scholarly journals Condensation of Rubisco into a proto-pyrenoid in higher plant chloroplasts

2020 ◽  
Author(s):  
Nicky Atkinson ◽  
Yuwei Mao ◽  
Kher Xing Chan ◽  
Alistair J. McCormick
Keyword(s):  
Higher Plants ◽  
Initial Step ◽  
Small Subunit ◽  
Operating Efficiency ◽  
Higher Plant ◽  
Bisphosphate Carboxylase ◽  
Spontaneous Condensation ◽  
Concentrating Mechanism ◽  
Chloroplast Stroma ◽  
Almost All

SummaryPhotosynthetic CO2 fixation in plants is limited by the inefficiency of the CO2-assimilating enzyme Rubisco (D-ribulose-1,5-bisphosphate carboxylase/ oxygenase)1–3. In plants possessing the C3 pathway, which includes most major staple crops, Rubisco is typically evenly distributed throughout the chloroplast stroma. However, in almost all eukaryotic algae Rubisco aggregates within a microcompartment known as the pyrenoid, in association with a CO2-concentrating mechanism that improves photosynthetic operating efficiency under conditions of low inorganic carbon4. Recent work has shown that the pyrenoid matrix is a phase-separated, liquid-like condensate5. In the alga Chlamydomonas reinhardtii, condensation is mediated by two components: Rubisco and the linker protein EPYC1 (Essential Pyrenoid Component 1)6,7. Here we show that expression of mature EPYC1 and a plant-algal hybrid Rubisco leads to spontaneous condensation of Rubisco into a single phase-separated compartment in Arabidopsis chloroplasts, with liquid-like properties similar to a pyrenoid matrix. The condensate displaces the thylakoid membranes and is enriched in hybrid Rubisco containing the algal Rubisco small subunit required for phase separation. Promisingly, photosynthetic CO2 fixation and growth is not impaired in stable transformants compared to azygous segregants. These observations represent a significant initial step towards enhancing photosynthesis in higher plants by introducing an algal CO2-concentrating mechanism, which is predicted to significantly increase the efficiency of photosynthetic CO2 uptake8,9.

scholarly journals Condensation of Rubisco into a proto-pyrenoid in higher plant chloroplasts

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Nicky Atkinson ◽  
Yuwei Mao ◽  
Kher Xing Chan ◽  
Alistair J. McCormick
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Recent Work ◽  
Single Phase ◽  
Inorganic Carbon ◽  
Higher Plants ◽  
Initial Step ◽  
Operating Efficiency ◽  
Higher Plant ◽  
Spontaneous Condensation ◽  
Concentrating Mechanism

AbstractPhotosynthetic CO2 fixation in plants is limited by the inefficiency of the CO2-assimilating enzyme Rubisco. In most eukaryotic algae, Rubisco aggregates within a microcompartment known as the pyrenoid, in association with a CO2-concentrating mechanism that improves photosynthetic operating efficiency under conditions of low inorganic carbon. Recent work has shown that the pyrenoid matrix is a phase-separated, liquid-like condensate. In the alga Chlamydomonas reinhardtii, condensation is mediated by two components: Rubisco and the linker protein EPYC1 (Essential Pyrenoid Component 1). Here, we show that expression of mature EPYC1 and a plant-algal hybrid Rubisco leads to spontaneous condensation of Rubisco into a single phase-separated compartment in Arabidopsis chloroplasts, with liquid-like properties similar to a pyrenoid matrix. This work represents a significant initial step towards enhancing photosynthesis in higher plants by introducing an algal CO2-concentrating mechanism, which is predicted to significantly increase the efficiency of photosynthetic CO2 uptake.

scholarly journals Substrate- and species-specific processing enzymes for chloroplast precursor proteins

1994 ◽  
Vol 300 (3) ◽  
pp. 787-792 ◽  
Author(s):  
Q Su ◽  
A Boschetti
Keyword(s):  
Higher Plants ◽  
Gel Filtration ◽  
Molecular Size ◽  
Small Subunit ◽  
Bisphosphate Carboxylase ◽  
Chlamydomonas Reinhardii ◽  
Processing Enzymes ◽  
Precursor Proteins ◽  
Species Specific ◽  
Correct Size

Using different precursors of chloroplast proteins and stromal extracts from both Chlamydomonas reinhardii and pea chloroplasts, we analysed the specificity of stroma-localized processing peptidases. By gel filtration of a stromal extract from isolated Chlamydomonas chloroplasts, fractions could be separated containing enzymic activities for processing the precursors of the small subunit of ribulose-1,5-bisphosphate carboxylase (pSS) and of the protein OEE1 from the photosynthetic water-splitting complex (pOEE1). The enzymes differed not only in molecular size, but also in their sensitivity to inhibitors and in their pH optima. Obviously, in the stroma of Chlamydomonas chloroplasts different peptidases exist for processing of pSS and pOEE1, the latter being converted into an intermediate-sized form, iOEE1, which was found to be further processed to mature OEE1 by a thylakoid-associated protease. To study the species-specificity of the stromal peptidases, stromal extracts from Chlamydomonas and pea chloroplasts were incubated with pSS from either of these organisms. In the heterologous combinations, the precursors were partly hydrolysed, but not to the correct size. In importation assays, pSS from pea (but also the precursor of the ribosomal protein L12 from spinach) could not enter into chloroplasts from Chlamydomonas. In contrast, the algal pSS was imported into chloroplasts from pea, although it was not processed to mature SS. Our results indicate that the importation machinery and the pSS-processing enzymes in higher plants and green algae have different specificities and that in Chlamydomonas several stromal peptidases for different precursor proteins exist.

Photoregulation of the biosynthesis of ribulose bisphosphate carboxylase

Development ◽  
1984 ◽  
Vol 83 (Supplement) ◽  
pp. 163-178
Author(s):  
R. John Ellis ◽  
Thomas F. Gallagher ◽  
Gareth I. Jenkins ◽  
C. Ruth Lennox
Keyword(s):  
Chloroplast Development ◽  
Higher Plants ◽  
Large Subunit ◽  
Nuclear Genome ◽  
Small Subunit ◽  
Ribulose Bisphosphate Carboxylase ◽  
Ribulose Bisphosphate ◽  
Bisphosphate Carboxylase ◽  
Subunit Mrna ◽  
Parallel Increase

Chloroplast development in higher plants is light dependent, and is accompanied by the synthesis of chlorophyll and the accumulation of many chloroplast polypeptides. There is a 100-fold greater content of the photosynthetic enzyme, ribulose-1,5-bisphosphate carboxylase-oxygenase, in light-grown seedlings of Pisum sativum than in dark-grown seedlings. Following the illumination of dark-grown seedlings, there is a parallel increase in the content of both the mRNA and the polypeptide of the small subunit of the carboxylase; this subunit is a product of the nuclear genome. The increases in the mRNA and the polypeptide of the large subunit, which is a product of the chloroplast genome, show less synchronicity. Studies with isolated leaf nuclei show that the increase in small subunit mRNA is mediated primarily at the level of transcription. Three distinct effects of light on transcription of small subunit genes have been found; a rapid (∼1 h) burst, followed by a decline, when etiolated plants are first exposed to light; a slow (∼36h) development of the competence to transcribe rapidly after the initial burst; rapid (∼20 min) switches in both directions when fully greened plants are exposed to light—dark transitions.

scholarly journals Degradation of the soybean ribulose-1,5-bisphosphate carboxylase small-subunit mRNA, SRS4, initiates with endonucleolytic cleavage.

1995 ◽  
Vol 15 (12) ◽  
pp. 6641-6652 ◽  
Author(s):  
M M Tanzer ◽  
R B Meagher
Keyword(s):  
Consensus Sequence ◽  
Random Order ◽  
Initial Step ◽  
Preliminary Evidence ◽  
Small Subunit ◽  
S1 Nuclease ◽  
Bisphosphate Carboxylase ◽  
Endonucleolytic Cleavage

The degradation of the soybean SRS4 mRNA, which encodes the small subunit of ribulose-1,5-bisphosphate carboxylase, yields a set of proximal (5' intact) and distal (3' intact) products both in vivo and in vitro. These products are generated by endonucleolytic cleavages that occur essentially in a random order, although some products are produced more rapidly than others. Comparison of sizes of products on Northern (RNA) blots showed that the combined sizes of pairs of proximal and distal products form contiguous full-length SRS4 mRNAs. When the 3' ends of the proximal products and the 5' ends of the distal products were mapped by S1 nuclease and primer extension assays, respectively, both sets of ends mapped to the same sequences within the SRS4 mRNA. A small in vitro-synthesized RNA fragment containing one cleavage site inhibited cleavage of all major sites, equivalently consistent with one enzymatic activity generating the endonucleolytic cleavage products. These products were rich in GU nucleotides, but no obvious consensus sequence was found among several cleavage sites. Preliminary evidence suggested that secondary structure could play a role in site selection. The structures of the 5' ends of the proximal products and the 3' ends of the distal products were examined. Proximal products were found with approximately equal frequency in both m7G cap(+) and m7G cap(-) fractions, suggesting that the endonucleolytic cleavage events occurred independently of the removal of the 5' cap structure. Distal products were distributed among fractions with poly(A) tails ranging from undetectable to greater than 100 nucleotides in length, suggesting that the endonucleolytic cleavage events occurred independently of poly(A) tail shortening. Together, these data support a stochastic endonuclease model in which an endonucleolytic cleavage event is the initial step in SRS4 mRNA degradation.

scholarly journals Structure of Rubisco fromArabidopsis thalianain complex with 2-carboxyarabinitol-1,5-bisphosphate

2018 ◽  
Vol 74 (1) ◽  
pp. 1-9 ◽  
Author(s):  
Karin Valegård ◽  
Dirk Hasse ◽  
Inger Andersson ◽  
Laura H. Gunn
Keyword(s):  
Crystal Structure ◽  
Model Organism ◽  
Carbon Assimilation ◽  
Small Subunit ◽  
Higher Plant ◽  
Bisphosphate Carboxylase ◽  
Structural Framework ◽  
Transition State Analogue ◽  
Subunit Isoforms ◽  
Photosynthetic Carbon Assimilation

The crystal structure of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) fromArabidopsis thalianais reported at 1.5 Å resolution. In light of the importance ofA. thalianaas a model organism for understanding higher plant biology, and the pivotal role of Rubisco in photosynthetic carbon assimilation, there has been a notable absence of anA. thalianaRubisco crystal structure.A. thalianaRubisco is an L8S8hexadecamer comprising eight plastome-encoded catalytic large (L) subunits and eight nuclear-encoded small (S) subunits.A. thalianaproduces four distinct small-subunit isoforms (RbcS1A, RbcS1B, RbcS2B and RbcS3B), and this crystal structure provides a snapshot ofA. thalianaRubisco containing the low-abundance RbcS3B small-subunit isoform. Crystals were obtained in the presence of the transition-state analogue 2-carboxy-D-arabinitol-1,5-bisphosphate.A. thalianaRubisco shares the overall fold characteristic of higher plant Rubiscos, but exhibits an interesting disparity between sequence and structural relatedness to other Rubisco isoforms. These results provide the structural framework to understandA. thalianaRubisco and the potential catalytic differences that could be conferred by alternativeA. thalianaRubisco small-subunit isoforms.

scholarly journals Electron flow to oxygen in higher plants and algae: rates and control of direct photoreduction (Mehler reaction) and rubisco oxygenase

2000 ◽  
Vol 355 (1402) ◽  
pp. 1433-1446 ◽  
Author(s):  
Murray R. Badger ◽  
Susanne von Caemmerer ◽  
Sari Ruuska ◽  
Hiromi Nakano
Keyword(s):  
Electron Transport ◽  
Energy Dissipation ◽  
Higher Plants ◽  
Cam Plants ◽  
Mehler Reaction ◽  
Bisphosphate Carboxylase ◽  
Ps I ◽  
Concentrating Mechanism ◽  
Photosynthetic Carbon ◽  
Strong Control

Linear electron transport in chloroplasts produces a number of reduced components associated with photosystem I (PS I) that may subsequently participate in reactions that reduce O 2 . The two primary reactions that have been extensively studied are: first, the direct reduction of O 2 to superoxide by reduced donors associated with PS I (the Mehler reaction), and second, the rubisco oxygenase (ribulose 1,5-bisphosphate carboxylase oxygenase EC 4.1.1.39) reaction and associated peroxisomal and mitochondrial reactions of the photorespiratory pathway. This paper reviews a number of recent and past studies with higher plants, algae and cyanobacteria that have attempted to quantify O 2 fluxes under various conditions and their contributions to a number of roles, including photon energy dissipation. In C 3 and Crassulacean acid metabolism (CAM) plants, a Mehler O 2 uptake reaction is unlikely to support a significant flow of electron transport (probably less than 10%). In addition, if it were present it would appear to scale with photosynthetic carbon oxidation cycle (PCO) and photosynthetic carbon reduction cycle (PCR) activity. This is supported by studies with antisense tobacco plants with reduced rubisco at low and high temperatures and high light, as well as studies with potatoes, grapes and madrone during water stress. The lack of significant Mehler in these plants directly argues for a strong control of Mehler reaction in the absence of ATP consumption by the PCR and PCO cycles. The difference between C 3 and C 4 plants is primarily that the level of light-dependent O 2 uptake is generally much lower in C 4 plants and is relatively insensitive to the external CO 2 concentration. Such a major difference is readily attributed to the operation of the C 4 CO 2 concentrating mechanism. Algae show a range of lightdependent O 2 uptake rates, similar to C 4 plants. As in C 4 plants, the O 2 uptake appears to be largely insensitive to CO 2 , even in species that lack a CO 2 concentrating mechanism and under conditions that are clearly limiting with respect to inorganic carbon supply. A part explanation for this could be that many algal rubsicos have considerably different oxygenase kinetic properties and exhibit far less oxygenase activity in air. This would lead to the conclusion that perhaps a greater proportion of the observed O 2 uptake may be due to a Mehler reaction and less to rubisco, compared with C 3 plants. In contrast to algae and higher plants, cyanobacteria appear to have a high capacity for Mehler O 2 uptake, which appears to be not well coupled or limited by ATP consumption. It is likely that in all higher plants and algae, which have a well-developed non-photochemical quenching mechanism, non-radiative energy dissipation is the major mechanism for dissipating excess photons absorbed by the light-harvesting complexes under stressful conditions. However, for cyanobacteria, with a lack of significant nonphotochemical quenching, the situation may well be different.

scholarly journals Rubisco activase requires residues in the large subunit N terminus to remodel inhibited plant Rubisco

2020 ◽  
Vol 295 (48) ◽  
pp. 16427-16435
Author(s):  
Jediael Ng ◽  
Zhijun Guo ◽  
Oliver Mueller-Cajar
Keyword(s):  
Higher Plants ◽  
Large Subunit ◽  
Rubisco Activase ◽  
Higher Plant ◽  
Bisphosphate Carboxylase ◽  
Interaction Sites ◽  
Dead End ◽  
Highly Sensitive ◽  
N Terminus ◽  
Sugar Phosphates

The photosynthetic CO2 fixing enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) forms dead-end inhibited complexes while binding multiple sugar phosphates, including its substrate ribulose 1,5-bisphosphate. Rubisco can be rescued from this inhibited form by molecular chaperones belonging to the ATPases associated with diverse cellular activities (AAA+ proteins) termed Rubisco activases (Rcas). The mechanism of green-type Rca found in higher plants has proved elusive, in part because until recently higher-plant Rubiscos could not be expressed recombinantly. Identifying the interaction sites between Rubisco and Rca is critical to formulate mechanistic hypotheses. Toward that end here we purify and characterize a suite of 33 Arabidopsis Rubisco mutants for their ability to be activated by Rca. Mutation of 17 surface-exposed large subunit residues did not yield variants that were perturbed in their interaction with Rca. In contrast, we find that Rca activity is highly sensitive to truncations and mutations in the conserved N terminus of the Rubisco large subunit. Large subunits lacking residues 1–4 are functional Rubiscos but cannot be activated. Both T5A and T7A substitutions result in functional carboxylases that are poorly activated by Rca, indicating the side chains of these residues form a critical interaction with the chaperone. Many other AAA+ proteins function by threading macromolecules through a central pore of a disc-shaped hexamer. Our results are consistent with a model in which Rca transiently threads the Rubisco large subunit N terminus through the axial pore of the AAA+ hexamer.

scholarly journals A Nuclear-coded Chloroplastic Inner Envelope Membrane Protein Uses a Soluble Sorting Intermediate upon Import into the Organelle

1997 ◽  
Vol 137 (6) ◽  
pp. 1279-1286 ◽  
Author(s):  
Jens Lübeck ◽  
Lisa Heins ◽  
Jürgen Soll
Keyword(s):  
Protein Import ◽  
Proximal Part ◽  
Small Subunit ◽  
Intermembrane Space ◽  
Envelope Membrane ◽  
Bisphosphate Carboxylase ◽  
Hybrid Proteins ◽  
Chloroplast Stroma ◽  
Inner Envelope

The chloroplastic inner envelope protein of 110 kD (IEP110) is part of the protein import machinery in the pea. Different hybrid proteins were constructed to assess the import and sorting pathway of IEP110. The IEP110 precursor (pIEP110) uses the general import pathway into chloroplasts, as shown by the mutual exchange of presequences with the precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase (pSSU). Sorting information to the chloroplastic inner envelope is contained in an NH2-proximal part of mature IEP110 (110N). The NH2-terminus serves to anchor the protein into the membrane. Large COOH-terminal portions of this protein (80–90 kD) are exposed to the intermembrane space in situ. Successful sorting and integration of IEP110 and the derived constructs into the inner envelope are demonstrated by the inaccessability of processed mature protein to the protease thermolysin but accessibility to trypsin, i.e., the imported protein is exposed to the intermembrane space. A hybrid protein consisting of the transit sequence of SSU, the NH2-proximal part of mature IEP110, and mature SSU (tpSSU-110N-mSSU) is completely imported into the chloroplast stroma, from which it can be recovered as soluble, terminally processed 110NmSSU. The soluble 110N-mSSU then enters a reexport pathway, which results not only in the insertion of 110N-mSSU into the inner envelope membrane, but also in the extrusion of large portions of the protein into the intermembrane space. We conclude that chloroplasts possess a protein reexport machinery for IEPs in which soluble stromal components interact with a membrane-localized translocation machinery.

Changes in protein and gene expression during induction of the CO2-concentrating mechanism in wild-type and mutant Chlamydomonas

1991 ◽  
Vol 69 (5) ◽  
pp. 1008-1016 ◽  
Author(s):  
Martin H. Spalding ◽  
Thomas L. Winder ◽  
James C. Anderson ◽  
Anne M. Geraghty ◽  
Laura F. Marek
Keyword(s):  
Transcript Abundance ◽  
Small Subunit ◽  
Wild Type ◽  
Bisphosphate Carboxylase ◽  
Phosphoglycolate Phosphatase ◽  
Concentrating Mechanism ◽  
Expression Of Genes ◽  
Genes Encoding ◽  
In The Wild ◽  
Inorganic Carbon Transport

Several changes occur in wild-type Chlamydomonas reinhardtii upon exposure to limiting CO2, including induction of several polypeptides. Polypeptide induction was previously shown to correlate with appearance of the active CO2-concentrating mechanism (CCM) of this alga. In this paper induction of polypeptides by limiting CO2 was investigated in mutants with lesions in the CCM. Mutants with lesions in the ca-1 and pmp-1 loci exhibited alterations in polypeptide induction, but it was concluded that the alterations probably do not represent their primary genetic lesions. Other changes that occur in this alga in response to limiting CO2 were also investigated. Based on a lack of significant change in the transcript abundance of ribulose-1,5-bisphosphate carboxylase/oxygenase large and small subunit genes in the wild type, it was concluded that the previously reported transient decline in synthesis of both subunits is controlled at the translational level. A transient increase in the activity of the photorespiratory enzyme phosphoglycolate phosphatase was observed in the wild type but not in a mutant, cia-5, that lacks induction of the CCM. In addition, changes in expression of genes encoding periplasmic carbonic anhydrase, a 36-kDa membrane-associated protein and a chlorophyll-binding protein occurred in the wild type but not in cia-5 in response to limiting CO2. The absence of these changes in cia-5 was attributed to a lack of either the signal itself or transduction of the signal responsible for adaptation to limiting CO2, which led to speculation that a larger range of responses are regulated by the same signal than was previously recognized. Key words: photosynthesis, photorespiration, algae, inorganic carbon transport, transcription, translation.

scholarly journals Catalytic by-product formation and ligand binding by ribulose bisphosphate carboxylases from different phylogenies

2006 ◽  
Vol 399 (3) ◽  
pp. 525-534 ◽  
Author(s):  
F. Grant Pearce
Keyword(s):  
Rhodospirillum Rubrum ◽  
Higher Plants ◽  
Product Formation ◽  
Enzyme Dynamics ◽  
Ribulose Bisphosphate ◽  
Higher Plant ◽  
Bisphosphate Carboxylase ◽  
Green Algal ◽  
Activated State ◽  
By Products

During catalysis, all Rubisco (D-ribulose-1,5-bisphosphate carboxylase/oxygenase) enzymes produce traces of several by-products. Some of these by-products are released slowly from the active site of Rubisco from higher plants, thus progressively inhibiting turnover. Prompted by observations that Form I Rubisco enzymes from cyanobacteria and red algae, and the Form II Rubisco enzyme from bacteria, do not show inhibition over time, the production and binding of catalytic by-products was measured to ascertain the underlying differences. In the present study we show that the Form IB Rubisco from the cyanobacterium Synechococcus PCC6301, the Form ID enzyme from the red alga Galdieria sulfuraria and the low-specificity Form II type from the bacterium Rhodospirillum rubrum all catalyse formation of by-products to varying degrees; however, the by-products are not inhibitory under substrate-saturated conditions. Study of the binding and release of phosphorylated analogues of the substrate or reaction intermediates revealed diverse strategies for avoiding inhibition. Rubisco from Synechococcus and R. rubrum have an increased rate of inhibitor release. G. sulfuraria Rubisco releases inhibitors very slowly, but has an increased binding constant and maintains the enzyme in an activated state. These strategies may provide information about enzyme dynamics, and the degree of enzyme flexibility. Our observations also illustrate the phylogenetic diversity of mechanisms for regulating Rubisco and raise questions about whether an activase-like mechanism should be expected outside the green-algal/higher-plant lineage.

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