Synthesis, assembly and degradation of thylakoid membrane proteins

The thylakoid membrane of chloroplasts contains four major protein complexes, involved in the photosynthetic electron transfer chain and in ATP synthesis. These complexes are built from a large number of polypeptide subunits encoded either in the nuclear or in the plastid genome. In this review, we are considering the mechanism that couples assembly (association of the polypeptides with each other and with their cofactors) with the upstream and downstream steps of the biogenetic pathway, translation and proteolytic degradation. We present the contrasting images of assembly that have emerged from a variety of approaches (studies of photosynthesis mutants, developmental studies and direct biochemical analysis of the kinetics of assembly). We develop the concept of control by epistasy of synthesis, through which the translation of certain subunits is controlled by the state of assembly of the complex and address the question of its mechanisms. We describe additional factors that assist in the integration and assembly of thylakoid membrane proteins.     

cis- and trans-Acting determinants for translation of psbD mRNA in Chlamydomonas reinhardtii

Chloroplast translation is mediated by nucleus-encoded factors that interact with distinct cis-acting RNA elements. A U-rich sequence within the 5' untranslated region of the psbD mRNA has previously been shown to be required for its translation in Chlamydomonas reinhardtii. By using UV cross-linking assays, we have identified a 40-kDa RNA binding protein, which binds to the wild-type psbD leader, but is unable to recognize a nonfunctional leader mutant lacking the U-rich motif. RNA binding is restored in a chloroplast cis-acting suppressor. The functions of several site-directed psbD leader mutants were analyzed with transgenic C. reinhardtii chloroplasts and the in vitro RNA binding assay. A clear correlation between photosynthetic activity and the capability to bind RNA by the 40-kDa protein was observed. Furthermore, the data obtained suggest that the poly(U) region serves as a molecular spacer between two previously characterized cis-acting elements, which are involved in RNA stabilization and translation. RNA-protein complex formation depends on the nuclear Nac2 gene product that is part of a protein complex required for the stabilization of the psbD mRNA. The sedimentation properties of the 40-kDa RNA binding protein suggest that it interacts directly with this Nac2 complex and, as a result, links processes of chloroplast RNA metabolism and translation.     

Translation in chloroplasts

The discovery that chloroplasts have semi-autonomous genetic systems has led to many insights into the biogenesis of these organelles and their evolution from free-living photosynthetic bacteria. Recent developments of our understanding of the molecular mechanisms of translation in chloroplasts suggest selective pressures that have maintained the 100-200 genes of the ancestral endosymbiont in chloroplast genomes. The ability to introduce modified genes into chloroplast genomes by homologous recombination and the recent development of an in vitro chloroplast translation system have been exploited for analyses of the cis-acting requirements for chloroplast translation. Trans-acting translational factors have been identified by genetic and biochemical approaches. Several studies have suggested that chloroplast mRNAs are translated in association with membranes.     

Chloroplast translation regulation

Chloroplast gene expression is primarily controlled during the translation of plastid mRNAs. Translation is regulated in response to a variety of biotic and abiotic factors, and requires a coordinate expression with the nuclear genome. The translational apparatus of chloroplasts is related to that of bacteria, but has adopted novel mechanisms in order to execute the specific roles that this organelle performs within a eukaryotic cell. Accordingly, plastid ribosomes contain a number of chloroplast-unique proteins and domains that may function in translational regulation. Chloroplast translation regulation involves cis-acting RNA elements (located in the mRNA 5′ UTR) as well as a set of corresponding trans-acting protein factors. While regulation of chloroplast translation is primarily controlled at the initiation steps through these RNA-protein interactions, elongation steps are also targets for modulating chloroplast gene expression. Translation of chloroplast mRNAs is regulated in response to light, and the molecular mechanisms underlying this response involve changes in the redox state of key elements related to the photosynthetic electron chain, fluctuations of the ADP/ATP ratio and the generation of a proton gradient. Photosynthetic complexes also experience assembly-related autoinhibition of translation to coordinate the expression of different subunits of the same complex. Finally, the localization of all these molecular events among the different chloroplast subcompartments appear to be a crucial component of the regulatory mechanisms of chloroplast gene expression.     

Altered mRNA binding activity and decreased translational initiation in a nuclear mutant lacking translation of the chloroplast psbA mRNA.

Translational regulation has been identified as one of the key steps in chloroplast-encoded gene expression. Genetic and biochemical analysis with Chlamydomonas reinhardtii has implicated nucleus-encoded factors that interact specifically with the 5' untranslated region of chloroplast mRNAs to mediate light-activated translation. F35 is a nuclear mutation in C. reinhardtii that specifically affects translation of the psbA mRNA (encoding D1, a core polypeptide of photosystem II), causing a photosynthetic deficiency in the mutant strain. The F35 mutant has reduced ribosome association of the psbA mRNA as a result of decreased translation initiation. This reduction in ribosome association correlates with a decrease in the stability of the mRNA. Binding activity of the psbA specific protein complex to the 5' untranslated region of the mRNA is diminished in F35 cells, and two members of this binding complex (RB47 and RB55) are reduced compared with the wild type. These data suggest that alteration of members of the psbA mRNA binding complex in F35 cells results in a reduction in psbA mRNA-protein complex formation, thereby causing a decrease in translation initiation of this mRNA.     

紫茎泽兰的光合作用特征及其生态学意义

本文研究了紫茎泽兰(Eupatorium adenophorum Spreng)光合作用强度的变化规律及其与环境主要生态因子的关系,比较了它与某些农作物叶片净光合速率的差异,得到如下结果: 1.紫茎泽兰是一种阳性偏阴的C_3类草本植物,其光合作用的光饱和点约为40000 lx,光补偿点约为700 lx,且具有80 ppm左右的CO_2补偿点。 2.紫茎泽兰的最大净光合速率能达到23毫克CO_2/平方分米·时左右,叶片净光合速率的日变化规律呈双峰曲线型(主峰在10时左右,次峰在16时左右)。在一年中有较长的时间,它的光合速率保持着较高的水平。 3.生长在一般菜园土上的紫茎泽兰,当土壤含水量降至17％左右时,叶片光合速率接近0:而且,受过干旱处理的紫茎泽兰植株,在恢复供水后的第三天,其光合速率只达到原来的53％。 根据以上结果,结合受紫茎泽兰危害地区干湿季分明的特点,提出干季是防除紫茎泽兰的最佳季节。     

Initiation codon mutations in the Chlamydomonas chloroplast petD gene result in temperature-sensitive photosynthetic growth.

The chloroplast petD gene encodes subunit IV of the cytochrome b6/f complex and is required for photosynthetic electron transport. We have created Chlamydomonas strains in which the initiation codon of the petD gene has been changed to AUU or AUC. These mutants can grow photosynthetically at room temperature, but not at 35 degrees C. The accumulation of subunit IV during photosynthetic or heterotrophic growth at room temperature is reduced to 10-20% of the wild-type level; petD mRNA abundance is reduced to approximately 50% of the wild-type amount. Pulse labeling experiments indicate that at room temperature, subunit IV translation proceeds at 10-20% of the wild-type rate. Cells grown heterotrophically at 35 degrees C accumulate < 5% as much subunit IV as wild-type cells grown under the same conditions, and < 1% as much subunit IV as wild-type cells grown at room temperature. We conclude that translation initiation in these mutants is inefficient, leading to decreased translation and accumulation of subunit IV. At 35 degrees C, translational inefficiency leads directly or indirectly to insufficient accumulation of subunit IV to support photosynthetic growth.     

Chloroplast biogenesis of photosystem II cores involves a series of assembly-controlled steps that regulate translation

The biogenesis of photosystem II, one of the major photosynthetic protein complexes, involves a cascade of assembly-governed regulation of translation of its major chloroplast-encoded subunits. In Chlamydomonas reinhardtii, the presence of the reaction center subunit D2 is required for the expression of the other reaction center subunit D1, while the presence of D1 is required for the expression of the core antenna subunit apoCP47. Using chimeric genes expressed in the chloroplast, we demonstrate that the decreased synthesis of D1 or apoCP47 in the absence of protein assembly is due to a genuine downregulation of translation. This regulation is mediated by the 5' untranslated region of the corresponding mRNA and originates from negative feedback exerted by the unassembled D1 or apoCP47 polypeptide. However, autoregulation of translation of subunit D1 is not implicated in the recovery from photoinhibition, which involves an increased translation of psbA mRNA in response to the degradation of photodamaged D1. De novo synthesis and repair of photosystem II complexes are independently controlled.     

Photosynthetic Control of Arabidopsis Leaf Cytoplasmic Translation Initiation by Protein Phosphorylation

Photosynthetic CO₂ assimilation is the carbon source for plant anabolism, including amino acid production and protein synthesis. The biosynthesis of leaf proteins is known for decades to correlate with photosynthetic activity but the mechanisms controlling this effect are not documented. The cornerstone of the regulation of protein synthesis is believed to be translation initiation, which involves multiple phosphorylation events in Eukaryotes. We took advantage of phosphoproteomic methods applied to Arabidopsis thaliana rosettes harvested under controlled photosynthetic gas-exchange conditions to characterize the phosphorylation pattern of ribosomal proteins (RPs) and eukaryotic initiation factors (eIFs). The analyses detected 14 and 11 new RP and eIF phosphorylation sites, respectively, revealed significant CO₂-dependent and/or light/dark phosphorylation patterns and showed concerted changes in 13 eIF phosphorylation sites and 9 ribosomal phosphorylation sites. In addition to the well-recognized role of the ribosomal small subunit protein RPS6, our data indicate the involvement of eIF3, eIF4A, eIF4B, eIF4G and eIF5 phosphorylation in controlling translation initiation when photosynthesis varies. The response of protein biosynthesis to the photosynthetic input thus appears to be the result of a complex regulation network involving both stimulating (e.g. RPS6, eIF4B phosphorylation) and inhibiting (e.g. eIF4G phosphorylation) molecular events.     

Light-activated translation of chloroplast mRNAs

The integrated regulation of mRNA stability, processing and translation facilitates the expression of several chloroplast genes, particularly in response to changes in illumination. Nuclear and chloroplast-encoded factors that mediate the expression of specific chloroplast messages have been characterized from green algae and plants. Recent studies suggest that the chloroplast might have recruited eukaryotic proteins, which are usually found in the cytoplasm or the endoplasmic reticulum, to couple the level of photosynthetic activity to gene expression via translational activation. Consequently, elements required for translational initiation of chloroplast messages differ from their prokaryotic ancestors. These results suggest that chloroplast translational regulation is a hybrid between prokaryotic and eukaryotic systems.     

In vitro synthesis, assembly and function of a photosynthetic membrane protein

Cell-free translation of Chlamydomonas reinhardtii RNA in the presence of photosynthetic membranes resulted in association of the herbicide binding (Qb) protein with membranes. Incubation of recovered membranes with high salt did not extract the polypeptide from membranes. Tryptic digestion of in vivo labeled membranes or membranes recovered from in vitro translation mixtures showed that Qb had similar orientation. In vitro translation in the presence of chloroplast membranes from cells exposed to high light intensity restored the membrane associated kinase activity lost by photoinhibition. Thus, in vitro synthesis resulted in functional integration of the Qb protein within the photosynthetic membrane.     

A Study on the Relationship Between the Net Photosynthetic Rate of Young European Beech and Main Climatic Factors Under Natural Conditions

Young European beech lives under the blank of European beech mature forest. Its photosynthetic rate was significantly affected by climatic factors. This paper deals with the effect of climatic factors on diurnal variations of the net photosynthetic rate,photosynthetic capacity,lightphocosynthesis curve and temperature effect of net photosynthetic rate under different radiations. The results are as follows:the climatic factors have greater influence on diurnal variations of the net photosynthetic rate. The influence coefficient of radiation,temperature and relative humidity on photosynthetic capacity are 0. 51, 0. 22. and 0. 17,respectively. The light-photosynthesis curve is Y= 1. 49273–1. 58993 × 0. 09328(0.01x). The optimum combination of environmental conditions of photosynthetic rate under natural conditions is:the radiation is more than 45 μE · m-2 · s-1 and meantime the temperature is between 10℃ and 20℃.     

Biogenesis of the chloroplast-encoded D1 protein: regulation of translation elongation, insertion, and assembly into photosystem II

Regulation of translation elongation, membrane insertion, and assembly of the chloroplast-encoded D1 protein of photosystem II (PSII) was studied using a chloroplast translation system in organello. Translation elongation of D1 protein was found to be regulated by (1) a redox component that can be activated not only by photosynthetic electron transfer but also by reduction with DTT; (2) the trans-thylakoid proton gradient, which is absolutely required for elongation of D1 nascent chains on the thylakoid membrane; and (3) the thiol reactants N-ethylmaleimide (NEM) and iodosobenzoic acid (IBZ), which inhibit translation elongation with concomitant accumulation of distinct D1 pausing intermediates. These results demonstrate that D1 translation elongation and membrane insertion are tightly coupled and highly regulated processes in that proper insertion is a prerequisite for translation elongation of D1. Cotranslational and post-translational assembly steps of D1 into PSII reaction center and core complexes occurred independently of photosynthetic electron transfer or trans-thylakoid proton gradient but were strongly affected by the thiol reactants DTT, NEM, and IBZ. These compounds reduced the stability of the early PSII assembly intermediates, hampered the C-terminal processing of the precursor of D1, and prevented the post-translational reassociation of CP43, indicating a strong dependence of the D1 assembly steps on proper redox conditions and the formation of disulfide bonds.     

eIF4B,eIF4G and RNA regulate eIF4A activity in translation initiation by modulating the eIF4A conformational cycle

Eukaryotic translation initiation factor eIF4A is a DEAD-box helicase that resolves secondary structure elements in the 5''-UTR of mRNAs during ribosome scanning. Its RNA-stimulated ATPase and ATP-dependent helicase activities are enhanced by other translation initiation factors, but the underlying mechanisms are unclear. DEAD-box proteins alternate between open and closed conformations during RNA unwinding. The transition to the closed conformation is linked to duplex destabilization. eIF4A is a special DEAD-box protein that can adopt three different conformations, an open state in the absence of ligands, a half-open state stabilized by the translation initiation factor eIF4G and a closed state in the presence of eIF4G and eIF4B. We show here that eIF4A alone does not measurably sample the closed conformation. The translation initiation factors eIF4B and eIF4G accelerate the eIF4A conformational cycle. eIF4G increases the rate of closing more than the opening rate, and eIF4B selectively increases the closing rate. Strikingly, the rate constants and the effect of eIF4B are different for different RNAs, and are related to the presence of single-stranded regions. Modulating the kinetics of the eIF4A conformational cycle is thus central for the multi-layered regulation of its activity, and for its role as a regulatory hub in translation initiation.     

Effect of high atmospheric CO2 concentration on δ13 of algae

Precambrian reduced carbon is more depleted in¹³C than what would be expected from the carbon isotopic composition of modern marine algae and algal mats. Since the photosynthetic carbon fixation by algae is the most likely source of the reduced carbon, the depletion has been considered an anomaly.We examined factors that might have contributed to the carbon isotope fractionation from inorganic sources through algae to organic matter in a sedimentary rock, and related laboratory obtainable data to those from Precambrian rocks. Laboratory culture experiments were then performed with nine strains of algae at various concentrations of carbon dioxide, and the result was interpreted according to the relationship.It indicated that the depletion could be understood in terms of a combined effect of fractionation factors, most depletion occurring at the fractionation during the photosynthetic carbon fixation. It also suggested that all but one algal strain incorporated bicarbonate as the source of carbon for its growth. The exception was a thermophilic, acidophilic alga, which must have used carbon dioxide as the carbon source.The present study suggests that Precambrian atmosphere was enriched in carbon dioxide roughly two orders of magnitude more than its present atmospheric level.     

The hypusine-containing translation factor eIF5A

Abstract

In addition to the small and large ribosomal subunits, aminoacyl-tRNAs, and an mRNA, cellular protein synthesis is dependent on translation factors. The eukaryotic translation initiation factor 5A (eIF5A) and its bacterial ortholog elongation factor P (EF-P) were initially characterized based on their ability to stimulate methionyl-puromycin (Met-Pmn) synthesis, a model assay for protein synthesis; however, the function of these factors in cellular protein synthesis has been difficult to resolve. Interestingly, a conserved lysine residue in eIF5A is post-translationally modified to hypusine and the corresponding lysine residue in EF-P from at least some bacteria is modified by the addition of a β-lysine moiety. In this review, we provide a summary of recent data that have identified a novel role for the translation factor eIF5A and its hypusine modification in the elongation phase of protein synthesis and more specifically in stimulating the production of proteins containing runs of consecutive proline residues.     

生态沟渠铜钱草光合日变化的研究

以生态沟渠铜钱草为材料,采用便携式CID-340光合仪对铜钱草成熟叶片净光合速率(Pn)以及胞间CO2浓度(Ci)、光合有效辐射(PAR)、气孔导度(Gs)、气温(Gs)、叶温(Tl))和蒸腾速率(Tr)等影响因子进行测定,以探讨其光合生理生态特性,旨在为修复沟渠湿地提供一定的理论依据。结果表明:(1)铜钱草叶片净光合速率(Pn)日变化曲线呈双峰型,主峰(19.32μmol.m-2.s-1)出现在15:00左右,次峰(16.21μmol.m-2.s-1)出现在11:00,中午出现光合"午休"现象。(2)用逐步多元回归方法得到净光合速率日变化与主要生理生态因子的回归方程为:Pn=-5.45613+0.006797PAR+0.050099Gs(复相关系数0.868)。逐步回归结果表明Pn受PAR和Gs的影响较大。偏相关分析和通径分析的结果表明PAR、Gs对铜钱草Pn日变化有重要影响,是影响铜钱草Pn的主要因子,影响大小的顺序为:Gs>PAR。