Role of pyrenoids in the CO2-concentrating mechanism: comparative morphology, physiology and molecular phylogenetic analysis of closely related strains of Chlamydomonas and Chloromonas (Volvocales)

The morphology of the pyrenoid and the physiology of the CO₂-concentrating mechanism (CCM) were investigated in Chlamydomonas (Cd.) mutabilis Gerloff UTEX 578, Cd. radiata Deason et Bold UTEX 966, Cd. augustae Skuja UTEX 1969, Cd. macrostellata Lund SAG 72.81, Cd. bipapillata Bourrelly SAG 11-47, and Chloromonas (Cr.) insignis Gerloff et Ettl NIES-447, all of which are closely related phylogenetically to the pyrenoid-less strains of Chloromonas. In the chloroplasts of Cd. mutabilis UTEX 578, Cd. radiata UTEX 966, Cd. augustae UTEX 1969, and Cd. macrostellata SAG 72.81, a typical, spheroidal, electron-dense pyrenoid matrix surrounded by starch granules was present, and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) molecules were highly concentrated in the pyrenoid matrix. On the other hand, while the pyrenoid matrix of Cr. insignis NIES-447 was electron-dense that of Cd. bipapillata SAG 11-47 was not, and neither was surrounded by starch granules. The pyrenoid matrices of these two species exhibited a higher concentration of Rubisco molecules than the thylakoid region (thylakoid and stroma) of the chloroplasts; however, the densities of Rubisco molecules in these pyrenoid matrices were low compared with those of the other four Chlamydomonas strains examined in this study and that of Cd. reinhardtii Dangeard. In all six strains examined, the presence of the CCM was indicated by relatively high photosynthetic affinities for CO₂ (low values of K_0.5(CO₂)). However, differences in the inorganic carbon (Ci) pools were recognized in relation to the differences in pyrenoid morphology among the strains. In the typical pyrenoid-containing strains. Cd. mutabilis UTEX 578 and Cd. radiata UTEX 966, the ratio of internal to external inorganic carbon was about 20, while in Cr. insignis NIES-447 and Cd. bipapillata SAG 11-47 the ratio was only 2–3 similar to the two pyrenoid-less, CCM-containing strains of Chloromonas previously examined (E. Morita et al., 1998, Planta 204: 269–276). It is thus speculated that the presence of typical pyrenoids with a high concentration of Rubisco molecules is related to the formation of large Ci pools in the CCM. Detailed phylogenetic relationships among these Chlamydomonas/Chloromonas strains and the pyrenoid-less Chloromonas strains previously investigated were inferred based on the sequence of rbcL, the gene for the large subunit of Rubisco. Two monophyletic groups were resolved with high bootstrap values. Based on the tree topology resolved, it was inferred that loss of the typical pyrenoids accompanied by a decrease in intracellular Ci pools might have taken place independently in the two groups. Received: 21 August 1998 / Accepted: 30 November 1998     

A 38-kilodalton low-CO2-inducible polypeptide is associated with the pyrenoid in Chlorella vulgaris

In the green alga Chlorella vulgaris UAM 101, a CO₂-concentrating mechanism (CCM) is induced when cells are transferred from high (5%) to low (0.03%) CO₂ concentrations. The induction of the CCM is correlated with de-novo synthesis of several polypeptides that remain to be identified. The internal carbonic anhydrase (CA; EC 4.2.1.1) activity increased 6- to 7-fold within 6 h of acclimation to air. When crude homogenates were further separated into soluble and insoluble fractions, nearly all of the CA activity was associated with the membrane fraction. Immunoblot analysis of cell homogenates probed with antibodies raised against the 37-kDa subunit of periplasmic CA of Chlamydomonas reinhardtii showed a cross-reaction with a single 38-kDa polypeptide in both high- and low-CO₂-grown cells. The up-regulation of the expression of the 38-kDa polypeptide was closely correlated with the increase in internal CA activity. Furthermore, its subcellular location was also correlated with the distribution of the activity. Immunoblot analysis of pyrenoid fractions showed that the 38-kDa polypeptide was concentrated in the pyrenoids from low-CO₂-grown cells but was not present in pyrenoids from high-CO₂-grown cells. In addition, immunogold labeling experiments showed that the protein was mainly associated with membranes crossing the pyrenoid, while it was absent from the pyrenoid matrix. These studies have identified a putative intracellular CA polypeptide associated with the pyrenoid in Chlorella vulgaris, suggesting that this structure may play an important role in the operation of the CCM and the acclimation to low CO₂ conditions. Received: 16 July 1997 / Accepted: 26 April 1998     

Acclimation of the summer annual species, Lolium temulentum, to CO2 enrichment

Lolium temulentum L. Ba 3081 was grown hydroponically in air (350 μmol mol⁻¹ CO₂) and elevated CO₂ (700 μmol mol⁻¹ CO₂) at two irradiances (150 and 500 μmol m⁻² s⁻¹) for 35 days at which point the plants were harvested. Elevated CO₂ did not modify relative growth rate or biomass at either irradiance. Foliar carbon-to-nitrogen ratios were decreased at elevated CO₂ and plants had a greater number of shorter tillers, particularly at the lower growth irradiance. Both light-limited and light-saturated rates of photosynthesis were stimulated. The amount of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) protein was increased at elevated CO₂, but maximum extractable Rubisco activities were not significantly increased. A pronounced decrease in the Rubisco activation state was found with CO₂ enrichment, particularly at the higher growth irradiance. Elevated-CO₂-induced changes in leaf carbohydrate composition were small in comparison to those caused by changes in irradiance. No CO₂-dependent effects on fructan biosynthesis were observed. Leaf respiration rates were increased by 68% in plants grown with CO₂ enrichment and low light. We conclude that high CO₂ will only result in increased biomass if total light input favourably increases the photosynthesis-to-respiration ratio. At low irradiances, biomass is more limited by increased rates of respiration than by CO₂-induced enhancement of photosynthesis. Received: 23 February 1999 / Accepted: 15 June 1999     

Photosynthetic CO2-use efficiency in lichens and their isolated photobionts: the possible role of a CO2-concentrating mechanism

The CO₂ dependence of net CO₂ assimilation was examined in a number of green algal and cyanobacterial lichens with the aim of screening for the algal/cyanobacterial CO₂-concentrating mechanism (CCM) in these symbiotic organisms. For the lichens Peltigera aphthosa (L.) Willd., P. canina (L.) Willd. and P. neopolydactyla (Gyeln.) Gyeln., the photosynthetic performance was also compared between intact thalli and their respective photobionts, the green alga Coccomyxa PA, isolated from Peltigera aphthosa and the cyanobacterium Nostoc PC, isolated from Peltigera canina. More direct evidence for the operation of a CCM was obtained by monitoring the effects of the carbonic-anhydrase inhibitors acetazolamide and ethoxyzolamide on the photosynthetic CO₂use efficiency of the photobionts. The results strongly indicate the operation of a CCM in all cyanobacterial lichens investigated and in cultured cells of Nostoc PC, similar to that described for free-living species of cyanobacteria. The green algal lichens were divided into two groups, one with a low and the other with a higher CO₂-use efficiency, indicative of the absence of a CCM in the former. The absence of a CCM in the low-affinity lichens was related to the photobiont, because free-living cells of Coccomyxa PA also apparently lacked a CCM. As a result of the postulated CCM, cyanobacterial Peltigera lichens have higher rates of net photosynthesis at normal CO₂ compared with Peltigera aphthosa. It is proposed that this increased photosynthetic capacity may result in a higher production potential, provided that photosynthesis is limited by CO₂ under natural conditions.     

羧酶体结构及其CO_2浓缩机制研究进展

羧酶体(Carboxysome)是高效的固碳微体,在CO2浓缩机制(CO2-concentrating mechanism,CCM)中发挥重要作用。在蓝藻及某些化能自养菌中,羧酶体作为类细胞器包裹1,5-二磷酸核酮糖羧化酶/加氧酶(RubisCO)和碳酸酐酶(Carbonic anhydrase,CA),它与无机碳转运蛋白共同在胞质中积累HCO3–,通过增加RubisCO周围的CO2浓度来提高固碳效率。随着羧酶体结构和功能的阐明,异源表达羧酶体已成功实现,并且已鉴定出编码羧酶体壳蛋白及内部组分的基因。首先简要介绍羧酶体的发现和种类,然后系统分析其结构及在CCM机制中的作用,并对其在代谢工程上的广阔应用前景进行了展望。     

Association of carbonic anhydrase with a Calvin cycle enzyme complex in Nicotiana tabacum

Chloroplast-localized carbonic anhydrase (CA; EC 4.2.1.1), an enzyme which catalyzes the reversible hydration of CO₂, appears to be associated with other enzymes of the Calvin cycle in a large multienzyme complex. Gel-filtration fast protein liquid chromatography (FPLC) of soluble proteins obtained by osmotic lysis of tobacco (Nicotiana tabacum L. cv. Carlson) chloroplasts results in the co-elution of a protein complex of greater than 600 kDa which includes CA, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), phosphoribulokinase (PRK), and ribose-5-phosphate isomerase. Anion-exchange FPLC of chloroplast extracts indicates that there is an association of CA with other proteins that modifies its elution profile in a NaCl gradient, and that Rubisco co-elutes with the fractions containing CA. Following a protocol described by Süss et al. (1993, Proc Natl Acad Sci USA 90: 5514–5518), limited protease treatment of chloroplast extracts was used to show that the association of PRK with other chloroplast proteins appears to protect a number of lysine and arginine residues which may be involved in specific protein-protein interactions. A similar treatment of CA indicates some protection of these residues when CA is associated with other chloroplast polypeptides but the level of protection is not as profound as that exhibited by PRK. In concert with previously published immunolocalization studies, these data indicate that CA may be associated with Rubisco at the stromal periphery of a Calvin cycle enzyme complex in which PRK is more centrally located and associated with thylakoid membranes. Received: 2 June 1997 / Accepted: 28 June 1997     

Identification and localization of a thylakoid-bound carbonic anhydrase from the green algae Tetraedron minimum (Chlorophyta) and Chlamydomonas noctigama (Chlorophyta)

In order to broaden our understanding of the eukaryotic CO₂-concentrating mechanism the occurrence and localization of a thylakoid-associated carbonic anhydrase (EC 4.2.1.1) were studied in the green algae Tetraedron minimum and Chlamydomonas noctigama. Both algae induce a CO₂-concentrating mechanism when grown under limiting CO₂ conditions. Using mass-spectrometric measurements of ¹⁸O exchange from doubly labelled CO₂, the presence of a thylakoid-associated carbonic anhydrase was confirmed for both species. From purified thylakoid membranes, photosystem I (PSI), photosystem II (PSII) and the light-harvesting complex of the photosynthetic apparatus were isolated by mild detergent gel. The protein fractions were identified by 77 K fluorescence spectroscopy and immunological studies. A polypeptide was found to immunoreact with an antibody raised against thylakoid carbonic anhydrase (CAH3) from Chlamydomonas reinhardtii. It was found that this polypeptide was mainly associated with PSII, although a certain proportion was also connected to light harvesting complex II. This was confirmed by activity measurements of carbonic anhydrase in isolated bands extracted from the mild detergent gel. The thylakoid carbonic anhydrase isolated from T. minimum had an isoelectric point between 5.4 and 4.8. Together the results are consistent with the hypothesis that thylakoid carbonic anhydrase resides within the lumen where it is associated with the PSII complex. Received: 13 May 2000 / Accepted: 16 August 2000     

Evidence for the functioning of photosynthetic CO2-concentrating mechanisms in lichens containing green algal and cyanobacterial photobionts

The photosynthetic properties of a range of lichens containing both green algal (11 species) and cyanobacterial (6 species) photobionts were examined with the aim of determining if there was clear evidence for the operation of a CO₂-concentrating mechanism (CCM) within the photobionts. Using a CO₂-gas-exchange system, which allowed resolution of fast transients, evidence was obtained for the existence of an inorganic carbon pool which accumulated in the light and was released in the dark. The pool was large (500–1000 nmol · mg Chl) in cyanobacterial lichens and about tenfold smaller in green algal lichens. In Hypogymnia physodes (L.) Nyl., which contains the green alga Trebouxia jamesii, a small inorganic carbon pool was rapidly formed in the light. Carbon dioxide was released from this pool into the gas phase upon darkening within about 20 s when photosynthesis was inhibited by the carbon-reduction-cycle inhibitor glycolaldehyde. In the absence of this inhibitor, release appeared to be obscured by carboxylation of ribulose bisphosphate. The kinetics of CO₂ uptake and release were monophasic. The operation of an active CCM could be distinguished from passive accumulation and release accompanying the reversible light-dependent alkalization of the stroma by the presence of saturation characteristics with respect to external CO₂. In Peltigera canina (L.) Willd., which contains the cyanobacterium Nostoc sp., a larger CO₂ pool was taken up over a longer period in the light and the release of this pool in the dark was slow, lasting 3–5 min. This pool also accumulated in the presence of glycolaldehyde, and under these conditions the CO₂ release was biphasic. In both species, photosynthesis at low CO₂ was inhibited by the carbonic-anhydrase inhibitor ethoxyzolamide (EZ). Inhibition could be reversed fully or to a considerable extent by high CO₂. In Peltigera, EZ decreased both the accumulation of the CO₂ pool by the CCM and the rate of photosynthesis. Free-living cultures of Nostoc sp. showed a similar effect of EZ on photosynthesis, although it was more dramatic than that seen with the lichen thalli. In contrast, in Hypogymnia, EZ actually increased the size of the CO₂ pool, although it inhibited photosynthesis. This effect was also seen when glycolaldehyde was present together with EZ. Surprisingly, EZ did not alter the kinetics of either CO₂ uptake or release. Taken together, the evidence indicates the operation in cyanobacterial lichens of a CCM which is capable of considerable elevation of internal CO₂ and is similar to that reported for free-living cyanobacteria. The CCM of green algal lichens accumulates much less CO₂ and is probably less effective than that which operates in cyanobacterial lichens.     

The carboxylase activity of Rubisco and the photosynthetic performance in aquatic plants

Summary Activated carboxylase activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), as well as photosynthetic rates were measured for 42 species of freshwater and marine macrophytes. While the carboxylase activity varied greatly among the species investigated (0.2–12.5 mol CO₂ mg^–1 chlorophyll min^–1), the submersed freshwater plants showed significantly lower activities than emergent, floating leaved or secondary submersed forms. The variability in photosynthetic rates correlated with the carboxylase activity only for the marine macroalgae, and their photosynthesis to carboxylase activity ratios were close to 1. These plants also had a consistently high inorganic carbon transport capability, and it is suggested that ribulose-1,5-bisphosphate carboxylase/oxygenase activity is an important internal factor regulating the photosynthetic capacity within this plant group where, apparently, the internal CO₂ concentration is high and photorespiration is suppressed. Among the freshwater forms, it appears that their much lower inorganic carbon transport ability, rather than their carboxylase activity, limits the photosynthetic process.     

Effect of heat stress on the inhibition and recovery of the ribulose-1,5-bisphosphate carboxylase/oxygenase activation state

Experiments were conducted to determine the relative contributions of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) activation state vis-à-vis Rubisco activase and metabolite levels to the inhibition of cotton (Gossypium hirsutum L.) photosynthesis by heat stress. Exposure of leaf tissue in the light to temperatures of 40 or 45 °C decreased the activation state of Rubisco to levels that were 65 or 10%, respectively, of the 28 °C control. Ribulose-1,5-bisphosphate (RuBP) levels increased in heat-stressed leaves, whereas the 3-phosphoglyceric acid pool was depleted. Heat stress did not affect Rubisco per se, as full activity could be restored by incubation with CO₂ and Mg²⁺. Inhibition and recovery of Rubisco activation state and carbon dioxide exchange rate (CER) were closely related under moderate heat stress (up to 42.5 °C). Moderate heat stress had negligible effect on Fv/Fm, the maximal quantum yield of photosystem II. In contrast, severe heat stress (45 °C) caused significant and irreversible damage to Rubisco activation, CER, and Fv/Fm. The rate of Rubisco activation after alleviating moderate heat stress was comparable to that of controls, indicating rapid reversibility of the process. However, moderate heat stress decreased both the rate and final extent of CER activation during dark-to-light transition. Treatment of cotton leaves with methyl viologen or an oxygen-enriched atmosphere reduced the effect of heat stress on Rubisco inactivation. Both treatments also reduced tissue RuBP levels, indicating that the amount of RuBP present during heat stress may influence the degree of Rubisco inactivation. Under both photorespiratory and non-photorespiratory conditions, the inhibition of the CER during heat stress could be completely reversed by increasing the internal partial pressure of CO₂ (Ci). However, the inhibition of the CER by nigericin, a K⁺ ionophore, was not reversible when the Ci was increased at ambient or high temperature. Our results indicate that inhibition of photosynthesis by moderate heat stress is not caused by inhibition of the capacity for RuBP regeneration. We conclude that heat stress inhibits Rubisco activation via a rapid and direct effect on Rubisco activase, possibly by perturbing Rubisco activase subunit interactions with each other or with Rubisco. Received: 25 February 2000 / Accepted: 13 May 2000     

Photosynthetic carbon acquisition in the lichen photobionts Coccomyxa and Trebouxia (Chlorophyta)

Processes involved in photosynthetic CO₂ acquisition were characterised for the isolated lichen photobiont Trebouxia erici (Chlorophyta, Trebouxiophyceae) and compared with Coccomyxa (Chlorophyta), a lichen photobiont without a photosynthetic CO₂-concentrating mechanism. Comparisons of ultrastructure and immuno-gold labelling of ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco; EC 4.1.1.39) showed that the chloroplast was larger in T. erici and that the majority of Rubisco was located in its centrally located pyrenoid. Coccomyxa had no pyrenoid and Rubisco was evenly distributed in its chloroplast. Both species preferred CO₂ rather than HCO₃^? as an external substrate for photosynthesis, but T. erici was able to use CO₂ concentrations below 10–12 μM more efficiently than Coccomyxa. In T. erici, the lipid-insoluble carbonic anhydrase (CA; EC 4.2.1.1) inhibitor acetazolamide (AZA) inhibited photosynthesis at CO₂ concentrations below 1 μM, while the lipid-soluble CA inhibitor ethoxyzolamide (EZA) inhibited CO₂-dependent O₂ evolution over the whole CO₂ range. EZA inhibited photosynthesis also in Coccomyxa, but to a much lesser extent below 10–12 μM CO₂. The internal CA activity of Trebouxia, per unit chlorophyll (Chl), was ca 10% of that of Coccomyxa. Internal CA activity was also detected in homogenates from T. erici and two Trebouxia-lichens (Lasallia hispanica and Cladina rangiferina). In all three, the predominating CA had α-type characteristics and was significantly inhibited by low concentrations of AZA, having an I₅₀ below 10–20 nM. In Coccomyxa a β-type CA predominates, which is much less sensitive to AZA. Thus, the two photobionts differed in three major characteristics with respect to CO₂ acquisition, the subcellular location of Rubisco, the relative requirement of CA and the biochemical characteristics of their predominating internal CA. These differences may be linked to the ability of Trebouxia to accumulate dissolved inorganic carbon internally, enhancing their CO₂ use efficiency at and below air-equilibrium concentrations (10–12 μM CO₂) in comparison with Coccomyxa.     

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 drelationship between CO2-assimilation rate,Rubisco carbamylation and Rubisco activase content in activase-deficient transgenic tobacco suggests a simple model of activase action

Transgenic tobacco (Nicotiana tabacum L. cv. W38) plants with an antisense gene directed against the mRNA of ribulose-1,5-bisphosphate carboxylase/ oxygenase (Rubisco) activase were used to examine the relationship between CO₂-assimilation rate, Rubisco carbamylation and activase content. Plants used were those members of the r₁ progeny of a primary transformant with two independent T-DNA inserts that could be grown without CO₂ supplementation. These plants had from < 1% to 20% of the activase content of control plants. Severe suppression of activase to amounts below 5% of those present in the controls was required before reductions in CO₂-assimilation rate and Rubisco carbamylation were observed, indicating that one activase tetramer is able to service as many as 200 Rubisco hexadecamers and maintain wild-type carbamylation levels in vivo. The reduction in CO₂-assimilation rate was correlated with the reduction in Rubisco carbamylation. The anti-activase plants had similar ribulose-1,5-bisphosphate pool sizes but reduced 3-phosphoglycerate pool sizes compared to those of control plants. Stomatal conductance was not affected by reduced activase content or CO₂-assimilation rate. A mathematical model of activase action is used to explain the observed hyperbolic dependence of Rubisco carbamylation on activase content.Abbreviations CA1P 2-carboxyarabinitol-1-phosphate - P_ip_a intercellular, ambient partial pressure of CO₂ - PGA 3-phospho-glycerate - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - SSU small subunit of Rubisco     

Physiological characteristics of the primitive CO2 concentrating mechanism in PEPC transgenic rice

C4 plants such as maize have CO2 concentrating mechanism and higher photosynthetic efficiency than C3 plants, especially under high light intensity, high temperature and drought conditions. In recent years, due to the rapid development of transgenic technique, different transgenic rice plants with high-level expression of C4 genes have been created by the successful introduction of genes encoding the key C4 photosynthetic path enzymes PEPC, PPDK and NADP-ME through agrobacteria-mediated…     

Temperature-conditional nuclear mutation of Chlamydomonas reinhardtii decreases the CO2/O2 specificity of chloroplast ribulosebisphosphate carboxylase/oxygenase

The Chlamydomonas reinhardtii (Dangeard) temperature-conditional mutant 68-11AR is phenotypically indistinguishable from the wild type at the permissive temperature (25°C), but has greatly reduced photosynthetic ability and requires acetate for growth at the restrictive temperature (35°C). The mutant strain is deficient in ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) holoenzyme when grown at 35°C. This decrease in the level of enzyme appears to be due to degradation of assembled holoenzyme rather than to a reduction in the synthesis of enzyme subunits. When grown at 25°C, the mutant has a substantial amount of Rubisco. Enzyme purified from 25°C-grown mutant cells was found to have a 16% decrease in the CO₂/O₂ specificity factor when compared to the wild-type enzyme. This alteration was accompanied by changes in the kinetic constants for both carboxylation and oxygenation. Although the Rubisco active site is located on the chloroplast-encoded large subunit, genetic analysis showed that the 68-11AR strain arose from a nucleargene mutation. The two nuclear genes that encode the Rubisco small subunits (rbcS1 and rbcS2) were cloned from mutant 68-11AR and completely sequenced, but no mutation was found. Analysis of restriction-fragment length polymorphisms also failed to detect linkage between mutant and rbcS gene loci. These results indicate that nuclear genes can influence Rubisco catalysis without necessarily encoding polypeptides that reside within the holoenzyme.Abbreviations and Symbols K _c Michaelis constant for CO₂ - K _o Michaelis constant for O₂ - mt mating type - pf paralyzed flagella - RFLP restriction-fragment length polymorphism - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose 1,5-bisphosphate - V _c V _max for carboxylation - V _o V _max for oxygenation - CO₂/O₂ specificity factor C. G. gratefully acknowledges fellowship support from the Consejo Superior de Investigaciones Cientificas (Spain). This work was supported by National Science Foundation grant MCB-9005547, and is published as Paper No. 10481, Journal Series, Nebraska Agricultural Research Division.     

Leaf photosynthesis, plant growth and nitrogen allocation in rice under different irradiances

The photosynthetic rates and various components of photosynthesis including ribulose-1,5-bisphosphate carboxylase (Rubisco; EC 4.1.1.39), chlorophyll (Chl), cytochrome (Cyt) f, and coupling factor 1 (CF₁) contents, and sucrose-phosphate synthase (SPS; EC 2.4.1.14) activity were examined in young, fully expanded leaves of rice (Oryza sativa L.) grown hydroponically under two irradiances, namely, 1000 and 350 μmol quanta · m⁻² · s⁻¹, at three N concentrations. The light-saturated rate of photosynthesis measured at 1800 μmol · m⁻² · s⁻¹ was almost the same for a given leaf N content irrespective of growth irradiance. Similarly, Rubisco content and SPS activity were not different for the same leaf N content between irradiance treatments. In contrast, Chl content was significantly greater in the plants grown at 350 μmol · m⁻² · s⁻¹, whereas Cyt f and CF₁ contents tended to be slightly smaller. However, these changes were not substantial, as shown by the fact that the light-limited rate of photosynthesis measured at 350 μmol · m⁻² · s⁻¹ was the same or only a little higher in the plants grown at 350 μmol · m⁻² · s⁻¹ and that CO₂-saturated photosynthesis did not differ between irradiance treatments. These results indicate that growth-irradiance-dependent changes in N partitioning in a leaf were far from optimal with respect to N-use efficiency of photosynthesis. In spite of the difference in growth irradiance, the relative growth rate of the whole plant did not differ between the treatments because there was an increase in the leaf area ratio in the low-irradiance-grown plants. This increase was associated with the preferential N-investment in leaf blades and the extremely low accumulation of starch and sucrose in leaf blades and sheaths, allowing a more efficient use of the fixed carbon. Thus, morphogenic responses at the whole-plant level may be more important for plants as an adaptation strategy to light environments than a response of N partitioning at the level of a single leaf. Received: 23 February 1997 / Accepted: 8 May 1997     

Inducibility of crassulacean acid metabolism (CAM) in Clusia species; physiological/biochemical characterisation and intercellular localization of carboxylation and decarboxylation processes in three species which exhibit different degrees of CAM

The biochemical basis for photosynthetic plasticity in tropical trees of the genus Clusia was investigated in three species that were from contrasting habitats and showed marked differences in their capacity for crassulacean acid metabolism (CAM). Physiological, anatomical and biochemical measurements were used to relate changes in the activities/amounts of key enzymes of C₃ and C₄ carboxylation to physiological performance under severe drought stress. On the basis of gas-exchange measurements and day/night patterns of organic acid turnover, the species were categorised as weak CAM-inducible (C.aripoensis Britt.), C₃-CAM intermediate (C. minor L.) and constitutive CAM (C.␣rosea Jacq. 9.). The categories reflect genotypic differences in physiological response to drought stress in terms of net carbon gain; in C. aripoensis net carbon gain was reduced by over 80% in drought-stressed plants whilst carbon gain was relatively unaffected after 10 d without water in C. rosea. In turn, genotypic differences in the capacity for CAM appeared to be directly related to the capacities/amounts of phosphoenolpyruvate carboxylase (PEPCase) and phosphoenolpyruvate carboxykinase (PEPCK) which increased in response to drought in both young and mature leaves. Whilst measured activities of PEPCase and PEPCK in well-watered plants of the C₃-CAM intermediate C. minor were 5–10 times in excess of that required to support the magnitude of organic acid turnover induced by drought, close correlations were observed between malate accumulation/PEPCase capacity and citrate decarboxylation/PEPCK capacity in all the species. Drought stress did not affect the amount of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) protein in any of the species but Rubisco activity was reduced by 35% in the weak CAM-inducible C. aripoensis. Similar amounts of glycine decarboxylase (GDC) protein were present in all three species regardless of the magnitude of CAM expression. Thus, the constitutive CAM species C. rosea did not appear to show reduced activity of this key enzyme of the photorespiratory pathway, which, in turn, may be related to the low internal conductance to CO₂ in this succulent species. Immuno-histochemical techniques showed that PEPCase, PEPCK and Rubisco were present in cells of the palisade and spongy parenchyma in leaves of species performing CAM. However, in leaves from well-watered plants of C. aripoensis which only performed C₃ photosynthesis, PEPCK was localized around latex-producing ducts. Differences in leaf anatomy between the species suggest that the association between mesophyll succulence and the capacity for CAM in these hemi-epiphytic stranglers has been selected for in arid environments. Received: 4 July 1997 / Accepted: 27 November 1997     

The in-vivo response of the ribulose-1,5-bisphosphate carboxylase activation state and the pool sizes of photosynthetic metabolites to elevated CO2 inPhaseolus vulgaris L.

The short-term, in-vivo response to elevated CO₂ of ribulose-1,5-bisphosphate carboxylase (RuBPCase, EC 4.1.1.39) activity, and the pool sizes of ribulose 1,5-bisphosphate, 3-phosphoglyceric acid, triose phosphates, fructose 1,6-bisphosphate, glucose 6-phosphate and fructose 6-phosphate in bean were studied. Increasing CO₂ from an ambient partial pressure of 360–1600 bar induced a substantial deactivation of RuBPCase at both saturating and subsaturating photon flux densities. Activation of RuBPCase declined for 30 min following the CO₂ increase. However, the rate of photosynthesis re-equilibrated within 6 min of the switch to high CO₂, indicating that RuBPCase activity did not limit photosynthesis at high CO₂. Following a return to low CO₂, RuBPCase activation increased to control levels within 10 min. The photosynthetic rate fell immediately after the return to low CO₂, and then increased in parallel with the increase in RuBPCase activation to the initial rate observed prior to the CO₂ increase. This indicated that RuBPCase activity limited photosynthesis while RuBPCase activation increased. Metabolite pools were temporarily affected during the first 10 min after either a CO₂ increase or decrease. However, they returned to their original level as the change in the activation state of RuBPCase neared completion. This result indicates that one role for changes in the activation state of RuBPCase is to regulate the pool sizes of photosynthetic intermediates.Abbreviations and symbols A net CO₂ assimilation rate - C_a ambient CO₂ partial pressure - C_i intercellular CO₂ partial pressure - CABP 2-carboxyarabinitol 1,5-bisphosphate - k_cat catalytic turnover rate per RuBPCase molecule - PFD photon flux density (400 to 700 nm on an area basis) - PGA 3-phosphoglyceric acid - P_i orthophosphate - RuBP ribulose 1,5-bisphosphate - RuBPCase ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39)