The small subunit of ribulose-1,5-bisphosphate carboxylase is plastid-encoded in the chlorophyll c-containing alga Cryptomonas Φ

The gene for the small subunit of ribulose-1,5-bisphosphate carboxylase (Rubisco) is located in the large single-copy region of the plastid genome of the chlorophyll c-containing alga Cryptomonas . The coding sequence is 417 base pairs long, encoding a protein of 139 amino acids, considerably longer than most other small subunit proteins. It is found 83 base pairs downstream from the gene for the large subunit and is cotranscribed with it. An 18 base pair perfect inverted repeat is located 8 base pairs beyond the termination codon. Sequence analysis shows the gene to be more closely related to cyanobacterial and cyanelle small-subunit genes than to those of green algae or land plants. This is the first reported sequence of a Rubisco small-subunit gene which is plastid-encoded and it exhibits a number of unique features. The derived amino acid sequence shows extensive similarity to a partial amino acid sequence from a brown alga, indicating that this gene will be of major interest as a probe for the small subunit genes in other algae and for determining possible evolutionary ancestors of algal plastids.     

MOLECULAR CHARACTERIZATION OF THE LECTIN,BRYOHEALIN, INVOLVED IN PROTOPLAST REGENERATION OF THE MARINE ALGA BRYOPSIS PLUMOSA (CHLOROPHYTA)1

When a coenocytic cell of the green alga Bryopsis plumosa (Hudson) C. Agardh was cut open and the cell contents expelled, the cell organelles agglutinated rapidly in seawater to form protoplasts. This process was mediated by a lectin, Bryohealin. The full sequence of the cDNA encoding Bryohealin was obtained, which consisted of 1,101 base pairs (bp), with 24 bp of 5′ untranslated region (UTR) and 201 bp of 3′ UTR. It had an open reading frame (ORF) of 771 bp encoding 257 amino acid residues. A signal peptide consisted of 22 amino acids presented before the start codon of Bryohealin, indicating that this lectin was a vacuolar (storage) protein. The C‐terminal sequence of Bryohealin was composed of antibiotic domains, suggesting that this lectin could perform two functions: (i) aggregation of cell organelles in seawater and (ii) protection from bacterial contamination for successful protoplast regeneration. The BLAST search result showed that Bryohealin had little sequence homology with any known plant lectins, but rather resembled animal lectins with fucolectin domains. The expression of recombinant Bryohealin (rBryohealin) was obtained in the Escherichia coli system.     

AMINO ACID SEQUENCE ANALYSIS OF THE SMALL SUBUNIT OF RIBULOSE BISPHOSPHATE CARBOXYLASE FROM FUCUS (PHAEOPHYCEAE)1

This paper reports for the first time amino acid sequence information for the small subunit of ribulose-1,5-bisphosphate carboxylase / oxygenase (Rubisco) from a non-green alga. N-terminal sequences are presented for the polypeptide from three species of the genus Fucus (Phaeophyceae). Although homologous to small subunit polypeptides from other organisms, the Fucus sequences exhibit a unique N-terminal section resembling neither cyanobacterial nor chlorophytic sequences. This difference may be a consequence of the plastid DNA coding arrangement for the small subunit in chromophytes, a situation reported for the related organism Olisthodiscus but not previously investigated at the amino acid sequence level.     

Synthesis of active Olisthodiscus luteus ribulose-1,5-bisphosphate carboxylase in Escherichia coli

The ribulose-1,5-bisphosphate carboxylase (Rubisco) large- and small-subunit genes are encoded on the chloroplast genome of the eukaryotic chromophytic alga Olisthodiscus luteus. Northern blot experiments indicate that both genes are co-transcribed into a single (>6 kb) mRNA molecule. Clones from the O. luteus rbc gene region were constructed with deleted 5 non-coding regions and placed under control of the lac promoter, resulting in the expression of high levels of O. luteus Rubisco large and small subunits in Escherichia coli. Sucrose gradient centrifugation of soluble extracts fractionated a minute amount of carboxylase activity that cosedimented with native hexadecameric O. luteus Rubisco. Most of the large subunit synthesized in E. coli appeared insoluble or formed an aggregate with the small subunit possessing an altered charge: mass ratio compared to the native holoenzyme. The presence in O. luteus of a polypeptide that has an identical molecular mass and cross reacts with antiserum generated against pea large-subunit binding protein may indicate that a protein of similar function is required for Rubisco assembly in O. luteus.     

NUCLEOTIDE SEQUENCE OF THE GENES FOR RIBULOSE-1,5-BISPHOSPHATE CARBOXYLASE/OXYGENASE LARGE SUBUNIT AND RIBOSOMAL PROTEIN S14 FROM A CHLORELLA-LIKE GREEN ALGA1

Two chloroplast genes were sequenced from an exsymbiotic strain of a eukaryotic, Chlorella-like green alga. The genes for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL) and the ribosomal protein S14 (rps14) were oriented in the same direction and were separated by 402 bp. The rbcLs of the exsymbiont and a free living Chlorella ellipsoidea were compared with other reported rbcL sequences. The rbcL gene of the exsymbiont is closely related to that of free-living Chlorella ellipsoidea. This is the first published report of an rps 14 gene sequence from an alga.     

Activation of interspecies-hybrid Rubisco enzymes to assess different models for the Rubisco–Rubisco activase interaction

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is prone to inactivation from non-productive binding of sugar-phosphates. Reactivation of Rubisco requires conformational remodeling by a specific chaperone, Rubisco activase. Rubisco activase from tobacco and other plants in the family Solanaceae is an inefficient activator of Rubisco from non-Solanaceae plants and from the green alga Chlamydomonas reinhardtii. To determine if the Rubisco small subunit plays a role in the interaction with Rubisco activase, a hybrid Rubisco (SSNT) composed of tobacco small subunits and Chlamydomonas large subunits was constructed. The SSNT hybrid, like other hybrid Rubiscos containing plant small subunits, supported photoautotrophic growth in Chlamydomonas, but growth in air was much slower than for cells containing wild-type Rubisco. The kinetic properties of the SSNT hybrid Rubisco were similar to the wild-type enzyme, indicating that the poor growth in air was probably caused by disruption of pyrenoid formation and the consequent impairment of the CO₂concentrating mechanism. Recombinant Rubisco activase from Arabidopsis activated the SSNT hybrid Rubisco and hybrid Rubiscos containing spinach and Arabidopsis small subunits at rates similar to the rates with wild-type Rubisco. However, none of the hybrid Rubiscos was activated by tobacco Rubisco activase. That replacement of Chlamydomonas small subunits with plant small subunits does not affect the species-specific interaction between Rubisco and Rubisco activase suggests that the association is not dominated by the small subunits that surround the Rubisco central solvent channel. Therefore, the geometry of a side-on binding mode is more consistent with the data than a top-on or ring-stacking binding mode.     

Rubisco but not Rubisco activase is clustered in the carboxysomes of the cyanobacterium Synechococcus sp. PCC 7942: Mud-induced carboxysomeless mutants

The Mud technology of Groisman and Casadaban was adapted to the cyanobacterium Synechococcus sp. PCC 7942. A new high-CO₂-requiring (hcr) mutant, hcr Mu28 was isolated following the integration of the Mud element 89 bp upstream of ORFI, at the 5′-flanking region of the rbc operon, which encodes RuBP carboxylase/oxygenase (Rubisco). The integration involved a 7 bp duplication that formed a direct repeat at the integration site, as previously shown in Escherichia coli The mutant was devoid of apparent carboxysome bodies, which are considered to be important for the availability of CO₂ for Rubisco. Immunolabelling studies demonstrated that Rubisco was distributed throughout hcr Mu28 cells, while in the wild type (WT) and in the carboxysome aberrant mutant hcr 0221, Rubisco was markedly associated with the carboxysomes. Rubisco activase, however, was evenly distributed throughout the cytosol of the hcr and WT cells, without any preferential association with the apparent carboxysomes.     

CLONING AND QUANTITATIVE ANALYSIS OF THE CARBONIC ANHYDRASE GENE FROM PORPHYRA YEZOENSIS1

Carbonic anhydrase (CA), an enzyme that catalyzes the interconversion of CO₂ and HCO₃^?, has a critical role in inorganic carbon acquisition in many kingdoms, including animals, plants, and bacteria. In this study, the full‐length cDNA of the CA gene from Porphyra yezoensis Ueda (denoted as PyCA) was cloned by using an expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE). The nucleotide sequence of PyCA consists of 1,153 bp, including a 5′ untranslated region (UTR) of 177 bp, a 3′ UTR of 151 bp, and an open reading frame (ORF) of 825 bp that can be translated into a 274‐amino‐acid putative peptide with a molecular mass (M) of 29.8 kDa and putative isoelectric point (pI) of 8.51. The predicted polypeptide has significant homology to the β‐CA from bacteria and unicellular algae, such as Porphyridium purpureum. The mRNA in filamentous thalli, leafy thalli, and conchospores was examined, respectively, by real‐time fluorescent quantitative PCR (qPCR), and the levels of PyCA are different at different stages of the life cycle. The lowest level of mRNA was observed in leafy thalli, and the level in filamentous thalli and in the conchospores was 4‐fold higher and 10‐fold higher, respectively.     

PRELIMINARY INVESTIGATIONS INTO THE STRUCTURE AND ACTIVITY OF RIBULOSE BISPHOSPHATE CARBOXYLASE FROM TWO PHOTOSYNTHETIC DINOFLAGELLATES1

Ribulose bisphosphate carboxylase / oxygenase (Rubisco) from the dinoflagellates Symbiodinium sp. Freudenthal and Amphidinium carterae Hulburt rapidly loses activity following cell lysis. Evidence presented indicates that this is not due to proteolysis. Using the tight binding inhibitor [¹⁴C] carboxyarabinitol bisphosphate as a marker, the Rubisco large subunit (LSu) from Symbiodinium sp. was purified. The subunit molecular weight was 56 kDa, while non-denaturing polyacrylamide gel electrophoresis indicated that the purified protein had a molecular weight significantly less than that expected of the intact hexadecameric protein. No trace of the small subunit was apparent. The initial loss of carboxylase activity following cell lysis may be due to instability of the quaternary structure of the enzyme. Antibodies prepared to the purified LSU cross-reacted with LSus from other dinoflagellates but not with the LSus of higher plants, diatoms, and other chromophytic algae. This suggests that the LSu of at least some dinoflagellates is antigenically different from that of other eukaryotes.     

Questions about the complexity of chloroplast ribulose-1,5-bisphosphate carboxylase/oxygenase

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) has played a central role in our understanding of chloroplast biogenesis and photosynthesis. In particular, its catalysis of the rate-limiting step of CO₂ fixation, and the mutual competition of CO₂ and O₂ at the active site, makes Rubisco a prime focus for genetically engineering an increase in photosynthetic productivity. Although it remains difficult to manipulate the chloroplast-encoded large subunit and nuclear-encoded small subunit of crop plants, much has been learned about the structure/function relationships of Rubisco by expressing prokaryotic genes in Escherichia coli or by exploiting classical genetics and chloroplast transformation of the green alga Chlamydomonas reinhardtii. However, the complexity of chloroplast Rubisco in land plants cannot be completely addressed with the existing model organisms. Two subunits encoded in different genetic compartments have coevolved in the formation of the Rubisco holoenzyme, but the function of the small subunit remains largely unknown. The subunits are posttranslationally modified, assembled via a complex process, and degraded in regulated ways. There is also a second chloroplast protein, Rubisco activase, that is responsible for removing inhibitory molecules from the large-subunit active site. Many of these complex interactions and processes display species specificity. This means that attempts to engineer or discover a better Rubisco may be futile if one cannot transfer the better enzyme to a compatible host. We must frame the questions that address this problem of chloroplast-Rubisco complexity. We must work harder to find the answers.     

Effects of Cerium on Key Enzymes of Carbon Assimilation of Spinach Under Magnesium Deficiency

The mechanism of the fact that cerium improves the photosynthesis of plants under magnesium deficiency is poorly understood. The main aim of the study was to determine the role of cerium in the amelioration of magnesium deficiency effects in CO₂ assimilation of spinach. Spinach plants were cultivated in Hoagland’s solution. They were subjected to magnesium deficiency and to cerium chloride administered in the magnesium-present Hoagland’s media and magnesium-deficient Hoagland’s media. The results showed that the chlorophyll synthesis and oxygen evolution was destroyed, and the activities of Rubisco carboxylasae and Rubisco activase and the expression of Rubisco large subunit (rbcL), Rubisco small subunit (rbcS), and Rubisco activase subunit (rca) were significantly inhibited, then plant growth was inhibited by magnesium deficiency. However, cerium promotes the chlorophyll synthesis, the activities of two key enzymes in CO₂ assimilation, and the expression of rbcL, rbcS, and rca, thus leading to the enhancement of spinach growth under magnesium-deficient conditions.     

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.     

Evolution of the Rubisco operon from prokaryotes to algae: Structure and analysis of the rbcS gene of the brown alga Pylaiella littoralis

The rbcS gene coding for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) of the brown alga Pylaiella littoralis is located within the plastid genome and is transcribed as a single polycistronic mRNA with the gene for the large subunit of Rubisco, rbcL. The structure of the Rubisco operon from P. littoralis was determined. Molecular phylogenies for rbcS and rbcL with a wide range of prokaryotes and eukaryotes were constructed which are congruent with recent evidence for polyphyletic plastid origins. Both rbcL and rbcS of the -purple bacterium Alcaligenes eutrophus clearly cluster with the rhodophyte and chromophyte proteins. The data suggest that the Rubisco operons of red algal and chromophytic plastids derive from -purple eubacterial antecedents, rather than the cyanobacterial lineage of eubacteria from which other of their genes derive. This implies a lateral transfer of Rubisco genes from -purple eubacterial ancestors to the cyanobacterial ancestor of rhodophyte and chromophyte plastids.     

Functional Hybrid Rubisco Enzymes with Plant Small Subunits and Algal Large Subunits: ENGINEERED rbcS cDNA FOR EXPRESSION IN CHLAMYDOMONAS*?

There has been much interest in the chloroplast-encoded large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) as a target for engineering an increase in net CO₂ fixation in photosynthesis. Improvements in the enzyme would lead to an increase in the production of food, fiber, and renewable energy. Although the large subunit contains the active site, a family of rbcS nuclear genes encodes the Rubisco small subunits, which can also influence the carboxylation catalytic efficiency and CO₂/O₂ specificity of the enzyme. To further define the role of the small subunit in Rubisco function, small subunits from spinach, Arabidopsis, and sunflower were assembled with algal large subunits by transformation of a Chlamydomonas reinhardtii mutant that lacks the rbcS gene family. Foreign rbcS cDNAs were successfully expressed in Chlamydomonas by fusing them to a Chlamydomonas rbcS transit peptide sequence engineered to contain rbcS introns. Although plant Rubisco generally has greater CO₂/O₂ specificity but a lower carboxylation V_max than Chlamydomonas Rubisco, the hybrid enzymes have 3–11% increases in CO₂/O₂ specificity and retain near normal V_max values. Thus, small subunits may make a significant contribution to the overall catalytic performance of Rubisco. Despite having normal amounts of catalytically proficient Rubisco, the hybrid mutant strains display reduced levels of photosynthetic growth and lack chloroplast pyrenoids. It appears that small subunits contain the structural elements responsible for targeting Rubisco to the algal pyrenoid, which is the site where CO₂ is concentrated for optimal photosynthesis.     

The 26- and 14-kDa phosphoproteins associated with spinach chloroplast envelope membranes are distinct membrane-bound pools of the light-harvesting complex of photosystem II and of the small subunit of ribulose-1,5-bisphosphate carboxylase-oxygenase

Two chloroplast envelope proteins from spinach (Spinacia oleracea L.) exhibiting relative molecular masses (M_rs) of 26 and 14 kDa are apparently phosphorylated by a unique Ca²⁺-dependent serine protein kinase. The activity of this enzyme shows the same sensitivity towards pH, Ca²⁺, Mg²⁺, H7 [1-(5-isoquinolinesulphonyl)-2-methylpiperazine] and ATP concentrations (Siegenthaler and Bovet 1993, Planta 190, 231–240). Autoradiographic analyses following two-dimensional-gel electrophoresis (isoelectric focusing and SDS-PAGE) associated with Western blotting experiments indicate that these two phosphoproteins appeared to be pools of the light-harvesting complex of photosystem II (LHCII) and of the ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) small subunit, respectively. Immunoprecipitation of envelope-phosphorylated proteins, using immunoglobulins (IgG) directed to the apoprotein of LHCII and to the holoenzyme of Rubisco confirmed that LHCII and the Rubisco small subunit effectively incorporated ³²P from (-³²P)ATP in isolated envelope membranes. We propose that, in agreement with the fact that protein import is driven by ATP, the phosphorylation of LHCII and the Rubisco small subunit could take place after the processing of precursor proteins and could be an obligatory step for their internalization into chloroplasts.Abbreviations 2D two dimensional - IEF isoelectric focusing - IgG immunoglobulin G - LHCII light-harvesting chlorophyll a/b proteins of PSII - LHCII A apoprotein a of LHCII - LHCIIB apoprotein b of LHCII - LS Rubisco large subunit - Mops (3-[N-morpholino]propanesulfonic acid) - M_r relative molecular mass - PI isoelectric point - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase - SS Rubisco small subunit The authors are grateful to Delphine Herrmann and Xavier Denys for their technical assistance. They also greatly thank Prof. R. J. Ellis and Dr. L. Barnett (Warwick University, UK) and Dr. P. Schürmann (University of Neuchâtel, Switzerland) for providing them with antibodies directed to the pea and spinach Rubisco holoenzymes and Dr. M. Spangfort (Lund University, Sweden) for his gift of the antibody directed to the pea LHCII apoprotein. This study was supported by the Swiss National Science Foundation. This work was part of a doctoral program carried out by L.B. in the Laboratoire de Physiologie végétale, Université de Neuchâtel, Switzerland.     

Isolation of a Protein Containing Covalently Linked Large and Small Subunits of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase from Botryococcus braunii

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) and a 66-kD protein were co-purified from solubilized microsomal preparations of the green alga Botryococcus braunii by Green A agarose, sucrose density gradient, MonoQ, and gel filtration. The 66-kD protein remained intact after 6 M urea treatment and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It could be detected in the soluble fraction of the cell-free extract but appeared to be more abundant in the microsomal preparations. It cross-reacted with antibodies raised against Rubisco holoenzyme, large and small subunits, indicating that the 66-kD protein contains both the large and the small subunits of Rubisco. The N-terminal amino acid sequence of this protein and that of a proteolytic fragment showed high homology with the mature Rubisco small subunits, and the sequence of another proteolytic fragment showed high homology with that of the Rubisco large subunit. It is concluded that the 66-kD protein is produced by cross-linking of large and small sub-units of Rubisco in the cell.