The CO2concentrating mechanism in cyanobactiria and microalgae

Over the past 10 years it has become clear that cyanobacteria and microalgae possess mechanisms for actively acquiring inorganic carbon from the external medium and are able to use this to elevate the CO₂ concentration around the active site of the primary photosynthetic carboxylating enzyme, ribulose bisphosphate carboxylase-oxygenase (Rubisco). This results in a vastly enhanced photosynthetic affinity for inorganic carbon (C_i) and improved photosynthetic efficiency. The CO₂ concentrating mechanism is dependent on the existence of membrane bound C_i transport systems, and a microenvironment within the cell where the accumulated C_i can be used to elevate CO₂ at the site of Rubisco. Evidence presented in this review suggests that in cyanobacteria this is achieved by the packaging of Rubisco and carbonic anhydrase (CA) into discrete structures, which are termed carboxysomes. Analogous structures in microalgae, termed pyrenoids, may perform a similar function. The recovery and analysis of high-CO₂-requiring mutants has greatly advanced our understanding of the mechanisms and genes underlying these systems, especially in cyanobacteria, and this review places particular emphasis on the contribution made by molecular genetic approaches.     

Enhancement of ethylene biosynthesis and germination with thidiazuron and some selected auxins in Striga asiatica seeds

Witchweed [ Striga asiatica (L.) Kuntze], an economically important parasitic weed on several poaceous crops, is difficult to control. In nature, germination and subsequent morphogenesis of Striga are cued to specific host-derived chemical signals. Seeds (approximately 2.4 mg) treated with thidiazuron (TDZ) or the auxins 2,4-dichlorophenoxy-acetic acid (2,4-D), 1-naphthalene acetic acid (NAA), or 2-(4-chloro- o -tolyloxy) propionic acid (MCPP) produced little ethylene (66-138 nl l⁻¹). Combinations of TDZ with the auxins increased ethylene production by 4- to 18-fold. Ethylene production was strongly inhibited (86–92%) by aminoethoxyvinylglycine (AVG), inhibitor of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase. Ethylene evolved from seeds treated with TDZ in combination with 2,4-D increased after a lag period and was promoted by a pretreatment in 2,4-D. TDZ or any of the auxins, at the rates tested, effected negligible to low levels of germination (0 to 16%), whereas mixtures of TDZ with the above auxins stimulated 38 to 84% germination. Test solutions containing TDZ and indole-3-acetic acid (IAA) were, however, less effective. TDZ/auxin-induced germination was inhibited by AVG and the ethylene action inhibitor silver thiosulfate (STS). The inhibitory effect of the former was reversed by treatment with ACC. In vitro studies revealed negligible germination (< 1%) on control medium. Seeds germinating on media containing TDZ alone developed into seedlings with distinct shoots and rudimentary roots. Seeds germinating on media containing 2,4-D, irrespective of TDZ concentration, were induced to form calli. The results are consistent with a model in which both germination and subsequent morphogenesis in Striga are associated with exogenous and endogenous phytohormones.     

Diversity in forms and functions of carbonic anhydrase in terrestrial higher plants

The review summarizes current data on the existence in terrestrial higher plants of several carbonic anhydrase forms differing in their properties, molecular structure, and intracellular localization. Possible functions of these carbonic anhydrases are discussed as well as specific features of carbon-concentrating mechanisms in phototrophic tissues of plants with C₃ and C₄ pathways of photosynthesis.     

微藻碳酸酐酶的特性及其环境调控

本文概述微藻碳酸酐酶的性质、种类、分布、分离纯化和环境调控的研究进展,并对未来有关微藻碳酸酐酶研究中需要探讨的问题作了展望。     

PHOTOSYNTHETIC INORGANIC CARBON ACQUISITION IN AN ACID‐TOLERANT,FREE‐LIVING SPECIES OF COCCOMYXA (CHLOROPHYTA)1

The processes of CO₂ acquisition were characterized for the acid‐tolerant, free‐living chlorophyte alga, CPCC 508. rDNA data indicate an affiliation to the genus Coccomyxa, but distinct from other known members of the genus. The alga grows over a wide range of pH from 3.0 to 9.0. External carbonic anhydrase (CA) was detected in cells grown above pH 5, with the activity increasing marginally from pH 7 to 9, but most of the CA activity was internal. The capacity for HCO₃^? uptake of cells treated with the CA inhibitor acetazolamide (AZA), was investigated by comparing the calculated rate of uncatalyzed CO₂ formation with the rate of photosynthesis. Active bicarbonate transport occurred in cells grown in media above pH 7.0. Monitoring CO₂ uptake and O₂ evolution by membrane‐inlet mass spectrometry demonstrated that air‐grown cells reduced the CO₂ concentration in the medium to an equilibrium concentration of 15 μM, but AZA‐treated cells caused a drop in extracellular CO₂ concentration to a compensation concentration of 27 μM at pH 8.0. CO₂‐pulsing experiments with cells in the light indicated that the cells do not actively take up CO₂. An internal pool of unfixed inorganic carbon was not detected at the CO₂ compensation concentration, probably because of the lack of active CO₂ uptake, but was detectable at times before compensation point was reached. These results indicate that this free‐living Coccomyxa possesses a CO₂‐concentrating mechanism (CCM) due to an active bicarbonate‐uptake system, unlike the Coccomyxa sp. occurring in symbiotic association with lichens.     

Carbonic anhydrases in plants and algae

Carbonic anhydrases catalyse the reversible hydration of CO₂, increasing the interconversion between CO₂ and HCO₃⁻ + H⁺ in living organisms. The three evolutionarily unrelated families of carbonic anhydrases are designated α-, β-and γ-CA. Animals have only the α-carbonic anhydrase type of carbonic anhydrase, but they contain multiple isoforms of this carbonic anhydrase. In contrast, higher plants, algae and cyanobacteria may contain members of all three CA families. Analysis of the Arabidopsis database reveals at least 14 genes potentially encoding carbonic anhydrases. The database also contains expressed sequence tags (ESTs) with homology to most of these genes. Clearly the number of carbonic anhydrases in plants is much greater than previously thought. Chlamydomonas, a unicellular green alga, is not far behind with five carbonic anhydrases already identified and another in the EST database. In algae, carbonic anhydrases have been found in the mitochondria, the chloroplast thylakoid, the cytoplasm and the periplasmic space. In C₃ dicots, only two carbonic anhydrases have been localized, one to the chloroplast stroma and one to the cytoplasm. A challenge for plant scientists is to identify the number, location and physiological roles of the carbonic anhydrases.     

Carbonic anhydrase activity and inorganic carbon fluxes in low- and high-C1 cells of Chlamydomonas reinhardtü and Scenedesmus obliquus

Carbonic anhydrase (CA) activity associated with high- and low-dissolved inorganic carbon (C₁) grown cells was examined in whole cells by measuring ¹⁸O exchange from doubly labeled CO₂ (¹³C¹⁸O¹⁸O). Both algal species showed the presence of extracellular (periplasmic) as well as intracellular CA activity, which were both greatly increased in low-C₁ cells. The periplasmic CA activity was at least 40-fold higher in lowcompared to high-C₁ cells in both C. reinhardtii and S. obliquus. while low-C₁ cells of S. obliquus showed the highest activity of internal CA. The CA inhibitor ethoxyzolamide showed a strong inhibition of the C₁ uptake process in both C. reinhardtii and S. obliquus as in cyanobacteria. which may indicate that the nature of the primary uptake process is similar in both green algae and cyanobacteria. By using a mass spectrometnc disequilibrium technique it was possible to separate the C₁ fluxes of net HCO^?₃-uptake and net CO₂-uptake during steady-state photosynthesis in high- and Sow-C₁ grown cells of Chlamydomonas reinhardtii (WT. 2137+) and Scenedesmus obliquus (WT. D3). It was found that both high- and low-C₁ cells of the two algae can utilize both CO₂ and HCO^?₃ for photosynthesis, although low-C₁ cells have a higher affinity for the uptake of both C₁ species. Induction at low-C₁ causes an increase in the affinity of both species for HCO^?₃ and CO₂; changes in net CO₂-uptake were, however, significantly greater.     

碳酸酐酶在中肋骨条藻光合作用中的作用

探讨了在正常空气条件下生长的中肋骨条藻(Skeletonema costatum)的碳酸酐酶(CA)在其光合固碳中的作用.在中肋骨条藻的胞内和胞外均有CA活性,但胞外CA活性很低.CA抑制剂AZ(乙酰唑磺胺)对中肋骨条藻的光合放氧速率没有明显影响,而CA抑制剂EZ(乙氧苯唑胺)对其光合放氧速率有强烈的抑制作用.EZ的抑制作用使细胞最大光合速率、饱和光强和无机碳亲和力下降,无机碳的补偿点和光呼吸提高,使强光下光抑制作用增强.这些结果表明:中肋骨条藻的胞外CA在其光合作用中所起的作用较小,而其胞内CA通过催化胞内碳库中的HCO-3快速转化成CO2,提高胞内CO2的有效供给,从而提高细胞光合固碳能力和对逆境(高O2、强光和低CO2)的适应能力.     

Crassulacean acid metabolism contributes significantly to the in situ carbon budget in a population of the invasive aquatic macrophyte Crassula helmsii

1. The ecophysiological significance of Crassulacean acid metabolism (CAM) in the invasive aquatic macrophyte Crassula helmsii was studied in an English soft‐water lake. The extent and the contribution of CAM to the carbon budget was examined in spring (April) and summer (July) along a depth gradient (0.5–2.2 m), covering the growth range of C. helmsii in the lake. 2. Significant in situ CAM activity (30–80 meq kg⁻¹ FW) was present in all specimens, although it decreased with depth and hence correlated with the decline in photon irradiance. Potential CAM activity (60–161 meq kg⁻¹ FW), measured after exposure to low concentrations of CO₂ in the day and high concentrations at night, were on average 2.7‐times greater than in situ CAM activity. Overall CAM activity increased from April to July, which is consistent with higher potential carbon limitation caused by increased temperature and light availability. 3. CAM activity in C. helmsii appeared to be carbon‐limited at night because night‐time carbon‐fixation increased at raised, compared to ambient, concentrations of CO₂. 4. The high in situ CAM activity in C. helmsii was reflected in the contribution of CAM to the total carbon budget which, independent of depth and season, ranged from 18% to 42%. The amount of CO₂ taken up in the night via CAM was 0.74 to 2.94 times the amount of CO₂ lost in respiration, thus emphasizing the importance of CAM in refixation of potentially lost respiratory CO₂. 5. The onset of decarboxylation in the morning appeared to be under circadian control as there was a delay of up to 5.5 h between the start of the light period and a decline in cell acidity level. 6. There was little variation in δ¹³C content (−21.69 to 23.49‰) with season or depth suggesting, along with the estimated contribution to the carbon‐budget, that CAM is important for the whole population of C. helmsii. CAM may confer a competitive advantage in relation to growth, which may be one of the reasons for the invasiveness of this species.     

Drought- and ABA-induced changes in photosynthesis of barley plants

The changes caused by drought stress and abscisic acid (ABA) on photosynthesis of barley plants (Hordeum vulgare. L. cv. Alfa) have been studied. Drought stress was induced by allowing the leaves to lose 12% of their fresh weight. Cycloheximide (CHI), an inhibitor of stress-induced ABA accumulation, was used to distinguish alterations in photosynthetic reactions that are induced after drought stress in response to elevated ABA levels from those that are caused directly by altered water relations. Four hoars after imposition of drought stress or 2 h after application of ABA, Ihe bulk of the leaf's ABA content measured by enzyme-amplified ELISA, increased 14- and 16-fold, respectively. CHI fully blocked the stress-induced ABA accumulation. Gas exchange measurements and analysis of enzyme activities were used to study the reactions of photosynthesis to drought stress and ABA. Leaf dehydration or ABA treatment led to a noticeable decrease in both the initial slope of the curves representing net photosynthetic rate versus intercellular CO₂ concentration and the maximal rate of photosynthesis; dehydration of CHI-treated plants showed much slower inhibition of the latter. The calculated values of the intercellular CO₂ concentration, CO₂ compensation point and maximal carboxylating efficiency of ribulose 1,5-bisphosphate (RuBP) carboxylase support the suggestion that biochemical factors are involved in the response of photosynthesis to ABA and drought stress. RuBP carboxylase activity was almost unaffected in ABA- and CHI-treated, non-stressed plants. A drop in enzyme activity was observed after leaf dehydration of the control and ABA-treated plants. When barley plants were supplied with ABA, the activity of carbonic anhydrase (CA, EC 4.2.2.1) increased more than 2-fold. Subsequent dehydration caused an over 1.5-fold increase in CA activity of the control plants and a more than 2.5-fold increase in ABA-treated plants. Dehydration of CHI-treated plants caused no change in enzyme activity. It is suggested that increased activity of CA is a photosynthetic response to elevated ABA concentration.     

PHOTOSYNTHETIC UTILIZATION OF INORGANIC CARBON IN THE ECONOMIC BROWN ALGA,HIZIKIA FUSIFORME (SARGASSACEAE) FROM THE SOUTH CHINA SEA1

The mechanism of inorganic carbon (C_i) acquisition by the economic brown macroalga, Hizikia fusiforme (Harv.) Okamura (Sargassaceae), was investigated to characterize its photosynthetic physiology. Both intracellular and extracellular carbonic anhydrase (CA) were detected, with the external CA activity accounting for about 5% of the total. Hizikia fusiforme showed higher rates of photosynthetic oxygen evolution at alkaline pH than those theoretically derived from the rates of uncatalyzed CO₂ production from bicarbonate and exhibited a high pH compensation point (pH 9.66). The external CA inhibitor, acetazolamide, significantly depressed the photosynthetic oxygen evolution, whereas the anion‐exchanger inhibitor 4,4′‐diisothiocyano‐stilbene‐2,2′‐disulfonate had no inhibitory effect on it, implying the alga was capable of using HCO₃^? as a source of C_i for its photosynthesis via the mediation of the external CA. CO₂ concentrations in the culture media affected its photosynthetic properties. A high level of CO₂ (10,000 ppmv) resulted in a decrease in the external CA activity; however, a low CO₂ level (20 ppmv) led to no changes in the external CA activity but raised the intracellular CA activity. Parallel to the reduction in the external CA activity at the high CO₂ was a reduction in the photosynthetic CO₂ affinity. Decreased activity of the external CA in the high CO₂ grown samples led to reduced sensitiveness of photosynthesis to the addition of acetazolamide at alkaline pH. It was clearly indicated that H. fusiforme, which showed CO₂‐limited photosynthesis with the half‐saturating concentration of C_i exceeding that of seawater, did not operate active HCO₃^? uptake but used it via the extracellular CA for its photosynthetic carbon fixation.     

Effect of external pH on inorganic carbon assimilation in unicellular marine green algae

Carbonic anhydrase (CA) induction has been studied in three marine green algae under acidic (pH 4.5) or alkaline (pH 8.0) conditions. An inhibition of the induction of the external CA in acidic conditions, similar to that observed in some freshwater green algae, could be observed in only Chlorella saccharophila. In the two other species, Chlorococcum littorale and Stichococcus bacillaris, no significant difference in CA induction was found under two pH conditions. The exact function of the external CA of C. saccharophila remains unclear, since cells grown under acidic conditions (under which this enzyme is repressed) possess the same abilities to use inorganic carbon (Ci) as alkaline‐grown cells. Internal pH values were not modified by the pH of the medium used to cultivate C. saccharophila. Regardless of the growth conditions, activities related to carbon fixation, that is, photosynthetic oxygen evolution, Ci uptake and assimilation were enhanced when the measurements were performed at acidic pH. This indicates that this marine alga is able to use CO₂ more efficiently than HCO₃^?. No evidence could be found for a specific Ci uptake and assimilation system in the acid‐grown cells.     

A NEW METHOD FOR ESTIMATING EXTERNAL,CARBONIC ANHYDRASE ACTMTY IN MACROALGAE1,2

The effects of external carbonic anhydrase (CA, E.C. 4.2.1.1) on HCO^?₃and CO₂use under disequilibnum conditions were examined in 14 species of macroalgae. CO₂ was added to the algae in synthetic seawater free from inorganic carbon and buffered to pH 8.7. This resulted in a transient O₂ evolution, which was similar for most of the species tested: an initial rapid increase in the rate was followed by a gradual decrease, approaching a steady state within 10 min. The initial high rate of O₂evolution was attributed to diffusive entry of CO₂into the cells and the steady state to use of HCO^?₃. An enhancement of CO₂diffusive entry was obtained with acetazolamide, an inhibitor of external CA activity. Using this enhancement, a variable was developed to quantify the degree to which CO₂entry into the cell was prevented by the external CA. This variable, CA (%), was used as a measure of the external CA activity of the alga. Comparative measurements of the external CA activity using a potentiomtric method revealed that the new method was able to detect low levels of external CA activity, where the potentiometric method failed. These two different methods could be used together to increase the reliability of the measurements. The new method was useful for red and brown macroalgae and for those green macroalgae that lacked direct HCO₃uptake. Thus prominent external CA activity was found for Valonia utricularis (green), Halopteris scoparia (brown), and Osmunda pinnatifida (red), where the potentiometric method failed, or nearly failed, to indicate any external CA activity. Direct uptake of HCO^?₃interfered with the new method and the degree of this interference was dependent on the magnitude of the uptake.     

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.     

Two modes of bicarbonate utilization in the marine green macroalga Ulva lactuca

The green marine macroalga Ulva lactuca L. was found to be able to utilize HCO₃^? from sea water in two ways. When grown in flowing natural sea water at 16°C under constant dim irradiance, photosynthesis at pH8.4 was suppressed by acetazolamide but unaffected by 4,4′-diisothiocyanostilbene-2,2′-disulphonate. These responses indicate that photosynthetic HCO₃^? utilization was via extracellular carbonic anhydrase (CA) -mediated dehydration followed by CO₂ uptake. The algae were therefore described as being in a ‘CA state’. If treated for more than 10 h in a sea water flow-through system at pH9.8, these thalli became insensitive to acetazolamide but sensitive to 4,4′-diisothiocyanostilbene-2,2′-disulphonate. This suggests the involvement of an anion exchanger (AE) in the direct uptake of HCO₃^?, and these plants were accordingly described as being in an ‘AE state’. Such thalli showed an approximately 10-fold higher apparent affinity for HCO₃^? (at pH9.4) than those in the ‘CA state’, while thalli of both states showed a very high apparent affinity for CO₂. These results suggest that the two modes of HCO₃^? utilization constitute two ways in which inorganic carbon may enter the Ulva lactuca cells, with the direct entry of HCO₃^?, characterizing the ‘AE state’, being inducible and possibly functioning as a complementary uptake system at high external pH values (e.g. under conditions conducive to high photosynthetic rates). Both mechanisms of entry appear to be connected to concentrating CO₂ inside the cell, probably via a separate mechanism operating intracellularly.     

不同光强下生长的几种亚热带森林树木的Rubisco羧化速率和碳酸酐酶的活性

9月和12月测定了生长于3种不同光强（100％、36％和16％的自然光）下生长的乔木荷树、黧蒴和灌木九节、罗伞盆栽幼苗叶片的Rubisco羧化速率（RCR）、碳酸酐酶（CA）活性和细胞间CO2浓度（Ci)。当生长光强降低时,4种供试植物的RCR和CA活性明显降低。9月时生长在16％自然光下荷树的RCR和CA比100％自然光者分别降低55％和50％,藜蒴则降低20％和35％,耐有的灌木树的降幅较小,仅为33％－38％（RCR）和22％－30％（CA）。12月的RCR和CA的水平较9月时低,翌年1月时自然林不同光强下生长的同类植物的RCA和CA随光强变化也有类似的趋势。RCR和CA活性呈正相关性,且两者与Ci呈弱负相关。推测高光强可能有利于激活Rubisco,促进CA内化的CO2→DlC（可溶性碳）→CO2活性和DlC的传输过程。     

干出和沉水不同条件下羊栖菜光合作用碳源获得机制比较

经济海洋褐藻羊栖菜(Hizikia fusiforme(Harv.)Okamura)低潮时常常周期性地暴露于空气中.为了认识这种海藻在潮汐循环背景下的光合特征,对其在高潮沉水和低潮干出不同条件下的光合作用碳素获得机制进行了比较.沉水时,羊栖菜主要利用海水中HCO3-作为外源无机碳源驱动光合作用;而在干出条件下,其光合作用的主要碳源为空气中的CO2.在这两种不同环境条件下,光合作用与pH值的关系不同:沉水状态时,羊栖菜在高pH值(10.0)下光合活性很弱;而在干出条件下,羊栖菜在高pH值时仍有较高的光合活性.然而,光合作用无论是在沉水还是在干出条件下,对外源碳源的获得都表现出对胞外碳酸酐酶(CA)强烈的依赖性,并且其光合速率都受周围环境中无机碳源水平的限制.此外,在沉水和干出两种环境条件下,羊栖菜光合作用都表现出对氧气的敏感性.这表明,在羊栖菜中,依赖胞外CA的碳源获得机制不能使细胞内CO2浓度提高到阻碍其光呼吸的程度.增加空气中或海水中无机碳的浓度,能促进羊栖菜的光合作用,进而增加这种海藻的水产养殖产量.     

Photosynthesis of Ulva fasciata. IV. pH, carbonic anhydrase and inorganic carbon conversions in the unstirred layer

Abstract. The common marine macroalga Ulva was found to have a surface pH of about 10 during photosynthesis. Under such a condition, the equilibrium CO₂ concentration within the unstirred layer would be below reported CO₂ compensation points, and dehydration of HCO₃ could not occur. Even at a compensation point approaching zero, uncatalysed rates of HCO₃ to CO₂ conversion within the unstirred layer volume could not support photosynthetic rates as measured under stirred conditions in the presence of a carbonic anhydrase inhibitor. Based on this, it is concluded that Ulva takes up HCO₃. It is likely that HCO₃ uptake leads to high internal CO₂ levels which, in turn, suppress photorespiration and thus cause this plant's efficient gas exchange features. Carbonic anhydrase activity was measurable only in plant extracts. However, inhibitor studies suggest the presence of a surface enzyme. The possible functions of extracellular carbonic anhydrase in Ulva are assessed in terms of CO₂ hydration during emergence and a possible HCO₃, transport system.