A three-step process of manganese acquisition and storage in the microalga Chlorella sorokiniana.

Metabolism of metals in microalgae and adaptation to metal excess are of significant environmental importance. We report a three-step mechanism that the green microalga Chlorella sorokiniana activates during the acquisition of and adaptation to manganese (Mn), which is both an essential trace metal and a pollutant of waters. In the early stage, Mn2+ was mainly bound to membrane phospholipids and phosphates in released mucilage. The outer cell wall was reorganized and lipids were accumulated, with a relative increase in lipid saturation. Intracellular redox settings were rapidly altered in the presence of Mn excess, with increased production of reactive oxygen species that resulted in lipid peroxidation and a decrease in the concentration of thiols. In the later stage, Mn2+ was chelated by polyphosphates and accumulated in the cells. The structure of the inner cell wall was modified and the redox milieu established a new balance. Polyphosphates serve as a transient Mn2+ storage ligand, as proposed previously. In the final stage, Mn was stored in multivalent Mn clusters that resemble the structure of the tetramanganese-calcium core of the oxygen-evolving complex. The present findings elucidate the bioinorganic chemistry and metabolism of Mn in microalgae, and may shed new light on water-splitting Mn clusters.

Tea plant roots respond to aluminum-induced mineral nutrient imbalances by transcriptional regulation of multiple cation and anion transporters.

BACKGROUND: Tea is one of the most popular non-alcoholic beverages in the world for its flavors and numerous health benefits. The tea tree (Camellia sinensis L.) is a well-known aluminum (Al) hyperaccumulator. However, it is not fully understood how tea plants have adapted to tolerate high concentrations of Al, which causes an imbalance of mineral nutrition in the roots. RESULTS: Here, we combined ionomic and transcriptomic profiling alongside biochemical characterization, to probe the changes of metal nutrients and Al responsive genes in tea roots grown under increasing concentrations of Al. It was found that a low level of Al (~ 0.4 mM) maintains proper nutrient balance, whereas a higher Al concentration (2.5 mM) compromised tea plants by altering micro- and macro-nutrient accumulation into roots, including a decrease in calcium (Ca), manganese (Mn), and magnesium (Mg) and an increase in iron (Fe), which corresponded with oxidative stress, cellular damage, and retarded root growth. Transcriptome analysis revealed more than 1000 transporter genes that were significantly changed in expression upon Al exposure compared to control (no Al) treatments. These included transporters related to Ca and Fe uptake and translocation, while genes required for N, P, and S nutrition in roots did not significantly alter. Transporters related to organic acid secretion, together with other putative Al-tolerance genes also significantly changed in response to Al. Two of these transporters, CsALMT1 and CsALS8, were functionally tested by yeast heterologous expression and confirmed to provide Al tolerance. CONCLUSION: This study shows that tea plant roots respond to high Al-induced mineral nutrient imbalances by transcriptional regulation of both cation and anion transporters, and therefore provides new insights into Al tolerance mechanism of tea plants. The altered transporter gene expression profiles partly explain the imbalanced metal ion accumulation that occurred in the Al-stressed roots, while increases to organic acid and Al tolerance gene expression partly explains the ability of tea plants to be able to grow in high Al containing soils. The improved transcriptomic understanding of Al exposure gained here has highlighted potential gene targets for breeding or genetic engineering approaches to develop safer tea products.

Mechanisms of detoxification of high copper concentrations by the microalga Chlorella sorokiniana.

Microalgae have evolved mechanisms to respond to changes in copper ion availability, which are very important for normal cellular function, to tolerate metal pollution of aquatic ecosystems, and for modulation of copper bioavailability and toxicity to other organisms. Knowledge and application of these mechanisms will benefit the use of microalgae in wastewater processing and biomass production, and the use of copper compounds in the suppression of harmful algal blooms. Here, using electron microscopy, synchrotron radiation-based Fourier transform infrared spectroscopy, electron paramagnetic resonance spectroscopy, and X-ray absorption fine structure spectroscopy, we show that the microalga Chlorella sorokiniana responds promptly to Cu2+ at high non-toxic concentration, by mucilage release, alterations in the architecture of the outer cell wall layer and lipid structures, and polyphosphate accumulation within mucilage matrix. The main route of copper detoxification is by Cu2+ coordination to polyphosphates in penta-coordinated geometry. The sequestrated Cu2+ was accessible and could be released by extracellular chelating agents. Finally, the reduction in Cu2+ to Cu1+ appears also to take place. These findings reveal the biochemical basis of the capacity of microalgae to adapt to high external copper concentrations and to serve as both, sinks and pools of environmental copper.

Increased metal tolerance and bioaccumulation of zinc and cadmium in Chlamydomonas reinhardtii expressing a AtHMA4 C-terminal domain protein.

The use of microalgal biomass for metal pollutant bioremediation might be improved by genetic engineering to modify the selectivity or capacity of metal biosorption. A plant cadmium (Cd) and zinc (Zn) transporter (AtHMA4) was used as a transgene to increase the ability of Chlamydomonas reinhardtii to tolerate 0.2 mM Cd and 0.3 mM Zn exposure. The transgenic cells showed increased accumulation and internalization of both metals compared to wild-type. AtHMA4 was expressed either as the full-length (FL) protein or just the C-terminal (CT) tail, which is known to have metal-binding sites. Similar Cd and Zn tolerance and accumulation was observed with expression of either the FL protein or CT domain, suggesting that enhanced metal tolerance was mainly due to increased metal binding rather than metal transport. The effectiveness of the transgenic cells was further examined by immobilization in calcium alginate to generate microalgal beads that could be added to a metal contaminated solution. Immobilization maintained metal tolerance, while AtHMA4-expressing cells in alginate showed a concentration-dependent increase in metal biosorption that was significantly greater than alginate beads composed of wild-type cells. This demonstrates that expressing AtHMA4 FL or CT has great potential as a strategy for bioremediation using microalgal biomass.

Macroalgae as spatial and temporal bioindicators of coastal metal pollution following remediation and diversion of acid mine drainage.

Acid mine drainage (AMD) is a significant contributor of metal pollution leading to ecosystem damage. Bioindicator organisms such as intertidal brown macroalgae have an important role in quantifying the risks of metal bioaccumulation in coastal locations exposed to AMD contamination. Measurement of As, Cd, Cu, Fe, Pb, and Zn accumulation was performed in Fucus serratus, Fucus vesiculosus and Ascophyllum nodosum sampled from two marine locations near to an abandoned Cu mine in Anglesey, Wales, UK. Transect samples were taken from a coastal location (Amlwch) that has seen a substantial increase in AMD contamination over 15 years, in comparison to a nearby estuarine location (Dulas Estuary leading to Dulas Bay) with a historic legacy of pollution. These were compared with samples from the same sites taken 30 years earlier. Some of the Dulas macroalgae samples had Cd, Cu and Zn concentrations that were above background but in general indicated a non-polluted estuary in comparison to substantial pollution over previous decades. In contrast, Fucus samples collected from directly below an AMD outflow at Amlwch showed extremely elevated metal bioaccumulation (>250 mg Fe g-1, >6 mg Cu g-1, >2 mg Zn g-1, >190 µg As g-1) and evidence of macroalgae toxicity, indicating severe pollution at this site. However, the pollution dispersed within 200 m of the outflow source. This study has demonstrated the efficiency of three brown macroalgae species as indicators for metal bioavailability at high spatial resolution and over time.

Two Glycerol-3-Phosphate Dehydrogenases from Chlamydomonas Have Distinct Roles in Lipid Metabolism.

The metabolism of glycerol-3-phosphate (G3P) is important for environmental stress responses by eukaryotic microalgae. G3P is an essential precursor for glycerolipid synthesis and the accumulation of triacylglycerol (TAG) in response to nutrient starvation. G3P dehydrogenase (GPDH) mediates G3P synthesis, but the roles of specific GPDH isoforms are currently poorly understood. Of the five GPDH enzymes in the model alga Chlamydomonas reinhardtii, GPD2 and GPD3 were shown to be induced by nutrient starvation and/or salt stress. Heterologous expression of GPD2, a putative chloroplastic GPDH, and GPD3, a putative cytosolic GPDH, in a yeast gpd1Δ mutant demonstrated the functionality of both enzymes. C. reinhardtii knockdown mutants for GPD2 and GPD3 showed no difference in growth but displayed significant reduction in TAG concentration compared with the wild type in response to phosphorus or nitrogen starvation. Overexpression of GPD2 and GPD3 in C. reinhardtii gave distinct phenotypes. GPD2 overexpression lines showed only subtle metabolic phenotypes and no significant alteration in growth. In contrast, GPD3 overexpression lines displayed significantly inhibited growth and chlorophyll concentration, reduced glycerol concentration, and changes to lipid composition compared with the wild type, including increased abundance of phosphatidic acids but reduced abundance of diglycerides, triglycerides, and phosphatidylglycerol lipids. This may indicate a block in the downstream glycerolipid metabolism pathway in GPD3 overexpression lines. Thus, lipid engineering by GPDH modification may depend on the activities of other downstream enzyme steps. These results also suggest that GPD2 and GPD3 GPDH isoforms are important for nutrient starvation-induced TAG accumulation but have distinct metabolic functions.

PSR1 Is a Global Transcriptional Regulator of Phosphorus Deficiency Responses and Carbon Storage Metabolism in Chlamydomonas reinhardtii.

Many eukaryotic microalgae modify their metabolism in response to nutrient stresses such as phosphorus (P) starvation, which substantially induces storage metabolite biosynthesis, but the genetic mechanisms regulating this response are poorly understood. Here, we show that P starvation-induced lipid and starch accumulation is inhibited in a Chlamydomonas reinhardtii mutant lacking the transcription factor Pi Starvation Response1 (PSR1). Transcriptomic analysis identified specific metabolism transcripts that are induced by P starvation but misregulated in the psr1 mutant. These include transcripts for starch and triacylglycerol synthesis but also transcripts for photosynthesis-, redox-, and stress signaling-related proteins. To further examine the role of PSR1 in regulating lipid and starch metabolism, PSR1 complementation lines in the psr1 strain and PSR1 overexpression lines in a cell wall-deficient strain were generated. PSR1 expression in the psr1 lines was shown to be functional due to rescue of the psr1 phenotype. PSR1 overexpression lines exhibited increased starch content and number of starch granules per cell, which correlated with a higher expression of specific starch metabolism genes but reduced neutral lipid content. Furthermore, this phenotype was consistent in the presence and absence of acetate. Together, these results identify a key transcriptional regulator in global metabolism and demonstrate transcriptional engineering in microalgae to modulate starch biosynthesis.

Spatial and temporal specificity of Ca2+ signalling in Chlamydomonas reinhardtii in response to osmotic stress.

Ca2+ -dependent signalling processes enable plants to perceive and respond to diverse environmental stressors, such as osmotic stress. A clear understanding of the role of spatiotemporal Ca2+ signalling in green algal lineages is necessary in order to understand how the Ca2+ signalling machinery has evolved in land plants. We used single-cell imaging of Ca2+ -responsive fluorescent dyes in the unicellular green alga Chlamydomonas reinhardtii to examine the specificity of spatial and temporal dynamics of Ca2+ elevations in the cytosol and flagella in response to salinity and osmotic stress. We found that salt stress induced a single Ca2+ elevation that was modulated by the strength of the stimulus and originated in the apex of the cell, spreading as a fast Ca2+ wave. By contrast, hypo-osmotic stress induced a series of repetitive Ca2+ elevations in the cytosol that were spatially uniform. Hypo-osmotic stimuli also induced Ca2+ elevations in the flagella that occurred independently from those in the cytosol. Our results indicate that the requirement for Ca2+ signalling in response to osmotic stress is conserved between land plants and green algae, but the distinct spatial and temporal dynamics of osmotic Ca2+ elevations in C. reinhardtii suggest important mechanistic differences between the two lineages.

The Plasmodium berghei Ca(2+)/H(+) exchanger, PbCAX, is essential for tolerance to environmental Ca(2+) during sexual development.

Ca(2+) contributes to a myriad of important cellular processes in all organisms, including the apicomplexans, Plasmodium and Toxoplasma. Due to its varied and essential roles, free Ca(2+) is tightly regulated by complex mechanisms. These mechanisms are therefore of interest as putative drug targets. One pathway in Ca(2+) homeostatic control in apicomplexans uses a Ca(2+)/H(+) exchanger (a member of the cation exchanger family, CAX). The P. falciparum CAX (PfCAX) has recently been characterised in asexual blood stage parasites. To determine the physiological importance of apicomplexan CAXs, tagging and knock-out strategies were undertaken in the genetically tractable T. gondii and P. berghei parasites. In addition, a yeast heterologous expression system was used to study the function of apicomplexan CAXs. Tagging of T. gondii and P. berghei CAXs (TgCAX and PbCAX) under control of their endogenous promoters could not demonstrate measureable expression of either CAX in tachyzoites and asexual blood stages, respectively. These results were consistent with the ability of parasites to tolerate knock-outs of the genes for TgCAX and PbCAX at these developmental stages. In contrast, PbCAX expression was detectable during sexual stages of development in female gametocytes/gametes, zygotes and ookinetes, where it was dispersed in membranous networks within the cytosol (with minimal mitochondrial localisation). Furthermore, genetically disrupted parasites failed to develop further from "round" form zygotes, suggesting that PbCAX is essential for ookinete development and differentiation. This impeded phenotype could be rescued by removal of extracellular Ca(2+). Therefore, PbCAX provides a mechanism for free living parasites to multiply within the ionic microenvironment of the mosquito midgut. Ca(2+) homeostasis mediated by PbCAX is critical and suggests plasmodial CAXs may be targeted in approaches designed to block parasite transmission.

Acclimation of microalgae to wastewater environments involves increased oxidative stress tolerance activity.

A wastewater environment can be particularly toxic to eukaryotic microalgae. Microalgae can adapt to these conditions but the specific mechanisms that allow strains to tolerate wastewater environments are unclear. Furthermore, it is unknown whether the ability to acclimate microalgae to tolerate wastewater is an innate or species-specific characteristic. Six different species of microalgae (Chlamydomonas debaryana, Chlorella luteoviridis, Chlorella vulgaris, Desmodesmus intermedius, Hindakia tetrachotoma and Parachlorella kessleri) that had never previously been exposed to wastewater conditions were acclimated over an 8-week period in secondary-treated municipal wastewater. With the exception of C. debaryana, acclimation to wastewater resulted in significantly higher growth rate and biomass productivity. With the exception of C. vulgaris, total chlorophyll content was significantly increased in all acclimated strains, while all acclimated strains showed significantly increased photosynthetic activity. The ability of strains to acclimate was species-specific, with two species, C. luteoviridis and P. kessleri, able to acclimate more efficiently to the stress than C. debaryana and D. intermedius. Metabolic fingerprinting of the acclimated and non-acclimated microalgae using Fourier transform infrared spectroscopy was able to differentiate strains on the basis of metabolic responses to the stress. In particular, strains exhibiting greater stress response and altered accumulation of lipids and carbohydrates could be distinguished. The acclimation to wastewater tolerance was correlated with higher accumulation of carotenoid pigments and increased ascorbate peroxidase activity.

Expression in yeast links field polymorphisms in PfATP6 to in vitro artemisinin resistance and identifies new inhibitor classes.

BACKGROUND: The mechanism of action of artemisinins against malaria is unclear, despite their widespread use in combination therapies and the emergence of resistance. RESULTS: Here, we report expression of PfATP6 (a SERCA pump) in yeast and demonstrate its inhibition by artemisinins. Mutations in PfATP6 identified in field isolates (such as S769N) and in laboratory clones (such as L263E) decrease susceptibility to artemisinins, whereas they increase susceptibility to unrelated inhibitors such as cyclopiazonic acid. As predicted from the yeast model, Plasmodium falciparum with the L263E mutation is also more susceptible to cyclopiazonic acid. An inability to knockout parasite SERCA pumps provides genetic evidence that they are essential in asexual stages of development. Thaperoxides are a new class of potent antimalarial designed to act by inhibiting PfATP6. Results in yeast confirm this inhibition. CONCLUSIONS: The identification of inhibitors effective against mutated PfATP6 suggests ways in which artemisinin resistance may be overcome.

Control of cyanobacterial growth with potassium; implications for bloom control in nuclear storage ponds.

Microbial blooms have been reported in the First Generation Magnox Storage Pond at the Sellafield Nuclear Facility. The pond is kept alkaline with NaOH to minimise fuel rod corrosion, however alkali-tolerant microbial blooms dominated by the cyanobacterium Pseudanabaena catenata are able to thrive in this hostile environment. This study assessed the impact of alternative alkali-dosing regimens (KOH versus NaOH treatment) on biomass accumulation, using a P. catenata dominated mixed culture, which is representative of the pond environment. Optical density was reduced by 40-67 % with KOH treatment over the 3-month chemostat experiment. Microbial community analysis and proteomics demonstrated that the KOH-dependent inhibition of cell growth was mostly specific to P. catenata. The addition of KOH to nuclear storage ponds may therefore help control growth of this pioneer photosynthetic organism due to its sensitivity to potassium, while maintaining the high pH needed to inhibit the corrosion of stored nuclear fuel.

Low-dose ionizing radiation generates a hormetic response to modify lipid metabolism in Chlorella sorokiniana.

Algal biomass is a viable source of chemicals and metabolites for various energy, nutritional, medicinal and agricultural uses. While stresses have commonly been used to induce metabolite accumulation in microalgae in attempts to enhance high-value product yields, this is often very detrimental to growth. Therefore, understanding how to modify metabolism without deleterious consequences is highly beneficial. We demonstrate that low-doses (1-5 Gy) of ionizing radiation in the X-ray range induces a non-toxic, hormetic response in microalgae to promote metabolic activation. We identify specific radiation exposure parameters that give reproducible metabolic responses in Chlorella sorokiniana caused by transcriptional changes. This includes up-regulation of >30 lipid metabolism genes, such as genes encoding an acetyl-CoA carboxylase subunit, phosphatidic acid phosphatase, lysophosphatidic acid acyltransferase, and diacylglycerol acyltransferase. The outcome is an increased lipid yield in stationary phase cultures by 25% in just 24 hours, without any negative effects on cell viability or biomass.

Natural wetlands are efficient at providing long-term metal remediation of freshwater systems polluted by acid mine drainage.

This study describes the first long-term (14-year) evaluation of the efficacy of an established (>100 years) natural wetland to remediate highly acidic mine drainage (AMD). Although natural wetlands are highly valued for their biodiversity, this study demonstrates that they also provide important ecosystem service functions through their ability to consistently and reliably improve water quality by mitigating AMD. The Afon Goch river flows from Parys Mountain copper mine via a natural wetland, and was the major source of Zn and Cu contamination to the Irish Sea. Prior to 2003 the wetland received severe acidic metal contamination and retained a large proportion of the contamination (55, 64, and 37% in dissolved Fe, Zn, and Cu) leading to a greatly reduced metal flow to the Irish Sea. Reduced wetland loadings midway through the sampling period led to a reduction of metals by 83-94% and a pH increase from 2.7 to 5.5, resulting in long-term improvements in the downstream benthic invertebrate community. High root metal accumulation by the dominant wetland plant species and the association of acidophilic bacteria in the wetland rhizosphere indicate that multiple interacting processes provide an efficient and self-sustaining system to remediate AMD.

The interplay between Cs and K in Pseudanabaena catenata; from microbial bloom control strategies to bioremediation options for radioactive waters.

Pseudanabaena dominates cyanobacterial blooms in the First-Generation Magnox Storage Pond (FGMSP) at a UK nuclear site. The fission product Cs is a radiologically significant radionuclide in the pond, and understanding the interactions between Cs and Pseudanabaena spp. is therefore important for determining facility management strategies, as well as improving understanding of microbiological responses to this non-essential chemical analogue of K. This study evaluated the fate of Cs following interactions with Pseudanabaena catenata, a laboratory strain most closely related to that dominating FGMSP blooms. Experiments showed that Cs (1 mM) exposure did not affect the growth of P. catenata, while a high concentration of K (5 mM) caused a significant reduction in cell yield. Scanning transmission X-ray microscopy elemental mapping identified Cs accumulation to discrete cytoplasmic locations within P. catenata cells, indicating a potential bioremediation option for Cs. Proteins related to stress responses and nutrient limitation (K, P) were stimulated by Cs treatment. Furthermore, selected K+ transport proteins were mis-regulated by Cs dosing, which indicates the importance of the K+ transport system for Cs accumulation. These findings enhance understanding of Cs fate and biological responses within Pseudanabaena blooms, and indicate that K exposure might provide a microbial bloom control strategy.

Identification of algal rich microbial blooms in the Sellafield Pile Fuel Storage Pond and the application of ultrasonic treatment to control the formation of blooms.

The presence of microorganisms in a range of nuclear facilities has been known for many years. In this study the microbial community inhabiting the Pile Fuel Storage Pond (PFSP), which is a legacy open-aired facility on the Sellafield nuclear site, Cumbria, UK, was determined to help target microbial bloom management strategies in this facility. The PFSP is currently undergoing decommissioning and the development of prolonged dense microbial blooms reduces the visibility within the water. Such impairment in the pond water visibility can lead to delays in pond operations, which also has financial implications. Efforts to control the microbial population within the PFSP are ongoing, with the installation of ultrasonic treatment units. Here next generation sequencing techniques focussing on broad targets for both eukaryotic and prokaryotic organisms were used to identify the microbial community. On-site monitoring of photosynthetic pigments indicated when microbial blooms formed and that eukaryotic algae were most likely to be responsible for these events. The sequencing data suggested that the blooms were dominated by members of the class Chrysophyceae, a group of golden algae, while evidence of cyanobacteria and other photosynthetic bacteria was limited, further supporting eukaryotic organisms causing the blooms. The results of sequencing data from 2018 was used to inform a change in the operational settings of the ultrasonic units, while monitoring of the microbial community and photosynthetic pigments trends was extended. Since the changes were made to the ultrasonic treatment, the visibility in the pond was significantly improved, with an absence of a spring bloom in 2020 and an overall reduction in the number of days lost due to microbial blooms annually. This work extends our knowledge of the diversity of microbes able to colonise nuclear fuel storage ponds, and also suggests that sequencing data can help to optimise the performance of ultrasonic treatments, to control algal proliferation in the PFSP facility and other inhospitable engineered systems.

Isolation and identification of arbuscular mycorrhizal fungi from an abandoned uranium mine and their role in soil-to-plant transfer of radionuclides and metals.

Arbuscular mycorrhizal fungi were recovered from soil samples from the naturally radioactive soil at the long-abandoned South Terras uranium mine in Cornwall, UK. Species of Rhizophagus, Claroideoglomus, Paraglomus, Septoglomus, and Ambispora were recovered, and pot cultures from all except Ambispora were established. Cultures were identified to species level using morphological observation and rRNA gene sequencing combined with phylogenetic analysis. These cultures were used in pot experiments designed with a compartmentalised system to assess the contribution of fungal hyphae to the accumulation of essential elements, such as copper and zinc, and non-essential elements, such as lead, arsenic, thorium, and uranium into root and shoot tissues of Plantago lanceolata. The results indicated that none of the treatments had any positive or negative impact on shoot and root biomass. However, Rhizophagus irregularis treatments showed higher accumulation of copper and zinc in shoots, while R. irregularis and Septoglomus constrictum enhanced arsenic accumulation in roots. Moreover, R. irregularis increased uranium concentration in roots and shoots of the P. lanceolata plant. This study provides useful insight into fungal-plant interactions that determine metal and radionuclide transfer from soil into the biosphere at contaminated sites such as mine workings.

A multiscale modelling approach for Haematococcus pluvialis cultivation under different environmental conditions.

Haematococcus pluvialis can produce significant amounts of industrially important compounds belonging to lipids and starch classes, including various specific pigments such as ß-carotene, lutein and astaxanthin, as well as lipids, carbohydrates and proteins. Their production can vary depending on environmental stress conditions like nutrient starvation. However, stress conditions lead also to undesired phenomena such as cell lysis, which is likely to be related to products loss. The microorganism develops towards smaller single cell volumes during the growth process, and eventually, more likely towards lysis when fission (i.e. cell division) slows down. The lysis process takes place simultaneously with nutrient depletion, so both growth and lysis are linked to the change of environmental conditions. In this work, we develop a novel multiscale segregated-structured model based on Population Balance Equations (PBEs) to describe the photoautotrophic growth of H.pluvialis, in particular cell growth, and lysis, making possible the description of the relationship between cell volume/transition, cell loss, and metabolic product availability. Cell volume is the internal coordinate of the population balance model, and its link with intrinsic concentrations is also presented. The model parameters are fitted against experimental data, extensive sensitivity analysis is performed and the model predictive capabilities are tested in terms of cell density distributions, as well as 0th and 1st order moments.

Phylogenetic analysis and structural prediction reveal the potential functional diversity between green algae SWEET transporters.

Sugar-Will-Eventually-be-Exported-Transporters (SWEETs) are an important family of sugar transporters that appear to be ubiquitous in all organisms. Recent research has determined the structure of SWEETs in higher plants, identified specific residues required for monosaccharide or disaccharide transport, and begun to understand the specific functions of individual plant SWEET proteins. However, in green algae (Chlorophyta) these transporters are poorly characterised. This study identified SWEET proteins from across representative Chlorophyta with the aim to characterise their phylogenetic relationships and perform protein structure modelling in order to inform functional prediction. The algal genomes analysed encoded between one and six SWEET proteins, which is much less than a typical higher plant. Phylogenetic analysis identified distinct clusters of over 70 SWEET protein sequences, taken from almost 30 algal genomes. These clusters remain separate from representative higher or non-vascular plant SWEETs, but are close to fungi SWEETs. Subcellular localisation predictions and analysis of conserved amino acid residues revealed variation between SWEET proteins of different clusters, suggesting different functionality. These findings also showed conservation of key residues at the substrate-binding site, indicating a similar mechanism of substrate selectivity and transport to previously characterised higher plant monosaccharide-transporting SWEET proteins. Future work is now required to confirm the predicted sugar transport specificity and determine the functional role of these algal SWEET proteins.

Cadmium exposure and phosphorus limitation increases metal content in the freshwater alga Chlamydomonas reinhardtii.

The characteristics of metal accumulation in freshwater microalgae are important to elucidate for a full understanding of metal cycling and toxicity in a freshwater system. This study has utilized an elemental profiling approach to investigate the impacts of Cd exposure and phosphorus (P) availability on metal accumulation after 7 days in batch culture-grown Chlamydomonas reinhardtii. Multivariate statistical analysis of the elemental data demonstrated distinct responses between both stresses. Sublethal concentrations of Cd (up to 15 µM) caused increased accumulation of Co. Cu, Fe, and Zn content also increased in response to enhanced Cd concentrations but only when P availability was low. While Cd exposure effected the accumulation of a few specific metals, P limitation increased the accumulation of all essential trace metals and macronutrients analyzed including Co, Fe, K, Na, and Zn but not Mn. The accumulation of Cd also markedly increased in response to P limitation. The impact of P availability on essential metal accumulation was the same when either inorganic P or an organic P source (glycerophosphate) was used. These results highlight the potential risks of metal toxicity for freshwater microalgae and aquatic food chains when P availability is limiting and which can be exacerbated by Cd pollution.