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     

Light-dependent fragmentation of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase in chloroplasts isolated from wheat leaves

The large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) is degraded into an N-terminal side fragment of 37 kDa and a C-terminal side fragment of 16 kDa by the hydroxyl radical in the lysates of chloroplasts in light (H. Ishida et al. 1997, Plant Cell Physiol 38: 471–479). In the present study, we demonstrate that this fragmentation of the LSU also occurs in the same manner in intact chloroplasts, and discuss the mechanisms of the fragmentation. The fragmentation of the LSU was observed when intact chloroplasts from wheat leaves were incubated under illumination in the presence of KCN or NaN₃, which is a potent inhibitor of active oxygen-scavenging enzyme(s). The properties, such as molecular masses and cross-reactivities against the site-specific anti-LSU antibodies, of the fragments found in the chloroplasts were the same as those found in the lysates. These results indicate that, as in the lysates, the fragmentation of the LSU in the intact chloroplasts was also caused by the hydroxyl radical generated in light. The fragmentation of the LSU was completely inhibited by 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU), and only partially inhibited by methyl viologen in the lysates. The addition of hydrogen peroxide to the lysates stimulated LSU fragmentation in light, but did not induce any fragmentation in darkness. Thus, we conclude that both production of hydrogen peroxide and generation of the reducing power at thylakoid membranes in light are essential requirements for fragmentation of the LSU. Received: 14 June 1997 / Accepted: 28 August 1997     

Construction of a Rubisco Fusion Gene and Its Expression in E. coli

A fusion gene was constructed with the Signal sequence of Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) small subunit from tomato and the coding region of Rubisco large subunit from spinach. The fusion gene was confirmed with restriction endonucleases and DNA sequencing analysis for the open reading frame. The chimeric gene was transferred to E. coli and its expression was induced by addition of IPTG. Expression of the Rubisco fusion gene was detected by Western blotting.     

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     

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     

Oxidative modification and breakdown of ribulose-1,5-bisphosphate carboxylase/oxygenase induced in Euglena gracitis by nitrogen starvation

When photoheterotrophic Euglena gracilis Z Pringsheim was subjected to nitrogen (N)-deprivation, the abundant photosynthetic enzyme ribulose-1,5-bis-phosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) was rapidly and selectively degraded. The breakdown began after a 4-h lag period and continued for a further 8 h at a steady rate. After 12 h of starvation, when the amount of Rubisco was reduced to 40%, the proteolysis of this enzyme slowed down while degradation of other proteins started at a similar pace. This resulted in a decline of culture growth, chloroplast disassembly — as witnessed by chlorophyll (Chl) loss — and cell bleaching. Experiments with spectinomycin, an inhibitor of chloroplastic translation, indicated that there was an absolute increase in the rate of Rubisco degradation in the N-deprived culture as compared with control conditions, where no significant carboxylase breakdown was detected. Oxidative aggregation of Rubisco (as detected by non-reductive electrophoresis) and association of the enzyme to membranes increased with time of N-starvation. Fluorescent labeling of oxidized cysteine (Cys) residues with monobromobimane indicated a progressive oxidation of Cys throughout the first hours of N-deprivation. It is concluded that Rubisco acts as an N store in Euglena, being first oxidized, and then degraded, during N-starvation. The mobilization of Rubisco allows sustained cell growth and division, at almost the same rate as the control (non-starved) culture, during 12 h of N-deprivation. Afterwards, breakdown is extended to other photosynthetic structures and the whole chloroplast is dismantled while cell growth is greatly reduced.Abbreviations Chl chlorophyll - Cys cysteine - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate We thank Drs. Pablo Vera and Ismael Rodrigo (Univ. Politécnica, Valencia, Spain) for advice and facilities in raising and collecting the anti-Rubisco serum. This work was supported by grants PB87-0353 and PB92-0821 of DGICYT and by a fellowship of the Spanish Ministerio de Educación y Ciencia (awarded to C.G.-F.).     

Presence of the CO2-concentrating mechanism in some species of the pyrenoid-less free-living algal genus Chloromonas (Volvocales, Chlorophyta)

Physiological and morphological characteristics related to the CO₂-concentrating mechanism (CCM) were examined in several species of the free-living, unicellular volvocalean genus Chloromonas (Chlorophyta), which differs morphologically from the genus Chlamydomonas only by lacking pyrenoids. The absence of pyrenoids in the chloroplasts of Chloromonas (Cr.) rosae UTEX 1337, Cr. serbinowii UTEX 492, Cr.␣clatharata UTEX 1970, Cr. rosae SAG 26.90, and Cr. palmelloides SAG 32.86 was confirmed by light and electron microscopy. In addition, immunogold electron microscopy demonstrated that ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) molecules were distributed almost evenly throughout the chloroplasts in all five Chloromonas strains. However, Chloromonas exhibited two types of physiological characteristics related to the CCM depending on the species or strains examined. Chloromonas rosae UTEX 1337 and Cr. serbinowii had high photosynthetic affinities for CO₂ in cells grown in culture medium bubbled with air (low-CO₂ cells), compared with those grown in medium bubbled with 5% CO₂ (high-CO₂ cells), indicating the presence of the low-CO₂-inducible CCM. In addition, these two Chloromonas strains exhibited low-CO₂-inducible carbonic anhydrase (CA; EC 4.2.1.1) activity and seemed to have small intracellular inorganic carbon pools. Therefore, it appears that Cr. rosae UTEX 1337 and Cr. serbinowii possess the CCM as in pyrenoid-containing microalgae such as Chlamydomonas reinhardtii. By contrast, Cr. clatharata, Cr. rosae SAG 26.90 and Cr. palmelloides showed low photosynthetic affinities for CO₂ when grown under both CO₂ conditions. Moreover, these three strains exhibited an apparent absence of intracellular inorganic carbon pools and lacked low-CO₂-inducible CA activity. Thus, Cr. clatharata, Cr. rosae SAG 26.90 and Cr. palmelloides, like other pyrenoid-less algae (lichen photobionts) reported previously, seem to lack the CCM. The present study is the first demonstration of the CCM in pyrenoid-less algae, indicating that pyrenoids or accumulation of Rubisco in the chloroplasts are not always essential for the CCM in algae. Focusing on this type of CCM in pyrenoid-less algae, the physiological and evolutionary significance of pyrenoid absence is discussed. Received: 1 May 1997 / Accepted: 11 September 1997     

Purification and properties of ribulose 1,5-bisphosphate carboxylase from sunflower leaves

Extracts from sunflower leaves possess a high ribulose-1,5-bisphosphate (RuBP) carboxylase capacity but this enzyme activity is not stable. A purification procedure, developed with preservation of carboxylase activity by MgSO₄, yielded purified RuBP carboxylase with high specific activity (40 nkat mg^-1 protein). Measurement of kinetic parameters showed high Km values (RuBP, HCO ₃ ^- ) and high Vmax of the reaction catalyzed by this sunflower enzyme; the results are compared with those obtained for soybean carboxylase. Enzyme characteristics are discussed in relation to stabilization and activation procedures and to the high photosynthesis rates of this C₃ species.     

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.     

Light-independent degradation of stromal proteins in intact chloroplasts isolated from Pisum sativum L. leaves: requirement for divalent cations

Intact chloroplasts were isolated from mature pea (Pisum sativum L.) leaves in order to study the degradation of several stromal proteins in organello. Changes in the abundances of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39), phosphoribulokinase (EC 2.7.1.19), glutamine synthetase (EC 6.3.1.2) and ferredoxin-dependent glutamine:α-ketoglutarate aminotransferase (glutamate synthase; EC 1.4.7.1) were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Coomassie-staining of the gels or immunoblotting using specific antibodies for the different enzymes. Degradation of several stromal proteins was strongly stimulated when intact chloroplasts were incubated in the light in the presence of dithiothreitol. Since free radicals may artificially accumulate in the chloroplast under such conditions and interfere with the stability of stromal proteins, the general relevance of these processes remains questionable. In the absence of light, proteolysis proceeded slowly in isolated chloroplasts and was not stimulated by dithiothreitol. Inhibition by ethylenediaminetetraacetic acid (EDTA), 1,10-phenanthroline or excess zinc ions as well as the requirement for divalent cations suggested that a zinc-containing metalloprotease participated in this process. Furthermore, light-independent degradation of ribulose-1,5-bisphosphate carboxylase/oxygenase and phosphoribulokinase was enhanced in chloroplasts isolated from leaves in which senescence was accelerated by nitrogen starvation. Our results indicate that light-independent stromal protein degradation in intact chloroplasts may be analogous to proteolysis that occurs in intact leaves during senescence. Received: 3 July 1997 / Accepted: 15 October 1997     

Degradation of active-oxygen-modified ribulose-1,5-bisphosphate carboxylase/oxygenase by chloroplastic proteases requires ATP-hydrolysis

Active oxygen (AO) species generated in plants under stress conditions trigger degradation of Rubisco (EC 4.1.1.39). To find out whether AO species activate proteases or make the protein susceptible to proteolysis, purified and ¹⁴C-labelled Rubisco protein was incubated with stromal preparations obtained from barley (Hordeum vulgare L.) leaves. The protein was degraded into distinct fragments only after a treatment with AO. This result shows that AO-treated Rubisco has been modified to become a substrate for stromal protease(s) and dismisses the possibility of protease activation. Upon degradation, distinct fragments accumulated with time. The fragmentation pattern was indistinguishable from that obtained with intact chloroplasts subjected to oxidative conditions (cf. M. Desimone et al., 1996, Plant Physiol 111: 789–796). Degradation required ATP-hydrolysis, since AMP, ADP or non-hydrolysable ATP-analogs did not support proteolysis. The ClpP-deficient stromal preparations degraded AO-modified Rubisco, making the involvement of the ClpC/P protease unlikely. Received: 1 September 1997 / Accepted 15 November 1997     

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     

Variation in cellular ribulose-1,5-bisphosphate-carboxylase content in leaves of Triticum genotypes at three levels of ploidy

Ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 1.1.39) (RuBPCase) was quantified using polyacrylamide-gel electrophoresis in whole 9-d-old first leaves of 14 genotypes of Triticum, and cellular RuBPCase levels calculated. Diploids, tetraploids and hexaploids were analysed and it was confirmed that the RuBPCase level per cell is closely related to ploidy in wheat. Inter-genotypic variation in RuBPCase levels per cell and per leaf were surveyed. It was found that the interactions between leaf size, cell size and RuBPCase levels result in small variations in RuBPCase levels per unit leaf area between genotypes.Abbreviation RuBPCase ribulose-1,5-bisphosphate carboxylase/oxygenase     

Characterization of the pyrenoid isolated from unicellular green algaChlamydomonas reinhardtii: Particulate form of RuBisCO protein

Summary The pyrenoid is a protein complex in the chloroplast stroma of eukaryotic algae. After the treatment with mercury chloride, pyrenoids were isolated by sucrose density gradient centrifugation from cell-wall less mutant cells, CW-15, as well as wild type cells, C-9, of unicellular green algaChlamydomonas reinhardtii. Pyrenoids were characterized as a fraction whose protein/chlorophyll ratio was very high, and also examined by Nomarski differential interference microscopy. Most of the components consisted of 55 kDa and 16 kDa polypeptides (11) which were immunologically identified as the large and small subunit of RuBisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase) protein, respectively. Some minor polypeptides were also detected. Substantial amount of RuBisCO protein is present as a particulate form in the pyrenoid in addition to the soluble form in algal chloroplast stroma.Abbreviations BPB bromophenol blue - DAB 3,3-diaminobenzidine - DTT dithiothreitol - ELISA enzyme-linked immunosorbent assay - High-CO₂ cells cells grown under air enriched with 4% CO₂ - Low-CO₂ cells cells grown under ordinary air (containing 0.04% CO₂) - NP-40 nonionic detergent (Nonidet) P-40 - PAGE polyacrylamide gel electrophoresis - PAP peroxidase-antiperoxidase conjugate - RuBisCO ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose-1,5-bisphosphate - SDS sodium dodecylsulfate     

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     

胶州湾浮游植物rbcL基因分子遗传多样性研究

利用聚合酶链式反应扩增胶州湾表层海水浮游植物核酮糖1,5-二磷酸羧化/氧化酶大亚基基因(rbcL)片段,建立了该基因片段变异类型文库.随机测定了28个rbcL片段序列,依此初步分析了胶州湾表层海水浮游植物rbcL基因分子遗传多样性.结果表明,春季胶州湾表层海水浮游植物优势种群为D类rbcL代表的浮游植物,其中隐藻占28.6%、Stramenopies占32.1%、定鞭藻占28.6%、红藻占3.6%.B类rbcL代表的浮游植物为绿藻,占7.1%.根据各操作分类单元丰度计算的分子遗传多样性指数为2.85,根据逆翻译成的氨基酸序列计算的序列多样性为0.20.     

Proteolysis of ribulose-1,5-bisphosphate carboxylase/oxygenase in leaves of Citrus explants throughout the annual cycle and its regulation by ethylene

Leaf senescence and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBP carboxylase, EC 4.1.1.39) degradation in orange [ Citrus sinensis (L.) Osbeck cv. Washington Navel] explants have been investigated. Explants consisted of a segment of stem (ca 15 cm) and 5 mature leaves. In vitro RuBP carboxylase degradation was determined by culturing the explants in water for different periods of time (3 days usually) and quantifying the two RuBP carboxylase subunits in the extracts following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In vitro RuBP carboxylase degradation was estimated by autodigestion of leaf extracts and SDS-PAGE. The extent of in vivo RuBP carboxylase degradation in explants cultured under 16 h light/8 h dark photoperiod varied throughout the year and showed a cyclic behaviour correlated with the growth cycle of Citrus. The highest proteolytic activity both in vivo and in vitro was found in explants made from April to August coinciding with the maximum vegetative growth period of the tree.
Leaf senescence and abscission could be retarded significantly at any time of the year by maintaining the explants continuously in the dark. Treatment of the explants in the dark with a continuous flow of ethylene enhanced both leaf abscission and rate of RuBP carboxylase degradation, proportionally to ethylene concentration (0.1-0.6 ppm). Ethylene-induced senescence of Citrus leaf explants in the dark appears to be a convenient model system to study the regulation of the proteolytic degradation of RuBP carboxylase.     

Effects of growth light and nitrogen nutrition on the organization of the photosynthetic apparatus in leaves of a C4 plant, Amaranthus cruentus

Properties of C4 photosynthesis were examined in Amaranthus cruentus L. (NAD-malic enzyme (ME) subtype, dicot) grown under different light and nitrogen (N) conditions, from the viewpoint of N investment into their photosynthetic components. In low-light (LL) leaves, chlorophyll content per leaf area was greater and chlorophyll alb ratio was lower than in high-light (HL) leaves. These indicate that LL leaves invest more N into their light-harvesting systems. However, this N investment did not contribute to the increase in the quantum yield of photosynthesis on the incident photon flux density (PFD) basis (Qi) in LL leaves. N allocation to ribulose 1,5-bisphosphate carboxylasel oxygenase (Rubisco) was significantly higher in HL-high N (HN) leaves than in other leaves. On the other hand, N allocation to C4 enzymes [phosphoenolpyruvate carboxylase (PEPC) and pyruvate Pi dikinase (PPDK)] was unaffected by the growth conditions. Maximum photosynthetic rates (Pmax) per Rubisco content were similar irrespective of the growth light treatments. Carbon isotope ratios (delta13 C) in the leaf dry matter were more negative in LL leaves than in HL leaves (LL = -19.3% per hundred, HL = -16.0% per hundred) and independent of leaf N. Vein density was highest in HL-HN leaves, and leaf thickness was unaffected by the growth light treatments. From these results, we conclude that A. cruentus leaves would not acclimate efficiently to low growth light.     

Proteolysis and transition-state-analogue binding of mutant forms of ribulose-1,5-bisphosphate carboxylase/oxygenase from Chlamydomonas reinhardtii

Trypsin digestion reduces the sizes of both the large and small subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) from the green alga Chlamydomonas reinhardtii. Incubation of either CO₂/Mg²⁺ -activated or nonactivated enzyme with the transition-state analogue carboxyarabinitol bisphosphate protects a trypsin-sensitive site of the large subunit, but not of the small subunit. Incubation of the nonactivated enzyme with ribulosebisphosphate (RuBP) provided the same degree of protection. Thus, the very tight binding that is a characteristic of the transitionstate analogue is apparently not required for the protection of the trypsin-sensitive site of the large subunit. Mutant enzymes that have reduced CO₂/O₂ specificities failed to bind carboxyarabinitol bisphosphate tightly. However, their large-subunit trypsin-sensitive sites could still be protected. The K _m values for RuBP were not significantly changed for the mutant enzymes, but the V _max values for carboxylation were reduced substantially. These results indicate that the failure of the mutant enzymes to bind the transition-state analogue tightly is primarily the consequence of an impairment in the second irreversible binding step. Thus, in all of the mutant enzymes, defects appear to exist in stabilizing the transition state of the carboxylation step, which is precisely the step proposed to influence the CO₂/O₂ specificity of Rubisco.Abbreviations and Symbols CABP 2-carboxyarabinitol 1,5-bisphosphate - enol-RuBP 2,3-enediolate of ribulose 1,5-bisphosphate - K _c K _m for CO₂ - K _o K _m for O₂ - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose 1,5-bisphosphate - V _c V _max for carboxylation - V _o V _max for oxygenation Paper No. 9313, Journal Series, Nebraska Agricultural Research DivisionThis work was supported by National Science Foundation grant DMB-8703820. We thank Drs. Archie Portis and Raymond Chollet for their helpful comments, and also thank Dr. Chollet for graciously providing CABP and [¹⁴C]CABP.