Role of galactolipid biosynthesis in coordinated development of photosynthetic complexes and thylakoid membranes during chloroplast biogenesis in Arabidopsis

The galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are the predominant lipids in thylakoid membranes and indispensable for photosynthesis. Among the three isoforms that catalyze MGDG synthesis in Arabidopsis thaliana, MGD1 is responsible for most galactolipid synthesis in chloroplasts, whereas MGD2 and MGD3 are required for DGDG accumulation during phosphate (Pi) starvation. A null mutant of Arabidopsis MGD1 (mgd1‐2), which lacks both galactolipids and shows a severe defect in chloroplast biogenesis under nutrient‐sufficient conditions, accumulated large amounts of DGDG, with a strong induction of MGD2/3 expression, during Pi starvation. In plastids of Pi‐starved mgd1‐2 leaves, biogenesis of thylakoid‐like internal membranes, occasionally associated with invagination of the inner envelope, was observed, together with chlorophyll accumulation. Moreover, the mutant accumulated photosynthetic membrane proteins upon Pi starvation, indicating a compensation for MGD1 deficiency by Pi stress‐induced galactolipid biosynthesis. However, photosynthetic activity in the mutant was still abolished, and light‐harvesting/photosystem core complexes were improperly formed, suggesting a requirement for MGDG for proper assembly of these complexes. During Pi starvation, distribution of plastid nucleoids changed concomitantly with internal membrane biogenesis in the mgd1‐2 mutant. Moreover, the reduced expression of nuclear‐ and plastid‐encoded photosynthetic genes observed in the mgd1‐2 mutant under Pi‐sufficient conditions was restored after Pi starvation. In contrast, Pi starvation had no such positive effects in mutants lacking chlorophyll biosynthesis. These observations demonstrate that galactolipid biosynthesis and subsequent membrane biogenesis inside the plastid strongly influence nucleoid distribution and the expression of both plastid‐ and nuclear‐encoded photosynthetic genes, independently of photosynthesis.     

植物叶绿体类囊体膜及膜蛋白研究进展

叶绿体是植物和真核藻类进行光合作用的场所。存在于叶绿体类囊体膜上的蛋白质复合物含有光反应所需的光合色素和电子传递链组分,在光合作用过程中,光化学反应发生在类囊体膜上。因此,类囊体膜是光能向化学能转化的主要场所,因而也一直是光合作用研究的热点。叶绿体类囊体膜的深入研究可以促进光合作用的分子机理研究。该文就叶绿体类囊体膜的三维构象及类囊体膜蛋白的组成和功能研究进行了综述。     

Novel light-regulated chloroplast thylakoid membrane protein

A 64 kilodalton chloroplast membrane polypeptide was dependent on growth irradiance with 10-fold greater quantities of the protein present in barley (Hordeum vulgare) grown under 500 micromoles of photons per square meter per second compared with growth at 50 micromoles per square meter per second. The concentration of the protein was sensitive to changes in irradiance, with a slow time course for the response (days) similar to other reported light acclimation processes. The polypeptide also was observed in maize (Zea mays), oats (Avena sativa), and wheat (Triticum aestivum), but not in soybean (Glycine max Merr). The 64 kilodalton polypeptide did not correspond to any thylakoid membrane protein with an assigned function, so its structural or regulatory role is not known.     

The chloroplast protein CPSAR1, dually localized in the stroma and the inner envelope membrane,is involved in thylakoid biogenesis

Thylakoid biogenesis is a crucial step for plant development involving the combined action of many cellular actors. CPSAR1 is shown here to be required for the normal organization of mature thylakoid stacks, and ultimately for embryo development. CPSAR1 is a chloroplast protein that has a dual localization in the stroma and the inner envelope membrane, according to microscopy studies and subfractionation analysis. CPSAR1 is close to the Obg nucleotide binding protein subfamily and displays GTPase activity, as demonstrated by in vitro assays. Disruption of the CPSAR1 gene via T‐DNA insertion results in the arrest of embryo development. In addition, transmission electron microscopy analysis indicates that mutant embryos are unable to develop thylakoid membranes, and remain white. Unstacked membrane structures resembling single lamellae accumulate in the stroma, and do not assemble into mature thylakoid stacks. CPSAR1 RNA interference induces partially developed thylakoids leading to pale‐green embryos. Altogether, the presented data demonstrate that CPSAR1 is a protein essential for the formation of normal thylakoid membranes, and suggest a possible involvement in the initiation of vesicles from the inner envelope membrane for the transfer of lipids to the thylakoids.     

Phosphorylation of thylakoid proteins during chloroplast biogenesis in greening etiolated and light-grown wheat leaves

Phosphorylation of polypeptides in isolated thylakoids was examined during chloroplast biogenesis in greening etiolated wheat leaves and 4 day-old wheat leaves grown under a diurnal light regime. At early stages of plastid development standard thylakoid preparations were heavily contaminated with nuclear proteins, which distorted the polypeptide phosphorylation profiles. Removal of contamination from membranes by sucrose density centrifugation demonstrated that the major membrane phosphoprotein in etioplasts was at 35 kDa. During etioplast greening a number of phosphoproteins appeared, of which the 25–27 kDa apoproteins of the light-harvesting chlorophylla/b protein complex associated with photosystem II (LHCII) became the most dominant. At the early stages of thylakoid development found at the base of the 4-day-old light grown leaf the LHCII apoproteins were evident as phosphoproteins; however the major phosphoprotein was polypeptide atca. 9kDA. Phosphorylation of both the LHCII apoproteins and the 9 kDa polypeptide in these thylakoids was not light-dependent. In the older thylakoids isolated from the leaf tip the LHCII apoproteins were the major phosphoproteins and their phosphorylation had become light-regulated; however phosphorylation of the 9 kDa polypeptide remained insensitive to light.     

Role of Arabidopsis CHL27 protein for photosynthesis, chloroplast development and gene expression profiling

In Chl biosynthesis, aerobic Mg-protoporphyrin IX monomethyl ester (MPE) cyclase is a key enzyme involved in the synthesis of protochlorophyllide a, and its membrane-bound component is known to be encoded by homologs of CHL27 in photosynthetic bacteria, green algae and plants. Here, we report that the Arabidopsis chl27-t knock-down mutant exhibits retarded growth and chloroplast developmental defects that are caused by damage to PSII reaction centers. The mutant contains a T-DNA insertion within the CHL27 promoter that dramatically reduces the CHL27 mRNA level. chl27-t mutant plants grew slowly with a pale green appearance, suggesting that they are defective in Chl biosynthesis. Chl fluorescence analysis showed significantly low photosynthetic activity in chl27-t mutants, indicating damage in their PSII reaction centers. The chl27-t mutation also conferred severe defects in chloroplast development, including the unstacking of thylakoid membranes. Microarray analysis of the chl27-t mutant showed repression of numerous nuclear genes involved in photosynthesis, including those encoding components of light-harvesting complex I (LHCI) and LHCII, and PSI and PSII, which accounts for the defects in photosynthetic activity and chloroplast development. In addition, the microarray data also revealed the significant repression of genes such as PORA and AtFRO6 for Chl biosynthesis and iron acquisition, respectively, and, furthermore, implied that there is cross-talk in the Chl biosynthetic pathway among the PORA, AtFRO6 and CHL27 proteins.     

Specific role of phosphatidylglycerol and functional overlaps with other thylakoid lipids in Arabidopsis chloroplast biogenesis

Key message

With phosphate deficiency, the role of phosphatidylglycerol is compensated by increased glycolipid content in thylakoid membrane biogenesis but not photosynthetic electron transport in Arabidopsis chloroplasts.

Abstract

In plants and cyanobacteria, anionic phosphatidylglycerol (PG) is the only major phospholipid in thylakoid membranes, where neutral galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are predominant. In addition to provide a lipid bilayer matrix, PG plays a specific role in photosynthetic electron transport. Non-phosphorous sulfoquinovosyldiacylglycerol (SQDG) is another anionic lipid in thylakoids; it substitutes for PG under phosphate (Pi) deficiency to maintain proper balance of anionic charge in thylakoid membranes. Although the crucial role of PG in photosynthesis has been deeply analyzed in cyanobacteria, its physiological function in seed plants other than photosynthesis remains unclear. To reveal specific roles of PG and functional overlaps with other thylakoid lipids, we characterized a PG-deficient Arabidopsis mutant (pgp1-2) under Pi-controlled conditions. Under Pi-sufficient conditions, the proportion of PG and other thylakoid lipids was decreased in pgp1-2, which led to severe disruption of thylakoid membrane biogenesis. Under Pi-deficient conditions, the proportion of all glycolipids in the mutant was greatly increased, with that of PG further decreased. In Pi-deficient pgp1-2, thylakoid membranes remarkably developed, which was accompanied by a change in nucleoid morphology and restored expression of nuclear- and plastid-encoded photosynthesis genes. Increase in glycolipid content with Pi deficiency may compensate for the loss of PG in terms of thylakoid membrane biogenesis. Although Pi deficiency increased chlorophyll and photosynthesis protein content in pgp1-2, it critically decreased photochemical activity in PSII. Further deprivation of PG in photosynthesis complexes may abolish the PSII activity in Pi-deficient pgp1-2, which suggests that glycolipids cannot replace PG in photosynthesis.

     

Maize mutants lacking chloroplast FtsY exhibit pleiotropic defects in the biogenesis of thylakoid membranes

A chloroplast signal recognition particle (SRP) that is related to the SRP involved in secretion in bacteria and eukaryotic cells is used for the insertion of light-harvesting chlorophyll proteins (LHCPs) into the thylakoid membranes. A conserved component of the SRP mechanism is a membrane-bound SRP receptor, denoted FtsY in bacteria. Plant genomes encode FtsY homologs that are targeted to the chloroplast (cpFtsY). To investigate the in vivo roles of cpFtsY, we characterized maize cpFtsY and maize mutants having a Mu transposon insertion in the corresponding gene (chloroplast SRP receptor1, or csr1). Maize cpFtsY accumulates to much higher levels in leaf tissue than in roots and stems. Interestingly, it is present at similar levels in etiolated and green leaf tissue and was found to bind the prolamellar bodies of etioplasts. A null cpFtsY mutant, csr1-1, showed a substantial loss of leaf chlorophyll, whereas a "leaky" allele, csr1-3, conditioned a more moderate chlorophyll deficiency. Both alleles caused the loss of various LHCPs and the thylakoid-bound photosynthetic enzyme complexes and were seedling lethal. By contrast, levels of the membrane-bound components of the thylakoid protein transport machineries were not altered. The thylakoid membranes in csr1-1 chloroplasts were unstacked and reduced in abundance, but the prolamellar bodies in mutant etioplasts appeared normal. These results demonstrate the essentiality of cpFtsY for the biogenesis not only of the LHCPs but also for the assembly of the other membrane-bound components of the photosynthetic apparatus.     

Response of soybean photosynthesis and chloroplast membrane function to canopy development and mutual shading

The effect of natural shading on photosynthetic capacity and chloroplast thylakoid membrane function was examined in soybean (Glycine max. cv Young) under field conditions using a randomized complete block design. Seedlings were thinned to 15 plants per square meter at 20 days after planting. Leaves destined to function in the shaded regions of the canopy were tagged during early expansion at 40 days after planting. To investigate the response of shaded leaves to an increase in available light, plants were removed from certain plots at 29 or 37 days after tagging to reduce the population from 15 to three plants per square meter and alter the irradiance and spectral quality of light. During the transition from a sun to a shade environment, maximum photosynthesis and chloroplast electron transport of control leaves decreased by two- to threefold over a period of 40 days followed by rapid senescence and abscission. Senescence and abscission of tagged leaves were delayed by more than 4 weeks in plots where plant populations were reduced to three plants per square meter. Maximum photosynthesis and chloroplast electron transport activity were stabilized or elevated in response to increased light when plant populations were reduced from 15 to three plants per square meter. Several chloroplast thylakoid membrane components were affected by light environment. Cytochrome f and coupling factor protein decreased by 40% and 80%, respectively, as control leaves became shaded and then increased when shaded leaves acclimated to high light. The concentrations of photosystem I (PSI) and photosystem II (PSII) reaction centers were not affected by light environment or leaf age in field grown plants, resulting in a constant PSII/PSI ratio of 1.6 ± 0.3. Analysis of the chlorophyll-protein composition revealed a shift in chlorophyll from PSI to PSII as leaves became shaded and a reversal of this process when shaded leaves were provided with increased light. These results were in contrast to those of soybeans grown in a growth chamber where the PSII/PSI ratio as well as cytochrome f and coupling factor protein levels were dependent on growth irradiance. To summarize, light environment regulated both the photosynthetic characteristics and the timing of senescence in soybean leaves grown under field conditions.     

EPR signals and orientation of cytochromes in the spinach chloroplast thylakoid membrane

The cytochromes in spinach chloroplasts were studied using EPR spectroscopy. In addition to the low-spin heme signals previously assigned, cytochrome f (g_z 3.51), high-potential cytochrome b-559 (g_z 3.08) and cytochrome b-559 converted to a low-potential form (g_z 2.94), a high-spin heme signal was induced by 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). However, this signal cannot be due to cytochrome b-563 in its native form. The orientation of the cytochromes in the thylakoid membrane was studied in magnetically oriented chloroplasts. Cytochrome b-559 in the native state and in the low-potential form was found to have its heme plane perpendicular to the membrane plane. The orientation was the same for cytochrome b-559 oxidized by low-temperature illumination, which suggests that also the reduced heme is oriented perpendicular to the membrane.     

Redox regulation in the chloroplast thylakoid lumen: a new frontier in photosynthesis research

Initially linked to photosynthesis, regulation by change in the redox state of thiol groups (S-S<-- -->2SH) is now known to occur throughout biology. Thus, in addition to serving important structural and catalytic functions, it is recognized that, in many cases, disulphide bonds can be broken and reformed for regulation. Several systems, each linking a hydrogen donor to an intermediary disulphide protein, act to effect changes that alter the activity of target proteins by change in the thiol redox state. Pertinent to the present discussion is the chloroplast ferredoxin/thioredoxin system, comprised of photoreduced ferredoxin, a thioredoxin, and the enzyme ferredoxin-thioredoxin reductase, that occur in the stroma. In this system, thioredoxin links the activity of enzymes to light: those enzymes functional in biosynthesis are reductively activated by light via thioredoxin (S-S-->2SH), whereas counterparts acting in degradation are deactivated under illumination conditions and are oxidatively activated in the dark (2SH-->S-S). Recent research has uncovered a new paradigm in which an immunophilin, FKBP13, and potentially other enzymes of the chloroplast thylakoid lumen are oxidatively activated in the light (2SH-->S-S). The present review provides a perspective on this recent work.     

Mutants of Chlamydomonas: tools to study thylakoid membrane structure, function and biogenesis.

The unicellular green alga Chlamydomonas reinhardtii is a model system for the study of photosynthesis and chloroplast biogenesis. C. reinhardtii has a photosynthesis apparatus similar to that of higher plants and it grows at rapid rate (generation time about 8 h). It is a facultative phototroph, which allows the isolation of mutants unable to perform photosynthesis and its sexual cycle allows a variety of genetic studies. Transformation of the nucleus and chloroplast genomes is easily performed. Gene transformation occurs mainly by homologous recombination in the chloroplast and heterologous recombination in the nucleus. Mutants are precious tools for studies of thylakoid membrane structure, photosynthetic function and assembly. Photosynthesis mutants affected in the biogenesis of a subunit of a protein complex usually lack the entire complex; this pleiotropic effect has been used in the identification of the other subunits, in the attribution of spectroscopic signals and also as a 'genetic cleaning' process which facilitates both protein complex purification, absorption spectroscopy studies or freeze-fracture analysis. The cytochrome b6f complex is not required for the growth of C. reinhardtii, unlike the case of photosynthetic prokaryotes in which the cytochrome complex is also part of the respiratory chain, and can be uniquely studied in Chlamydomonas by genetic approaches. We describe in greater detail the use of Chlamydomonas mutants in the study of this complex.     

The chloroplast thylakoid membrane system is a molecular conveyor belt

Light drives photosynthesis, but paradoxically light is also the most variable environmental factor influencing photosynthesis both qualitatively and quantitatively. The photosynthetic apparatus of higher plants is adaptable in the extreme, as exemplified by its capacity for acclimation to very bright sunny or deeply shaded conditions. It can also respond to rapid changes in light such as sunflecks. In this paper I offer a model that i) explains the thylakoid membrane organisation into grana stacks and stroma lamellae, ii) proposes a role for rapid D1 protein turnover and LHCII phosphorylation, and iii) suggests a mechanism for photoinhibition. I argue that the photosynthetic membrane system is dynamic in three dimensions, so much so that, in the light, it is in constant motion and operates in a manner somewhat analogous to a conveyor belt. D1 protein degradation is proposed to be the motor that drives this system. Photoinhibition is suggested to be due to the arrest of D1 protein turnover.     

CGL160-mediated recruitment of the coupling factor CF1 is required for efficient thylakoid ATP synthase assembly,photosynthesis, and chloroplast development in Arabidopsis

Chloroplast ATP synthases consist of a membrane-spanning coupling factor (CF_O) and a soluble coupling factor (CF₁). It was previously demonstrated that CONSERVED ONLY IN THE GREEN LINEAGE160 (CGL160) promotes the formation of plant CF_O and performs a similar function in the assembly of its c-ring to that of the distantly related bacterial Atp1/UncI protein. Here, we show that in Arabidopsis (Arabidopsis thaliana) the N-terminal portion of CGL160 (AtCGL160N) is required for late steps in CF₁-CF_O assembly. In plants that lacked AtCGL160N, CF₁-CF_O content, photosynthesis, and chloroplast development were impaired. Loss of AtCGL160N did not perturb c-ring formation, but led to a 10-fold increase in the numbers of stromal CF₁ subcomplexes relative to that in the wild type. Co-immunoprecipitation and protein crosslinking assays revealed an association of AtCGL160 with CF₁ subunits. Yeast two-hybrid assays localized the interaction to a stretch of AtCGL160N that binds to the DELSEED-containing CF₁-β subdomain. Since Atp1 of Synechocystis (Synechocystis sp. PCC 6803) could functionally replace the membrane domain of AtCGL160 in Arabidopsis, we propose that CGL160 evolved from a cyanobacterial ancestor and acquired an additional function in the recruitment of a soluble CF₁ subcomplex, which is critical for the modulation of CF₁-CF_O activity and photosynthesis.

The green-lineage specific N-terminal domain of CGL160 recruits coupling factor 1 and its lack affects chloroplast development, photosynthesis and thylakoid ATP synthase assembly in Arabidopsis.

IN A NUTSHELL Background: Thylakoid ATP synthases are impressive molecular engines that harness the light-driven proton gradient to generate ATP during photosynthesis. Their molecular mode of operation and atomic structure have been elucidated, but their assembly process is still under investigation. Specific auxiliary factors assist in ATP synthase assembly and prevent the accumulation of dead-end products or deleterious intermediates. CGL160 is one such factor and consists of a membrane and an N-terminal domain. The membrane domain of CGL160 is distantly related to bacterial Atp1 proteins, which are also present in cyanobacteria. Previous studies demonstrated that CGL160 promotes efficient formation of the membranous c-ring of thylakoid ATP synthases in Arabidopsis thaliana. Question: What is the function of the green lineage-specific N-terminal domain of CGL160 in thylakoid ATP synthase assembly, and what is the evolutionary relationship between CGL160 and Atp1? Findings: Here, we showed that the N-terminal domain of CGL160 is required for the late steps in thylakoid ATP synthase assembly and recruits the stromal ATP synthase intermediate coupling factor CF₁. The assembly step is critical for chloroplast development in the dark, ATP synthase activity, and photosynthesis in A. thaliana. We also revealed that Atp1 from the cyanobacterium Synechocystis spec PCC 6803 could functionally replace the membrane domain of CGL160 in A. thaliana. These results indicated that Atp1 operates in c-ring assembly in cyanobacteria and that CGL160 evolved from its cyanobacterial ancestor Atp1. However, CGL160 acquired an additional function in linking a soluble ATP synthase intermediate to a membranous subcomplex. Next steps: The next steps are to identify all auxiliary factors required for the assembly of thylakoid ATP synthases and to understand their precise function in ATP synthase formation. Detailed knowledge of the factors and the assembly process could provide elegant strategies for adjusting proton circuits and altering the ATP budget in crops or other photosynthetic organisms.     

AKR2A-mediated import of chloroplast outer membrane proteins is essential for chloroplast biogenesis

In plant cells, chloroplasts have essential roles in many biochemical reactions and physiological responses. Chloroplasts require numerous protein components, but only a fraction of these proteins are encoded by the chloroplast genome. Instead, most are encoded by the nuclear genome and imported into chloroplasts from the cytoplasm post-translationally. Membrane proteins located in the chloroplast outer envelope membrane (OEM) have a critical function in the import of proteins into the chloroplast. However, the biogenesis of chloroplast OEM proteins remains poorly understood. Here, we report that an Arabidopsis ankyrin repeat protein, AKR2A, plays an essential role in the biogenesis of the chloroplast OEM proteins. AKR2A binds to chloroplast OEM protein targeting signals, as well as to chloroplasts. It also displays chaperone activity towards chloroplast OEM proteins, and facilitates the targeting of OEP7 to chloroplasts in vitro. AKR2A RNAi in plants with an akr2b knockout background showed greatly reduced levels of chloroplast proteins, including OEM proteins, and chloroplast biogenesis was also defective. Thus, AKR2A functions as a cytosolic mediator for sorting and targeting of nascent chloroplast OEM proteins to the chloroplast.     

Incorporation of 32P into membrane phospholipids during chloroplast biogenesis

The composition of membrane phospholipids during chloroplast biogenesis was studied. The maximal level of phosphatidic acid was observed in the membrane fraction of proplastids. Phosphatidylglycerol was found to be the most abundant phospholipid component of grana thylakoids. The evidence from the in vivo experiments indicates that phosphatidic acid and phosphatidylglycerol incorporate the 32P label at a high rate at all stages of the chloroplast biogenesis. It is concluded that plastids are the site of the phosphatidylglycerol biosynthesis in the plant cell.     

Functional characterization of ObgC in ribosome biogenesis during chloroplast development

The Spo0B-associated GTP-binding protein (Obg) GTPase, essential for bacterial viability, is also conserved in eukaryotes, but its primary role in eukaryotes remains unknown. Here, our functional characterization of Arabidopsis and rice obgc mutants strongly underlines the evolutionarily conserved role of eukaryotic Obgs in organellar ribosome biogenesis. The mutants exhibited a chlorotic phenotype, caused by retarded chloroplast development. A plastid DNA macroarray revealed a plastid-encoded RNA polymerase (PEP) deficiency in an obgc mutant, caused by incompleteness of the PEP complex, as its western blot exhibited reduced levels of RpoA protein, a component of PEP. Plastid rRNA profiling indicated that plastid rRNA processing is defective in obgc mutants, probably resulting in impaired ribosome biogenesis and, in turn, in reduced levels of RpoA protein. RNA co-immunoprecipitation revealed that ObgC specifically co-precipitates with 23S rRNA in vivo. These findings indicate that ObgC functions primarily in plastid ribosome biogenesis during chloroplast development. Furthermore, complementation analysis can provide new insights into the functional modes of three ObgC domains, including the Obg fold, G domain and OCT.