Cardiopulmonary and metabolic responses during a 2-day CPET in myalgic encephalomyelitis/chronic fatigue syndrome: translating reduced oxygen consumption to impairment status to treatment considerations.

BACKGROUND: Post-exertional malaise (PEM), the hallmark symptom of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), represents a constellation of abnormal responses to physical, cognitive, and/or emotional exertion including profound fatigue, cognitive dysfunction, and exertion intolerance, among numerous other maladies. Two sequential cardiopulmonary exercise tests (2-d CPET) provide objective evidence of abnormal responses to exertion in ME/CFS but validated only in studies with small sample sizes. Further, translation of results to impairment status and approaches to symptom reduction are lacking. METHODS: Participants with ME/CFS (Canadian Criteria; n = 84) and sedentary controls (CTL; n = 71) completed two CPETs on a cycle ergometer separated by 24 h. Two-way repeated measures ANOVA compared CPET measures at rest, ventilatory/anaerobic threshold (VAT), and peak effort between phenotypes and CPETs. Intraclass correlations described stability of CPET measures across tests, and relevant objective CPET data indicated impairment status. A subset of case-control pairs (n = 55) matched for aerobic capacity, age, and sex, were also analyzed. RESULTS: Unlike CTL, ME/CFS failed to reproduce CPET-1 measures during CPET-2 with significant declines at peak exertion in work, exercise time, V ˙ e, V ˙ O2, V ˙ CO2, V ˙ T, HR, O2pulse, DBP, and RPP. Likewise, CPET-2 declines were observed at VAT for V ˙ e/ V ˙ CO2, PetCO2, O2pulse, work, V ˙ O2 and SBP. Perception of effort (RPE) exceeded maximum effort criteria for ME/CFS and CTL on both CPETs. Results were similar in matched pairs. Intraclass correlations revealed greater stability in CPET variables across test days in CTL compared to ME/CFS owing to CPET-2 declines in ME/CFS. Lastly, CPET-2 data signaled more severe impairment status for ME/CFS compared to CPET-1. CONCLUSIONS: Presently, this is the largest 2-d CPET study of ME/CFS to substantiate impaired recovery in ME/CFS following an exertional stressor. Abnormal post-exertional CPET responses persisted compared to CTL matched for aerobic capacity, indicating that fitness level does not predispose to exertion intolerance in ME/CFS. Moreover, contributions to exertion intolerance in ME/CFS by disrupted cardiac, pulmonary, and metabolic factors implicates autonomic nervous system dysregulation of blood flow and oxygen delivery for energy metabolism. The observable declines in post-exertional energy metabolism translate notably to a worsening of impairment status. Treatment considerations to address tangible reductions in physiological function are proffered. TRIAL REGISTRATION NUMBER: ClinicalTrials.gov, retrospectively registered, ID# NCT04026425, date of registration: 2019-07-17.

Absence of carbonic anhydrase in chloroplasts affects C3 plant development but not photosynthesis.

The enzyme carbonic anhydrase (CA), which catalyzes the interconversion of bicarbonate with carbon dioxide (CO2) and water, has been hypothesized to play a role in C3 photosynthesis. We identified two tobacco stromal CAs, ß-CA1 and ß-CA5, and produced CRISPR/Cas9 mutants affecting their encoding genes. While single knockout lines Δß-ca1 and Δß-ca5 had no striking phenotypic differences compared to wild type (WT) plants, Δß-ca1ca5 leaves developed abnormally and exhibited large necrotic lesions even when supplied with sucrose. Leaf development of Δß-ca1ca5 plants normalized at 9,000 ppm CO2 Leaves of Δß-ca1ca5 mutants and WT that had matured in high CO2 had identical CO2 fixation rates and photosystem II efficiency. Fatty acids, which are formed through reactions with bicarbonate substrates, exhibited abnormal profiles in the chloroplast CA-less mutant. Emerging Δß-ca1ca5 leaves produce reactive oxygen species in chloroplasts, perhaps due to lower nonphotochemical quenching efficiency compared to WT. Δß-ca1ca5 seedling germination and development is negatively affected at ambient CO2 Transgenes expressing full-length ß-CA1 and ß-CA5 proteins complemented the Δß-ca1ca5 mutation but inactivated (ΔZn-ßCA1) and cytoplasm-localized (Δ62-ßCA1) forms of ß-CA1 did not reverse the growth phenotype. Nevertheless, expression of the inactivated ΔZn-ßCA1 protein was able to restore the hypersensitive response to tobacco mosaic virus, while Δß-ca1 and Δß-ca1ca5 plants failed to show a hypersensitive response. We conclude that stromal CA plays a role in plant development, likely through providing bicarbonate for biosynthetic reactions, but stromal CA is not needed for maximal rates of photosynthesis in the C3 plant tobacco.

The RanBP2 zinc finger domains of chloroplast RNA editing factor OZ1 are required for protein-protein interactions and conversion of C to U.

In the chloroplast, organelle zinc finger 1 (OZ1) is a RanBP2-type zinc finger (Znf) protein required for many RNA editing events, a process by which specific cytosines are enzymatically converted to uracils as a correction mechanism for missense mutations in the organelle genomes. RNA editing is carried out by a large multi-protein complex called the 'editosome' that contains members of the pentatricopeptide repeat (PPR) protein family, the RNA editing factor interacting protein (also known as MORF) family and the organelle RNA-recognition motif (ORRM) family, in addition to OZ1. OZ1 is an 82-kDa protein with distinct domains, including a pair of Znf domains and a unique C-terminal region. To elucidate the functions of these domains, we have generated truncations of OZ1 for use in protein-protein interaction assays that identified the C-terminal region of OZ1, as well as the Znf domains as the primary interactors with PPR proteins, which are factors required for site-specificity and enzymatic editing. Expression of these OZ1 truncations in vivo showed that the Znf domains were required to restore chloroplast RNA editing in oz1 knockout plants. Mutation of key structural residues in the Znf domains showed that they are necessary for editing and required for interaction with ORRM1, a general editing factor with an RNA-binding domain. These functional characterizations of the Znfs and novel C-terminal domain contribute to our understanding of the model for the chloroplast plant editosome.

Proteomics and cytokine analyses distinguish myalgic encephalomyelitis/chronic fatigue syndrome cases from controls.

BACKGROUND: Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex, heterogenous disease characterized by unexplained persistent fatigue and other features including cognitive impairment, myalgias, post-exertional malaise, and immune system dysfunction. Cytokines are present in plasma and encapsulated in extracellular vesicles (EVs), but there have been only a few reports of EV characteristics and cargo in ME/CFS. Several small studies have previously described plasma proteins or protein pathways that are associated with ME/CFS. METHODS: We prepared extracellular vesicles (EVs) from frozen plasma samples from a cohort of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) cases and controls with prior published plasma cytokine and plasma proteomics data. The cytokine content of the plasma-derived extracellular vesicles was determined by a multiplex assay and differences between patients and controls were assessed. We then performed multi-omic statistical analyses that considered not only this new data, but extensive clinical data describing the health of the subjects. RESULTS: ME/CFS cases exhibited greater size and concentration of EVs in plasma. Assays of cytokine content in EVs revealed IL2 was significantly higher in cases. We observed numerous correlations among EV cytokines, among plasma cytokines, and among plasma proteins from mass spectrometry proteomics. Significant correlations between clinical data and protein levels suggest roles of particular proteins and pathways in the disease. For example, higher levels of the pro-inflammatory cytokines Granulocyte-Monocyte Colony-Stimulating Factor (CSF2) and Tumor Necrosis Factor (TNFα) were correlated with greater physical and fatigue symptoms in ME/CFS cases. Higher serine protease SERPINA5, which is involved in hemostasis, was correlated with higher SF-36 general health scores in ME/CFS. Machine learning classifiers were able to identify a list of 20 proteins that could discriminate between cases and controls, with XGBoost providing the best classification with 86.1% accuracy and a cross-validated AUROC value of 0.947. Random Forest distinguished cases from controls with 79.1% accuracy and an AUROC value of 0.891 using only 7 proteins. CONCLUSIONS: These findings add to the substantial number of objective differences in biomolecules that have been identified in individuals with ME/CFS. The observed correlations of proteins important in immune responses and hemostasis with clinical data further implicates a disturbance of these functions in ME/CFS.

A RanBP2-type zinc finger protein functions in intron splicing in Arabidopsis mitochondria and is involved in the biogenesis of respiratory complex I.

The RanBP2 zinc finger (Znf) domain is a prevalent domain that mediates protein interaction and RNA binding. In Arabidopsis, a clade of four RanBP2 Znf-containing proteins, named the Organelle Zinc (OZ) finger family, are known or predicted to be targeted to either the mitochondria or the plastids. Previously we reported that OZ1 is absolutely required for the editing of 14 sites in chloroplasts. We now have investigated the function of OZ2, whose null mutation is embryo lethal. We rescued the null mutant by expressing wild-type OZ2 under the control of the seed-specific ABSCISIC ACID-INSENSITIVE3 (ABI3) promoter. Rescued mutant plants exhibit severely delayed development and a distinctive morphological phenotype. Genetic and biochemical analyses demonstrated that OZ2 promotes the splicing of transcripts of several mitochondrial nad genes and rps3. The splicing defect of nad transcripts results in the destabilization of complex I, which in turn affects the respiratory ability of oz2 mutants, turning on the alternative respiratory pathway, and impacting the plant development. Protein-protein interaction assays demonstrated binding of OZ2 to several known mitochondrial splicing factors targeting the same splicing events. These findings extend the known functional repertoire of the RanBP2 zinc finger domain in nuclear splicing to include plant organelle splicing.

Urine Metabolomics Exposes Anomalous Recovery after Maximal Exertion in Female ME/CFS Patients.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating disease with unknown etiology or effective treatments. Post-exertional malaise (PEM) is a key symptom that distinguishes ME/CFS patients. Investigating changes in the urine metabolome between ME/CFS patients and healthy subjects following exertion may help us understand PEM. The aim of this pilot study was to comprehensively characterize the urine metabolomes of eight female healthy sedentary control subjects and ten female ME/CFS patients in response to a maximal cardiopulmonary exercise test (CPET). Each subject provided urine samples at baseline and 24 h post-exercise. A total of 1403 metabolites were detected via LC-MS/MS by Metabolon® including amino acids, carbohydrates, lipids, nucleotides, cofactors and vitamins, xenobiotics, and unknown compounds. Using a linear mixed effects model, pathway enrichment analysis, topology analysis, and correlations between urine and plasma metabolite levels, significant differences were discovered between controls and ME/CFS patients in many lipid (steroids, acyl carnitines and acyl glycines) and amino acid subpathways (cysteine, methionine, SAM, and taurine; leucine, isoleucine, and valine; polyamine; tryptophan; and urea cycle, arginine and proline). Our most unanticipated discovery is the lack of changes in the urine metabolome of ME/CFS patients during recovery while significant changes are induced in controls after CPET, potentially demonstrating the lack of adaptation to a severe stress in ME/CFS patients.

Altered Fatty Acid Oxidation in Lymphocyte Populations of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome.

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a disabling multisystem illness in which individuals are plagued with fatigue, inflammatory symptoms, cognitive dysfunction, and the hallmark symptom, post-exertional malaise. While the cause of this disease remains unknown, there is evidence of a potential infectious component that, along with patient symptoms and common onsets of the disease, implicates immune system dysfunction. To further our understanding of the state of ME/CFS lymphocytes, we characterized the role of fatty acids in isolated Natural Killer cells, CD4+ T cells, and CD8+ T cells in circulation and after overnight stimulation, through implicit perturbations to fatty acid oxidation. We examined samples obtained from at least 8 and as many as 20 subjects for immune cell fatty acid characterization in a variety of experiments and found that all three isolated cell types increased their utilization of lipids and levels of pertinent proteins involved in this metabolic pathway in ME/CFS samples, particularly during higher energy demands and activation. In T cells, we characterized the cell populations contributing to these metabolic shifts, which included CD4+ memory cells, CD4+ effector cells, CD8+ naïve cells, and CD8+ memory cells. We also discovered that patients with ME/CFS and healthy control samples had significant correlations between measurements of CD4+ T cell fatty acid metabolism and demographic data. These findings provide support for metabolic dysfunction in ME/CFS immune cells. We further hypothesize about the consequences that these altered fuel dependencies may have on T and NK cell effector function, which may shed light on the illness's mechanism of action.

Recovery from Exercise in Persons with Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS).

Background and Objectives: Post-exertional malaise (PEM) is the hallmark of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), but there has been little effort to quantitate the duration of PEM symptoms following a known exertional stressor. Using a Symptom Severity Scale (SSS) that includes nine common symptoms of ME/CFS, we sought to characterize the duration and severity of PEM symptoms following two cardiopulmonary exercise tests separated by 24 h (2-day CPET). Materials and Methods: Eighty persons with ME/CFS and 64 controls (CTL) underwent a 2-day CPET. ME/CFS subjects met the Canadian Clinical Criteria for diagnosis of ME/CFS; controls were healthy but not participating in regular physical activity. All subjects who met maximal effort criteria on both CPETs were included. SSS scores were obtained at baseline, immediately prior to both CPETs, the day after the second CPET, and every two days after the CPET-1 for 10 days. Results: There was a highly significant difference in judged recovery time (ME/CFS = 12.7 ± 1.2 d; CTL = 2.1 ± 0.2 d, mean ± s.e.m., Chi2 = 90.1, p < 0.0001). The range of ME/CFS patient recovery was 1-64 days, while the range in CTL was 1-10 days; one subject with ME/CFS had not recovered after one year and was not included in the analysis. Less than 10% of subjects with ME/CFS took more than three weeks to recover. There was no difference in recovery time based on the level of pre-test symptoms prior to CPET-1 (F = 1.12, p = 0.33). Mean SSS scores at baseline were significantly higher than at pre-CPET-1 (5.70 ± 0.16 vs. 4.02 ± 0.18, p < 0.0001). Pharmacokinetic models showed an extremely prolonged decay of the PEM response (Chi2 > 22, p < 0.0001) to the 2-day CPET. Conclusions: ME/CFS subjects took an average of about two weeks to recover from a 2-day CPET, whereas sedentary controls needed only two days. These data quantitate the prolonged recovery time in ME/CFS and improve the ability to obtain well-informed consent prior to doing exercise testing in persons with ME/CFS. Quantitative monitoring of PEM symptoms may provide a method to help manage PEM.

A procedure to introduce point mutations into the Rubisco large subunit gene in wild-type plants.

Photosynthetic inefficiencies limit the productivity and sustainability of crop production and the resilience of agriculture to future societal and environmental challenges. Rubisco is a key target for improvement as it plays a central role in carbon fixation during photosynthesis and is remarkably inefficient. Introduction of mutations to the chloroplast-encoded Rubisco large subunit rbcL is of particular interest for improving the catalytic activity and efficiency of the enzyme. However, manipulation of rbcL is hampered by its location in the plastome, with many species recalcitrant to plastome transformation, and by the plastid's efficient repair system, which can prevent effective maintenance of mutations introduced with homologous recombination. Here we present a system where the introduction of a number of silent mutations into rbcL within the model plant Nicotiana tabacum facilitates simplified screening via additional restriction enzyme sites. This system was used to successfully generate a range of transplastomic lines from wild-type N. tabacum with stable point mutations within rbcL in 40% of the transformants, allowing assessment of the effect of these mutations on Rubisco assembly and activity. With further optimization the approach offers a viable way forward for mutagenic testing of Rubisco function in planta within tobacco and modification of rbcL in other crops where chloroplast transformation is feasible. The transformation strategy could also be applied to introduce point mutations in other chloroplast-encoded genes.

Hybrid Cyanobacterial-Tobacco Rubisco Supports Autotrophic Growth and Procarboxysomal Aggregation.

Much of the research aimed at improving photosynthesis and crop productivity attempts to overcome shortcomings of the primary CO2-fixing enzyme Rubisco. Cyanobacteria utilize a CO2-concentrating mechanism (CCM), which encapsulates Rubisco with poor specificity but a relatively fast catalytic rate within a carboxysome microcompartment. Alongside the active transport of bicarbonate into the cell and localization of carbonic anhydrase within the carboxysome shell with Rubisco, cyanobacteria are able to overcome the limitations of Rubisco via localization within a high-CO2 environment. As part of ongoing efforts to engineer a ß-cyanobacterial CCM into land plants, we investigated the potential for Rubisco large subunits (LSU) from the ß-cyanobacterium Synechococcus elongatus (Se) to form aggregated Rubisco complexes with the carboxysome linker protein CcmM35 within tobacco (Nicotiana tabacum) chloroplasts. Transplastomic plants were produced that lacked cognate Se Rubisco small subunits (SSU) and expressed the Se LSU in place of tobacco LSU, with and without CcmM35. Plants were able to form a hybrid enzyme utilizing tobacco SSU and the Se LSU, allowing slow autotrophic growth in high CO2 CcmM35 was able to form large Rubisco aggregates with the Se LSU, and these incorporated small amounts of native tobacco SSU. Plants lacking the Se SSU showed delayed growth, poor photosynthetic capacity, and significantly reduced Rubisco activity compared with both wild-type tobacco and lines expressing the Se SSU. These results demonstrate the ability of the Se LSU and CcmM35 to form large aggregates without the cognate Se SSU in planta, harboring active Rubisco that enables plant growth, albeit at a much slower pace than plants expressing the cognate Se SSU.

Cytokine profiling of extracellular vesicles isolated from plasma in myalgic encephalomyelitis/chronic fatigue syndrome: a pilot study.

BACKGROUND: Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a debilitating disease of unknown etiology lasting for a minimum of 6 months but usually for many years, with features including fatigue, cognitive impairment, myalgias, post-exertional malaise, and immune system dysfunction. Dysregulation of cytokine signaling could give rise to many of these symptoms. Cytokines are present in both plasma and extracellular vesicles, but little investigation of EVs in ME/CFS has been reported. Therefore, we aimed to characterize the content of extracellular vesicles (EVs) isolated from plasma (including circulating cytokine/chemokine profiling) from individuals with ME/CFS and healthy controls. METHODS: We included 35 ME/CFS patients and 35 controls matched for age, sex and BMI. EVs were enriched from plasma by using a polymer-based precipitation method and characterized by Nanoparticle Tracking Analysis (NTA), Transmission Electron Microscopy (TEM) and immunoblotting. A 45-plex immunoassay was used to determine cytokine levels in both plasma and isolated EVs from a subset of 19 patients and controls. Linear regression, principal component analysis and inter-cytokine correlations were analyzed. RESULTS: ME/CFS individuals had significantly higher levels of EVs that ranged from 30 to 130 nm in size as compared to controls, but the mean size for total extracellular vesicles did not differ between groups. The enrichment of typical EV markers CD63, CD81, TSG101 and HSP70 was confirmed by Western blot analysis and the morphology assessed by TEM showed a homogeneous population of vesicles in both groups. Comparison of cytokine concentrations in plasma and isolated EVs of cases and controls yielded no significant differences. Cytokine-cytokine correlations in plasma revealed a significant higher number of interactions in ME/CFS cases along with 13 inverse correlations that were mainly driven by the Interferon gamma-induced protein 10 (IP-10), whereas in the plasma of controls, no inverse relationships were found across any of the cytokines. Network analysis in EVs from controls showed 2.5 times more significant inter-cytokine interactions than in the ME/CFS group, and both groups presented a unique negative association. CONCLUSIONS: Elevated levels of 30-130 nm EVs were found in plasma from ME/CFS patients and inter-cytokine correlations revealed unusual regulatory relationships among cytokines in the ME/CFS group that were different from the control group in both plasma and EVs. These disturbances in cytokine networks are further evidence of immune dysregulation in ME/CFS.

A faster Rubisco with potential to increase photosynthesis in crops.

In photosynthetic organisms, D-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the major enzyme assimilating atmospheric CO2 into the biosphere. Owing to the wasteful oxygenase activity and slow turnover of Rubisco, the enzyme is among the most important targets for improving the photosynthetic efficiency of vascular plants. It has been anticipated that introducing the CO2-concentrating mechanism (CCM) from cyanobacteria into plants could enhance crop yield. However, the complex nature of Rubisco's assembly has made manipulation of the enzyme extremely challenging, and attempts to replace it in plants with the enzymes from cyanobacteria and red algae have not been successful. Here we report two transplastomic tobacco lines with functional Rubisco from the cyanobacterium Synechococcus elongatus PCC7942 (Se7942). We knocked out the native tobacco gene encoding the large subunit of Rubisco by inserting the large and small subunit genes of the Se7942 enzyme, in combination with either the corresponding Se7942 assembly chaperone, RbcX, or an internal carboxysomal protein, CcmM35, which incorporates three small subunit-like domains. Se7942 Rubisco and CcmM35 formed macromolecular complexes within the chloroplast stroma, mirroring an early step in the biogenesis of cyanobacterial ß-carboxysomes. Both transformed lines were photosynthetically competent, supporting autotrophic growth, and their respective forms of Rubisco had higher rates of CO2 fixation per unit of enzyme than the tobacco control. These transplastomic tobacco lines represent an important step towards improved photosynthesis in plants and will be valuable hosts for future addition of the remaining components of the cyanobacterial CCM, such as inorganic carbon transporters and the ß-carboxysome shell proteins.

A protein with an unusually short PPR domain, MEF8, affects editing at over 60 Arabidopsis mitochondrial C targets of RNA editing.

An RNA-seq approach was used to investigate the role of a PLS-subfamily pentatricopeptide repeat protein, Mitochondrial Editing Factor 8 (MEF8), on editing in Arabidopsis mitochondria and plastids. MEF8 has an intact DYW domain, but possesses an unusually short PLS repeat region of only five repeats. The MEF8 T-DNA insertion (mef8) line exhibited reduced editing at 38 mitochondrial editing sites and increased editing at 24 sites; therefore the absence of MEF8 affects 11% of the mitochondrial editome. Notably, 60% of the matR transcripts' sites showed a decrease of editing extent in the mef8 mutant. An E549A substitution in the MEF8 protein replaced the putatively catalytic glutamate of the HXE motif in the DYW domain. Complementation with MEF8-E549A failed to restore editing at the main target sites but was able to restore editing at the matR transcript; it also decreased the editing extent of most of the C targets exhibiting an increase of editing extent in the mef8 mutant plant. Thus, MEF8 has two antagonistic effects on mitochondrial editing: stimulatory, which requires a catalytic glutamate for most of the targets except for the matR transcript, and inhibitory, for which glutamate is dispensable.

An Organelle RNA Recognition Motif Protein Is Required for Photosystem II Subunit psbF Transcript Editing.

Loss-of-function mutations in ORGANELLE RNA RECOGNITION MOTIF PROTEIN6 (ORRM6) result in the near absence of RNA editing of psbF-C77 and the reduction in accD-C794 editing in Arabidopsis (Arabidopsis thaliana). The orrm6 mutants have decreased levels of photosystem II (PSII) proteins, especially PsbF, lower PSII activity, pale green pigmentation, smaller leaf and plant sizes, and retarded growth. Stable expression of ORRM6 rescues the orrm6 editing defects and mutant phenotype. Unlike ORRM1, the other known ORRM plastid editing factor, ORRM6, does not contain RNA editing interacting protein/multiple organellar RNA editing factor (RIP/MORF) boxes, which are required for ORRM1 to interact with site-specific pentatricopeptide repeat protein editing factors. ORRM6 interacts with RIP1/MORF8, RIP2/MORF2, and RIP9/MORF9, known components of RNA editosomes. While some plastid RRM proteins are involved in other forms of RNA processing and translation, the primary function of ORRM6 is evidently to mediate psbF-C77 editing, like the essential site-specific pentatricopeptide repeat protein LOW PSII ACCUMULATION66. Stable expression in the orrm6 mutants of a nucleus-encoded, plastid-targeted PsbF protein from a psbF gene carrying a T at nucleotide 77 significantly increases leaf and plant sizes, chlorophyll content, and PSII activity. These transformants demonstrate that plastid RNA editing can be bypassed through the expression of nucleus-encoded, edited forms of plastid genes.

A zinc finger motif-containing protein is essential for chloroplast RNA editing.

C-to-U editing of transcripts in plant organelles is carried out by small (<400 kD) protein complexes called editosomes. Recognition of the proper C target for editing is mediated by pentatricopeptide repeat (PPR) containing proteins that recognize cis-elements. Members of two additional gene families, the RIP/MORF and ORRM families, have each been found to be required for editing of particular sets of Cs in mitochondria and/or chloroplasts. By co-immunoprecipitation of the chloroplast editing factor ORRM1, followed by mass spectrometry, we have now identified a member of the RanBP2 type zinc fingers (pFAM00641) protein family that is required for editing of 14 sites in chloroplasts and affects editing efficiency of another 16 chloroplast C targets. In yeast two-hybrid assays, OZ1 (Organelle Zinc finger 1) interacts with PPR site recognition factors whose cognate sites are affected when OZ1 is mutated. No interaction of OZ1 with the chloroplast editing factors RIP2 and RIP9 was detected; however, OZ1 interacts with ORRM1, which binds to RIP proteins, allowing us to build a model for the chloroplast RNA editosome. The RNA editosomes that act upon most chloroplast C targets are likely to contain a PPR protein recognition factor, either RIP2 or RIP9, ORRM1, and OZ1. The organelle zinc finger editing factor family (OZ) contains 4 members in Arabidopsis, three that are predicted to be targeted to chloroplasts and one to mitochondria. With the identification of OZ1, there are now 4 nuclear-encoded protein families known to be essential for plant organelle RNA editing.

Redesigning photosynthesis to sustainably meet global food and bioenergy demand.

The world's crop productivity is stagnating whereas population growth, rising affluence, and mandates for biofuels put increasing demands on agriculture. Meanwhile, demand for increasing cropland competes with equally crucial global sustainability and environmental protection needs. Addressing this looming agricultural crisis will be one of our greatest scientific challenges in the coming decades, and success will require substantial improvements at many levels. We assert that increasing the efficiency and productivity of photosynthesis in crop plants will be essential if this grand challenge is to be met. Here, we explore an array of prospective redesigns of plant systems at various scales, all aimed at increasing crop yields through improved photosynthetic efficiency and performance. Prospects range from straightforward alterations, already supported by preliminary evidence of feasibility, to substantial redesigns that are currently only conceptual, but that may be enabled by new developments in synthetic biology. Although some proposed redesigns are certain to face obstacles that will require alternate routes, the efforts should lead to new discoveries and technical advances with important impacts on the global problem of crop productivity and bioenergy production.

Towards engineering carboxysomes into C3 plants.

Photosynthesis in C3 plants is limited by features of the carbon-fixing enzyme Rubisco, which exhibits a low turnover rate and can react with O2 instead of CO2 , leading to photorespiration. In cyanobacteria, bacterial microcompartments, known as carboxysomes, improve the efficiency of photosynthesis by concentrating CO2 near the enzyme Rubisco. Cyanobacterial Rubisco enzymes are faster than those of C3 plants, though they have lower specificity toward CO2 than the land plant enzyme. Replacement of land plant Rubisco by faster bacterial variants with lower CO2 specificity will improve photosynthesis only if a microcompartment capable of concentrating CO2 can also be installed into the chloroplast. We review current information about cyanobacterial microcompartments and carbon-concentrating mechanisms, plant transformation strategies, replacement of Rubisco in a model C3 plant with cyanobacterial Rubisco and progress toward synthesizing a carboxysome in chloroplasts.

Transgenic tobacco plants with improved cyanobacterial Rubisco expression but no extra assembly factors grow at near wild-type rates if provided with elevated CO2.

Introducing a carbon-concentrating mechanism and a faster Rubisco enzyme from cyanobacteria into higher plant chloroplasts may improve photosynthetic performance by increasing the rate of CO2 fixation while decreasing losses caused by photorespiration. We previously demonstrated that tobacco plants grow photoautotrophically using Rubisco from Synechococcus elongatus, although the plants exhibited considerably slower growth than wild-type and required supplementary CO2 . Because of concerns that vascular plant assembly factors may not be adequate for assembly of a cyanobacterial Rubisco, prior transgenic plants included the cyanobacterial chaperone RbcX or the carboxysomal protein CcmM35. Here we show that neither RbcX nor CcmM35 is needed for assembly of active cyanobacterial Rubisco. Furthermore, by altering the gene regulatory sequences on the Rubisco transgenes, cyanobacterial Rubisco expression was enhanced and the transgenic plants grew at near wild-type growth rates, although still requiring elevated CO2 . We performed detailed kinetic characterization of the enzymes produced with and without the RbcX and CcmM35 cyanobacterial proteins. These transgenic plants exhibit photosynthetic characteristics that confirm the predicted benefits of introduction of non-native forms of Rubisco with higher carboxylation rate constants in vascular plants and the potential nitrogen-use efficiency that may be achieved provided that adequate CO2 is available near the enzyme.

RNA Recognition Motif-Containing Protein ORRM4 Broadly Affects Mitochondrial RNA Editing and Impacts Plant Development and Flowering.

Plant RNA editosomes modify cytidines (C) to uridines (U) at specific sites in plastid and mitochondrial transcripts. Members of the RNA-editing factor interacting protein (RIP) family and Organelle RNA Recognition Motif-containing (ORRM) family are essential components of the Arabidopsis (Arabidopsis thaliana) editosome. ORRM2 and ORRM3 have been recently identified as minor mitochondrial editing factors whose silencing reduces editing efficiency at ∼6% of the mitochondrial C targets. Here we report the identification of ORRM4 (for organelle RRM protein 4) as a novel, major mitochondrial editing factor that controls ∼44% of the mitochondrial editing sites. C-to-U conversion is reduced, but not eliminated completely, at the affected sites. The orrm4 mutant exhibits slower growth and delayed flowering time. ORRM4 affects editing in a site-specific way, though orrm4 mutation affects editing of the entire transcript of certain genes. ORRM4 contains an RRM domain at the N terminus and a Gly-rich domain at the C terminus. The RRM domain provides the editing activity of ORRM4, whereas the Gly-rich domain is required for its interaction with ORRM3 and with itself. The presence of ORRM4 in the editosome is further supported by its interaction with RIP1 in a bimolecular fluorescence complementation assay. The identification of ORRM4 as a major mitochondrial editing factor further expands our knowledge of the composition of the RNA editosome and reveals that adequate mitochondrial editing is necessary for normal plant development.