SAGA1 and SAGA2 promote starch formation around proto-pyrenoids in Arabidopsis chloroplasts.

The pyrenoid is a chloroplastic microcompartment in which most algae and some terrestrial plants condense the primary carboxylase, Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) as part of a CO2-concentrating mechanism that improves the efficiency of CO2 capture. Engineering a pyrenoid-based CO2-concentrating mechanism (pCCM) into C3 crop plants is a promising strategy to enhance yield capacities and resilience to the changing climate. Many pyrenoids are characterized by a sheath of starch plates that is proposed to act as a barrier to limit CO2 diffusion. Recently, we have reconstituted a phase-separated "proto-pyrenoid" Rubisco matrix in the model C3 plant Arabidopsis thaliana using proteins from the alga with the most well-studied pyrenoid, Chlamydomonas reinhardtii [N. Atkinson, Y. Mao, K. X. Chan, A. J. McCormick, Nat. Commun. 11, 6303 (2020)]. Here, we describe the impact of introducing the Chlamydomonas proteins StArch Granules Abnormal 1 (SAGA1) and SAGA2, which are associated with the regulation of pyrenoid starch biogenesis and morphology. We show that SAGA1 localizes to the proto-pyrenoid in engineered Arabidopsis plants, which results in the formation of atypical spherical starch granules enclosed within the proto-pyrenoid condensate and adjacent plate-like granules that partially cover the condensate, but without modifying the total amount of chloroplastic starch accrued. Additional expression of SAGA2 further increases the proportion of starch synthesized as adjacent plate-like granules that fully encircle the proto-pyrenoid. Our findings pave the way to assembling a diffusion barrier as part of a functional pCCM in vascular plants, while also advancing our understanding of the roles of SAGA1 and SAGA2 in starch sheath formation and broadening the avenues for engineering starch morphology.

The role of BST4 in the pyrenoid of Chlamydomonas reinhardtii.

In many eukaryotic algae, CO2 fixation by Rubisco is enhanced by a CO2-concentrating mechanism, which utilizes a Rubisco-rich organelle called the pyrenoid. The pyrenoid is traversed by a network of thylakoid-membranes called pyrenoid tubules, proposed to deliver CO2. In the model alga Chlamydomonas reinhardtii (Chlamydomonas), the pyrenoid tubules have been proposed to be tethered to the Rubisco matrix by a bestrophin-like transmembrane protein, BST4. Here, we show that BST4 forms a complex that localizes to the pyrenoid tubules. A Chlamydomonas mutant impaired in the accumulation of BST4 (bst4) formed normal pyrenoid tubules and heterologous expression of BST4 in Arabidopsis thaliana did not lead to the incorporation of thylakoids into a reconstituted Rubisco condensate. Chlamydomonas bst4 mutant did not show impaired growth at air level CO2. By quantifying the non-photochemical quenching (NPQ) of chlorophyll fluorescence, we show that bst4 displays a transiently lower thylakoid lumenal pH during dark to light transition compared to control strains. When acclimated to high light, bst4 had sustained higher NPQ and elevated levels of light-induced H2O2 production. We conclude that BST4 is not a tethering protein, but rather is an ion channel involved in lumenal pH regulation possibly by mediating bicarbonate transport across the pyrenoid tubules.

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

Photosynthetic 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.

The structural basis of Rubisco phase separation in the pyrenoid.

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.

The pyrenoidal linker protein EPYC1 phase separates with hybrid Arabidopsis-Chlamydomonas Rubisco through interactions with the algal Rubisco small subunit.

Photosynthetic efficiencies in plants are restricted by the CO2-fixing enzyme Rubisco but could be enhanced by introducing a CO2-concentrating mechanism (CCM) from green algae, such as Chlamydomonas reinhardtii (hereafter Chlamydomonas). A key feature of the algal CCM is aggregation of Rubisco in the pyrenoid, a liquid-like organelle in the chloroplast. Here we have used a yeast two-hybrid system and higher plants to investigate the protein-protein interaction between Rubisco and essential pyrenoid component 1 (EPYC1), a linker protein required for Rubisco aggregation. We showed that EPYC1 interacts with the small subunit of Rubisco (SSU) from Chlamydomonas and that EPYC1 has at least five SSU interaction sites. Interaction is crucially dependent on the two surface-exposed α-helices of the Chlamydomonas SSU. EPYC1 could be localized to the chloroplast in higher plants and was not detrimental to growth when expressed stably in Arabidopsis with or without a Chlamydomonas SSU. Although EPYC1 interacted with Rubisco in planta, EPYC1 was a target for proteolytic degradation. Plants expressing EPYC1 did not show obvious evidence of Rubisco aggregation. Nevertheless, hybrid Arabidopsis Rubisco containing the Chlamydomonas SSU could phase separate into liquid droplets with purified EPYC1 in vitro, providing the first evidence of pyrenoid-like aggregation for Rubisco derived from a higher plant.

A Rubisco-binding protein is required for normal pyrenoid number and starch sheath morphology in Chlamydomonas reinhardtii.

A phase-separated, liquid-like organelle called the pyrenoid mediates CO2 fixation in the chloroplasts of nearly all eukaryotic algae. While most algae have 1 pyrenoid per chloroplast, here we describe a mutant in the model alga Chlamydomonas that has on average 10 pyrenoids per chloroplast. Characterization of the mutant leads us to propose a model where multiple pyrenoids are favored by an increase in the surface area of the starch sheath that surrounds and binds to the liquid-like pyrenoid matrix. We find that the mutant's phenotypes are due to disruption of a gene, which we call StArch Granules Abnormal 1 (SAGA1) because starch sheath granules, or plates, in mutants lacking SAGA1 are more elongated and thinner than those of wild type. SAGA1 contains a starch binding motif, suggesting that it may directly regulate starch sheath morphology. SAGA1 localizes to multiple puncta and streaks in the pyrenoid and physically interacts with the small and large subunits of the carbon-fixing enzyme Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase), a major component of the liquid-like pyrenoid matrix. Our findings suggest a biophysical mechanism by which starch sheath morphology affects pyrenoid number and CO2-concentrating mechanism function, advancing our understanding of the structure and function of this biogeochemically important organelle. More broadly, we propose that the number of phase-separated organelles can be regulated by imposing constraints on their surface area.

Progress and challenges of engineering a biophysical CO2-concentrating mechanism into higher plants.

Growth and productivity in important crop plants is limited by the inefficiencies of the C3 photosynthetic pathway. Introducing CO2-concentrating mechanisms (CCMs) into C3 plants could overcome these limitations and lead to increased yields. Many unicellular microautotrophs, such as cyanobacteria and green algae, possess highly efficient biophysical CCMs that increase CO2 concentrations around the primary carboxylase enzyme, Rubisco, to enhance CO2 assimilation rates. Algal and cyanobacterial CCMs utilize distinct molecular components, but share several functional commonalities. Here we outline the recent progress and current challenges of engineering biophysical CCMs into C3 plants. We review the predicted requirements for a functional biophysical CCM based on current knowledge of cyanobacterial and algal CCMs, the molecular engineering tools and research pipelines required to translate our theoretical knowledge into practice, and the current challenges to achieving these goals.

Rubisco small subunits from the unicellular green alga Chlamydomonas complement Rubisco-deficient mutants of Arabidopsis.

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.

Elevation of CpG frequencies in influenza A genome attenuates pathogenicity but enhances host response to infection.

Previously, we demonstrated that frequencies of CpG and UpA dinucleotides profoundly influence the replication ability of echovirus 7 (Tulloch et al., 2014). Here, we show that that influenza A virus (IAV) with maximised frequencies of these dinucleotides in segment 5 showed comparable attenuation in cell culture compared to unmodified virus and a permuted control (CDLR). Attenuation was also manifested in vivo, with 10-100 fold reduced viral loads in lungs of mice infected with 200PFU of CpG-high and UpA-high mutants. However, both induced powerful inflammatory cytokine and adaptive (T cell and neutralising antibody) responses disproportionate to their replication. CpG-high infected mice also showed markedly reduced clinical severity, minimal weight loss and reduced immmunopathology in lung, yet sterilising immunity to lethal dose WT challenge was achieved after low dose (20PFU) pre-immunisation with this mutant. Increasing CpG dinucleotide frequencies represents a generic and potentially highly effective method for generating safe, highly immunoreactive vaccines.

Introducing an algal carbon-concentrating mechanism into higher plants: location and incorporation of key components.

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.

RNA virus attenuation by codon pair deoptimisation is an artefact of increases in CpG/UpA dinucleotide frequencies.

Mutating RNA virus genomes to alter codon pair (CP) frequencies and reduce translation efficiency has been advocated as a method to generate safe, attenuated virus vaccines. However, selection for disfavoured CPs leads to unintended increases in CpG and UpA dinucleotide frequencies that also attenuate replication. We designed and phenotypically characterised mutants of the picornavirus, echovirus 7, in which these parameters were independently varied to determine which most influenced virus replication. CpG and UpA dinucleotide frequencies primarily influenced virus replication ability while no fitness differences were observed between mutants with different CP usage where dinucleotide frequencies were kept constant. Contrastingly, translation efficiency was unaffected by either CP usage or dinucleotide frequencies. This mechanistic insight is critical for future rational design of live virus vaccines and their safety evaluation; attenuation is mediated through enhanced innate immune responses to viruses with elevated CpG/UpA dinucleotide frequencies rather the viruses themselves being intrinsically defective.

The influence of CpG and UpA dinucleotide frequencies on RNA virus replication and characterization of the innate cellular pathways underlying virus attenuation and enhanced replication.

Most RNA viruses infecting mammals and other vertebrates show profound suppression of CpG and UpA dinucleotide frequencies. To investigate this functionally, mutants of the picornavirus, echovirus 7 (E7), were constructed with altered CpG and UpA compositions in two 1.1-1.3 Kbase regions. Those with increased frequencies of CpG and UpA showed impaired replication kinetics and higher RNA/infectivity ratios compared with wild-type virus. Remarkably, mutants with CpGs and UpAs removed showed enhanced replication, larger plaques and rapidly outcompeted wild-type virus on co-infections. Luciferase-expressing E7 sub-genomic replicons with CpGs and UpAs removed from the reporter gene showed 100-fold greater luminescence. E7 and mutants were equivalently sensitive to exogenously added interferon-ß, showed no evidence for differential recognition by ADAR1 or pattern recognition receptors RIG-I, MDA5 or PKR. However, kinase inhibitors roscovitine and C16 partially or entirely reversed the attenuated phenotype of high CpG and UpA mutants, potentially through inhibition of currently uncharacterized pattern recognition receptors that respond to RNA composition. Generating viruses with enhanced replication kinetics has applications in vaccine production and reporter gene construction. More fundamentally, the findings introduce a new evolutionary paradigm where dinucleotide composition of viral genomes is subjected to selection pressures independently of coding capacity and profoundly influences host-pathogen interactions.

Identification of genes involved in the response of Arabidopsis to simultaneous biotic and abiotic stresses.

In field conditions, plants may experience numerous environmental stresses at any one time. Research suggests that the plant response to multiple stresses is different from that for individual stresses, producing nonadditive effects. In particular, the molecular signaling pathways controlling biotic and abiotic stress responses may interact and antagonize one another. The transcriptome response of Arabidopsis (Arabidopsis thaliana) to concurrent water deficit (abiotic stress) and infection with the plant-parasitic nematode Heterodera schachtii (biotic stress) was analyzed by microarray. A unique program of gene expression was activated in response to a combination of water deficit and nematode stress, with 50 specifically multiple-stress-regulated genes. Candidate genes with potential roles in controlling the response to multiple stresses were selected and functionally characterized. RAPID ALKALINIZATION FACTOR-LIKE8 (AtRALFL8) was induced in roots by joint stresses but conferred susceptibility to drought stress and nematode infection when overexpressed. Constitutively expressing plants had stunted root systems and extended root hairs. Plants may produce signal peptides such as AtRALFL8 to induce cell wall remodeling in response to multiple stresses. The methionine homeostasis gene METHIONINE GAMMA LYASE (AtMGL) was up-regulated by dual stress in leaves, conferring resistance to nematodes when overexpressed. It may regulate methionine metabolism under conditions of multiple stresses. AZELAIC ACID INDUCED1 (AZI1), involved in defense priming in systemic plant immunity, was down-regulated in leaves by joint stress and conferred drought susceptibility when overexpressed, potentially as part of abscisic acid-induced repression of pathogen response genes. The results highlight the complex nature of multiple stress responses and confirm the importance of studying plant stress factors in combination.

The interaction of plant biotic and abiotic stresses: from genes to the field.

Plant responses to different stresses are highly complex and involve changes at the transcriptome, cellular, and physiological levels. Recent evidence shows that plants respond to multiple stresses differently from how they do to individual stresses, activating a specific programme of gene expression relating to the exact environmental conditions encountered. Rather than being additive, the presence of an abiotic stress can have the effect of reducing or enhancing susceptibility to a biotic pest or pathogen, and vice versa. This interaction between biotic and abiotic stresses is orchestrated by hormone signalling pathways that may induce or antagonize one another, in particular that of abscisic acid. Specificity in multiple stress responses is further controlled by a range of molecular mechanisms that act together in a complex regulatory network. Transcription factors, kinase cascades, and reactive oxygen species are key components of this cross-talk, as are heat shock factors and small RNAs. This review aims to characterize the interaction between biotic and abiotic stress responses at a molecular level, focusing on regulatory mechanisms important to both pathways. Identifying master regulators that connect both biotic and abiotic stress response pathways is fundamental in providing opportunities for developing broad-spectrum stress-tolerant crop plants.

Influence of combined biotic and abiotic stress on nutritional quality parameters in tomato (Solanum lycopersicum).

Induction of abiotic stress in tomato plants has been proposed as a mechanism for improving the nutritional quality of fruits. However, the occurrence of biotic stress can interfere with normal abiotic stress responses. In this study, the combined effect of water stress and infection with plant-parasitic nematodes on the nutritional quality of tomato was investigated. Plants were exposed to one or both stresses, and the levels of phenolic compounds, carotenoids, and sugars in fruits were analyzed as well as physiological responses. Levels of carotenoids lycopene and ß-carotene were lower in water-stressed tomatoes but exhibited a different response pattern under combined stress. Nematode stress was associated with increased flavonoid levels, albeit with reduced yields, while chlorogenic acid was increased by nematodes, water stress, and the combined stress. Sugar levels were higher only in tomatoes exposed to both stresses. These results emphasize the importance of studying plant stress factors in combination.