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1.
Funct Plant Biol ; 48(7): 732-742, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34099101

RESUMO

The distributions of CAM and C3 epiphytic bromeliads across an altitudinal gradient in western Panama were identified from carbon isotope (δ13C) signals, and epiphyte water balance was investigated via oxygen isotopes (δ18O) across wet and dry seasons. There were significant seasonal differences in leaf water (δ18Olw), precipitation, stored 'tank' water and water vapour. Values of δ18Olw were evaporatively enriched at low altitude in the dry season for the C3 epiphytes, associated with low relative humidity (RH) during the day. Crassulacean acid metabolism (CAM) δ18Olw values were relatively depleted, consistent with water vapour uptake during gas exchange under high RH at night. At high altitude, cloudforest locations, C3 δ18Olw also reflected water vapour uptake by day. A mesocosm experiment with Tillandsia fasciculata (CAM) and Werauhia sanguinolenta (C3) was combined with simulations using a non-steady-state oxygen isotope leaf water model. For both C3 and CAM bromeliads, δ18Olw became progressively depleted under saturating water vapour by day and night, although evaporative enrichment was restored in the C3 W. sanguinolenta under low humidity by day. Source water in the overlapping leaf base 'tank' was also modified by evaporative δ18O exchanges. The results demonstrate how stable isotopes in leaf water provide insights for atmospheric water vapour exchanges for both C3 and CAM systems.


Assuntos
Vapor , Água , Isótopos de Oxigênio , Panamá , Folhas de Planta
2.
Sci Adv ; 6(46)2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33177094

RESUMO

Approximately one-third of the Earth's photosynthetic CO2 assimilation occurs in a pyrenoid, an organelle containing the CO2-fixing enzyme Rubisco. How constituent proteins are recruited to the pyrenoid and how the organelle's subcompartments-membrane tubules, a surrounding phase-separated Rubisco matrix, and a peripheral starch sheath-are held together is unknown. Using the model alga Chlamydomonas reinhardtii, we found that pyrenoid proteins share a sequence motif. We show that the motif is necessary and sufficient to target proteins to the pyrenoid and that the motif binds to Rubisco, suggesting a mechanism for targeting. The presence of the Rubisco-binding motif on proteins that localize to the tubules and on proteins that localize to the matrix-starch sheath interface suggests that the motif holds the pyrenoid's three subcompartments together. Our findings advance our understanding of pyrenoid biogenesis and illustrate how a single protein motif can underlie the architecture of a complex multilayered phase-separated organelle.

3.
Nat Plants ; 6(12): 1480-1490, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33230314

RESUMO

Approximately one-third of global CO2 fixation occurs in a phase-separated algal organelle called the pyrenoid. The existing data suggest that the pyrenoid forms by the phase separation of the CO2-fixing enzyme Rubisco with a linker protein; however, the molecular interactions underlying this phase separation remain unknown. Here we present the structural basis of the interactions between Rubisco and its intrinsically disordered linker protein Essential Pyrenoid Component 1 (EPYC1) in the model alga Chlamydomonas reinhardtii. We find that EPYC1 consists of five evenly spaced Rubisco-binding regions that share sequence similarity. Single-particle cryo-electron microscopy of these regions in complex with Rubisco indicates that each Rubisco holoenzyme has eight binding sites for EPYC1, one on each Rubisco small subunit. Interface mutations disrupt binding, phase separation and pyrenoid formation. Cryo-electron tomography supports a model in which EPYC1 and Rubisco form a codependent multivalent network of specific low-affinity bonds, giving the matrix liquid-like properties. Our results advance the structural and functional understanding of the phase separation underlying the pyrenoid, an organelle that plays a fundamental role in the global carbon cycle.


Assuntos
Chlamydomonas reinhardtii/química , Chlamydomonas reinhardtii/metabolismo , Estrutura Molecular , Fotossíntese/fisiologia , Ribulose-Bifosfato Carboxilase/química , Ribulose-Bifosfato Carboxilase/metabolismo
4.
New Phytol ; 227(3): 810-823, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32249430

RESUMO

Green algae expressing a carbon-concentrating mechanism (CCM) are usually associated with a Rubisco-containing micro-compartment, the pyrenoid. A link between the small subunit (SSU) of Rubisco and pyrenoid formation in Chlamydomonas reinhardtii has previously suggested that specific RbcS residues could explain pyrenoid occurrence in green algae. A phylogeny of RbcS was used to compare the protein sequence and CCM distribution across the green algae and positive selection in RbcS was estimated. For six streptophyte algae, Rubisco catalytic properties, affinity for CO2 uptake (K0.5 ), carbon isotope discrimination (δ13 C) and pyrenoid morphology were compared. The length of the ßA-ßB loop in RbcS provided a phylogenetic marker discriminating chlorophyte from streptophyte green algae. Rubisco kinetic properties in streptophyte algae have responded to the extent of inducible CCM activity, as indicated by changes in inorganic carbon uptake affinity, δ13 C and pyrenoid ultrastructure between high and low CO2 conditions for growth. We conclude that the Rubisco catalytic properties found in streptophyte algae have coevolved and reflect the strength of any CCM or degree of pyrenoid leakiness, and limitations to inorganic carbon in the aquatic habitat, whereas Rubisco in extant land plants reflects more recent selective pressures associated with improved diffusive supply of the terrestrial environment.


Assuntos
Chlamydomonas reinhardtii , Clorófitas , Carbono , Dióxido de Carbono , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Clorófitas/metabolismo , Fotossíntese , Filogenia , Ribulose-Bifosfato Carboxilase/metabolismo
5.
Nat Genet ; 51(4): 627-635, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30886426

RESUMO

Photosynthetic organisms provide food and energy for nearly all life on Earth, yet half of their protein-coding genes remain uncharacterized1,2. Characterization of these genes could be greatly accelerated by new genetic resources for unicellular organisms. Here we generated a genome-wide, indexed library of mapped insertion mutants for the unicellular alga Chlamydomonas reinhardtii. The 62,389 mutants in the library, covering 83% of nuclear protein-coding genes, are available to the community. Each mutant contains unique DNA barcodes, allowing the collection to be screened as a pool. We performed a genome-wide survey of genes required for photosynthesis, which identified 303 candidate genes. Characterization of one of these genes, the conserved predicted phosphatase-encoding gene CPL3, showed that it is important for accumulation of multiple photosynthetic protein complexes. Notably, 21 of the 43 higher-confidence genes are novel, opening new opportunities for advances in understanding of this biogeochemically fundamental process. This library will accelerate the characterization of thousands of genes in algae, plants, and animals.


Assuntos
Chlamydomonas reinhardtii/genética , Clorófitas/genética , Eucariotos/genética , Mutação/genética , Fotossíntese/genética , Biblioteca Gênica , Genoma/genética , Estudo de Associação Genômica Ampla/métodos , Genômica/métodos , Análise de Sequência de DNA/métodos
6.
J Exp Bot ; 68(14): 3739-3749, 2017 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-28911054

RESUMO

The confinement of Rubisco in a chloroplast microcompartment, or pyrenoid, is a distinctive feature of most microalgae, and contributes to perhaps ~30 Pg of carbon fixed each year, yet our understanding of pyrenoid composition, regulation, and function remains fragmentary. Recently, significant progress in understanding the pyrenoid has arisen from studies using mutant lines, mass spectrometric analysis of isolated pyrenoids, and advanced ultrastructural imaging of the microcompartment in the model alga Chlamydomonas. The emergence of molecular details in other lineages provides a comparative framework for this review, and evidence that most pyrenoids function similarly, even in the absence of a common ancestry. The objective of this review is to explore pyrenoid diversity throughout key algal lineages and discuss whether common ultrastructural and cellular features are indicative of common functional processes. By characterizing pyrenoid origins in terms of mechanistic and structural parallels, we hope to provide key unanswered questions which will inform future research directions.


Assuntos
Chlamydomonas , Cloroplastos/metabolismo , Cloroplastos/ultraestrutura , Microalgas , Alga Marinha , Chlamydomonas/metabolismo , Chlamydomonas/ultraestrutura , Microalgas/metabolismo , Microalgas/ultraestrutura , Ribulose-Bifosfato Carboxilase/metabolismo , Alga Marinha/metabolismo , Alga Marinha/ultraestrutura
7.
J Exp Bot ; 68(14): 3903-3913, 2017 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-28911055

RESUMO

The pyrenoid of the unicellular green alga Chlamydomonas reinhardtii is a microcompartment situated in the centre of the cup-shaped chloroplast, containing up to 90% of cellular Rubisco. Traversed by a network of dense, knotted thylakoid tubules, the pyrenoid has been proposed to influence thylakoid biogenesis and ultrastructure. Mutants that are unable to assemble a pyrenoid matrix, due to expressing a vascular plant version of the Rubisco small subunit, exhibit severe growth and photosynthetic defects and have an ineffective carbon-concentrating mechanism (CCM). The present study set out to determine the cause of photosynthetic limitation in these pyrenoid-less lines. We tested whether electron transport and light use were compromised as a direct structural consequence of pyrenoid loss or as a metabolic effect downstream of lower CCM activity and resulting CO2 limitation. Thylakoid organization was unchanged in the mutants, including the retention of intrapyrenoid-type thylakoid tubules, and photosynthetic limitations associated with the absence of the pyrenoid were rescued by exposing cells to elevated CO2 levels. These results demonstrate that Rubisco aggregation in the pyrenoid functions as an essential element for CO2 delivery as part of the CCM, and does not play other roles in maintenance of photosynthetic membrane energetics.


Assuntos
Dióxido de Carbono/metabolismo , Chlamydomonas reinhardtii/metabolismo , Cloroplastos/metabolismo , Fotossíntese , Tilacoides/metabolismo , Chlamydomonas reinhardtii/genética
9.
J Exp Bot ; 68(14): 3891-3902, 2017 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-28520898

RESUMO

Carbon-concentrating mechanisms (CCMs) enable efficient photosynthesis and growth in CO2-limiting environments, and in eukaryotic microalgae localisation of Rubisco to a microcompartment called the pyrenoid is key. In the model green alga Chlamydomonas reinhardtii, Rubisco preferentially relocalises to the pyrenoid during CCM induction and pyrenoid-less mutants lack a functioning CCM and grow very poorly at low CO2. The aim of this study was to investigate the CO2 response of pyrenoid-positive (pyr+) and pyrenoid-negative (pyr-) mutant strains to determine the effect of pyrenoid absence on CCM induction and gene expression. Shotgun proteomic analysis of low-CO2-adapted strains showed reduced accumulation of some CCM-related proteins, suggesting that pyr- has limited capacity to respond to low-CO2 conditions. Comparisons between gene transcription and protein expression revealed potential regulatory interactions, since Rubisco protein linker (EPYC1) protein did not accumulate in pyr- despite increased transcription, while elements of the LCIB/LCIC complex were also differentially expressed. Furthermore, pyr- showed altered abundance of a number of proteins involved in primary metabolism, perhaps due to the failure to adapt to low CO2. This work highlights two-way regulation between CCM induction and pyrenoid formation, and provides novel candidates for future studies of pyrenoid assembly and CCM function.


Assuntos
Proteínas de Algas/genética , Carbono/metabolismo , Chlamydomonas reinhardtii/genética , Cloroplastos/metabolismo , Expressão Gênica , Fotossíntese , Proteínas de Algas/metabolismo , Chlamydomonas reinhardtii/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Análise de Sequência de DNA
10.
New Phytol ; 214(2): 655-667, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28084636

RESUMO

Introducing components of algal carbon concentrating mechanisms (CCMs) into higher plant chloroplasts could increase photosynthetic productivity. A key component is the Rubisco-containing pyrenoid that is needed to minimise CO2 retro-diffusion for CCM operating efficiency. Rubisco in Arabidopsis was re-engineered to incorporate sequence elements that are thought to be essential for recruitment of Rubisco to the pyrenoid, namely the algal Rubisco small subunit (SSU, encoded by rbcS) or only the surface-exposed algal SSU α-helices. Leaves of Arabidopsis rbcs mutants expressing 'pyrenoid-competent' chimeric Arabidopsis SSUs containing the SSU α-helices from Chlamydomonas reinhardtii can form hybrid Rubisco complexes with catalytic properties similar to those of native Rubisco, suggesting that the α-helices are catalytically neutral. The growth and photosynthetic performance of complemented Arabidopsis rbcs mutants producing near wild-type levels of the hybrid Rubisco were similar to those of wild-type controls. Arabidopsis rbcs mutants expressing a Chlamydomonas SSU differed from wild-type plants with respect to Rubisco catalysis, photosynthesis and growth. This confirms a role for the SSU in influencing Rubisco catalytic properties.


Assuntos
Arabidopsis/genética , Chlamydomonas/enzimologia , Teste de Complementação Genética , Mutação/genética , Subunidades Proteicas/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismo , Sequência de Aminoácidos , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Biocatálise , Clorofila/metabolismo , Fluorescência , Regulação da Expressão Gênica de Plantas , Isoenzimas/metabolismo , Fenótipo , Fotossíntese , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Subunidades Proteicas/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribulose-Bifosfato Carboxilase/química
11.
Proc Natl Acad Sci U S A ; 113(21): 5958-63, 2016 May 24.
Artigo em Inglês | MEDLINE | ID: mdl-27166422

RESUMO

Biological carbon fixation is a key step in the global carbon cycle that regulates the atmosphere's composition while producing the food we eat and the fuels we burn. Approximately one-third of global carbon fixation occurs in an overlooked algal organelle called the pyrenoid. The pyrenoid contains the CO2-fixing enzyme Rubisco and enhances carbon fixation by supplying Rubisco with a high concentration of CO2 Since the discovery of the pyrenoid more that 130 y ago, the molecular structure and biogenesis of this ecologically fundamental organelle have remained enigmatic. Here we use the model green alga Chlamydomonas reinhardtii to discover that a low-complexity repeat protein, Essential Pyrenoid Component 1 (EPYC1), links Rubisco to form the pyrenoid. We find that EPYC1 is of comparable abundance to Rubisco and colocalizes with Rubisco throughout the pyrenoid. We show that EPYC1 is essential for normal pyrenoid size, number, morphology, Rubisco content, and efficient carbon fixation at low CO2 We explain the central role of EPYC1 in pyrenoid biogenesis by the finding that EPYC1 binds Rubisco to form the pyrenoid matrix. We propose two models in which EPYC1's four repeats could produce the observed lattice arrangement of Rubisco in the Chlamydomonas pyrenoid. Our results suggest a surprisingly simple molecular mechanism for how Rubisco can be packaged to form the pyrenoid matrix, potentially explaining how Rubisco packaging into a pyrenoid could have evolved across a broad range of photosynthetic eukaryotes through convergent evolution. In addition, our findings represent a key step toward engineering a pyrenoid into crops to enhance their carbon fixation efficiency.


Assuntos
Dióxido de Carbono/metabolismo , Chlamydomonas reinhardtii/enzimologia , Organelas/enzimologia , Ribulose-Bifosfato Carboxilase/metabolismo , Chlamydomonas reinhardtii/genética , Organelas/genética , Ribulose-Bifosfato Carboxilase/genética
12.
Curr Opin Plant Biol ; 31: 181-8, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27194106

RESUMO

Many algae use a biophysical carbon concentrating mechanism for active accumulation and retention of inorganic carbon within chloroplasts, with CO2 fixation by RuBisCO within a micro-compartment, the pyrenoid. Engineering such mechanisms into higher plant chloroplasts is a possible route to augment RuBisCO operating efficiency and photosynthetic rates. Significant progress has been made recently in characterising key algal transporters and identifying factors responsible for the aggregation of RuBisCO into the pyrenoid. Several transporters have now also been successfully incorporated into higher plant chloroplasts. Consistent with the predictions from modelling, regulation of higher plant plastidic carbonic anhydrases and some form of RuBisCO aggregation will be needed before the mechanism delivers potential benefits. Key research priorities include a better understanding of the regulation of the algal carbon concentrating mechanism, advancing the fundamental characterisation of known components, evaluating whether higher plant chloroplasts can accommodate a pyrenoid, and, ultimately, testing transgenic lines under realistic growth conditions.


Assuntos
Dióxido de Carbono/metabolismo , Plantas/metabolismo , Cloroplastos/metabolismo , Fotossíntese/fisiologia , Ribulose-Bifosfato Carboxilase/metabolismo
13.
Plant Biotechnol J ; 14(5): 1302-15, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26538195

RESUMO

Many eukaryotic green algae possess biophysical carbon-concentrating mechanisms (CCMs) that enhance photosynthetic efficiency and thus permit high growth rates at low CO2 concentrations. They are thus an attractive option for improving productivity in higher plants. In this study, the intracellular locations of ten CCM components in the unicellular green alga Chlamydomonas reinhardtii were confirmed. When expressed in tobacco, all of these components except chloroplastic carbonic anhydrases CAH3 and CAH6 had the same intracellular locations as in Chlamydomonas. CAH6 could be directed to the chloroplast by fusion to an Arabidopsis chloroplast transit peptide. Similarly, the putative inorganic carbon (Ci) transporter LCI1 was directed to the chloroplast from its native location on the plasma membrane. CCP1 and CCP2 proteins, putative Ci transporters previously reported to be in the chloroplast envelope, localized to mitochondria in both Chlamydomonas and tobacco, suggesting that the algal CCM model requires expansion to include a role for mitochondria. For the Ci transporters LCIA and HLA3, membrane location and Ci transport capacity were confirmed by heterologous expression and H(14) CO3 (-) uptake assays in Xenopus oocytes. Both were expressed in Arabidopsis resulting in growth comparable with that of wild-type plants. We conclude that CCM components from Chlamydomonas can be expressed both transiently (in tobacco) and stably (in Arabidopsis) and retargeted to appropriate locations in higher plant cells. As expression of individual Ci transporters did not enhance Arabidopsis growth, stacking of further CCM components will probably be required to achieve a significant increase in photosynthetic efficiency in this species.


Assuntos
Proteínas de Algas/metabolismo , Carbono/metabolismo , Chlamydomonas reinhardtii/genética , Proteínas de Algas/genética , Arabidopsis/citologia , Arabidopsis/genética , Arabidopsis/metabolismo , Dióxido de Carbono/metabolismo , Anidrases Carbônicas , Chlamydomonas reinhardtii/citologia , Chlamydomonas reinhardtii/metabolismo , Cloroplastos/metabolismo , Regulação da Expressão Gênica de Plantas , Genes Reporter , Mutação , Fotossíntese , Plantas Geneticamente Modificadas , Proteínas Recombinantes de Fusão , Nicotiana/citologia , Nicotiana/genética , Nicotiana/metabolismo , Transgenes
14.
Plant Physiol ; 166(2): 1073-82, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25106822

RESUMO

In the model green alga Chlamydomonas reinhardtii, a carbon-concentrating mechanism (CCM) is induced under low CO2 in the light and comprises active inorganic carbon transport components, carbonic anhydrases, and aggregation of Rubisco in the chloroplast pyrenoid. Previous studies have focused predominantly on asynchronous cultures of cells grown under low versus high CO2. Here, we have investigated the dynamics of CCM activation in synchronized cells grown in dark/light cycles compared with induction under low CO2. The specific focus was to undertake detailed time course experiments comparing physiology and gene expression during the dark-to-light transition. First, the CCM could be fully induced 1 h before dawn, as measured by the photosynthetic affinity for inorganic carbon. This occurred in advance of maximum gene transcription and protein accumulation and contrasted with the coordinated induction observed under low CO2. Between 2 and 1 h before dawn, the proportion of Rubisco and the thylakoid lumen carbonic anhydrase in the pyrenoid rose substantially, coincident with increased CCM activity. Thus, other mechanisms are likely to activate the CCM before dawn, independent of gene transcription of known CCM components. Furthermore, this study highlights the value of using synchronized cells during the dark-to-light transition as an alternative means of investigating CCM induction.


Assuntos
Carbono/metabolismo , Chlamydomonas reinhardtii/fisiologia , Escuridão , Luz , Proteínas de Plantas/metabolismo , Chlamydomonas reinhardtii/genética , Chlamydomonas reinhardtii/metabolismo , Expressão Gênica , Genes de Plantas , Proteínas de Plantas/biossíntese
15.
Proc Natl Acad Sci U S A ; 109(47): 19474-9, 2012 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-23112177

RESUMO

The pyrenoid is a subcellular microcompartment in which algae sequester the primary carboxylase, ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). The pyrenoid is associated with a CO(2)-concentrating mechanism (CCM), which improves the operating efficiency of carbon assimilation and overcomes diffusive limitations in aquatic photosynthesis. Using the model alga Chlamydomonas reinhardtii, we show that pyrenoid formation, Rubisco aggregation, and CCM activity relate to discrete regions of the Rubisco small subunit (SSU). Specifically, pyrenoid occurrence was shown to be conditioned by the amino acid composition of two surface-exposed α-helices of the SSU: higher plant-like helices knock out the pyrenoid, whereas native algal helices establish a pyrenoid. We have also established that pyrenoid integrity was essential for the operation of an active CCM. With the algal CCM being functionally analogous to the terrestrial C(4) pathway in higher plants, such insights may offer a route toward transforming algal and higher plant productivity for the future.


Assuntos
Chlamydomonas/enzimologia , Organelas/metabolismo , Ribulose-Bifosfato Carboxilase/química , Ribulose-Bifosfato Carboxilase/metabolismo , Sequência de Aminoácidos , Processos Autotróficos/efeitos dos fármacos , Carbono/metabolismo , Dióxido de Carbono/farmacologia , Chlamydomonas/efeitos dos fármacos , Chlamydomonas/crescimento & desenvolvimento , Chlamydomonas/ultraestrutura , Deleção de Genes , Cinética , Dados de Sequência Molecular , Organelas/ultraestrutura , Oxigênio/metabolismo , Fenótipo , Fotossíntese/efeitos dos fármacos , Estrutura Secundária de Proteína , Spinacia oleracea/efeitos dos fármacos , Spinacia oleracea/enzimologia , Relação Estrutura-Atividade
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