function. CpSRP54 constitutes a conserved multidomain protein, comprising a GTPase (NG) and a methionine-rich (M) domain linked by a flexible region. It is further characterized by a plastid-specific C-terminal tail region containing the cpSRP43-binding motif. To characterize the physiological role of the various regions of cpSRP54 in thylakoid membrane protein transport, we generated Arabidopsis thaliana cpSRP54 knockout (ffc1-2) lines producing truncated cpSRP54 variants or a GTPase point mutation variant. Phenotypic characterization of the complementation lines demonstrated that the C-terminal tail region of cpSRP54 plays an important role exclusively in posttranslational LHCP transport. Furthermore, we show that the GTPase activity of cpSRP54 plays an essential role in the transport pathways for both nuclear- as well as plastid-encoded proteins. In addition, our data revealed that plants expressing cpSRP54 without the C-terminal region exhibit a strongly increased accumulation of a photosystem I assembly intermediate.

Loss of CpFTSY Reduces Photosynthetic Performance and Affects Insertion of PsaC of PSI in Diatoms.

The chloroplast signal recognition particle (CpSRP) receptor (CpFTSY) is a component of the CpSRP pathway that post-translationally targets light-harvesting complex proteins (LHCPs) to the thylakoid membranes in plants and green algae containing chloroplasts derived from primary endosymbiosis. In plants, CpFTSY also plays a major role in the co-translational incorporation of chloroplast-encoded subunits of photosynthetic complexes into the thylakoids. This role has not been demonstrated in green algae. So far, its function in organisms with chloroplasts derived from secondary endosymbiotic events has not been elucidated. Here, we report the generation and characterization of mutants lacking CpFTSY in the diatom Phaeodactylum tricornutum. We found that this protein is not involved in inserting LHCPs into thylakoid membranes, indicating that the post-translational part of the CpSRP pathway is not active in this group of microalgae. The lack of CpFTSY caused an increased level of photoprotection, low electron transport rates, inefficient repair of photosystem II (PSII), reduced growth, a strong decline in the PSI subunit PsaC and upregulation of proteins that might compensate for a non-functional co-translational CpSRP pathway during light stress conditions. The phenotype was highly similar to the one described for diatoms lacking another component of the co-translational CpSRP pathway, the CpSRP54 protein. However, in contrast to cpsrp54 mutants, only one thylakoid membrane protein, PetD of the Cytb6f complex, was downregulated in cpftsy. Our results point to a minor role for CpFTSY in the co-translational CpSRP pathway, suggesting that other mechanisms may partially compensate for the effect of a disrupted CpSRP pathway.

Functional studies of CpSRP54 in diatoms show that the mechanism of thylakoid protein insertion differs from that in plants and green algae.

The chloroplast signal recognition particle 54 kDa (CpSRP54) protein is a member of the CpSRP pathway known to target proteins to thylakoid membranes in plants and green algae. Loss of CpSRP54 in the marine diatom Phaeodactylum tricornutum lowers the accumulation of a selection of chloroplast-encoded subunits of photosynthetic complexes, indicating a role in the co-translational part of the CpSRP pathway. In contrast to plants and green algae, absence of CpSRP54 does not have a negative effect on the content of light-harvesting antenna complex proteins and pigments in P. tricornutum, indicating that the diatom CpSRP54 protein has not evolved to function in the post-translational part of the CpSRP pathway. Cpsrp54 KO mutants display altered photophysiological responses, with a stronger induction of photoprotective mechanisms and lower growth rates compared to wild type when exposed to increased light intensities. Nonetheless, their phenotype is relatively mild, thanks to the activation of mechanisms alleviating the loss of CpSRP54, involving upregulation of chaperones. We conclude that plants, green algae, and diatoms have evolved differences in the pathways for co-translational and post-translational insertion of proteins into the thylakoid membranes.

Fine mapping of CscpFtsY, a gene conferring the yellow leaf phenotype in cucumber (Cucumis sativus L.).

BACKGROUND: Leaf color mutants are ideal materials to study pigment metabolism and photosynthesis. Leaf color variations are mainly affected by chlorophylls (Chls) and carotenoid contents and chloroplast development in higher plants. However, the regulation of chlorophyll metabolism remains poorly understood in many plant species. The chloroplast signal-recognition particle system is responsible for the insertion of the light-harvesting chlorophyll a/b proteins (LHCPs) to thylakoid membranes, which controls the chloroplast development as well as the regulation of Chls biosynthesis post-translationally in higher plants. RESULTS: In this study, the yellow leaf cucumber mutant, named yl, was found in an EMS-induced mutant library, which exhibited a significantly reduced chlorophyll content, abnormal chloroplast ultrastructure and decreased photosynthetic capacity. Genetic analysis demonstrated that the phenotype of yl was controlled by a recessive nuclear gene. Using BSA-seq technology combined with the map-based cloning method, we narrowed the locus to a 100 kb interval in chromosome 3. Linkage analysis and allelism test validated the candidate SNP residing in CsaV3_3G009150 encoding one homolog of chloroplast signal-recognition particle (cpSRP) receptor in Arabidopsis, cpFtsY, could be responsible for the yellow leaf phenotype of yl. The relative expression of CscpFtsY was significantly down-regulated in different organs except for the stem, of yl compared with that in the wild type (WT). Subcellular localization result showed that CscpFtsY located in the chloroplasts of mesophyll cells. CONCLUSIONS: The yl mutant displayed Chls-deficient, impaired chloroplast ultrastructure with intermittent grana stacks and significantly decreased photosynthetic capacity. The isolation of CscpFtsY in cucumber could accelerate the progress on chloroplast development by cpSRP-dependant LHCP delivery system and regulation of Chls biosynthesis in a post-translational way.

Molecular mechanism of SRP-dependent light-harvesting protein transport to the thylakoid membrane in plants.

The light-harvesting chlorophyll a/b binding proteins (LHCP) belong to a large family of membrane proteins. They form the antenna complexes of photosystem I and II and function in light absorption and transfer of the excitation energy to the photosystems. As nuclear-encoded proteins, the LHCPs are imported into the chloroplast and further targeted to their final destination-the thylakoid membrane. Due to their hydrophobicity, the formation of the so-called 'transit complex' in the stroma is important to prevent their aggregation in this aqueous environment. The posttranslational LHCP targeting mechanism is well regulated through the interaction of various soluble and membrane-associated protein components and includes several steps: the binding of the LHCP to the heterodimeric cpSRP43/cpSRP54 complex to form the soluble transit complex; the docking of the transit complex to the SRP receptor cpFtsY and the Alb3 translocase at the membrane followed by the release and integration of the LHCP into the thylakoid membrane in a GTP-dependent manner. This review summarizes the molecular mechanisms and dynamics behind the posttranslational LHCP targeting to the thylakoid membrane of Arabidopsis thaliana.

Loss of CpSRP54 function leads to a truncated light-harvesting antenna size in Chlamydomonas reinhardtii.

The Chlamydomonas reinhardtii truncated light-harvesting antenna 4 (tla4) DNA transposon mutant has a pale green phenotype, a lower chlorophyll (Chl) per cell and a higher Chl a/b ratio in comparison with the wild type. It required a higher light intensity for the saturation of photosynthesis and displayed a greater per chlorophyll light-saturated rate of oxygen evolution than the wild type. The Chl antenna size of the photosystems in the tla4 mutant was only about 65% of that measured in the wild type. Molecular genetic analysis revealed that a single plasmid DNA insertion disrupted two genes on chromosome 11 of the mutant. A complementation study identified the "chloroplast signal recognition particle 54" gene (CpSRP54), as the lesion causing the tla4 phenotype. Disruption of this gene resulted in partial failure to assemble and, therefore, lower levels of light-harvesting Chl-binding proteins in the C. reinhardtii thylakoids. A comparative in silico 3-D structure-modeling analysis revealed that the M-domain of the CpSRP54 of C. reinhardtii possesses a more extended finger loop structure, due to different amino acid composition, as compared to that of the Arabidopsis CpSRP54. The work demonstrated that CpSRP54 deletion in microalgae can serve to generate tla mutants with a markedly smaller photosystem Chl antenna size, improved solar energy conversion efficiency, and photosynthetic productivity in high-density cultures under bright sunlight conditions.

Chloroplast PetD protein: evidence for SRP/Alb3-dependent insertion into the thylakoid membrane.

BACKGROUND: In thylakoid membrane, each monomer of the dimeric complex of cytochrome b 6 f is comprised of eight subunits that are both nucleus- and plastid-encoded. Proper cytochrome b 6 f complex integration into the thylakoid membrane requires numerous regulatory factors for coordinated transport, insertion and assembly of the subunits. Although, the chloroplast-encoded cytochrome b 6 f subunit IV (PetD) consists of three transmembrane helices, the signal and the mechanism of protein integration into the thylakoid membrane have not been identified. RESULTS: Here, we demonstrate that the native PetD subunit cannot incorporate into the thylakoid membranes spontaneously, but that proper integration occurs through the post-translational signal recognition particle (SRP) pathway. Furthermore, we show that PetD insertion into thylakoid membrane involves the coordinated action of cpFTSY, cpSRP54 and ALB3 insertase. CONCLUSIONS: PetD subunit integration into the thylakoid membrane is a post-translational and an SRP-dependent process that requires the formation of the cpSRP-cpFtsY-ALB3-PetD complex. This data provides a new insight into the molecular mechanisms by which membrane proteins integration into the thylakoid membrane is accomplished and is not limited to PetD.

Simultaneous CAS9 editing of cpSRP43, LHCA6, and LHCA7 in Picochlorum celeri lowers chlorophyll levels and improves biomass productivity.

High cellular pigment levels in dense microalgal cultures contribute to excess light absorption. To improve photosynthetic yields in the marine microalga Picochlorum celeri, CAS9 gene editing was used to target the molecular chaperone cpSRP43. Depigmented strains (>50% lower chlorophyll) were generated, with proteomics showing attenuated levels of most light harvesting complex (LHC) proteins. Gene editing generated two types of cpSRP43 transformants with distinct lower pigment phenotypes: (i) a transformant (Δsrp43) with both cpSRP43 diploid alleles modified to encode non-functional polypeptides and (ii) a transformant (STR30309) with a 3 nt in-frame insertion in one allele at the CAS9 cut site (non-functional second allele), leading to expression of a modified cpSRP43. STR30309 has more chlorophyll than Δsrp43 but substantially less than wild type. To further decrease light absorption by photosystem I in STR30309, CAS9 editing was used to stack in disruptions of both LHCA6 and LHCA7 to generate STR30843, which has higher (5-24%) productivities relative to wild type in solar-simulating bioreactors. Maximal productivities required frequent partial harvests throughout the day. For STR30843, exemplary diel bioreactor yields of ~50 g m-2 day-1 were attained. Our results demonstrate diel productivity gains in P. celeri by lowering pigment levels.

PALE-GREEN LEAF 1, a rice cpSRP54 protein, is essential for the assembly of the PSI-LHCI supercomplex.

Although photosynthetic multiprotein complexes have received major attention, our knowledge about the assembly of these proteins into functional complexes in plants is still limited. In the present study, we have identified a chlorophyll-deficient mutant, pale-green leaf 1 (pgl1), in rice that displays abnormally developed chloroplasts. Map-based cloning of this gene revealed that OsPGL1 encodes a chloroplast targeted protein homologous to the 54-kDa subunit of the signal recognition particle (cpSRP54). Immunoblot analysis revealed that the accumulation of the PSI core proteins PsaA and PsaB, subunits from the ATP synthase, cytochrome, and light-harvesting complex (LHC) is dramatically reduced in pgl1. Blue native gel analysis of thylakoid membrane proteins showed the existence of an extra band in the pgl1 mutant, which located between the dimeric PSII/PSI-LHCI and the monomeric PSII. Immunodetection after 2D separation indicated that the extra band consists of the proteins from the PSI core complex. Measurements of chlorophyll fluorescence at 77 K further confirmed that PSI, rather than PSII, was primarily impaired in the pgl1 mutant. These results suggest that OsPGL1 might act as a molecular chaperone that is required for the efficient assembly and specific integration of the peripheral LHCI proteins into the PSI core complex in rice.

Protein Targeting Into the Thylakoid Membrane Through Different Pathways.

In higher plants, chloroplasts are essential semi-autonomous organelles with complex compartments. As part of these sub-organellar compartments, the sheet-like thylakoid membranes contain abundant light-absorbing chlorophylls bound to the light-harvesting proteins and to some of the reaction center proteins. About half of the thylakoid membrane proteins are encoded by nuclear genes and synthesized in the cytosol as precursors before being imported into the chloroplast. After translocation across the chloroplast envelope by the Toc/Tic system, these proteins are subsequently inserted into or translocated across the thylakoid membranes through distinct pathways. The other half of thylakoid proteins are encoded by the chloroplast genome, synthesized in the stroma and integrated into the thylakoid through a cotranslational process. Much progress has been made in identification and functional characterization of new factors involved in protein targeting into the thylakoids, and new insights into this process have been gained. In this review, we introduce the distinct transport systems mediating the translocation of substrate proteins from chloroplast stroma to the thylakoid membrane, and present the recent advances in the identification of novel components mediating these pathways. Finally, we raise some unanswered questions involved in the targeting of chloroplast proteins into the thylakoid membrane, along with perspectives for future research.

The BF4 and p71 antenna mutants from Chlamydomonas reinhardtii.

Two pale green mutants of the green alga Chlamydomonas reinhardtii, which have been used over the years in many photosynthesis studies, the BF4 and p71 mutants, were characterized and their mutated gene identified in the nuclear genome. The BF4 mutant is defective in the insertase Alb3.1 whereas p71 is defective in cpSRP43. The two mutants showed strikingly similar deficiencies in most of the peripheral antenna proteins associated with either photosystem I or photosystem 2. As a result the two photosystems have a reduced antenna size with photosystem 2 being the most affected. Still up to 20% of the antenna proteins remain in these strains, with the heterodimer Lhca5/Lhca6 showing a lower sensitivity to these mutations. We discuss these phenotypes in light of those of other allelic mutants that have been described in the literature and suggest that eventhough the cpSRP route serves as the main biogenesis pathway for antenna proteins, there should be an escape pathway which remains to be genetically identified.

The chloroplast signal recognition particle (CpSRP) pathway as a tool to minimize chlorophyll antenna size and maximize photosynthetic productivity.

The concept of the Truncated Light-harvesting chlorophyll Antenna (TLA) size, as a tool by which to maximize sunlight utilization and photosynthetic productivity in microalgal mass cultures or high-density plant canopies, is discussed. TLA technology is known to improve sunlight-to-product energy conversion efficiencies and is hereby exemplified by photosynthetic productivity estimates of wild type and a TLA strain under simulated mass culture conditions. Recent advances in the generation of TLA-type mutants by targeting genes of the chloroplast signal-recognition particle (CpSRP) pathway, affecting the thylakoid membrane assembly of light-harvesting proteins, are also summarized. Two distinct CpSRP assembly pathways are recognized, one entailing post-translational, the other a co-translational mechanism. Differences between the post-translational and co-translational integration mechanisms are outlined, as these pertain to the CpSRP-mediated assembly of thylakoid membrane protein complexes in higher plants and green microalgae. The applicability of the CpSRP pathway genes in efforts to generate TLA-type strains with enhanced solar energy conversion efficiency in photosynthesis is evaluated.