Soft X-ray spectro-tomography study of cyanobacterial biomineral nucleation

Quantitative three-dimensional (3D) chemical mapping using angle-scan spectro-tomography in a scanning transmission (soft) X-ray microscope (STXM) has been used for the first time to characterize the early stages of CaCO₃ biomineral nucleation on the surface of planktonic freshwater cyanobacterial cells of the strain Synechococcus leopoliensis PCC 7942. The apparatus for STXM angle-scan tomography is described. Aspects of sample preparation, sample mounting and data acquisition and quantitative analysis and interpretation are discussed in detail. Angle-scan tomography and chemically selective 3D imaging at multiple photon energies has been combined with a complete 2D spectromicroscopic characterization of the biochemical and mineralogical composition. This has provided detailed insights into the mechanisms of mineral nucleation, leading to development of a detailed model of CaCO₃ nucleation by the cyanobacterial strain S. leopoliensis PCC 7942. It shows that Ca is absorbed by the extracellular polymeric substances (EPS) of the cyanobacteria and that CaCO₃ with aragonite-like short-range order is precipitated rather homogeneously within the EPS. The precipitation of the thermodynamically more stable calcite polymorph then starts at Ca-rich hot spots within the EPS and close to the cyanobacteria.     

Calcification in aquatic plants

Abstract. The CaCO₃ deposits of aquatic plants may be intra-, inter- and extracellular. Calcification is mainly the result of photosynthetic CO₂ or HCO⁻₃ assimilation. This raises the local pH and CO²⁻₃ concentration resulting from shifts in the dissolved inorganic carbon equilibrium, due to either net CO₂ depletion as in Halimeda or localized OH⁻ efflux (or H⁺ influx) as in Chara. The plant cell wall may be important in CaCO₃ nucleation by acting as an epitaxial substratum or template, or by creating a microenvironment enriched in Ca²⁺ compared to Mg²⁺. Hypotheses on the reason for the lack of calcification in many aquatic plants are presented.     

Bacterially mediated precipitation in marine stromatolites

Stromatolites are laminated, lithified (CaCO₃) sedimentary deposits formed by precipitation and/or sediment accretion by cyanobacterial–bacterial mat communities. Stromatolites have been associated with these communities as far back as the Precambrian era some 2+ billion years ago. The means by which microbial communities mediate the precipitation processes have remained unclear, and are the subject of considerable debate and speculation. Two alternative explanations for microbially mediated precipitation include: (i) cyanobacterial photosynthesis increases pH in a system supersaturated in respect of CaCO₃, resulting in CaCO₃ precipitation and then laminated lithification, and (ii) decomposition of cyanobacterial extracellular organic matter (e.g. sheaths, mucilage and organic acids) by microheterotrophs leads to release of organic-bound Ca²⁺ ions and CaCO₃ precipitation. We evaluated these explanations by examining metabolically active, lithifying stromatolitic mat communities from Highborne Cay, Bahamas, using microautoradiography. Microautoradiographic detection of ¹⁴CO₂ fixation and ³H organic matter ( d -glucose and an amino acid mixture) utilization by photosynthetically active cyanobacteria and microheterotrophs, combined with community-level uptake experiments, indicate that bacteria, rather than cyanobacteria are the dominant sites of CaCO₃ deposition. In the oligotrophic waters in which stromatolites exist, microheterotrophs are reliant on the photosynthetic community as a main source of organic matter. Therefore, autotrophic production indirectly controls microbially mediated precipitation and stromatolite formation in these shallow marine environments.     

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.     

Inorganic carbon limitation of photosynthesis in lake phytoplankton

1. Inorganic carbon availability influences species composition of phytoplankton in acidic and highly alkaline lakes, whereas the overall influence on community photosynthesis and growth is subject to debate.
2. The influence of total dissolved inorganic carbon (DIC) and free CO₂ on community photosynthesis was studied in six Danish lakes during the summer of 1995. The lakes were selected to ensure a wide range of chlorophyll a concentrations (1–120 μg l^–1), pH (5.6–9.6) and DIC concentration (0.02–2.5 m m ). Photosynthesis experiments were performed using the ¹⁴C technique in CO₂-manipulated water samples, either by changing the pH or by adding/removing CO₂.
3. Lake waters were naturally CO₂ supersaturated during most of the experimental period and inorganic carbon limitation of photosynthetic rates did not occur under ambient conditions. However, photosynthesis by phytoplankton in lakes with low and intermediate DIC concentrations was seriously restricted when CO₂ concentrations declined. Similarly, photosynthesis was limited by low CO₂ concentrations during phytoplankton blooms in the hardwater alkaline lakes.     

Physiological effects of sulphur dioxide. 1. The effect of SO2 on photosynthesis and stomatal regulation of Vicia faba L.

Abstract. The effect of short-term SO₂ fumigation on photosynthesis and transpiration of Vicia faba L. was measured at different irradiances and SO₂ concentrations. At high irradiances photosynthetic rates were reduced when leaves were exposed to SO₂ and the magnitude of the reduction was linearly related to the rate of SO₂ uptake through the stomata. Photosynthetic rates stabilized within 2 h after the start of fumigation.
The effect of SO₂ on photosynthesis was measured at different CO₂ concentrations to analyse the contribution of stomatal and non-stomatal factors to photosynthetic inhibition. Mesophyll resistance to CO₂ diffusion increased as a result of SO₂ exposure and caused a rapid reduction in photosynthesis after the start of fumigation. Stomatal resistance was not affected directly by SO₂ fumigation, but indirectly as a result of a feedback loop between net photosynthesis and internal CO₂ concentration.
Analysis of gas-exchange measurements in biochemical terms indicated that photosynthetic inhibition during SO₂ exposure can be explained by a stronger reduction in the affinity of RBP carboxylase/oxygenase for CO₂ than for O₂.     

Does photosynthetic acclimation to elevated CO2 increase photosynthetic nitrogen-use efficiency? A study of three native UK grassland species in open-top chambers

1. The photosynthetic response to elevated CO₂ and nutrient stress was investigated in Agrostis capillaris, Lolium perenne and Trifolium repens grown in an open-top chamber facility for 2 years under two nutrient regimes. Acclimation was evaluated by measuring the response of light-saturated photosynthesis to changes in the substomatal CO₂ concentration.
2. Growth at elevated CO₂ resulted in reductions in apparent Rubisco activity in vivo in all three species, which were associated with reductions of total leaf nitrogen content on a unit area basis for A. capillaris and L. perenne . Despite this acclimation, photosynthesis was significantly higher at elevated CO₂ for T. repens and A. capillaris , the latter exhibiting the greatest increase of carbon uptake at the lowest nutrient supply.
3. The photosynthetic nitrogen-use efficiency (the rate of carbon assimilation per unit leaf nitrogen) increased at elevated CO₂, not purely owing to higher values of photosynthesis at elevated CO₂, but also as a result of lower leaf nitrogen contents.
4. Contrary to most previous studies, this investigation indicates that elevated CO₂ can stimulate photosynthesis under a severely limited nutrient supply. Changes in photosynthetic nitrogen-use efficiency may be a critical determinant of competition within low nutrient ecosystems and low input agricultural systems.     

Growth in elevated CO2 enhances temperature response of photosynthesis in wheat

The temperature dependence of C₃ photosynthesis may be altered by the growth environment. The effects of long-term growth in elevated CO₂ on photosynthesis temperature response have been investigated in wheat ( Triticum aestivum L.) grown in controlled chambers with 370 or 700 μmol mol⁻¹ CO₂ from sowing through to anthesis. Gas exchange was measured in flag leaves at ear emergence, and the parameters of a biochemical photosynthesis model were determined along with their temperature responses. Elevated CO₂ slightly decreased the CO₂ compensation point and increased the rate of respiration in the light and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) V_cmax, although the latter effect was reversed at 15°C. With elevated CO₂, J_max decreased in the 15–25°C temperature range and increased at 30 and 35°C. The temperature response (activation energy) of V_cmax and J_max increased with growth in elevated CO₂. CO₂ enrichment decreased the ribulose 1,5-bisphosphate (RuBP)-limited photosynthesis rates at lower temperatures and increased Rubisco- and RuBP-limited rates at higher temperatures. The results show that the photosynthesis temperature response is enhanced by growth in elevated CO₂. We conclude that if temperature acclimation and factors such as nutrients or water availability do not modify or negate this enhancement, the effects of future increases in air CO₂ on photosynthetic electron transport and Rubisco kinetics may improve the photosynthetic response of wheat to global warming.     

CARBONIC ANHYDRASE ACTIVITY IN THE BLOOM-FORMING DINOFLAGELLATE PERIDINIUM GATUNENSE

The response of carbonic anhydrase (CA) activity in Peridinium gatunense Nygaard, the natural bloom-forming dinoflagellate in Lake Kinneret, to diel and seasonal variations in environmental conditions was characterized under controlled laboratory experiments. Simulated diel cycles demonstrated large changes in the ambient concentration of dissolved CO₂ and parallel changes in CA activity. The CA activity depended on the total concentrations of inorganic carbon (C₁) and in particular on the dissolved CO₂. Lowering the C₁ concentrations resulted in a large increase in CA activity within several hours. Light and photosynthesis were both required for the induction of CA activity. Under CO₂ -limited conditions, the dependence of the photosynthetic rate on CA (estimated from the ratio of photosynthetic rates in the presence or absence of CA inhibitors) was greater in P. gatunense than in other eukaryotic microalgae. This points to the ecological significance of CA in photosynthetic carbon uptake mechanisms of a large, dominant alga in a natural ecosystem .     

The molecular and biochemical analyses of CO2-concentrating mechanisms in cyanobacteria and microalgae

Abstract. The supply of dissolved inorganic carbon for photosynthesis in the aquatic environment is much more variable than that experienced by terrestrial plants. In response to this variability, cyanobacteria and many species of microalgae acclimate to rapid changes in the availability of dissolved inorganic carbon by the induction of high affinity/high capacity CO₂-concentrating systems (CCMs). Biochemical and molecular analyses of the acclimation response have recently identified several components that are required for efficient operation of the CCMs and CO₂ assimilation. This has been accomplished using in vivo labelling studies, and characterization of high inorganic carbon (C_i) requiring cyanobacterial and algal mutants. The identification and regulation of expression of polypeptides synthesized in response to limiting C_i concentrations, and the proposed role of the carboxysome and the pyrenoid in the functioning of the CCMs are examined.     

Carbon dioxide-concentrating mechanism and the development of extracellular carbonic anhydrase in the marine picoeukaryote Micromonas pusilla

A range of marine photosynthetic picoeukaryote phytoplankton species grown in culture were screened for the presence of extracellular carbonic anhydrase (CA_ext), a key enzyme in inorganic carbon acquisition under carbon- limiting conditions in some larger marine phytoplankton species. Of the species tested, extracellular carbonic anhydrase was detected only in Micromonas pusilla Butcher. The rapid, light-dependent development of CA_ext when cells were transferred from carbon-replete to carbon-limiting conditions was regulated by the available free- CO₂ concentration and not by total dissolved inorganic carbon. Kinetic studies provided support for a CO₂- concentrating mechanism in that the K _0.5[CO₂] (i.e. the CO₂ concentration required for the half-maximal rate of photosynthesis) was substantially lower than the K _m[CO₂] of Rubisco from related taxa, whilst the intracellular carbon pool was at least seven fold greater than the extracellular DIC concentration, for extracellular DIC values 1.0 m m .
It is proposed that when the flux of CO₂ into the cell is insufficient to support the photosynthetic rate at an optimum photon irradiance, the development of CA_ext increases the availability of CO₂ at the plasma membrane. This ensures rapid acclimation to environmental change and provides an explanation for the central role of M. pusilla as a carbon sink in oligotrophic environments.     

What physiological acclimation supports increased growth at high CO2 conditions?

Chlamydomonas acidophila Negoro is a green algal species abundant in acidic waters (pH 2–3.5), in which inorganic carbon is present only as CO₂. Previous studies have shown that aeration with CO₂ increased its maximum growth rate, suggesting CO₂ limitation under natural conditions. To unravel the underlying physiological mechanisms at high CO₂ conditions that enables increased growth, several physiological characteristics from high- and low-CO₂-acclimated cells were studied: maximum quantum yield, photosynthetic O₂ evolution (P_max), affinity constant for CO₂ by photosynthesis (K_0.5,p), a CO₂-concentrating mechanism (CCM), cellular Rubisco content and the affinity constant of Rubisco for CO₂ (K_0.5,r). The results show that at high CO₂ concentrations, C. acidophila had a higher K_0.5,p, P_max, maximum quantum yield, switched off its CCM and had a lower Rubisco content than at low CO₂ conditions. In contrast, the K_0.5,r was comparable under high and low CO₂ conditions. It is calculated that the higher P_max can already explain the increased growth rate in a high CO₂ environment. From an ecophysiological point of view, the increased maximum growth rate at high CO₂ will likely not be realised in the field because of other population regulating factors and should be seen as an acclimation to CO₂ and not as proof for a CO₂ limitation.     

Carbonic anhydrases in higher plants and aquatic microorganisms

At physiological pH-values CO₂ and HCO⁻₃are the dominant inorganic carbon species and the interconversion between both is catalyzed by carbonic anhydrase (EC 4.2.1.1). This enzyme is widely distributed among photosynthetic organisms. In the first part of the review, the similarities and the differences of carbonic anhydrases from plants and animals are briefly described. In the second part recent advances in molecular biology to understand the structure of carbonic anhydrase from higher terrestrial plants as well as its involvement in photosynthetic CO₂ fixation are summarized. Lastly, the review deals with the presence of carbonic anhydrase in aquatic organisms including cyanobacteria, microalgae, macroalgae and angiosperms. Evidence for the presence of extracellular and intracellular isozymes in these organisms are discussed. The properties and function(s) of carbonic anhydrase during the operation of the inorganic carbon concentrating mechanism are also described.     

Increasing growth temperature reduces the stimulatory effect of elevated CO2 on photosynthesis or biomass in two perennial species

We examined how anticipated changes in CO₂ concentration and temperature interacted to alter plant growth, harvest characteristics and photosynthesis in two cold-adapted herbaceous perennials, alfalfa ( Medicago sativa L. cv. Arc) and orchard grass ( Dactylis glomerata L. cv. Potomac). Plants were grown at two CO₂ concentrations (362 [ambient] and 717 [elevated] μmol mol⁻¹ CO₂) and four constant day/night temperatures of 15, 20, 25 and 30°C in controlled environmental chambers. Elevated CO₂ significantly increased total plant biomass and protein over a wide range of temperatures in both species. Stimulation of photosynthetic rate, however, was eliminated at the highest growth temperature in M. sativa and relative stimulation of plant biomass and protein at high CO₂ declined as temperature increased in both species. Lack of a synergistic effect between temperature and CO₂ was unexpected since elevated CO₂ reduces the amount of carbon lost via photorespiration and photorespiration increases with temperature. Differences between anticipated stimulatory effects of CO₂ and temperature and whole plant single and leaf measurements are discussed. Data from this study suggest that stimulatory effects of atmospheric CO₂ on growth and photosynthesis may decline with anticipated increases in global temperature, limiting the degree of carbon storage in these two perennial species.     

Influence of elevated CO2 and nitrogen supply on the carbon assimilation performance and cell composition of the unicellular alga Dunaliella viridis

The carbon assimilation efficiency and the internal composition of the chlorophyte Dunaliella viridis have been studied under conditions of current (0.035%) and enriched (1%) levels of CO₂, with and without N limitation (supplied as nitrate). Results show that both photosynthesis and growth rates are enhanced by high CO₂, but the strategy of acclimation also involves the light harvesting machinery and the nutritional metabolism in an N supply dependent manner. D. viridis carried out a qualitative rather than a quantitative acclimation of the light harvesting system leading to increased PSII quantum yields. Total internal C decreased as a consequence of either active growth or organic carbon release to the external medium. The latter process allowed photosynthetic electron transport to proceed at higher rates than under normal CO₂ conditions, and maintained the internal C:N balance in a narrow range (under N sufficiency). N limitation generally prevented the effects of high CO₂, with some exceptions such as the photosynthetic O₂ evolution rate.     

Elevated CO2 and ozone reduce nitrogen acquisition by Pinus halepensis from its mycorrhizal symbiont

The effects of 700 μmol mol⁻¹ CO₂ and 200 nmol mol⁻¹ ozone on photosynthesis in Pinus halepensis seedlings and on N translocation from its mycorrhizal symbiont, Paxillus involutus, were studied under nutrient-poor conditions. After 79 days of exposure, ozone reduced and elevated CO₂ increased net assimilation rate. However, the effect was dependent on daily accumulated exposure. No statistically significant differences in total plant mass accumulation were observed, although ozone-treated plants tended to be smaller. Changes in atmospheric gas concentrations induced changes in allocation of resources: under elevated ozone, shoots showed high priority over roots and had significantly elevated N concentrations. As a result of different shoot N concentration and net carbon assimilation rates, photosynthetic N use efficiency was significantly increased under elevated CO₂ and decreased under ozone. The differences in photosynthesis were mirrored in the growth of the fungus in symbiosis with the pine seedlings. However, exposure to CO₂ and ozone both reduced the symbiosis-mediated N uptake. The results suggest an increased carbon cost of symbiosis-mediated N uptake under elevated CO₂, while under ozone, plant N acquisition is preferentially shifted towards increased root uptake.     

Increased photosynthetic capacity of Scirpus olneyi after 4 years of exposure to elevated CO2

Abstract. While a short-term exposure to elevated atmospheric CO₂ induces a large increase in photosynthesis in many plants, long-term growth in elevated CO₂ often results in a smaller increase due to reduced photosynthetic capacity. In this study, it was shown that, for a wild C₃ species growing in its natural environment and exposed to elevated CO₂ for four growing seasons, the photosynthetic capacity has actually increased by 31%. An increase in photosynthetic capacity has been observed in other species growing in the field, which suggests that photosynthesis of certain field grown plants will continue to respond to elevated levels of atmospheric CO₂     

Effect of elevated CO2, temperature and drought on photosynthesis of nodulated alfalfa during a cutting regrowth cycle

Rising atmospheric CO₂ may increase potential net leaf photosynthesis under short-term exposure, but this response decreases under long-term exposure because plants acclimate to elevated CO₂ concentrations through a process known as downregulation. One of the main factors that may influence this phenomenon is the balance between sources and sinks in the plant. The usual method of managing a forage legume like alfalfa requires the cutting of shoots and subsequent regrowth, which alters the source/sink ratio and thus photosynthetic behaviour. The aim of this study was to determine the effect of CO₂ (ambient, around 350 vs. 700 µmol mol⁻¹), temperature (ambient vs. ambient + 4° C) and water availability (well-irrigated vs. partially irrigated) on photosynthetic behaviour in nodulated alfalfa before defoliation and after 1 month of regrowth. At the end of vegetative normal growth, plants grown under conditions of elevated CO₂ showed photosynthetic acclimation with lower photosynthetic rates, V_cmax and ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) activity. This decay was probably a consequence of a specific rubisco protein reduction and/or inactivation. In contrast, high CO₂ during regrowth did not change net photosynthetic rates or yield differences in V_cmax or rubisco total activity. This absence of photosynthetic acclimation was directly associated with the new source-sink status of the plants during regrowth. After cutting, the higher root/shoot ratio in plants and remaining respiration can function as a strong sink for photosynthates, avoiding leaf sugar accumulation, the negative feed-back control of photosynthesis, and as a consequence, photosynthetic downregulation.     

Modelling environmental controls on ecosystem photosynthesis and the carbon isotope composition of ecosystem-respired CO2 in a coastal Douglas-fir forest

We developed and applied an ecosystem-scale model that calculated leaf CO₂ assimilation, stomatal conductance, chloroplast CO₂ concentration and the carbon isotope composition of carbohydrate formed during photosynthesis separately for sunlit and shaded leaves within multiple canopy layers. The ecosystem photosynthesis model was validated by comparison to leaf-level gas exchange measurements and estimates of ecosystem-scale photosynthesis from eddy covariance measurements made in a coastal Douglas-fir forest on Vancouver Island. A good agreement was also observed between modelled and measured δ ¹³C values of ecosystem-respired CO₂ ( δ _R). The modelled δ _R values showed strong responses to variation in photosynthetic photon flux density (PPFD), air temperature, vapour pressure deficit (VPD) and available soil moisture in a manner consistent with leaf-level studies of photosynthetic ¹³C discrimination. Sensitivity tests were conducted to evaluate the effect of (1) changes in the lag between the time of CO₂ fixation and the conversion of organic matter back to CO₂; (2) shifts in the proportion of autotrophic and heterotrophic respiration; (3) isotope fractionation during respiration; and (4) environmentally induced changes in mesophyll conductance, on modelled δ _R values. Our results indicated that δ _R is a good proxy for canopy-level C _c/ C _a and ¹³C discrimination during photosynthetic gas exchange, and therefore has several applications in ecosystem physiology.     

Photosynthesis, carbohydrate levels and chlorophyll fluorescence-estimated intercellular CO2 in water-stressed Casuarina equisetifolia Forst. & Forst.

The effect of long-term water stress on photosynthetic carbon metabolism in Casuarina equisetifolia Forst. & Forst. was analysed by measuring CO₂ assimilation, stomatal conductance, the quantum yield of photosystem II ( Φ _PSII), enzyme activities, and the levels of photosynthetic intermediates and carbohydrates. CO₂ assimilation decreased under water stress while the intercellular CO₂ concentration ( C _i) as estimated by gas exchange measurements remained high. However, the estimates of C _i from measurements of Φ _PSII suggest that the decrease in photosynthesis can be explained in terms of stomatal closure. Water stress decreased total stromal fructose-1,6-bisphosphatase activity and did not alter the activities and activation states of ribulose bisphosphate carboxylase oxygenase and NADP-dependent malate dehydrogenase (NADP-MDH). The concentration of photosynthetic metabolites, glucose, fructose and sucrose decreased, whereas starch concentrations increased under drought conditions.