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.

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.

Organic complexation of U(VI) in reducing soils at a natural analogue site: Implications for uranium transport.

Understanding the long-term fate, stability, and bioavailability of uranium (U) in the environment is important for the management of nuclear legacy sites and radioactive wastes. Analysis of U behavior at natural analogue sites permits evaluation of U biogeochemistry under conditions more representative of long-term equilibrium. Here, we have used bulk geochemical and microbial community analysis of soils, coupled with X-ray absorption spectroscopy and µ-focus X-ray fluorescence mapping, to gain a mechanistic understanding of the fate of U transported into an organic-rich soil from a pitchblende vein at the UK Needle's Eye Natural Analogue site. U is highly enriched in the Needle's Eye soils (∼1600 mg kg-1). We show that this enrichment is largely controlled by U(VI) complexation with soil organic matter and not U(VI) bioreduction. Instead, organic-associated U(VI) seems to remain stable under microbially-mediated Fe(III)-reducing conditions. U(IV) (as non-crystalline U(IV)) was only observed at greater depths at the site (>25 cm); the soil here was comparatively mineral-rich, organic-poor, and sulfate-reducing/methanogenic. Furthermore, nanocrystalline UO2, an alternative product of U(VI) reduction in soils, was not observed at the site, and U did not appear to be associated with Fe-bearing minerals. Organic-rich soils appear to have the potential to impede U groundwater transport, irrespective of ambient redox conditions.

Multiple environmental factors influence 238U, 232Th and 226Ra bioaccumulation in arbuscular mycorrhizal-associated plants.

Ecological consequences of low-dose radioactivity from natural sources or radioactive waste are important to understand but knowledge gaps still remain. In particular, the soil transfer and bioaccumulation of radionuclides into plant roots is poorly studied. Furthermore, better knowledge of arbuscular mycorrhizal (AM) fungi association may help understand the complexities of radionuclide bioaccumulation within the rhizosphere. Plant bioaccumulation of uranium, thorium and radium was demonstrated at two field sites, where plant tissue concentrations reached up to 46.93 µg g-1 238U, 0.67 µg g-1 232Th and 18.27 kBq kg-1 226Ra. High root retention of uranium was consistent in all plant species studied. In contrast, most plants showed greater bioaccumulation of thorium and radium into above-ground tissues. The influence of specific soil parameters on root radionuclide bioaccumulation was examined. Total organic carbon significantly explained the variation in root uranium concentration, while other soil factors including copper concentration, magnesium concentration and pH significantly correlated with root concentrations of uranium, radium and thorium, respectively. All four orders of Glomeromycota were associated with root samples from both sites and all plant species studied showed varying association with AM fungi, ranging from zero to >60% root colonisation by fungal arbuscules. Previous laboratory studies using single plant-fungal species association had found a positive role of AM fungi in root uranium transfer, but no significant correlation between the amount of fungal infection and root uranium content in the field samples was found here. However, there was a significant negative correlation between AM fungal infection and radium accumulation. This study is the first to examine the role of AM fungi in radionuclide soil-plant transfer at a community level within the natural environment. We conclude that biotic factors alongside various abiotic factors influence the soil-plant transfer of radionuclides and future mechanistic studies are needed to explain these interactions in more detail.

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.

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.

Using FTIR spectroscopy for rapid determination of lipid accumulation in response to nitrogen limitation in freshwater microalgae.

In this study Fourier transform infrared micro-spectroscopy (FTIR) was used to determine lipid and carbohydrate content over time in the freshwater microalgae Chlamydomonas reinhardtii and Scenedesmus subspicatus grown in batch culture in limiting concentrations of nitrogen (N). Both algae exhibited restricted cell division and increased cell size following N-limitation. FTIR spectra of cells in N-limited media showed increasing lipid:amide I and carbohydrate:amide I ratios over time. The use of lipid- and starch-staining dyes confirmed that the observed ratio changes were due to increased lipid and carbohydrate synthesis. These results demonstrate rapid metabolic responses of C. reinhardtii and S. subspicatus to changing nutrient availability, and indicate the efficiency of FTIR as a reliable method for high-throughput determination of lipid induction.

A role for the AtMTP11 gene of Arabidopsis in manganese transport and tolerance.

The Arabidopsis AtMTP family of genes encode proteins of the cation diffusion facilitator (CDF) family, with several members having roles in metal tolerances. Four of the 11 proteins in the family form a distinct cluster on a phylogenetic tree and are closely related to ShMTP8, a CDF identified in the tropical legume Stylosanthes hamata that is implicated in the transport of Mn(2+) into the vacuole as a tolerance mechanism. Of these four genes, AtMTP11 was the most highly expressed member of the Arabidopsis subgroup. When AtMTP11 was expressed in Saccharomyces cerevisiae, it conferred Mn(2+) tolerance and transported Mn(2+) by a proton-antiport mechanism. A mutant of Arabidopsis with a disrupted AtMTP11 gene (mtp11) was found to have increased sensitivity to Mn(2+) but not to Cu(2+) or Zn(2+). At a non-toxic but sufficient Mn(2+) supply (basal), the mutant accumulated more Mn(2+) than the wild type, but did not show any obvious deleterious effects on growth. When grown with Mn(2+) supplies that ranged from basal to toxic, the mutant accumulated Mn(2+) concentrations in shoots similar to those in wild-type plants, despite showing symptoms of Mn(2+) toxicity. AtMTP11 fused to green fluorescent protein co-localized with a reporter specific for pre-vacuolar compartments. These findings provide evidence for Mn(2+)-specific transport activity by AtMTP11, and implicate the pre-vacuolar compartments in both Mn(2+) tolerance and Mn(2+) homeostasis mechanisms of Arabidopsis.

Characterization of CAX4, an Arabidopsis H(+)/cation antiporter.

Ion compartmentalization is essential for plant growth and development. The Arabidopsis open reading frames for CAX1, CAX2, and CAX3 (cation exchangers 1, 2, and 3) were previously identified as transporters that may modulate ion fluxes across the vacuolar membrane. To understand the diversity and role of H(+)/cation transporters in controlling plant ion levels, another homolog of the CAX genes, CAX4, was cloned from an Arabidopsis cDNA library. CAX4 is 53% identical to CAX1 at the amino acid level, 42% identical to CAX2, and 54% identical to CAX3. CAX4 transcripts appeared to be expressed at low levels in all tissues and levels of CAX4 RNA increased after Mn(2+), Na(+), and Ni(2+) treatment. An N-terminal CAX4-hemagglutinin fusion appeared to localize to both yeast and plant vacuolar membranes. When expressed in yeast, CAX4, like CAX3, failed to suppress the Ca(2+) sensitivity of yeast strains deficient in vacuolar Ca(2+) transport. Several modifications to CAX4 allowed the protein to transport Ca(2+). Addition of amino acids to the N terminus of CAX4 and CAX3 caused both transporters to suppress the sensitivity of yeast strains deficient in vacuolar Ca(2+) transport. These findings suggest that CAX transporters may modulate their ion transport properties through alterations at the N terminus.