Immunolocalization and distribution of form ii rubisco in the pyrenoid and chloroplast stroma of amphidinium carterae and form i rubisco in the symbiont-derived plastids of peridinium foliaceum (dinophyceae)

Chloroplasts of peridinin-containing dinoflagellates have recently been shown to contain Form II Rubisco, which consists of large subunits only and is coded by nuclear genes. We have used immunoelectron microscopy to determine the distribution of Form II and Form I Rubisco in dinoflagellates. In sections of Amphidinium carterae Hulburt, the pyrenoid was intensely labeled and the rest of the chloroplast moderately labeled by antisera to Form II Rubisco from the purple non-sulfur bacterium Rhodospirillum rubrum and the symbiotic dinoflagellate Symbiodinium sp. No labeling was observed when sections were exposed to antiserum against Form I Rubisco of the haptophyte alga Isochrysis galbana. In contrast, cell sections of the dinoflagellate Peridinium foliaceum (Stein) Biecheler, whose chloroplasts belong to a diatom endosymbiont, showed no labeling with the two antisera against Form II Rubisco, but heavy pyrenoid labeling was present after treatment with antiserum against Form I Rubisco of I. galbana. The same immunolabeling results were obtained with the free-living diatom Phaeodactylum tricornutum Bohlin. Volumetric analysis of the distribution of Form II Rubisco in the chloroplast of A. carterae showed that, in cells grown under moderate photon irradiance, 72.9% of the plastid's Rubisco was localized in the pyrenoid, whereas in cells grown under low irradiance only 37.0% of the Rubisco was found in the pyrenoid. This light-induced concentration of Rubisco in the pyrenoid suggests that a CO₂–concentrating mechanism may elevate CO₂ within the pyrenoid, favoring the efficient fixation of CO₂ by pyrenoid Rubisco.     

The induction of the CO2 concentrating mechanism in a starch-less mutant of Chlamydomonas reinhardtii

In Chlamydomonas reinhardtii the formation of a starch sheath surrounding the pyrenoid is observed when cells grown under high CO₂ (5% CO₂ in air) are transferred to low CO₂ (0.03%) conditions. Formation of the starch sheath occurs coincidentally with induction of the CO₂ concentrating mechanism and with de novo synthesis of 5 polypeptides with molecular masses of 21, 36, 37, 42–44 kDa. We studied the effect of CO₂ concentrations on photosynthesis, ultrastructure and protein synthesis in Chlamydomonas reinhardtii cw-15 (wild phenotype for photosynthesis) and in the starch-less mutant BAFJ -6, with the aim to clarify the role of the pyrenoid starch sheath in the operation of the CO₂ concentrating mechanism and whether these low CO₂-inducible polypeptides are involved in the formation of starch sheath. When wild type and starch-less mutant cells were transferred from high to low CO₂, the CO₂ requirement for half-maximal rates of photosynthesis decreased from 40 μM to 2 μM CO₂. ³⁵SO₄^2- labeling analyses showed that the starch-less mutant induced the 5 low CO₂-inducible polypeptides. These observations suggest that the starch-less mutant was able to induce a fully active CO₂ concentrating mechanism. Since the starch-less mutant did not form a pyrenoid starch sheath, we suggest that the starch sheath is not involved in the operation of the CO₂ concentrating mechanism and that none of these 5 low CO₂-inducible proteins is involved in the formation of the starch sheath in Chlamydomonas .     

Regulation of the CO2-concentrating mechanism in Chlorella vulgaris UAM 101 by glucose

In the green alga Chlorella vulgaris UAM 101, a CO₂-concentrating mechanism is induced when the cells are growing under low CO₂ conditions. We have investigated the effect of glucose on the induction of this mechanism. Cells adapted to low CO₂ in the presence of glucose showed a reduced ability to transport and fix external inorganic carbon. This reduction was correlated with a decrease in internal carbonic anhydrase activity. 3- O -methyl-glucose, a nonmetabolizable analog of glucose, caused a more dramatic repression of these phenomena. Immunoblot analyses of total cell protein of Chlorella vulgaris UAM 101 against large subunit of ribulose-1.5-bisphosphate carboxylase/oxygenase and ribulose 1.5-bisphosphate-carboxylase/oxygenase activase polyclonal antibodies showed that the expression of these two polypeptides was affected by neither CO₂ level, nor glucose or 3- O -methyl-glucose. Ultrastructure studies showed that the low CO₂-induced development of the pyrenoid was also affected by glucose. Immunocytochemical data demonstrated that ribulose-1.5-bisphosphate carboxylase/oxygenase was exclusively located in the pyrenoid matrix. This localization and the density of labeling of the pyrenoid region were affected by neither CO₂ level nor the presence of glucose.     

The Nostoc-Gunnera symbiosis: carbon fixation and translocation

The in vitro specific activity of ribulose-1,5-bisphosphate carboxylase (Rubisco; EC 4. 1. 1. 39) and the dark and light in vivo CO₂ fixation activities were determined in the cyanobiont of Gunnera . Compared to the free-living isolate Nostoc PCC 9231, the in vitro Rubisco activity was high, while the in vivo CO₂ fixation was very low. Light did not significantly influence CO₂ fixation if the cyanobiont was left in the sliced Gunnera tissues, while a small light stimulation was found for CO₂ fixation of the freshly-isolated cyanobiont. The adjacent non-infected Gunnera tissue showed a very low CO₂ fixation. A rapid translocation of fixed ¹⁴CO₂ from leaves towards apical parts of the plant was apparent, in particular to the symbiotic tissue. The ¹⁴C label appeared mainly in soluble form in this tissue and was rapidly catabolised as shown by ¹⁴C chase experiments. Also, short-term experiments revealed that maximum ¹⁴C accumulation occurred in the symbiotic tissue showing the highest rates of nitrogen fixation (Söderbäck et al. 1990), about 10–15 mm from the plant apex. The data were taken to indicate that there is a modification in the photosynthetic light reaction of the cyanobiont and that the cyanobiont lives heterotrophically in the dark on photo-synthate rapidly delivered from nearby leaves of the host plant.     

In Situ Association of Calvin Cycle Enzymes,Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase Activase,Ferredoxin-NADP+ Reductase,and Nitrite Reductase with Thylakoid and Pyrenoid Membranes of Chlamydomonas reinhardtii Chloroplasts as Revealed by Immunoelectron Microscopy

The in situ localization of the chloroplast enzymes ribulose-1,5-bisphosphate carboxylase (Rubisco), Rubisco activase, ribose-5-phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, aldolase, nitrite reductase, ferredoxin-NADP+ reductase, and H+-ATP synthase was studied by immunoelectron microscopy in Chlamydomonas reinhardtii. Immunogold labeling revealed that, despite Rubisco in the pyrenoid matrix, Calvin cycle enzymes, Rubisco activase, nitrite reductase, ferredoxin-NADP+ reductase, and H+-ATP synthase are associated predominantly with chloroplast thylakoid membranes and the inner surface of the pyrenoid membrane. This is in accord with previous enzyme localization studies in higher plants (K.H. Suss, C. Arkona, R. Manteuffel, K. Adler [1993] Proc Natl Acad Sci USA 90: 5514-5518). Pyrenoid tubules do not contain these enzymes. The pyrenoid matrix consists of Rubisco but is devoid of the other photosynthetic enzymes investigated. Evidence for the occurrence of two Rubisco forms differing in their spatial localization has also been obtained: Rubisco form I appears to be membrane associated like other Calvin cycle components, whereas Rubisco form II is confined to the pyrenoid matrix. It is proposed that enzyme form I represents an active Rubisco when assembled into Calvin cycle enzyme complexes, whereas Rubisco form II may be part of a CO2-concentrating mechanism. Pyrenoidal Calvin cycle complexes are thought to be highly active in CO2 fixation and important for the synthesis of starch around the pyrenoid.     

Direct and indirect effects of Cd2+ on photosynthesis in sugar beet (Beta vulgaris)

Sugar-beet plants ( Beta vulgaris L. cv. Monohill) were cultivated for 4 weeks in a complete nutrient solution. Indirect effects of cadmium were studied by adding 5, 10 or 20 μ M CdCl₂ to the culture medium while direct effects were determined by adding 1, 5, 20, 50 or 2 000 μ M CdCl₂ to the assay media. The photosynthetic properties were characterized by measurement of CO₂ fixation in intact plants, fluorescence emission by intact leaves and isolated chloroplasts, photosystem (PS) I and PSII mediated electron transport of isolated chloroplasts, and CO₂-dependent O₂ evolution by protoplasts. When directly applied to isolated leaves, protoplasts and chloroplasts. Cd²⁺ impeded CO₂ fixation without affecting the rates of electron transport of PSI or PSII or the rate of dark respiration. When Cd²⁺ was applied through the culture medium the capacity for, and the maximal quantum yield of CO₂ assimilation by intact plants both decreased. This was associated with: (1) decreased total as well as effective chlorophyll content (PSII antennae size), (2) decreased coupling of electron transport in isolated chloroplasts, (3) perturbed carbon reduction cycle as indicated by fluorescence measurements. Also, protoplasts isolated from leaves of Cd²⁺-cultivated plants showed an increased rate of dark respiration.     

Gas exchange in intact isolated chloroplasts from Chlamydomonas reinhardtii during starch degradation in the dark

During starch degradation in intact isolated chloroplasts from Chlamydomonas reinhardtii gas exchange was studied with a mass spectrometer. Oxygen uptake by intact chloroplasts in the dark never exceeded 1.5% of the starch degradation rate [maximum 15 nmol O₂ (mg Chl)⁻¹ h⁻¹ consumed. 1 000 nmol glucose (mg Chl)⁻¹h⁻¹ degraded]. Evolution of CO₂ under aerobic conditions [9.8–28 nmol (mg Chl)⁻¹ h⁻¹] was stimulated by addition of 0.1–0.5 m M oxaloacetate [393–425 nmol CO₂ (mg Chl)⁻¹ h⁻¹]. Pyridoxal phosphate (5 m M ) inhibited starch degradation by more than 80%, but had no effect on O₂ uptake. Starch degradation rates and CO₂ evolution did not differ under acrobic and anaerobic conditions. Increasing P_i in the reaction medium from 0.5 m M to 5.0 m M stimulated starch degradation by 230 and 260% under aerobic and anaerobic conditions, respectively. A rapid autooxidation of reduced ferredoxin was observed in a reconstituted system consisting of purified Chlamydomonas ferredoxin, purified Chlamydomonas NADP-ferredoxin oxidoreductase (EC 1.6.7.1) and NADPH. Addition of isolated thylakoids from C. reinhardtii did not affect the rate of O₂ uptake. Our results clearly indicate the absence of any oxygen requirement during starch degradation in isolated chloroplasts.     

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.     

Adjustments of net photosynthesis in Solanum tuberosum in response to reciprocal changes in ambient and elevated growth CO2 partial pressures

Single leaf photosynthetic rates and various leaf components of potato ( Solanum tuberosum L.) were studied 1–3 days after reciprocally transferring plants between the ambient and elevated growth CO₂ treatments. Plants were raised from individual tuber sections in controlled environment chambers at either ambient (36 Pa) or elevated (72 Pa) CO₂. One half of the plants in each growth CO₂ treatment were transferred to the opposite CO₂ treatment 34 days after sowing (DAS). Net photosynthesis (P_n) rates and various leaf components were then measured 34, 35 and 37 DAS at both 36 and 72 Pa CO₂. Three-day means of single leaf P_n rates, leaf starch, glucose, initial and total Rubisco activity, Rubisco protein, chlorophyll ( a + b ), chlorophyll ( a/b ), α -amino N, and nitrate levels differed significantly in the continuous ambient and elevated CO₂ treatments. Acclimation of single leaf P_n rates was partially to completely reversed 3 days after elevated CO₂-grown plants were shifted to ambient CO₂, whereas there was little evidence of photosynthetic acclimation 3 days after ambient CO₂-grown plants were shifted to elevated CO₂. In a four-way comparison of the 36, 72, 36 to 72 (shifted up) and 72 to 36 (shifted down) Pa CO₂ treatments 37 DAS, leaf starch, soluble carbohydrates, Rubisco protein and nitrate were the only photosynthetic factors that differed significantly. Simple and multiple regression analyses suggested that negative changes of P_n in response to growth CO₂ treatment were most closely correlated with increased leaf starch levels.     

Does down‐regulation of photosynthetic capacity by elevated CO2 depend on N supply in Dactylis glomerata?

Dactylis glomerata was grown hydroponically in a controlled environment at ambient (360 μl l⁻¹) or elevated (680 μl l⁻¹) CO₂ and four concentrations of nitrogen (0.15, 0.6, 1.5 and 6.0 m M NO₃⁻), to test the hypothesis that reduction of photosynthetic capacity at elevated [CO₂] is dependent on N availability and mediated by a build-up of non-structural carbohydrates. Photosynthetic capacity of the youngest fully expanded leaf (leaf 5, 2 days after full expansion) was reduced in CO₂-enriched plants at low, but not high N supply and so the stimulation of net photosynthesis by CO₂ enhancement was less at low than at high N supply. CO₂ enrichment resulted in a decrease in ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) content on a leaf area basis at 0.6 and 1.5 m M NO₃⁻, but not at 0.15 and 6.0 m M NO₃⁻, and had no effect on the total N content of the leaf on an area basis. However, decreases in Rubisco content could be primarily accounted for by a decrease in total N content of leaves, independent of [CO₂]. A doubling of the Rubisco content by increasing the N supply beyond 0.6 m M had only a marginal effect on the maximum carboxylation velocity in vivo, suggesting that the fraction of inactive Rubisco increased with increasing N supply. Although CO₂-enriched plants accumulated more non-structural carbohydrates in the leaf, the reduction of photosynthetic capacity at low N supply was not mediated simply by a build-up of carbohydrates. In D . glomerata , the photosynthetic capacity was mainly determined by the total N content of the leaf.     

Light dependent activation of CO2 assimilation and the ratio of Rubisco activase to Rubisco during leaf aging of rice (Oryza sativa)

The effects of the ratio of Rubisco activase to Rubisco (activase/Rubisco ratio) on light dependent activation of CO₂ assimilation were investigated during leaf aging of rice. Changes of photosynthetic CO₂ gas exchange rates in relation to step increases of light intensity from two photon flux densities of 60 µmol m⁻² s⁻¹ (low initial PFD) and 500 µmol m⁻² s⁻¹ (high initial PFD) to saturated PFD of 1 800 µmol m⁻² s⁻¹ were measured. These photosynthetic activation processes were considered to be limited by the Rubisco activation rate when analyzed by the relaxation method. The relaxation time of low initial PFD gradually declined from 3 to 33 days after leaf emergence and showed high and negative correlation to the activase/Rubisco ratio. The initial rate of Rubisco activation under low initial PFD linearly correlated to the amounts of Rubisco activase, whereas these were almost constant from 3 to 23 days after leaf emergence. But these correlations could not be recognized in the case of high initial PFD. Moreover, the relaxation times were more sensitive to intercellular CO₂ concentration (C_i) under high initial PFD than under low initial PFD, especially, at C_i below 300 µl l⁻¹. These results suggest the involvement of the activase/Rubisco ratio in the photosynthetic activation under relatively low initial PFD, and the limitation of photosynthetic activation under relatively high initial PFD by Rubisco carbamylation during leaf aging of rice.     

The impact of elevated ozone and carbon dioxide on young Acer saccharum seedlings

The effects of high O₃ (200 nl l⁻¹ during the light period) and high CO₂ (650 μl l⁻¹ CO₂, 24 h a day) alone and in combination were studied on 45-day-old sugar maple ( Acer saccharum Marsh.) seedlings for 61 days in growth chambers. After 2 months of treatment under the environmental conditions of the experiment, sugar maple seedlings did not show a marked response to the elevated CO₂ treatment: the effect of high CO₂ on biomass was only detected in the leaves which developed during the treatment, and assimilation rate was not increased. Under high O₃ at ambient CO₂, assimilation rate at days 41 and 55 and Rubisco content at day 61 decreased in the first pair of leaves; total biomass was reduced by 43%. In these seedlings large increases (more than 2-fold) in glucose 6-phosphate dehydrogenase (G6PDH, EC 1.1.1.49) activity and in anaplerotic CO₂ fixation by phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) were observed, suggesting that an enhanced reducing power and carbon skeleton production was needed for detoxification and repair of oxidative damage. Under high O₃ at elevated CO₂, a stimulation of net CO₂ assimilation was observed after 41 days but was no longer observed at day 55. However, at day 61, the total biomass was only reduced by 21% and stimulation of G6PDH and PEPC was less pronounced than under high O₃ at ambient CO₂. This suggests that high CO₂ concentration protects, to some extent, against O₃ by providing additional carbon and energy through increased net assimilation.     

Inhibition of photosynthesis by heat stress: the activation state of Rubisco as a limiting factor in photosynthesis

Although the catalytic activity of Rubisco increases with temperature, the low affinity of the enzyme for CO₂ and its dual nature as an oxygenase limit the possible increase in net photosynthesis with temperature. For cotton, comparisons of measured rates of net photosynthesis with predicted rates that take into account limitations imposed by the kinetic properties of Rubisco indicate that direct inhibition of photosynthesis occurs at temperatures higher than about 30°C. Inhibition of photosynthesis by moderate heat stress (i.e. 30–42°C) is generally attributed to reduced rates of RuBP regeneration caused by disruption of electron transport activity, and specifically inactivation of the oxygen evolving enzymes of photosystem II. However, measurements of chlorophyll fluorescence and metabolite levels at air-levels of CO₂ indicate that electron transport activity is not limiting at temperatures that inhibit CO₂ fixation. Instead, recent evidence shows that inhibition of net photosynthesis correlates with a decrease in the activation state of Rubisco in both C₃ and C₄ plants and that this decrease in the amount of active Rubisco can fully account for the temperature response of net photosynthesis. Biochemically, the decrease in Rubisco activation can be attributed to: (1) more rapid de-activation of Rubisco caused by a faster rate of dead-end product formation; and (2) slower re-activation of Rubisco by activase. The net result is that as temperature increases activase becomes less effective in keeping Rubisco catalytically competent. In this opinionated review, we discuss how these processes limit photosynthetic performance under moderate heat stress.     

Acclimation of Arabidopsis thaliana to long-term CO2 enrichment and nitrogen supply is basically a matter of growth rate adjustment

The long-term response of Arabidopsis thaliana to increasing CO₂ was evaluated in plants grown in 800 μl l⁻¹ CO₂ from sowing and maintained, in hydroponics, on three nitrogen supplies: "low,""medium" and "high." The global response to high CO₂ and N-supply was evaluated by measuring growth parameters in parallel with photosynthetic activity, leaf carbohydrates, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) messenger RNA and protein, stomatal conductance (g_s) and density. CO₂ enrichment was found to stimulate biomass production, whatever the N-supply. This stimulation was transient on low N-supply and persisted throughout the whole vegetative growth only in high N-supply. Acclimation on low N–high CO₂ was not associated with carbohydrate accumulation or with a strong reduction in Rubisco amount or activity. At high N-supply, growth stimulation by high CO₂ was mainly because of the acceleration of leaf production and expansion while other parameters such as specific leaf area, root/shoot ratio and g_s appeared to be correlated with total leaf area. Our results thus suggest that, in strictly controlled and stable growing conditions, acclimation of A. thaliana to long-term CO₂ enrichment is mostly controlled by growth rate adjustment.     

Measurements of photosynthesis and respiration in plants

Methods for measuring the rates of photosynthesis and respiration in plants are reviewed. Closed systems that involve manometric techniques, ¹⁴CO₂ fixation, O₂ electrodes and other methods for measuring dissolved and gas phase O₂ are described. These methods typically provide time-integrated rate measurements, and limitations to their use are discussed. Open gas exchange systems that use infra-red CO₂ gas analysers and differential O₂ analysers for measuring instantaneous rates of CO₂ and O₂ exchange are described. Important features of the analysers, design features of gas exchange systems, and sources of potential error are considered. The analysis of chlorophyll fluorescence parameters for estimating the quantum yield for O₂ evolution and CO₂ fixation is described in relation to new fluorescence imaging systems for large scale screening of photosynthetic phenotypes, and the microimaging of individual chloroplasts.     

Biosynthesis of aromatic amino acids by highly purified spinach chloroplasts – Compartmentation and regulation of the reactions

The ability of chloroplasts to synthesize aromatic amino acids from CO₂ was investigated using highly purified, intact spinach ( Spinacia oleracea L. cv. Viking II) chloroplasts and ¹⁴CO₂. Incorporation of ¹⁴C into aromatic amino acids was very low, however, and this was assumed to be due to lack of phosphoenolpyruvate (PEP), one of the substrates for the shikimate/arogenate pathway leading to aromatic amino acids in chloroplasts. Therefore, the glycolytic enzymes phosphoglycerate mutase (EC 2.7.5.3) and enolase (EC 4.2.1.11) were added to the ¹⁴CO₂ fixation medium in order to convert labelled 3-phosphoglycerate exported from the intact chloroplasts to 2-phosphoglycerate and PEP. In this way a part of the glycolytic pathway was reconstituted outside the chloroplasts to substitute for the cytoplasm lost on isolation. The presence of both enzymes in the medium increased incorporation of ¹⁴C into Tyr and Phe more than ten-fold and incorporation into Trp about two-fold, while total ¹³CO₂ fixation rates were not affected. Our results suggest that chloroplasts do not contain phosphoglycerate mutase or enolase, and that, in vivo, PEP is synthesized in the cytoplasm and imported to the chloroplast stroma for the biosynthesis of aromatic amino acids. The biosynthesis of all three aromatic amino acids was under feedback control. Using expected physiological concentrations (below 100 μ M ), each of the aromatic amino acids exerted a strict feedback inhibition of its own biosynthesis only.     

PYRUVATE METABOLISM IN THE LOBSTER NERVE AS AFFECTED BY THE PARTIAL PRESSURE OF CARBON DIOXIDE: OBSERVATIONS ON THE SYNTHESIS OF ACETYLCHOLINE AND ON METABOLIC COMPARTMENTATION

Abstract— In the lobster nerve the fixation of CO, at various levels of _pCO2 was studied by the incorporation of [l-¹⁴C]pyruvate. Incorporation of ¹⁴C was solely dependent on CO₂ fixation since the C-1 was decarboxylated in the formation of acetyl-CoA. Paired-nerve studies with [2-¹⁴C]pyruvate afforded a study of pyruvate metabolism in the lobster nerve. [I¹⁴C]Pyruvate was incorporated to nearly the same extent at all levels of pCO₂ including zero pCO₂, a finding that suggested metabolic recycling of CO₂. The magnitude of the metabolic recycling of C-1 of pyruvate or pyruvate dismutation was estimated to be nearly 20 per cent of total CO₂ fixation. Re-evaluation of the relative contributions of the CO₂ fixation. and acetyl-CoA pathways on the basis of more extensive data gave a ratio of 2:3.
The pCO₂ affected synthesis of ACh and the level of citrate. With increasing pCO₂, the specific radioactivity of ACh decreased much more than the content of ACh. The decrease in the specific radioactivity of ACh but not that of citrate further suggested metabolic compartmentation. The implication of these findings is discussed.
Alanine functioned as a metabolic sink for the incorporated pyruvate. Pyruvate levels were estimated to be approximately 0.1 nmol/mg of protein.     

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.     

Photosynthetic capacity in relation to nitrogen content and its partitioning in lichens with different photobionts

We tested the hypothesis that lichen species with a photosynthetic CO₂-concentrating mechanism (CCM) use nitrogen more efficiently in photosynthesis than species without this mechanism. Total ribulose bisphosphate carboxylase-oxygenase (Rubisco; EC 4.1.1.39) and chitin (the nitrogenous component of fungal cell walls), were quantified and related to photosynthetic capacity in eight lichens. The species represented three modes of CO₂ acquisition and two modes of nitrogen acquisition, and included one cyanobacterial ( Nostoc ) lichen with a CCM and N₂ fixation, four green algal ( Trebouxia ) lichens with a CCM but without N₂ fixation and three lichens with green algal primary photobionts ( Coccomyxa or Dictyochloropsis ) lacking a CCM. The latter have N₂-fixing Nostoc in cephalodia. When related to thallus dry weight, total thallus nitrogen varied 20-fold, chitin 40-fold, Chl a 5-fold and Rubisco 4-fold among the species. Total nitrogen was lowest in three of the four Trebouxia lichens and highest in the bipartite cyanobacterial lichen. Lichens with the lowest nitrogen invested a larger proportion of this into photosynthetic components, while the species with high nitrogen made relatively more chitin. As a result, the potential photosynthetic nitrogen use efficiency was negatively correlated to total thallus nitrogen for this range of species. The cyanobacterial lichen had a higher photosynthetic capacity in relation to both Chl a and Rubisco compared with the green algal lichens. For the range of green algal lichens both Chl a and Rubisco contents were linearly related to photosynthetic capacity, so the data did not support the hypothesis of an enhanced photosynthetic nitrogen use efficiency in green-algal lichens with a CCM.