Rubisco Evolution in C4 Eudicots: An Analysis of Amaranthaceae Sensu Lato

Background

Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) catalyses the key reaction in the photosynthetic assimilation of CO₂. In C₄ plants CO₂ is supplied to Rubisco by an auxiliary CO₂-concentrating pathway that helps to maximize the carboxylase activity of the enzyme while suppressing its oxygenase activity. As a consequence, C₄ Rubisco exhibits a higher maximum velocity but lower substrate specificity compared with the C₃ enzyme. Specific amino-acids in Rubisco are associated with C₄ photosynthesis in monocots, but it is not known whether selection has acted on Rubisco in a similar way in eudicots.

Methodology/Principal Findings

We investigated Rubisco evolution in Amaranthaceae sensu lato (including Chenopodiaceae), the third-largest family of C₄ plants, using phylogeny-based maximum likelihood and Bayesian methods to detect Darwinian selection on the chloroplast rbcL gene in a sample of 179 species. Two Rubisco residues, 281 and 309, were found to be under positive selection in C₄ Amaranthaceae with multiple parallel replacements of alanine by serine at position 281 and methionine by isoleucine at position 309. Remarkably, both amino-acids have been detected in other C₄ plant groups, such as C₄ monocots, illustrating a striking parallelism in molecular evolution.

Conclusions/Significance

Our findings illustrate how simple genetic changes can contribute to the evolution of photosynthesis and strengthen the hypothesis that parallel amino-acid replacements are associated with adaptive changes in Rubisco.     

Molecular evolution of rbcL in three gymnosperm families: identifying adaptive and coevolutionary patterns

Background

The chloroplast-localized ribulose-1, 5-biphosphate carboxylase/oxygenase (Rubisco), the primary enzyme responsible for autotrophy, is instrumental in the continual adaptation of plants to variations in the concentrations of CO₂. The large subunit (LSU) of Rubisco is encoded by the chloroplast rbcL gene. Although adaptive processes have been previously identified at this gene, characterizing the relationships between the mutational dynamics at the protein level may yield clues on the biological meaning of such adaptive processes. The role of such coevolutionary dynamics in the continual fine-tuning of RbcL remains obscure.

Results

We used the timescale and phylogenetic analyses to investigate and search for processes of adaptive evolution in rbcL gene in three gymnosperm families, namely Podocarpaceae, Taxaceae and Cephalotaxaceae. To understand the relationships between regions identified as having evolved under adaptive evolution, we performed coevolutionary analyses using the software CAPS. Importantly, adaptive processes were identified at amino acid sites located on the contact regions among the Rubisco subunits and on the interface between Rubisco and its activase. Adaptive amino acid replacements at these regions may have optimized the holoenzyme activity. This hypothesis was pinpointed by evidence originated from our analysis of coevolution that supported the correlated evolution between Rubisco and its activase. Interestingly, the correlated adaptive processes between both these proteins have paralleled the geological variation history of the concentration of atmospheric CO₂.

Conclusions

The gene rbcL has experienced bursts of adaptations in response to the changing concentration of CO₂ in the atmosphere. These adaptations have emerged as a result of a continuous dynamic of mutations, many of which may have involved innovation of functional Rubisco features. Analysis of the protein structure and the functional implications of such mutations put forward the conclusion that this evolutionary scenario has been possible through a complex interplay between adaptive mutations, often structurally destabilizing, and compensatory mutations. Our results unearth patterns of evolution that have likely optimized the Rubisco activity and uncover mutational dynamics useful in the molecular engineering of enzymatic activities.

Reviewers

This article was reviewed by Prof. Christian Blouin (nominated by Dr W Ford Doolittle), Dr Endre Barta (nominated by Dr Sandor Pongor), and Dr Nicolas Galtier.     

Changes in the turnover of Rubisco and levels of mRNAs of rbcL and rbcS in rice leaves from emergence to senescence

Changes in the amount of ribulose 1,5‐bisphosphate carboxylase/oxygenase (Rubisco; EC 4·1·1·39) synthesized and degraded and the levels of rbcL and rbcS mRNAs were examined in the eighth leaf blades of rice from emergence to senescence. Synthesis of Rubisco was very active during leaf expansion, became quite low at the time of full expansion and then declined further during senescence. The changes in the levels of rbcL and rbcS mRNAs co‐ordinated approximately with those in the amount of Rubisco synthesized. Thus, it is suggested that the amount of Rubisco synthesized is determined primarily by the levels of rbcL and rbcS mRNAs during the life span of the leaves. Degradation of Rubisco started just before the time of full expansion and became far more active than its synthesis during senescence. Since the synthesis of Rubisco during senescence scarcely contributed to its amount, it can be concluded that the degradation of Rubisco is the major determinant for the amount of Rubisco in senescent leaves. The decline in the level of rbcL mRNA occurred much earlier in the developmental stage and proceeded at a much faster rate than that of rbcL DNA, indicating that the level of rbcL DNA is not a major determinant for the level of rbcL mRNA in senescent leaves of rice.     

Molecular Adaptation of rbcL in the Heterophyllous Aquatic Plant Potamogeton

Background

Heterophyllous aquatic plants show marked phenotypic plasticity. They adapt to environmental changes by producing different leaf types: submerged, floating and terrestrial leaves. By contrast, homophyllous plants produce only submerged leaves and grow entirely underwater. Heterophylly and submerged homophylly evolved under selective pressure modifying the species-specific optima for photosynthesis, but little is known about the evolutionary outcome of habit. Recent evolutionary analyses suggested that rbcL, a chloroplast gene that encodes a catalytic subunit of RuBisCO, evolves under positive selection in most land plant lineages. To examine the adaptive evolutionary process linked to heterophylly or homophylly, we analyzed positive selection in the rbcL sequences of ecologically diverse aquatic plants, Japanese Potamogeton.

Principal Findings

Phylogenetic and maximum likelihood analyses of codon substitution models indicated that Potamogeton rbcL has evolved under positive Darwinian selection. The positive selection has operated specifically in heterophyllous lineages but not in homophyllous ones in the branch-site models. This suggests that the selective pressure on this chloroplast gene was higher for heterophyllous lineages than for homophyllous lineages. The replacement of 12 amino acids occurred at structurally important sites in the quaternary structure of RbcL, two of which (residue 225 and 281) were identified as potentially under positive selection.

Conclusions/Significance

Our analysis did not show an exact relationship between the amino acid replacements and heterophylly or homophylly but revealed that lineage-specific positive selection acted on the Potamogeton rbcL. The contrasting ecological conditions between heterophyllous and homophyllous plants have imposed different selective pressures on the photosynthetic system. The increased amino acid replacement in RbcL may reflect the continuous fine-tuning of RuBisCO under varying ecological conditions.     

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.     

Restoration of the wild-type locus in an RuBP carboxylase/oxygenase mutant of Synechocystis PCC 6803 via targeted gene recombination.

Summary The interaction between homologous DNA sequences, distant from each other in the chromosome, was examined in the cyanobacterium Synechocystis PCC 6803. Most of the rbcL gene encoding the large subunit of ribulose bisphosphate carboxylase/oxygenase (Rubisco) was duplicated in the genome by a targeted insertion of a 3-truncated gene copy into the psbA-I locus. Both rbcL genes, in the psbA-I region and at the rbc locus, were non-functional; The former due to the 3 truncation, and the latter due to a deletion in the 5-region (creating a 5 truncation) and a mutation associated with an insertion of the Rhodospirillum rubrum rbc gene, yielding a high-CO₂-requiring mutant (cyanorubrum). The 3 and the 5 truncated rbcL genes were linked to chloramphenicol and kanamycin resistance markers, respectively. Decreasing the kanamycin selective pressure concomitantly with exposure of the double resistance mutant to air, resulted in air-growing colonies. Analysis of their genomes, Rubisco proteins, and their ultrastructure revealed: 1) Reconstitution of a full-length cyanobacterial rbcL gene at the rbc locus; 2) simultaneous synthesis of the cyanobacterial (L₈S₈) and R. rubrum L₂) enzymes in meroploids containing both mutated and reconstituted rbcL genes; 3) reappearance of carboxysomes. Our results indicate extensive recombinatorial interactions between the homologous sequences at both loci leading to reconstitution of the cyanobacterial rbcL gene.     

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.     

CO2 assimilation, ribulose-1,5-bisphosphate carboxylase/oxygenase, carbohydrates and photosynthetic electron transport probed by the JIP-test, of tea leaves in response to phosphorus supply

Background  

Although the effects of P deficiency on tea (Camellia sinensis (L.) O. Kuntze) growth, P uptake and utilization as well as leaf gas exchange and Chl a fluorescence have been investigated, very little is known about the effects of P deficiency on photosynthetic electron transport, photosynthetic enzymes and carbohydrates of tea leaves. In this study, own-rooted 10-month-old tea trees were supplied three times weekly for 17 weeks with 500 mL of nutrient solution at a P concentration of 0, 40, 80, 160, 400 or 1000 μM. This objective of this study was to determine how P deficiency affects CO₂ assimilation, Rubisco, carbohydrates and photosynthetic electron transport in tea leaves to understand the mechanism by which P deficiency leads to a decrease in CO₂ assimilation.     

Construction of a Synechocystic PCC6803 mutant suitable for the study of variant hexadecameric ribulose bisphosphate carboxylase/oxygenase enzymes

The cyanobacterium Synechocystis PCC6803 was chosen as a target organism for construction of a suitable photosynthetic host to enable selection of variant plant-like ribulose bisphosphate carboxylase/oxygenase (Rubisco) enzymes. The DNA region containing the operon encoding Rubisco (rbc) was cloned, sequenced and used for the construction of a transformation vector bearing flanking sequences to the rbc genes. This vector was utilized for the construction of a cyanobacterial rbc null mutant in which the entire sequence comprising both rbc genes, was replaced by the Rhodospirillum rubrum rbcL gene linked to a chloramphenicol resistance gene. Chloramphenicol-resistant colonies, Syn6803rbc, were detected within 8 days when grown under 5% CO₂ in air. These transformants were unable to grow in air (0.03% CO₂). Analysis of their genome and Rubisco protein confirmed the site of the mutation at the rbc locus, and indicated that the mutation had segregated throughout all of the chromosome copies, consequently producing only the bacterial type of the enzyme. In addition, no carboxysome structures could be detected in the new mutant. Successful restoration of the wild-type rbc locus, using vectors bearing the rbc operon flanked by additional sequences at both termini, could only be achieved upon incubating the transformed cells under 5% CO₂ in air prior to their transferring to air. The yield of restored transformants was proportionally related to the length of those sequences flanking the rbc operon which participate in the homologous recombination. The Syn6803rbc mutant is amenable for the introduction of in vitro mutagenized rbc genes into the rbc locus, aiming at the genetic modification of the hexadecameric type Rubisco.Abbreviations Cm^r chloramphenicol resistance - Km^r kanamycin resistance - HCR high CO₂ requirer - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase - SSC sodium chloride and sodium citrate - wt wild-type     

Effects of nitrogen nutrition on the relationships between the levels of rbcS and rbcL mRNAs and the amount of ribulose 1·5-bisphosphate carboxylase/oxygenase synthesized in the eighth leaves of rice from emergence through senescence

Soon after the emergence of the eighth leaf blades, rice plants were grown with two (1 and 4 mm ) levels of nitrogen (N) supply, and the relationships between the levels of rbcS and rbcL mRNAs, the amount of ribulose 1·5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.1.39) synthesized and the N influx were examined in the eighth leaf blades from emergence through senescence. The levels of both rbcS and rbcL mRNAs, the amount of Rubisco synthesized and the N influx were greater for the 4 mm N treatment than for the 1 mm N treatment throughout the experiment. The amount of Rubisco synthesized was well correlated with the levels of both mRNAs during leaf expansion, but not after the completion of leaf expansion in both N treatments. The ratio of the amount of Rubisco synthesized to the levels of both mRNAs dramatically declined after full expansion. On the other hand, the amount of Rubisco synthesized was well correlated with the N influx in both N treatments. These results indicate that the N influx, namely, N availability, rather than the levels of rbcS and rbcL mRNAs, is more closely related to the amount of Rubisco synthesized in the leaf blade of rice throughout the lifespan of a leaf.     

Evaluating the feasibility of using candidate DNA barcodes in discriminating species of the large Asteraceae family

Background  

Five DNA regions, namely, rbcL, matK, ITS, ITS2, and psbA-trnH, have been recommended as primary DNA barcodes for plants. Studies evaluating these regions for species identification in the large plant taxon, which includes a large number of closely related species, have rarely been reported.     

Accumulation and processing of a recombinant protein designed as a cleavable fusion to the endogenous Rubisco LSU protein in Chlamydomonas chloroplast

Background  

Expression of recombinant proteins in green algal chloroplast holds substantial promise as a platform for the production of human therapeutic proteins. A number of proteins have been expressed in the chloroplast of Chlamydomonas reinhardtii, including complex mammalian proteins, but many of these proteins accumulate to significantly lower levels than do endogenous chloroplast proteins. We examined if recombinant protein accumulation could be enhanced by genetically fusing the recombinant reporter protein, luciferase, to the carboxy-terminal end of an abundant endogenous protein, the large subunit of ribulose bisphosphate carboxylase (Rubisco LSU). Additionally, as recombinant proteins fused to endogenous proteins are of little clinical or commercial value, we explored the possibility of engineering our recombinant protein to be cleavable from the endogenous protein in vivo. This strategy would obviate the need for further in vitro processing steps in order to produce the desired recombinant protein. To achieve this, a native protein-processing site from preferredoxin (preFd) was placed between the Rubisco LSU and luciferase coding regions in the fusion protein construct.     

Proteome analysis of Aspergillus niger: Lactate added in starch-containing medium can increase production of the mycotoxin fumonisin B2 by modifying acetyl-CoA metabolism

Background  

Aspergillus niger is a filamentous fungus found in the environment, on foods and feeds and is used as host for production of organic acids, enzymes and proteins. The mycotoxin fumonisin B₂ was recently found to be produced by A. niger and hence very little is known about production and regulation of this metabolite. Proteome analysis was used with the purpose to reveal how fumonisin B₂ production by A. niger is influenced by starch and lactate in the medium.     

Microbial ribulose 1,5-bisphosphate carboxylase/oxygenase: A different perspective

Marine and terrestrial photosynthetic and chemoautotrophic microorganisms assimilate considerable amounts of carbon dioxide. Like green plastids, the predominant means by which this process occurs is via the Calvin-Benson-Bassham reductive pentose phosphate pathway, where ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) plays a paramount role. Recent findings indicate that this enzyme is subject to diverse means of control, including specific and elaborate means to guarantee its high rate and extent of synthesis. In addition, powerful and specific means to regulate Rubisco activity is a characteristic feature of many microbial systems. In many respects, the diverse properties of microbial Rubisco enzymes suggest interesting strategies to elucidate the molecular basis of CO₂/O₂ specificity, the holy grail of Rubisco biochemistry. These systems thus provide, as the title suggests, different perspectives to this fundamental problem. These include vast possibilities for imaginative biological selection using metabolically versatile organisms with well-defined genetic transfer capabilities to solve important issues of Rubisco specificity and molecular control. This review considers the major issues of Rubisco biochemistry and regulation in photosynthetic microoganisms including proteobacteria, cyanobacteria, marine nongreen algae, as well as other interesting prokaryotic and eukaryotic microbial systems recently shown to possess this enzyme.     

Signatures of selection in loci governing major colour patterns in <Emphasis Type="Italic">Heliconius</Emphasis> butterflies and related species

Background  

Protein-coding change is one possible genetic mechanism underlying the evolution of adaptive wing colour pattern variation in Heliconius butterflies. Here we determine whether 38 putative genes within two major Heliconius patterning loci, HmYb and HmB, show evidence of positive selection. Ratios of nonsynonymous to synonymous nucleotide changes (ω) were used to test for selection, as a means of identifying candidate genes within each locus that control wing pattern.     

Similar rates of protein adaptation in <Emphasis Type="Italic">Drosophila miranda</Emphasis> and <Emphasis Type="Italic">D. melanogaster</Emphasis>, two species with different current effective population sizes

Background  

Adaptive protein evolution is common in several Drosophila species investigated. Some studies point to very weak selection operating on amino-acid mutations, with average selection intensities on the order of N _e s ~ 5 in D. melanogaster and D. simulans. Species with lower effective population sizes should undergo less adaptation since they generate fewer mutations and selection is ineffective on a greater proportion of beneficial mutations.     

Selection dramatically reduces effective population size in HIV-1 infection

Background  

In HIV-1 evolution, a 100–100,000 fold discrepancy between census size and effective population size (N _e ) has been noted. Although it is well known that selection can reduce N _e , high in vivo mutation and recombination rates complicate attempts to quantify the effects of selection on HIV-1 effective size.