Temperature sensitivity of carbon concentrating mechanisms in the diatom Phaeodactylum tricornutum.

Marine diatoms are key primary producers across diverse habitats in the global ocean. Diatoms rely on a biophysical carbon concentrating mechanism (CCM) to supply high concentrations of CO2 around their carboxylating enzyme, RuBisCO. The necessity and energetic cost of the CCM are likely to be highly sensitive to temperature, as temperature impacts CO2 concentration, diffusivity, and the kinetics of CCM components. Here, we used membrane inlet mass spectrometry (MIMS) and modeling to capture temperature regulation of the CCM in the diatom Phaeodactylum tricornutum (Pt). We found that enhanced carbon fixation rates by Pt at elevated temperatures were accompanied by increased CCM activity capable of maintaining RuBisCO close to CO2 saturation but that the mechanism varied. At 10 and 18 °C, diffusion of CO2 into the cell, driven by Pt's 'chloroplast pump' was the major inorganic carbon source. However, at 18 °C, upregulation of the chloroplast pump enhanced (while retaining the proportion of) both diffusive CO2 and active HCO3- uptake into the cytosol, and significantly increased chloroplast HCO3- concentrations. In contrast, at 25 °C, compared to 18 °C, the chloroplast pump had only a slight increase in activity. While diffusive uptake of CO2 into the cell remained constant, active HCO3- uptake across the cell membrane increased resulting in Pt depending equally on both CO2 and HCO3- as inorganic carbon sources. Despite changes in the CCM, the overall rate of active carbon transport remained double that of carbon fixation across all temperatures tested. The implication of the energetic cost of the Pt CCM in response to increasing temperatures was discussed.

Effects of RuBisCO and CO2 concentration on cyanobacterial growth and carbon isotope fractionation.

Carbon isotope biosignatures preserved in the Precambrian geologic record are primarily interpreted to reflect ancient cyanobacterial carbon fixation catalyzed by Form I RuBisCO enzymes. The average range of isotopic biosignatures generally follows that produced by extant cyanobacteria. However, this observation is difficult to reconcile with several environmental (e.g., temperature, pH, and CO2 concentrations), molecular, and physiological factors that likely would have differed during the Precambrian and can produce fractionation variability in contemporary organisms that meets or exceeds that observed in the geologic record. To test a specific range of genetic and environmental factors that may impact ancient carbon isotope biosignatures, we engineered a mutant strain of the model cyanobacterium Synechococcus elongatus PCC 7942 that overexpresses RuBisCO across varying atmospheric CO2 concentrations. We hypothesized that changes in RuBisCO expression would impact the net rates of intracellular CO2 fixation versus CO2 supply, and thus whole-cell carbon isotope discrimination. In particular, we investigated the impacts of RuBisCO overexpression under changing CO2 concentrations on both carbon isotope biosignatures and cyanobacterial physiology, including cell growth and oxygen evolution rates. We found that an increased pool of active RuBisCO does not significantly affect the 13 C/12 C isotopic discrimination (εp ) at all tested CO2 concentrations, yielding εp of ≈ 23‰ for both wild-type and mutant strains at elevated CO2 . We therefore suggest that expected variation in cyanobacterial RuBisCO expression patterns should not confound carbon isotope biosignature interpretation. A deeper understanding of environmental, evolutionary, and intracellular factors that impact cyanobacterial physiology and isotope discrimination is crucial for reconciling microbially driven carbon biosignatures with those preserved in the geologic record.

Resurrected Rubisco suggests uniform carbon isotope signatures over geologic time.

The earliest geochemical indicators of microbes-and the enzymes that powered them-extend back ∼3.8 Ga on Earth. Paleobiologists often attempt to understand these indicators by assuming that the behaviors of extant microbes and enzymes are uniform with those of their predecessors. This consistency in behavior seems at odds with our understanding of the inherent variability of living systems. Here, we examine whether a uniformitarian assumption for an enzyme thought to generate carbon isotope indicators of biological activity, RuBisCO, can be corroborated by independently studying the history of changes recorded within RuBisCO's genetic sequences. We resurrected a Precambrian-age RuBisCO by engineering its ancient DNA inside a cyanobacterium genome and measured the engineered organism's fitness and carbon-isotope-discrimination profile. Results indicate that Precambrian uniformitarian assumptions may be warranted but with important caveats. Experimental studies illuminating early innovations are crucial to explore the molecular foundations of life's earliest traces.

It's what's inside that matters: physiological adaptations of high-latitude marine microalgae to environmental change.

Marine microalgae within seawater and sea ice fuel high-latitude ecosystems and drive biogeochemical cycles through the fixation and export of carbon, uptake of nutrients, and production and release of oxygen and organic compounds. High-latitude marine environments are characterized by cold temperatures, dark winters and a strong seasonal cycle. Within this environment a number of diverse and dynamic habitats exist, particularly in association with the formation and melt of sea ice, with distinct microalgal communities that transition with the season. Algal physiology is a crucial component, both responding to the dynamic environment and in turn influencing its immediate physicochemical environment. As high-latitude oceans shift into new climate regimes the analysis of seasonal responses may provide insights into how microalgae will respond to long-term environmental change. This review discusses recent developments in our understanding of how the physiology of high-latitude marine microalgae is regulated over a polar seasonal cycle, with a focus on ice-associated (sympagic) algae. In particular, physiologies that impact larger scale processes will be explored, with an aim to improve our understanding of current and future ecosystems and biogeochemical cycles.

Use of exogenous glycine betaine and its precursor choline as osmoprotectants in Antarctic sea-ice diatoms1.

Wide salinity ranges experienced during the seasonal freeze and melt of sea ice likely constrain many biological processes. Microorganisms generally protect against fluctuating salinities through the uptake, production, and release of compatible solutes. Little is known, however, about the use or fate of glycine betaine (GBT hereafter), one of the most common compatible solutes, in sea-ice diatoms confronted with shifts in salinity. We quantified intracellular concentrations and used [14 C]-labeled compounds to track the uptake and fate of the nitrogen-containing osmolyte GBT and its precursor choline in three Antarctic sea-ice diatoms Nitzschia lecointei, Navicula cf. perminuta, and Fragilariopsis cylindrus at -1°C. Experiments show that these diatoms have effective transporters for GBT, but take up lesser amounts of choline. Neither compound was respired. Uptake of GBT protected cells against hyperosmotic shock and corresponded with reduced production of extracellular polysaccharides in N. lecointei cells, which released 85% of the retained GBT following hypoosmotic shock. The ability of sea-ice diatoms to rapidly scavenge and release compatible solutes is likely an important strategy for survival during steep fluctuations in salinity. The release and recycling of compatible solutes may play an important role in algal-bacterial interactions and nitrogen cycling within the semi-enclosed brines of sea ice.

The role of Rubisco kinetics and pyrenoid morphology in shaping the CCM of haptophyte microalgae.

The haptophyte algae are a cosmopolitan group of primary producers that contribute significantly to the marine carbon cycle and play a major role in paleo-climate studies. Despite their global importance, little is known about carbon assimilation in haptophytes, in particular the kinetics of their Form 1D CO2-fixing enzyme, Rubisco. Here we examine Rubisco properties of three haptophytes with a range of pyrenoid morphologies (Pleurochrysis carterae, Tisochrysis lutea, and Pavlova lutheri) and the diatom Phaeodactylum tricornutum that exhibit contrasting sensitivities to the trade-offs between substrate affinity (Km) and turnover rate (kcat) for both CO2 and O2. The pyrenoid-containing T. lutea and P. carterae showed lower Rubisco content and carboxylation properties (KC and kCcat) comparable with those of Form 1D-containing non-green algae. In contrast, the pyrenoid-lacking P. lutheri produced Rubisco in 3-fold higher amounts, and displayed a Form 1B Rubisco kCcat-KC relationship and increased CO2/O2 specificity that, when modeled in the context of a C3 leaf, supported equivalent rates of photosynthesis to higher plant Rubisco. Correlation between the differing Rubisco properties and the occurrence and localization of pyrenoids with differing intracellular CO2:O2 microenvironments has probably influenced the divergent evolution of Form 1B and 1D Rubisco kinetics.

The potential for co-evolution of CO2-concentrating mechanisms and Rubisco in diatoms.

Diatoms are a diverse group of unicellular algae that contribute significantly to global photosynthetic carbon fixation and export in the modern ocean, and are an important source of microfossils for paleoclimate reconstructions. Because of their importance in the environment, diatoms have been a focus of study on the physiology and ecophysiology of carbon fixation, in particular their CO2-concentrating mechanisms (CCMs) and Rubisco characteristics. While carbon fixation in diatoms is not as well understood as in certain model aquatic photoautotrophs, a greater number of species have been examined in diatoms. Recent work has highlighted a large diversity in the function, physiology, and kinetics of both the CCM and Rubisco between different diatom species. This diversity was unexpected since it has generally been assumed that CCMs and Rubiscos were similar within major algal lineages as the result of selective events deep in evolutionary history, and suggests a more recent co-evolution between the CCM and Rubisco within diatoms. This review explores our current understanding of the diatom CCM and highlights the diversity of both the CCM and Rubisco kinetics. We will suggest possible environmental, physiological, and evolutionary drivers for the co-evolution of the CCM and Rubisco in diatoms.

Rubisco Extraction and Purification from Diatoms.

This protocol describes a method to extract ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) from diatoms (Bacillariophyta) to determine catalytic performance. This protocol has been adapted from use in cyanobacteria and higher plants (Andrews, 1988; Whitney and Sharwood, 2007). First part (steps A1-A3) of the extraction provides a crude extract of Rubisco that is sufficient for carboxylation assays to measure the Michaelis constant for CO2 (KC) and the catalytic turnover rate ( kcat c ). However, the further purification steps outlined (steps B1-B4) are needed for measurements of Rubisco CO2/O2 Specificity (SC/O, [ Kane et al., 1994 ]).

Large variation in the Rubisco kinetics of diatoms reveals diversity among their carbon-concentrating mechanisms.

While marine phytoplankton rival plants in their contribution to global primary productivity, our understanding of their photosynthesis remains rudimentary. In particular, the kinetic diversity of the CO2-fixing enzyme, Rubisco, in phytoplankton remains unknown. Here we quantify the maximum rates of carboxylation (k cat (c)), oxygenation (k cat (o)), Michaelis constants (K m) for CO2 (K C) and O2 (K O), and specificity for CO2 over O2 (SC/O) for Form I Rubisco from 11 diatom species. Diatom Rubisco shows greater variation in K C (23-68 µM), SC/O (57-116mol mol(-1)), and K O (413-2032 µM) relative to plant and algal Rubisco. The broad range of K C values mostly exceed those of C4 plant Rubisco, suggesting that the strength of the carbon-concentrating mechanism (CCM) in diatoms is more diverse, and more effective than previously predicted. The measured k cat (c) for each diatom Rubisco showed less variation (2.1-3.7s(-1)), thus averting the canonical trade-off typically observed between K C and k cat (c) for plant Form I Rubisco. Uniquely, a negative relationship between K C and cellular Rubisco content was found, suggesting variation among diatom species in how they allocate their limited cellular resources between Rubisco synthesis and their CCM. The activation status of Rubisco in each diatom was low, indicating a requirement for Rubisco activase. This work highlights the need to better understand the correlative natural diversity between the Rubisco kinetics and CCM of diatoms and the underpinning mechanistic differences in catalytic chemistry among the Form I Rubisco superfamily.

Slow carboxylation of Rubisco constrains the rate of carbon fixation during Antarctic phytoplankton blooms.

High-latitude oceans are areas of high primary production despite temperatures that are often well below the thermal optima of enzymes, including the key Calvin Cycle enzyme, Ribulose 1,5 bisphosphate carboxylase oxygenase (Rubisco). We measured carbon fixation rates, protein content and Rubisco abundance and catalytic rates during an intense diatom bloom in the Western Antarctic Peninsula (WAP) and in laboratory cultures of a psychrophilic diatom (Fragilariopsis cylindrus). At -1°C, the Rubisco turnover rate, kcat (c) , was 0.4 C s(-1) per site and the half saturation constant for CO2 was 15 µM (vs c. 3 C s(-1) per site and 50 µM at 20°C). To achieve high carboxylation rates, psychrophilic diatoms increased Rubisco abundance to c. 8% of biomass (vs c. 0.6% at 20°C), along with their total protein content, resulting in a low carbon : nitrogen ratio of c. 5. In psychrophilic diatoms, Rubisco must be almost fully active and near CO2 saturation to achieve carbon fixation rates observed in the WAP. Correspondingly, total protein concentrations were close to the highest ever measured in phytoplankton and likely near the maximum possible. We hypothesize that this high protein concentration, like that of Rubisco, is necessitated by slow enzyme rates, and that carbon fixation rates in the WAP are near a theoretical maximum.

Gross and net production during the spring bloom along the Western Antarctic Peninsula.

This study explores some of the physiological mechanisms responsible for high productivity near the shelf in the Western Antarctic Peninsula despite a short growing season and cold temperature. We measured gross and net primary production at Palmer Station during the summer of 2012/2013 via three different techniques: incubation with H2 (18) O; incubation with (14) CO2 ; and in situ measurements of O2 /Ar and triple oxygen isotope. Additional laboratory experiments were performed with the psychrophilic diatom Fragilariopsis cylindrus. During the spring bloom, which accounted for more than half of the seasonal gross production at Palmer Station, the ratio of net-to-gross production reached a maximum greater than c. 60%, among the highest ever reported. The use of multiple techniques showed that these high ratios resulted from low heterotrophic respiration and very low daylight autotrophic respiration. Laboratory experiments revealed a similar ratio of net-to-gross O2 production in F. cylindrus and provided the first experimental evidence for an important level of cyclic electron flow (CEF) in this organism. The low ratio of community respiration to gross primary production observed during the bloom at Palmer Station may be characteristic of high latitude coastal ecosystems and partially supported by a very active CEF in psychrophilic phytoplankton.

Low temperature reduces the energetic requirement for the CO2 concentrating mechanism in diatoms.

The goal of this study is to investigate the CO2 concentrating mechanism (CCM) of the dominant phytoplankton species during the growing season at Palmer station in the Western Antarctic Peninsula. Key CCM parameters (cellular half-saturation constants for CO2 fixation, carbonic anhydrase activity, CO2 /HCO3 (-) uptake, δ(13) Corg ) in natural phytoplankton assemblages were determined. Those results, together with additional measurements on CO2 membrane permeability from Fragilariopsis cylindrus laboratory cultures, were used to develop a numerical model of the CCM of cold water diatoms. The field data demonstrate that the dominant species throughout the season possess an effective CCM, which achieves near saturation of CO2 for fixation. The model provides a means to examine the role of eCA activity and HCO3 (-) /CO2 uptake in the functioning of the CCM. According to the model, the increase in δ(13) Corg during the bloom results chiefly from decreasing ambient CO2 concentration (which reduces the gross diffusive flux across the membrane) rather than a shift in inorganic carbon uptake from CO2 to HCO3 (-) . The CCM of diatoms in the Western Antarctic Peninsula functions with a relatively small expenditure of energy, resulting chiefly from the low half-saturation constant for Rubisco at cold temperatures.

The minimal CO2-concentrating mechanism of Prochlorococcus spp. MED4 is effective and efficient.

As an oligotrophic specialist, Prochlorococcus spp. has streamlined its genome and metabolism including the CO2-concentrating mechanism (CCM), which serves to elevate the CO2 concentration around Rubisco. The genomes of Prochlorococcus spp. indicate that they have a simple CCM composed of one or two HCO3(-) pumps and a carboxysome, but its functionality has not been examined. Here, we show that the CCM of Prochlorococcus spp. is effective and efficient, transporting only two molecules of HCO3(-) per molecule of CO2 fixed. A mechanistic, numerical model with a structure based on the CCM components present in the genome is able to match data on photosynthesis, CO2 efflux, and the intracellular inorganic carbon pool. The model requires the carboxysome shell to be a major barrier to CO2 efflux and shows that excess Rubisco capacity is critical to attaining a high-affinity CCM without CO2 recovery mechanisms or high-affinity HCO3(-) transporters. No differences in CCM physiology or gene expression were observed when Prochlorococcus spp. was fully acclimated to high-CO2 (1,000 µL L(-1)) or low-CO2 (150 µL L(-1)) conditions. Prochlorococcus spp. CCM components in the Global Ocean Survey metagenomes were very similar to those in the genomes of cultivated strains, indicating that the CCM in environmental populations is similar to that of cultured representatives.

Rubisco is a small fraction of total protein in marine phytoplankton.

Ribulose 1,5 bisphosphate carboxylase oxygenase (Rubisco) concentrations were quantified as a proportion of total protein in eight species of microalgae. This enzyme has been assumed to be a major fraction of total protein in phytoplankton, as has been demonstrated in plants, potentially constituting a large sink for cellular nitrogen. Representative microalgae were grown in batch and continuous cultures under nutrient-replete, nitrogen (N)-limited, or phosphorus (P)-limited conditions with varying CO(2). Quantitative Western blots were performed using commercially available global antibodies and protein standards. Field incubations with natural populations of organisms from the coast of California were conducted under both nutrient-replete and N-limited conditions with varying CO(2). In all experiments, Rubisco represented < 6% of total protein. In nutrient-replete exponentially growing batch cultures, concentrations ranged from 2% to 6%, while in nutrient-limited laboratory and field cultures, concentrations were < 2.5%. Rubisco generally decreased with increasing CO(2) and with decreasing growth rates. Based on a calculation of maximum Rubisco activity, these results suggest that phytoplankton contain the minimum concentration of enzyme necessary to support observed growth rates. Unlike in plants, Rubisco does not account for a major fraction of cellular N in phytoplankton.