Identifying the mechanistic basis to nitrogen responsiveness in two contrasting Setaria italica accessions.

Nitrogen (N) is a macronutrient limiting crop productivity with varied requirements across species and genotypes. Understanding the mechanistic basis of N responsiveness by comparing contrasting genotypes could inform the development and selection of varieties with lower N demands, or inform agronomic practices to sustain yields with lower N inputs. Given the established role of millets in ensuring climate-resilient food and nutrition security, we investigated the physiological and genetic basis of nitrogen responsiveness in foxtail millet (Setaria italica L.). We had previously identified genotypic variants linked to N responsiveness, and here, we dissect the mechanistic basis of the trait by examining the physiological and molecular behaviour of N responsive (NRp-SI58) and non-responsive (NNRp-SI114) accessions at high and low N. Under high N, NRp-SI58 allocates significantly more biomass to nodes, internodes and roots, more N to developing grains, and is more effective at remobilising flag leaf N compared to NNRp-SI114. Post anthesis flag leaf gene expression suggests that differences in N induce much higher transcript abundance in NNRp-SI114 than NRp-SI58, a large proportion of which are potentially regulated by APETALA2 (AP2) transcription factors. Overall, the study provides novel insights into the regulation and manipulation of N responsiveness in S. italica.

Prospects and perspectives: inferring physiological and regulatory targets for CAM from molecular and modelling approaches.

BACKGROUND AND SCOPE: This review summarizes recent advances in our understanding of Crassulacean Acid Metabolism (CAM) by integrating evolutionary, ecological, physiological, metabolic and molecular perspectives. A number of key control loops which moderate the expression of CAM phases, and their metabolic and molecular control, are explored. These include nocturnal stomatal opening, activation of phosphoenolpyruvate carboxylase by a specific protein kinase, interactions with circadian clock control, as well as daytime decarboxylation and activation of Rubisco. The vacuolar storage and release of malic acid and the interplay between the supply and demand for carbohydrate reserves are also key metabolic control points. FUTURE OPPORTUNITIES: We identify open questions and opportunities, with experimentation informed by top-down molecular modelling approaches allied with bottom-up mechanistic modelling systems. For example, mining transcriptomic datasets using high-speed systems approaches will help to identify targets for future genetic manipulation experiments to define the regulation of CAM (whether circadian or metabolic control). We emphasize that inferences arising from computational approaches or advanced nuclear sequencing techniques can identify potential genes and transcription factors as regulatory targets. However, these outputs then require systematic evaluation, using genetic manipulation in key model organisms over a developmental progression, combining gene silencing and metabolic flux analysis and modelling to define functionality across the CAM day-night cycle. From an evolutionary perspective, the origins and function of CAM succulents and responses to water deficits are set against the mesophyll and hydraulic limitations imposed by cell and tissue succulence in contrasting morphological lineages. We highlight the interplay between traits across shoots (3D vein density, mesophyll conductance and cell shrinkage) and roots (xylem embolism and segmentation). Thus, molecular, biophysical and biochemical processes help to curtail water losses and exploit rapid rehydration during restorative rain events. In the face of a changing climate, we hope such approaches will stimulate opportunities for future research.

Epiphytic CAM bromeliads indicate vulnerability of tropical forest communities to climate change.

BACKGROUND AND SCOPE: Vascular epiphytes have a variety of mechanisms to trap and retain water, including crassulacean acid metabolism (CAM). Niche segregation was investigated for epiphytic bromeliads on the tropical Caribbean island of Trinidad, where habitats range from lowland deciduous forests to high-rainfall montane tropical forests, ~1000 m in elevation. METHODS: Four tank-impounding bromeliad epiphytes in the genus Aechmea (Ae. aquilega, Ae. fendleri, Ae. nudicaulis and Ae. dichlamydea) with CAM were mapped across their distinct geographical and elevational zonations in northern Trinidad and Tobago. Species distribution modelling was used to determine environmental limitations for each species. Anatomical and physiological measurements included leaf succulence traits, gas exchange and CAM activity; hydraulic conductance and vulnerability; stomatal sensitivity and quantum yield responses to nocturnal temperature and long-term water deficits. KEY RESULTS: A total of 2876 field observations identified the transitions between the lowland Ae. aquilega and montane Ae. fendleri, occurring >500 m a.s.l. at the drier western end of the Northern Mountain Range and at progressively lower elevations towards the wetter, eastern region. Anatomical and physiological sensitivities of gas exchange, CAM activity and water use, and responses to elevated nocturnal temperatures and drought, were markedly different for Ae. fendleri compared with Ae. aquilega or the ubiquitous Ae. nudicaulis. CONCLUSIONS: The species distribution model highlighted the susceptibility of Ae. fendleri to a changing climate. For each species, physiological and anatomical traits were tailored to environmental tolerances, consistent with specialist or generalist niche preferences. Using Intergovernmental Panel on Climate Change scenarios, we predict that rapid rainfall and temperature changes will lead to the loss of Ae. fendleri and associated lower (and upper) montane forest communities from Trinidad, seriously impacting both biodiversity and critical ecosystem functions here and in other tropical island habitats. Epiphytic bromeliads act as markers for threatened communities, and their physiological tolerances represent key indicators of climate change impacts.

Stable isotope signals provide seasonal climatic markers for moss functional groups.

Living moss biomass and archival peat deposits represent key indicators of present and past climatic conditions, but prediction of future climatic impacts requires appropriate marker species to be characterized under a range of contemporary conditions. Stable isotope signals in high latitude moss deposits offer potential climatic proxies. Seasonal changes in δ13C and δ18O of organic material (cellulose) in representative functional groups, and associated photosynthetic activity (as chlorophyll fluorescence) have been compared across East Anglia, UK, as a function of tissue water content. Representative species from contrasting acid bog, heathland, and fen woodland habitats were selected for monthly sampling of recent growth tissues between spring 2017 and autumn 2018, with isotopic signals in purified cellulose compared with tissue water, precipitation, and nearby groundwater signals. Sphagnum and Polytrichum groups, which tend to dominate peat formation, provided contrasting and complementary indicators of seasonal variations in carbon assimilation. Cellulose δ18O signals from Sphagnum spp. demonstrate seasonal variations in source precipitation inputs; carbon isotope signals in Polytrichum spp. indicate evaporative demand and photosynthetic limitation.

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.

Model approaches to advance crassulacean acid metabolism system integration.

This review summarises recent progress in understanding crassulacean acid metabolism (CAM) systems and the integration of internal and external stimuli to maximise water-use efficiency. Complex CAM traits have been reduced to their minimum and captured as computational models, which can now be refined using recently available data from transgenic manipulations and large-scale omics studies. We identify three key areas in which an appropriate choice of modelling tool could help capture relevant comparative molecular data to address the evolutionary drivers and plasticity of CAM. One focus is to identify the environmental and internal signals that drive inverse stomatal opening at night. Secondly, it is important to identify the regulatory processes required to orchestrate the diel pattern of carbon fluxes within mesophyll layers. Finally, the limitations imposed by contrasting succulent systems and associated hydraulic conductance components should be compared in the context of water-use and evolutionary strategies. While network analysis of transcriptomic data can provide insights via co-expression modules and hubs, alternative forms of computational modelling should be used iteratively to define the physiological significance of key components and informing targeted functional gene manipulation studies. We conclude that the resultant improvements of bottom-up, mechanistic modelling systems can enhance progress towards capturing the physiological controls for phylogenetically diverse CAM systems in the face of the recent surge of information in this omics era.

Orchestral manoeuvres in the light: crosstalk needed for regulation of the Chlamydomonas carbon concentration mechanism.

The inducible carbon concentration mechanism (CCM) in Chlamydomonas reinhardtii has been well defined from a molecular and ultrastructural perspective. Inorganic carbon transport proteins, and strategically located carbonic anhydrases deliver CO2 within the chloroplast pyrenoid matrix where Rubisco is packaged. However, there is little understanding of the fundamental signalling and sensing processes leading to CCM induction. While external CO2 limitation has been believed to be the primary cue, the coupling between energetic supply and inorganic carbon demand through regulatory feedback from light harvesting and photorespiration signals could provide the original CCM trigger. Key questions regarding the integration of these processes are addressed in this review. We consider how the chloroplast functions as a crucible for photosynthesis, importing and integrating nuclear-encoded components from the cytoplasm, and sending retrograde signals to the nucleus to regulate CCM induction. We hypothesize that induction of the CCM is associated with retrograde signals associated with photorespiration and/or light stress. We have also examined the significance of common evolutionary pressures for origins of two co-regulated processes, namely the CCM and photorespiration, in addition to identifying genes of interest involved in transcription, protein folding, and regulatory processes which are needed to fully understand the processes leading to CCM induction.

Rubisco and carbon-concentrating mechanism co-evolution across chlorophyte and streptophyte green algae.

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.

Unexpected Connections between Humidity and Ion Transport Discovered Using a Model to Bridge Guard Cell-to-Leaf Scales.

Stomatal movements depend on the transport and metabolism of osmotic solutes that drive reversible changes in guard cell volume and turgor. These processes are defined by a deep knowledge of the identities of the key transporters and of their biophysical and regulatory properties, and have been modeled successfully with quantitative kinetic detail at the cellular level. Transpiration of the leaf and canopy, by contrast, is described by quasilinear, empirical relations for the inputs of atmospheric humidity, CO2, and light, but without connection to guard cell mechanics. Until now, no framework has been available to bridge this gap and provide an understanding of their connections. Here, we introduce OnGuard2, a quantitative systems platform that utilizes the molecular mechanics of ion transport, metabolism, and signaling of the guard cell to define the water relations and transpiration of the leaf. We show that OnGuard2 faithfully reproduces the kinetics of stomatal conductance in Arabidopsis thaliana and its dependence on vapor pressure difference (VPD) and on water feed to the leaf. OnGuard2 also predicted with VPD unexpected alterations in K+ channel activities and changes in stomatal conductance of the slac1 Cl- channel and ost2 H+-ATPase mutants, which we verified experimentally. OnGuard2 thus bridges the micro-macro divide, offering a powerful tool with which to explore the links between guard cell homeostasis, stomatal dynamics, and foliar transpiration.

Bryophyte stable isotope composition, diversity and biomass define tropical montane cloud forest extent.

Liverworts and mosses are a major component of the epiphyte flora of tropical montane forest ecosystems. Canopy access was used to analyse the distribution and vertical stratification of bryophyte epiphytes within tree crowns at nine forest sites across a 3400 m elevational gradient in Peru, from the Amazonian basin to the high Andes. The stable isotope compositions of bryophyte organic material (13C/12C and 18O/16O) are associated with surface water diffusive limitations and, along with C/N content, provide a generic index for the extent of cloud immersion. From lowland to cloud forest δ13C increased from -33‰ to -27‰, while δ18O increased from 16.3‰ to 18.0‰. Epiphytic bryophyte and associated canopy soil biomass in the cloud immersion zone was estimated at up to 45 t dry mass ha-1, and overall water holding capacity was equivalent to a 20 mm precipitation event. The study emphasizes the importance of diverse bryophyte communities in sequestering carbon in threatened habitats, with stable isotope analysis allowing future elevational shifts in the cloud base associated with changes in climate to be tracked.

A repeat protein links Rubisco to form the eukaryotic carbon-concentrating organelle.

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.

Global Sensitivity Analysis of OnGuard Models Identifies Key Hubs for Transport Interaction in Stomatal Dynamics.

The physical requirement for charge to balance across biological membranes means that the transmembrane transport of each ionic species is interrelated, and manipulating solute flux through any one transporter will affect other transporters at the same membrane, often with unforeseen consequences. The OnGuard systems modeling platform has helped to resolve the mechanics of stomatal movements, uncovering previously unexpected behaviors of stomata. To date, however, the manual approach to exploring model parameter space has captured little formal information about the emergent connections between parameters that define the most interesting properties of the system as a whole. Here, we introduce global sensitivity analysis to identify interacting parameters affecting a number of outputs commonly accessed in experiments in Arabidopsis (Arabidopsis thaliana). The analysis highlights synergies between transporters affecting the balance between Ca2+ sequestration and Ca2+ release pathways, notably those associated with internal Ca2+ stores and their turnover. Other, unexpected synergies appear, including with the plasma membrane anion channels and H+-ATPase and with the tonoplast TPK K+ channel. These emergent synergies, and the core hubs of interaction that they define, identify subsets of transporters associated with free cytosolic Ca2+ concentration that represent key targets to enhance plant performance in the future. They also highlight the importance of interactions between the voltage regulation of the plasma membrane and tonoplast in coordinating transport between the different cellular compartments.

Economic and hydraulic divergences underpin ecological differentiation in the Bromeliaceae.

Leaf economic and hydraulic theories have rarely been applied to the ecological differentiation of speciose herbaceous plant radiations. The role of character trait divergences and network reorganization in the differentiation of the functional types in the megadiverse Neotropical Bromeliaceae was explored by quantifying a range of leaf economic and hydraulic traits in 50 diverse species. Functional types, which are defined by combinations of C3 or Crassulacean acid metabolism (CAM) photosynthesis, terrestrial or epiphytic habits, and non-specialized, tank-forming or atmospheric morphologies, segregated clearly in trait space. Most classical leaf economic relationships were supported, but they were weakened by the presence of succulence. Functional types differed in trait-network architecture, suggesting that rewiring of trait-networks caused by innovations in habit and photosynthetic pathway is an important aspect of ecological differentiation. The hydraulic data supported the coupling of leaf hydraulics and gas exchange, but not the hydraulic safety versus efficiency hypothesis, and hinted at an important role for the extra-xylary compartment in the control of bromeliad leaf hydraulics. Overall, our findings highlight the fundamental importance of structure-function relationships in the generation and maintenance of ecological diversity.

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.

An Optimal Frequency in Ca2+ Oscillations for Stomatal Closure Is an Emergent Property of Ion Transport in Guard Cells.

Oscillations in cytosolic-free Ca(2+) concentration ([Ca(2+)]i) have been proposed to encode information that controls stomatal closure. [Ca(2+)]i oscillations with a period near 10 min were previously shown to be optimal for stomatal closure in Arabidopsis (Arabidopsis thaliana), but the studies offered no insight into their origins or mechanisms of encoding to validate a role in signaling. We have used a proven systems modeling platform to investigate these [Ca(2+)]i oscillations and analyze their origins in guard cell homeostasis and membrane transport. The model faithfully reproduced differences in stomatal closure as a function of oscillation frequency with an optimum period near 10 min under standard conditions. Analysis showed that this optimum was one of a range of frequencies that accelerated closure, each arising from a balance of transport and the prevailing ion gradients across the plasma membrane and tonoplast. These interactions emerge from the experimentally derived kinetics encoded in the model for each of the relevant transporters, without the need of any additional signaling component. The resulting frequencies are of sufficient duration to permit substantial changes in [Ca(2+)]i and, with the accompanying oscillations in voltage, drive the K(+) and anion efflux for stomatal closure. Thus, the frequency optima arise from emergent interactions of transport across the membrane system of the guard cell. Rather than encoding information for ion flux, these oscillations are a by-product of the transport activities that determine stomatal aperture.

Specialized stomatal humidity responses underpin ecological diversity in C3 bromeliads.

The Neotropical Bromeliaceae display an extraordinary level of ecological variety, with species differing widely in habit, photosynthetic pathway and growth form. Divergences in stomatal structure and function, hitherto understudied in treatments of bromeliad evolutionary physiology, could have been critical to the generation of variety in ecophysiological strategies among the bromeliads. Because humidity is a key factor in bromeliad niches, we focussed on stomatal responses to vapour pressure deficit (VPD). We measured the sensitivity of stomatal conductance and assimilation rate to VPD in eight C3 bromeliad species of contrasting growth forms and ecophysiological strategies and parameterised the kinetics of stomatal responses to a step change in VPD. Notably, three tank-epiphyte species displayed low conductance, high sensitivity and fast kinetics relative to the lithophytes, while three xeromorphic terrestrial species showed high conductance and sensitivity but slow stomatal kinetics. An apparent feedforward response of transpiration to VPD occurred in the tank epiphytes, while water-use efficiency was differentially impacted by stomatal closure depending on photosynthetic responses. Differences in stomatal responses to VPD between species of different ecophysiological strategies are closely linked to modifications of stomatal morphology, which we argue has been a pivotal component of the evolution of high diversity in this important plant family.

The algal pyrenoid: key unanswered questions.

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.

Pyrenoid loss impairs carbon-concentrating mechanism induction and alters primary metabolism in Chlamydomonas reinhardtii.

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.

Pyrenoid loss in Chlamydomonas reinhardtii causes limitations in CO2 supply, but not thylakoid operating efficiency.

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.