CO2浓度对条浒苔Rubisco酶聚集蛋白核的影响

以生长快速、细胞具多个蛋白核的大型海藻条浒苔作为材料研究CO2浓度对条浒苔Rubisco酶在蛋白核和叶绿体基质之间迁移的影响.应用金标免疫电镜分子定位技术对Rubisco酶集中蛋白核程度进行数值化分析.电镜下可观察到标记Rubisco的金颗粒大部分集中分布在蛋白核中.根据Morita(1997)提出的方法,设定PR-ratio值(蛋白核内分布的Rubisco酶总量与蛋白核外类囊体基质中的Rubisco酶总量之比)作为衡量Rubisco集中蛋白核程度的分析指标.不同CO2浓度对于Kubisco酶分布的长期影响和短期影响研究均显示CO2浓度升高时,Rubisco倾向于向叶绿体基质中扩散;CO2浓度较低或无CO2培养时,Kubisco酶不断向蛋白核中集中.研究结果显示,蛋白核可能在光合作用和CCM机制中具有重要作用.     

The evolution of inorganic carbon concentrating mechanisms in photosynthesis

Inorganic carbon concentrating mechanisms (CCMs) catalyse the accumulation of CO(2) around rubisco in all cyanobacteria, most algae and aquatic plants and in C(4) and crassulacean acid metabolism (CAM) vascular plants. CCMs are polyphyletic (more than one evolutionary origin) and involve active transport of HCO(3)(-), CO(2) and/or H(+), or an energized biochemical mechanism as in C(4) and CAM plants. While the CCM in almost all C(4) plants and many CAM plants is constitutive, many CCMs show acclimatory responses to variations in the supply of not only CO(2) but also photosynthetically active radiation, nitrogen, phosphorus and iron. The evolution of CCMs is generally considered in the context of decreased CO(2) availability, with only a secondary role for increasing O(2). However, the earliest CCMs may have evolved in oxygenic cyanobacteria before the atmosphere became oxygenated in stromatolites with diffusion barriers around the cells related to UV screening. This would decrease CO(2) availability to cells and increase the O(2) concentration within them, inhibiting rubisco and generating reactive oxygen species, including O(3).     

Energy costs of carbon dioxide concentrating mechanisms in aquatic organisms

Minimum energy (as photon) costs are predicted for core reactions of photosynthesis, for photorespiratory metabolism in algae lacking CO₂ concentrating mechanisms (CCMs) and for various types of CCMs; in algae, with CCMs; allowance was made for leakage of CO₂ from the internal pool. These predicted values are just compatible with the minimum measured photon costs of photosynthesis in microalgae and macroalgae lacking or expressing CCMs. More energy-expensive photorespiration, for example for organisms using Rubiscos with lower CO₂–O₂ selectivity coefficients, would be less readily accommodated within the lowest measured photon costs of photosynthesis by algae lacking CCMs. The same applies to the cases of CCMs with higher energy costs of active transport of protons or inorganic carbon species, or greater allowance for significant leakage from the accumulated intracellular pool of CO₂. High energetic efficiency can involve a higher concentration of catalyst to achieve a given rate of reaction, adding to the resource costs of growth. There are no obvious mechanistic interpretations of the occurrence of CCMs algae adapted to low light and low temperatures using the rationales adopted for the occurrence of C₄ photosynthesis in terrestrial flowering plants. There is an exception for cyanobacteria with low-selectivity Form IA or IB Rubiscos, and those dinoflagellates with low-selectivity Form II Rubiscos, for which very few natural environments have high enough CO₂:O₂ ratios to allow photosynthesis in the absence of CCMs.     

The roles of carbonic anhydrases in photosynthetic CO2 concentrating mechanisms

Cyanobacteria, algae, aquatic angiosperms and higher plants have all developed their own unique versions of photosynthetic CO₂ concentrating mechanisms (CCMs) to aid Rubisco in efficient CO₂ capture. An important aspect of all CCMs is the critical roles that the specialised location and function that various carbonic anhydrase enzymes play in the overall process, participating the interconversion of CO₂ and HCO₃ ⁻ species both inside and outside the cell. This review examines what we currently understand about the nature of the carbonic anhydrase enzymes, their localisation and roles in the various CCMs that have been studied in detail. This revised version was published online in August 2006 with corrections to the Cover Date.     

Inorganic carbon concentrating mechanisms in relation to the biology of algae

Significant advances have recently been made in our understanding of the mechanism of inorganic carbon transport in algae and, especially, cyanobacteria with inorganic carbon concentrating mechanisms (CCMs). Furthermore, the role of CCMs in increasing the rate of photosynthesis in air-equilibrated solutions is also quite well understood. However, less often considered is how the presence (or absence) of a CCM relates to the biology of algae. This mini-review relates the occurrence of algal CCMs to phylogeny, life form, life history, and interactions with other organisms. While some patterns can be seen, the occurrence of CCMs in relation to the overall biology of the algae needs more investigation. This revised version was published online in August 2006 with corrections to the Cover Date.     

CO2 concentrating mechanisms in cyanobacteria: molecular components,their diversity and evolution

Cyanobacteria have evolved an extremely effective single-cell CO(2) concentrating mechanism (CCM). Recent molecular, biochemical and physiological studies have significantly extended current knowledge about the genes and protein components of this system and how they operate to elevate CO(2) around Rubisco during photosynthesis. The CCM components include at least four modes of active inorganic carbon uptake, including two bicarbonate transporters and two CO(2) uptake systems associated with the operation of specialized NDH-1 complexes. All these uptake systems serve to accumulate HCO(3)(-) in the cytosol of the cell, which is subsequently used by the Rubisco-containing carboxysome protein micro-compartment within the cell to elevate CO(2) around Rubisco. A specialized carbonic anhydrase is also generally present in this compartment. The recent availability of at least nine cyanobacterial genomes has made it possible to begin to undertake comparative genomics of the CCM in cyanobacteria. Analyses have revealed a number of surprising findings. Firstly, cyanobacteria have evolved two types of carboxysomes, correlated with the form of Rubisco present (Form 1A and 1B). Secondly, the two HCO(3)(-) and CO(2) transport systems are distributed variably, with some cyanobacteria (Prochlorococcus marinus species) appearing to lack CO(2) uptake systems entirely. Finally, there are multiple carbonic anhydrases in many cyanobacteria, but, surprisingly, several cyanobacterial genomes appear to lack any identifiable CA genes. A pathway for the evolution of CCM components is suggested.     

Inorganic carbon concentrating mechanisms in free-living and symbiotic dinoflagellates and chromerids

Photosynthetic dinoflagellates are ecologically and biogeochemically important in marine and freshwater environments. However, surprisingly little is known of how this group acquires inorganic carbon or how these diverse processes evolved. Consequently, how CO₂ availability ultimately influences the success of dinoflagellates over space and time remains poorly resolved compared to other microalgal groups. Here we review the evidence. Photosynthetic core dinoflagellates have a Form II RuBisCO (replaced by Form IB or Form ID in derived dinoflagellates). The in vitro kinetics of the Form II RuBisCO from dinoflagellates are largely unknown, but dinoflagellates with Form II (and other) RuBisCOs have inorganic carbon concentrating mechanisms (CCMs), as indicated by in vivo internal inorganic C accumulation and affinity for external inorganic C. However, the location of the membrane(s) at which the essential active transport component(s) of the CCM occur(s) is (are) unresolved; isolation and characterization of functionally competent chloroplasts would help in this respect. Endosymbiotic Symbiodiniaceae (in Foraminifera, Acantharia, Radiolaria, Ciliata, Porifera, Acoela, Cnidaria, and Mollusca) obtain inorganic C by transport from seawater through host tissue. In corals this transport apparently provides an inorganic C concentration around the photobiont that obviates the need for photobiont CCM. This is not the case for tridacnid bivalves, medusae, or, possibly, Foraminifera. Overcoming these long-standing knowledge gaps relies on technical advances (e.g., the in vitro kinetics of Form II RuBisCO) that can functionally track the fate of inorganic C forms.     

Heat sensitivity of Rubisco,Rubisco activase and Rubisco binding protein in higher plants

During the past few years the investigations concerning Rubisco and the changes of its activity and properties at elevated temperature were reconsidered with special reference to the important role of Rubisco activase and Rubisco binding protein. The major changes in Rubisco, Rubisco activase and Rubisco binding protein reported recently are presented in this review. New information on these proteins, including their changes under heat stress conditions, is discussed together with open questions.     

Role of carboxysomes in cyanobacterial CO2 assimilation: CO2 concentrating mechanisms and metabolon implications

Many carbon-fixing organisms have evolved CO₂ concentrating mechanisms (CCMs) to enhance the delivery of CO₂ to RuBisCO, while minimizing reactions with the competitive inhibitor, molecular O₂. These distinct types of CCMs have been extensively studied using genetics, biochemistry, cell imaging, mass spectrometry, and metabolic flux analysis. Highlighted in this paper, the cyanobacterial CCM features a bacterial microcompartment (BMC) called ‘carboxysome’ in which RuBisCO is co-encapsulated with the enzyme carbonic anhydrase (CA) within a semi-permeable protein shell. The cyanobacterial CCM is capable of increasing CO₂ around RuBisCO, leading to one of the most efficient processes known for fixing ambient CO₂. The carboxysome life cycle is dynamic and creates a unique subcellular environment that promotes activity of the Calvin–Benson (CB) cycle. The carboxysome may function within a larger cellular metabolon, physical association of functionally coupled proteins, to enhance metabolite channelling and carbon flux. In light of CCMs, synthetic biology approaches have been used to improve enzyme complex for CO₂ fixations. Research on CCM-associated metabolons has also inspired biologists to engineer multi-step pathways by providing anchoring points for enzyme cascades to channel intermediate metabolites towards valuable products.     

Drought stress effects on Rubisco in wheat: changes in the Rubisco large subunit

The leaf protein pattern from drought-tolerant and drought-sensitive wheat varieties subjected to severe soil drought but with the possibility for recover from stress was studied by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). The spots representing Rubisco large subunit (RLS) were identified using polyclonal antibodies against Rubisco and immunoblotting. Some qualitative and quantitative differences in the 2D-PAGE protein map of wheat varieties were revealed under drought conditions. Three days recovery of wheat plants were not enough for restoring RLS quantity to the level of controls after 7 days drought, especially in the drought-sensitive variety Miziya. There are contradictory data in the literature concerning increased or diminished RLS level in drought stressed plants. A comparison of RLS after SDS-PAGE and 2D-PAGE was made. The revealed protein pattern depended on the presence or absence of protease inhibitors in the extraction buffer, on the procedure of extraction, and on the degree of stress.     

Carbon dioxide concentrating mechanism in Chlamydomonas reinhardtii: inorganic carbon transport and CO2 recapture

Many microalgae are capable of acclimating to CO(2) limited environments by operating a CO(2) concentrating mechanism (CCM), which is driven by various energy-coupled inorganic carbon (Ci; CO(2) and HCO(3)(-)) uptake systems. Chlamydomonas reinhardtii (hereafter, Chlamydomonas), a versatile genetic model organism, has been used for several decades to exemplify the active Ci transport in eukaryotic algae, but only recently have many molecular details behind these Ci uptake systems emerged. Recent advances in genetic and molecular approaches, combined with the genome sequencing of Chlamydomonas and several other eukaryotic algae have unraveled some unique characteristics associated with the Ci uptake mechanism and the Ci-recapture system in eukaryotic microalgae. Several good candidate genes for Ci transporters in Chlamydomonas have been identified, and a few specific gene products have been linked with the Ci uptake systems associated with the different acclimation states. This review will focus on the latest studies on characterization of functional components involved in the Ci uptake and the Ci-recapture in Chlamydomonas.     

Novel mechanisms in murine nitrofen-induced pulmonary hypoplasia: FGF-10 rescue in culture

We evaluated the role of the key pulmonary morphogenetic gene fibroblast growth factor-10 (Fgf10) in murine nitrofen-induced primary lung hypoplasia, which is evident before the time of diaphragm closure. In situ hybridization and competitive RT-PCR revealed a profound disturbance in the temporospatial pattern as well as a 10-fold decrease in mRNA expression level of Fgf10 but not of the inducible inhibitor murine Sprouty2 (mSpry2) after nitrofen treatment. Exogenous FGF-10 increased branching not only of control lungs [13% (right) and 27% (left); P < 0.01] but also of nitrofen-exposed lungs [23% (right) and 77% (left); P < 0.01]. Expression of mSpry2 increased 10-fold with FGF-10 in both nitrofen-treated and control lungs, indicating intact downstream FGF signaling mechanisms after nitrofen treatment. We conclude that nitrofen inhibits Fgf10 expression, which is essential for lung growth and branching. Exogenous FGF-10 not only stimulates FGF signaling, marked by increased mSpry2 expression, in both nitrofen-treated and control lungs but also substantially rescues nitrofen-induced lung hypoplasia in culture.     

Algal and aquatic plant carbon concentrating mechanisms in relation to environmental change

Carbon dioxide concentrating mechanisms (also known as inorganic carbon concentrating mechanisms; both abbreviated as CCMs) presumably evolved under conditions of low CO(2) availability. However, the timing of their origin is unclear since there are no sound estimates from molecular clocks, and even if there were, there are no proxies for the functioning of CCMs. Accordingly, we cannot use previous episodes of high CO(2) (e.g. the Palaeocene-Eocene Thermal Maximum) to indicate how organisms with CCMs responded. Present and predicted environmental change in terms of increased CO(2) and temperature are leading to increased CO(2) and HCO(3)(-) and decreased CO(3)(2-) and pH in surface seawater, as well as decreasing the depth of the upper mixed layer and increasing the degree of isolation of this layer with respect to nutrient flux from deeper waters. The outcome of these forcing factors is to increase the availability of inorganic carbon, photosynthetic active radiation (PAR) and ultraviolet B radiation (UVB) to aquatic photolithotrophs and to decrease the supply of the nutrients (combined) nitrogen and phosphorus and of any non-aeolian iron. The influence of these variations on CCM expression has been examined to varying degrees as acclimation by extant organisms. Increased PAR increases CCM expression in terms of CO(2) affinity, whilst increased UVB has a range of effects in the organisms examined; little relevant information is available on increased temperature. Decreased combined nitrogen supply generally increases CO(2) affinity, decreased iron availability increases CO(2) affinity, and decreased phosphorus supply has varying effects on the organisms examined. There are few data sets showing interactions amongst the observed changes, and even less information on genetic (adaptation) changes in response to the forcing factors. In freshwaters, changes in phytoplankton species composition may alter with environmental change with consequences for frequency of species with or without CCMs. The information available permits less predictive power as to the effect of the forcing factors on CCM expression than for their overall effects on growth. CCMs are currently not part of models as to how global environmental change has altered, and is likely to further alter, algal and aquatic plant primary productivity.     

Carbon concentration mechanisms in photosynthetic microorganisms

Unicellular green algae and cyanobacteria have mechanism to actively concentrate dissolved inorganic carbon into the cells, only if they are grown with air levels of CO2. The carbon concentration mechanisms are commonly known as "CCM" or "DIC-pumps". The DIC-pumps are environmental adaptation that function to actively transport and accumulate inorganic carbon (HCO3- and CO2; Ci) within the cell and then uses this Ci pool to actively increase the concentration of CO2 at the site of ribulose bisphosphate carboxylase-oxygenase (Rubisco), the primary CO2-fixing enzyme. The current working model for dissolved inorganic carbon concentration mechanism in unicellular green algae includes several isoforms of carbonic anhydrase (CA), and ATPase driven active transporters at the plasmalemma and at the inner chloroplast envelopes. In the past fifteen years, significant progress has been made in isolating and characterizing the various isoforms of carbonic anhydrase at the biochemical and molecular level. However, we have an inadequate understanding of active transporters that are located on the plasmalemma and at the chloroplast envelopes. In this mini-review we focus on certain aspects of the induction, function and significance of the dissolved inorganic carbon concentration mechanisms in aquatic photosynthetic microorganisms.     

Regulation of Rubisco activase and its interaction with Rubisco

The large, alpha-isoform of Rubisco activase confers redox regulation of the ATP/ADP response of the ATP hydrolysis and Rubisco activation activities of the multimeric activase holoenzyme complex. The alpha-isoform has a C-terminal extension that contains the redox-sensitive cysteine residues and is characterized by a high content of acidic residues. Cross-linking and site-directed mutagenesis studies of the C-terminal extension that have provided new insights into the mechanism of redox regulation are reviewed. Also reviewed are new details about the interaction between activase and Rubisco and the likely mechanism of 'activation' that resulted from mutagenesis in a 'Sensor 2' domain of activase that AAA(+) proteins often use for substrate recognition. Two activase residues in this domain were identified that are involved in Rubisco recognition. The results directly complement earlier studies that identified critical residues for activase recognition in the large subunit of Rubisco.     

Light Activation of Rubisco by Rubisco Activase and Thylakoid Membranes

A reconstituted system comprising ribulose bisphosphate carboxylase/oxygenase(rubisco), rubisco activase, washed thylakoid membranes, andATP was used to demonstrate a light-dependent stimulation ofrubisco activation. ATP, ribulose bisphosphate, H⁺, and Mg²⁺concentrations are normally light-dependent variables in thechloroplast but were maintained at pre-determined levels. Resultsindicated that rubisco activase and washed thylakoid membranesare sufficient to catalyze light stimulation of rubisco activationwith the reconstituted system, and that rubisco activase isrequired for this light stimulation. The washed thylakoid membranesdid not exhibit rubisco activase activity, nor was rubisco activaseprotein detected immunologically. Light-dependent activationof rubisco in the reconstituted system was similar in whole-chainand PS I electron transport reactions, and saturated at approximately100 µmol photons m^–2 s^–1. ¹ Present address: Department of Biological Sciences, LouisianaTech University, Ruston, LA 71272, U.S.A.     

CO2‐concentrating mechanisms in three southern hemisphere strains of Emiliania huxleyi

Rising global CO₂ is changing the carbonate chemistry of seawater, which is expected to influence the way phytoplankton acquire inorganic carbon. All phytoplankton rely on ribulose‐bisphosphate carboxylase oxygenase (RUBISCO) for assimilation of inorganic carbon in photosynthesis, but this enzyme is inefficient at present day CO₂ levels. Many algae have developed a range of energy demanding mechanisms, referred to as carbon concentrating mechanisms (CCMs), which increase the efficiency of carbon acquisition. We investigated CCM activity in three southern hemisphere strains of the coccolithophorid Emiliania huxleyi W. W. Hay & H. P. Mohler. Both calcifying and non‐calcifying strains showed strong CCM activity, with HCO₃^? as a preferred source of photosynthetic carbon in the non‐calcifying strain, but a higher preference for CO₂ in the calcifying strains. All three strains were characterized by the presence of pyrenoids, external carbonic anhydrase (CA) and high affinity for CO₂ in photosynthesis, indicative of active CCMs. We postulate that under higher CO₂ levels cocco‐lithophorids will be able to down‐regulate their CCMs, and re‐direct some of the metabolic energy to processes such as calcification. Due to the expected rise in CO₂ levels, photosynthesis in calcifying strains is expected to benefit most, due to their use of CO₂ for carbon uptake. The non‐calcifying strain, on the other hand, will experience only a 10% increase in HCO₃^?, thus making it less responsive to changes in carbonate chemistry of water.     

Rubisco Activity: Effects of Drought Stress

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activityis modulated in vivo either by reaction with CO₂ and Mg²⁺ tocarbamylate a lysine residue in the catalytic site, or by thebinding of inhibitors within the catalytic site. Binding ofinhibitors blocks either activity or the carbamylation of thelysine residue that is essential for activity. At night, inmany species, 2-carboxyarabinitol-1-phosphate (CA1P) is formedwhich binds tightly to Rubisco, inhibiting catalytic activity.Recent work has shown that tight-binding inhibitors can alsodecrease Rubisco activity in the light and contribute to theregulation of Rubisco activity. Here we determine the influencethat such inhibitors of Rubisco exert on catalytic activityduring drought stress. In tobacco plants, ‘total Rubiscoactivity’, i.e. the activity following pre-incubationwith CO₂ and Mg²⁺, was positively correlated with leaf relativewater content. However, ‘total Rubisco activity’in extracts from leaves with low water potential increased markedlywhen tightly bound inhibitors were removed, thus increasingthe number of catalytic sites available. This suggests thatin tobacco the decrease of Rubisco activity under drought stressis not primarily the result of changes in activation by CO₂and Mg²⁺ but due rather to the presence of tight-binding inhibitors.The amounts of inhibitor present in leaves of droughted tobaccobased on the decrease in Rubisco activity per mg soluble proteinwere usually much greater than the amounts of the known inhibitors(CA1P and ‘daytime inhibitor’) that can be recoveredin acid extracts. Alternative explanations for the differencebetween maximal and total activities are discussed.     

Rubisco regulation: a role for inhibitors

In photosynthesis Rubisco catalyses the assimilation of CO(2) by the carboxylation of ribulose-1,5-bisphosphate. However, the catalytic properties of Rubisco are not optimal for current or projected environments and limit the efficiency of photosynthesis. Rubisco activity is highly regulated in response to short-term fluctuations in the environment, although such regulation may not be optimally poised for crop productivity. The regulation of Rubisco activity in higher plants is reviewed here, including the role of Rubisco activase, tight binding inhibitors, and the impact of abiotic stress upon them.