Phenotypic and physiological responses to salt exposure in Sorghum reveal diversity among domesticated landraces.

PREMISE: Soil salinity negatively impacts plant function, development, and yield. To overcome this impediment to agricultural productivity, variation in morphological and physiological response to salinity among genotypes of important crops should be explored. Sorghum bicolor is a staple crop that has adapted to a variety of environmental conditions and contains a significant amount of standing genetic diversity, making it an exemplary species to study variation in salinity tolerance. METHODS: Twenty-one diverse Sorghum accessions were treated with nonsaline water or 75 mM sodium chloride. Salinity tolerance was assessed via changes in biomass between control and salt-treated individuals. Accessions were first rank-ordered for salinity tolerance, and then individuals spanning a wide range of responses were analyzed for foliar proline and ion accumulation. Tolerance rankings were then overlaid on a neighbor-joining tree. RESULTS: We found that, while proline is often a good indicator of osmotic adjustment and is historically associated with increased salt tolerance in many species, proline accumulation in sorghum reflects a stress response injury rather than acclimation. When combining ion profiles with stress tolerance indices, the variation observed in tolerance was not a sole result of Na+ accumulation, but rather reflected accession-specific mechanisms. CONCLUSIONS: We identified significant variation in salinity tolerance among Sorghum accessions that may be a result of the domestication history of Sorghum. When we compared our results with known phylogenetic relationships within sorghum, the most parsimonious explanation for our findings is that salinity tolerance was acquired early during domestication and subsequently lost in accessions growing in areas varying in soil salinity.

Pseudomonas fluorescens increases mycorrhization and modulates expression of antifungal defense response genes in roots of aspen seedlings.

BACKGROUND: Plants, fungi, and bacteria form complex, mutually-beneficial communities within the soil environment. In return for photosynthetically derived sugars in the form of exudates from plant roots, the microbial symbionts in these rhizosphere communities provide their host plants access to otherwise inaccessible nutrients in soils and help defend the plant against biotic and abiotic stresses. One role that bacteria may play in these communities is that of Mycorrhizal Helper Bacteria (MHB). MHB are bacteria that facilitate the interactions between plant roots and symbiotic mycorrhizal fungi and, while the effects of MHB on the formation of plant-fungal symbiosis and on plant health have been well documented, the specific molecular mechanisms by which MHB drive gene regulation in plant roots leading to these benefits remain largely uncharacterized. RESULTS: Here, we investigate the effects of the bacterium Pseudomonas fluorescens SBW25 (SBW25) on aspen root transcriptome using a tripartite laboratory community comprised of Populus tremuloides (aspen) seedlings and the ectomycorrhizal fungus Laccaria bicolor (Laccaria). We show that SBW25 has MHB activity and promotes mycorrhization of aspen roots by Laccaria. Using transcriptomic analysis of aspen roots under multiple community compositions, we identify clusters of co-regulated genes associated with mycorrhization, the presence of SBW25, and MHB-associated functions, and we generate a combinatorial logic network that links causal relationships in observed patterns of gene expression in aspen seedling roots in a single Boolean circuit diagram. The predicted regulatory circuit is used to infer regulatory mechanisms associated with MHB activity. CONCLUSIONS: In our laboratory conditions, SBW25 increases the ability of Laccaria to form ectomycorrhizal interactions with aspen seedling roots through the suppression of aspen root antifungal defense responses. Analysis of transcriptomic data identifies that potential molecular mechanisms in aspen roots that respond to MHB activity are proteins with homology to pollen recognition sensors. Pollen recognition sensors integrate multiple environmental signals to down-regulate pollenization-associated gene clusters, making proteins with homology to this system an excellent fit for a predicted mechanism that integrates information from the rhizosphere to down-regulate antifungal defense response genes in the root. These results provide a deeper understanding of aspen gene regulation in response to MHB and suggest additional, hypothesis-driven biological experiments to validate putative molecular mechanisms of MHB activity in the aspen-Laccaria ectomycorrhizal symbiosis.

Expression analysis and functional characterization of two PHT1 family phosphate transporters in ryegrass.

MAIN CONCLUSION: The phosphate transporters LpPHT1;1 and LpPHT1;4 have different roles in phosphate uptake and translocation in ryegrass under P stress condition. The phosphate transporter 1 (PHT1) family are integral membrane proteins that operate in phosphate uptake, distribution and remobilization within plants. In this study, we report on the functional characterization and expression of two PHT1 family members from ryegrass plants (Lolium perenne L.) and determine their roles in the specificity of Pi transport. The expression level of LpPHT1;4 was strongly influenced by phosphorus (P) status, being higher under P-starvation condition. In contrast, the expression level of LpPHT1;1 was not correlated with P supply. Yeast mutant complementation assays showed that LpPHT1;4 can complement the growth defect of the yeast mutant Δpho84 under Pi-deficient conditions, whereas the yeast mutant expressing LpPHT1;1 was not able to restore growth. Phylogenetic and molecular analyses indicated high sequence similarity to previously identified PHT1s from other species in the Poaceae. These results suggest that LpPHT1;1 may function as a low-affinity Pi transporter, whereas LpPHT1;4 could acts as a high-affinity Pi transporter to maintain Pi homeostasis under stress conditions in ryegrass plants. This study will form the basis for the long-term goal of improving the phosphate use efficiency of ryegrass plants.

Comparative Phytonutrient Analysis of Broccoli By-Products: The Potentials for Broccoli By-Product Utilization.

The phytonutrient concentrations of broccoli (Brassica oleracea var. italica) florets, stems, and leaves were compared to evaluate the value of stem and leaf by-products as a source of valuable nutrients. Primary metabolites, including amino acids, organic acids, and sugars, as well as glucosinolates, carotenoids, chlorophylls, vitamins E and K, essential mineral elements, total phenolic content, antioxidant activity, and expression of glucosinolate biosynthesis and hydrolysis genes were quantified from the different broccoli tissues. Broccoli florets had higher concentrations of amino acids, glucoraphanin, and neoglucobrassicin compared to other tissues, whereas leaves were higher in carotenoids, chlorophylls, vitamins E and K, total phenolic content, and antioxidant activity. Leaves were also good sources of calcium and manganese compared to other tissues. Stems had the lowest nitrile formation from glucosinolate. Each tissue exhibited specific core gene expression profiles supporting glucosinolate metabolism, with different gene homologs expressed in florets, stems, and leaves, which suggests that tissue-specific pathways function to support primary and secondary metabolic pathways in broccoli. This comprehensive nutrient and bioactive compound profile represents a useful resource for the evaluation of broccoli by-product utilization in the human diet, and as feedstocks for bioactive compounds for industry.

Soluble soil aluminum alters the relative uptake of mineral nitrogen forms by six mature temperate broadleaf tree species: possible implications for watershed nitrate retention.

Increased availability of monomeric aluminum (Al3+) in forest soils is an important adverse effect of acidic deposition that reduces root growth and inhibits nutrient uptake. There is evidence that Al3+ exposure interferes with NO3- uptake. If true for overstory trees, the reduction in stand demand for NO3- could increase NO3- discharge in stream water. These effects may also differ between species that tolerate different levels of soil acidity. To examine these ideas, we measured changes in relative uptake of NO3- and NH4+ by six tree species in situ under increased soil Al3+ using a 15N-labeling technique, and measured soluble soil Al levels in a separate whole-watershed acidification experiment in the Fernow Experimental Forest (WV). When exposed to added Al3+, the proportion of inorganic N acquired as NO3- dropped 14% across species, but we did not detect a reduction in overall N uptake, nor did tree species differ in this response. In the long-term acidification experiment, we found that soluble soil Al was mostly in the free Al3+ form, and the concentration of Al3+ was ~65 µM higher (~250%) in the mineral soil of the acidified watershed vs. an untreated watershed. Thus, increased levels of soil Al3+ under acidic deposition cause a reduction in uptake of NO3- by mature trees. When our 15N uptake results were applied to the watershed acidification experiment, they suggest that increased Al3+ exposure could reduce tree uptake of NO3- by 7.73 kg N ha-1 year-1, and thus increase watershed NO3- discharge.

The influence of phosphorus availability and Laccaria bicolor symbiosis on phosphate acquisition, antioxidant enzyme activity, and rhizospheric carbon flux in Populus tremuloides.

Many forest tree species are dependent on their symbiotic interaction with ectomycorrhizal (ECM) fungi for phosphorus (P) uptake from forest soils where P availability is often limited. The ECM fungal association benefits the host plant under P limitation through enhanced soil exploration and increased P acquisition by mycorrhizas. To study the P starvation response (PSR) and its modification by ECM fungi in Populus tremuloides, a comparison was made between nonmycorrhizal (NM) and mycorrhizal with Laccaria bicolor (Myc) seedlings grown under different concentrations of phosphate (Pi) in sand culture. Although differences in growth between NM and Myc plants were small, Myc plants were more effective at acquiring P from low Pi treatments, with significantly lower k m values for root and leaf P accumulation. Pi limitation significantly increased the activity of catalase, ascorbate peroxidase, and guaiacol-dependent peroxidase in leaves and roots to greater extents in NM than Myc P. tremuloides. Phosphoenolpyruvate carboxylase activity also increased in NM plants under P limitation, but was unchanged in Myc plants. Formate, citrate, malonate, lactate, malate, and oxalate and total organic carbon exudation by roots was stimulated by P limitation to a greater extent in NM than Myc plants. Colonization by L. bicolor reduced the solution Pi concentration thresholds where PSR physiological changes occurred, indicating that enhanced Pi acquisition by P. tremuloides colonized by L. bicolor altered host P homeostasis and plant stress responses to P limitation. Understanding these plant-symbiont interactions facilitates the selection of more P-efficient forest trees and strategies for tree plantation production on marginal soils.

Development of soils and communities of plants and arbuscular mycorrhizal fungi on West Virginia surface mines.

Surface mining followed by reclamation to pasture is a major driver of land use and cover change in Appalachia. Prior research suggests that many aspects of ecosystem recovery are either slow or incomplete. We examined ecosystem structure-including soil physical and chemical properties, arbuscular mycorrhizal fungal (AMF) infectivity and community composition, and plant diversity and community composition-on a chronosequence of pasture-reclaimed surface mines and a non-mined pasture in northern West Virginia. Surface mining and reclamation dramatically altered ecosystem structure. Some aspects of ecosystem structure, including many measures of soil chemistry and infectivity of AMF, returned rapidly to levels found on the non-mined reference site. Other aspects of ecosystem structure, notably soil physical properties and AMF and plant communities, showed incomplete or no recovery over the short-to-medium term. In addition, invasive plants were prevalent on reclaimed mine sites. The results point to the need for investigation on how reclamation practices could minimize establishment of exotic invasive plant species and reduce the long-term impacts of mining on ecosystem structure and function.

A multi-omic survey of black cottonwood tissues highlights coordinated transcriptomic and metabolomic mechanisms for plant adaptation to phosphorus deficiency.

Introduction: Phosphorus (P) deficiency in plants creates a variety of metabolic perturbations that decrease photosynthesis and growth. Phosphorus deficiency is especially challenging for the production of bioenergy feedstock plantation species, such as poplars (Populus spp.), where fertilization may not be practically or economically feasible. While the phenotypic effects of P deficiency are well known, the molecular mechanisms underlying whole-plant and tissue-specific responses to P deficiency, and in particular the responses of commercially valuable hardwoods, are less studied. Methods: We used a multi-tissue and multi-omics approach using transcriptomic, proteomic, and metabolomic analyses of the leaves and roots of black cottonwood (Populus trichocarpa) seedlings grown under P-deficient (5 µM P) and replete (100 µM P) conditions to assess this knowledge gap and to identify potential gene targets for selection for P efficiency. Results: In comparison to seedlings grown at 100 µM P, P-deficient seedlings exhibited reduced dry biomass, altered chlorophyll fluorescence, and reduced tissue P concentrations. In line with these observations, growth, C metabolism, and photosynthesis pathways were downregulated in the transcriptome of the P-deficient plants. Additionally, we found evidence of strong lipid remodeling in the leaves. Metabolomic data showed that the roots of P-deficient plants had a greater relative abundance of phosphate ion, which may reflect extensive degradation of P-rich metabolites in plants exposed to long-term P-deficiency. With the notable exception of the KEGG pathway for Starch and Sucrose Metabolism (map00500), the responses of the transcriptome and the metabolome to P deficiency were consistent with one another. No significant changes in the proteome were detected in response to P deficiency. Discussion and conclusion: Collectively, our multi-omic and multi-tissue approach enabled the identification of important metabolic and regulatory pathways regulated across tissues at the molecular level that will be important avenues to further evaluate for P efficiency. These included stress-mediating systems associated with reactive oxygen species maintenance, lipid remodeling within tissues, and systems involved in P scavenging from the rhizosphere.

The role of arbuscular mycorrhizas in decreasing aluminium phytotoxicity in acidic soils: a review.

Soil acidity is an impediment to agricultural production on a significant portion of arable land worldwide. Low productivity of these soils is mainly due to nutrient limitation and the presence of high levels of aluminium (Al), which causes deleterious effects on plant physiology and growth. In response to acidic soil stress, plants have evolved various mechanisms to tolerate high concentrations of Al in the soil solution. These strategies for Al detoxification include mechanisms that reduce the activity of Al3+ and its toxicity, either externally through exudation of Al-chelating compounds such as organic acids into the rhizosphere or internally through the accumulation of Al-organic acid complexes sequestered within plant cells. Additionally, root colonization by symbiotic arbuscular mycorrhizal (AM) fungi increases plant resistance to acidity and phytotoxic levels of Al in the soil environment. In this review, the role of the AM symbiosis in increasing the Al resistance of plants in natural and agricultural ecosystems under phytotoxic conditions of Al is discussed. Mechanisms of Al resistance induced by AM fungi in host plants and variation in resistance among AM fungi that contribute to detoxifying Al in the rhizosphere environment are considered with respect to altering Al bioavailability.

Mitochondrial metabolism is central for response and resistance of Saccharomyces cerevisiae to exposure to a glyphosate-based herbicide.

Glyphosate-based herbicides, the most extensively used herbicides in the world, are available in an enormous number of commercial formulations with varying additives and adjuvants. Here, we study the effects of one such formulation, Credit41, in two genetically diverse yeast strains. A quantitative trait loci (QTL) analysis between a sensitive laboratory strain and a resistant strain linked mitochondrial function to Credit41 resistance. Two genes encoding mitochondrial proteins identified through the QTL analysis were HFA1, a gene that encodes a mitochondrial acetyl CoA carboxylase, and AAC3, which encodes a mitochondrial inner membrane ATP/ADP translocator. Further analysis of previously studied whole-genome sequencing data showed that, although each strain uses varying routes to attain glyphosate resistance, most strains have duplications of mitochondrial genes. One of the most well-studied functions of the mitochondria is the assembly of Fe-S clusters. In the current study, the expression of iron transporters in the transcriptome increased in cells resistant to Credit41. The levels of iron within the cell also increased in cells exposed to Credit41 but not pure glyphosate. Hence, the additives in glyphosate-based herbicides have a significant contribution to the negative effects of these commercial formulations on biological systems.

MCHM Acts as a Hydrotrope, Altering the Balance of Metals in Yeast.

While drugs and other industrial chemicals are routinely studied to assess risks, many widely used chemicals have not been thoroughly evaluated. One such chemical, 4-methylcyclohexane methanol (MCHM), is an industrial coal-cleaning chemical that contaminated the drinking water supply in Charleston, WV, USA in 2014. While a wide range of ailments was reported following the spill, little is known about the molecular effects of MCHM exposure. We used the yeast model to explore the impacts of MCHM on cellular function. Exposure to MCHM dramatically altered the yeast transcriptome and the balance of metals in yeast. Underlying genetic variation in the response to MCHM, transcriptomics and, mutant analysis uncovered the role of the metal transporters, Arn2 and Yke4, to MCHM response. Expression of Arn2, which is involved in iron uptake, was lower in MCHM-tolerant yeast and loss of Arn2 further increased MCHM tolerance. Genetic variation within Yke4, an ER zinc transporter, also mediated response to MCHM, and loss of Yke4 decreased MCHM tolerance. The addition of zinc to MCHM-sensitive yeast rescued growth inhibition. In vitro assays demonstrated that MCHM acted as a hydrotrope and prevented protein interactions, while zinc induced the aggregation of proteins. We hypothesized that MCHM altered the structures of extracellular domains of proteins, and the addition of zinc stabilized the structure to maintain metal homeostasis in yeast exposed to MCHM.

Rhizosphere carbon deposition, oxidative stress and nutritional changes in two poplar species exposed to aluminum.

Species and hybrids in the genus Populus have become the focus of investigation for use in biofuels production and their capacity to sequester carbon (C) in the environment. The identification of species resistant to marginal edaphic sites may be important in both of these endeavors. Plant growth, total dissolved organic carbon (TOC) and low molecular weight organic acid (OA) production, antioxidative enzyme activities and mineral content were assessed in Populus tremuloides L. and Populus trichocarpa Torr. & Gray seedlings under exposure to aluminum (Al). Both species were sensitive to Al, with significant reductions in shoot and root biomass at and above 50 microM Al. Exposure to Al induced 40-fold increases in TOC deposition in P. tremuloides and 100-fold increases in P. trichocarpa. In P. tremuloides, Al treatment induced root exudation of malic and citric acids, while Al increased exudation of citrate and oxalate in P. trichocarpa. Organic acids accounted for 20-64% of total C released upon Al exposure, with the proportion of OAs increasing in P. tremuloides and decreasing in P. trichocarpa. Dose-dependent responses of catalase and ascorbate peroxidase were observed in both root and leaf tissues, indicating that Al exposure induced oxidative stress in poplar. Treatment at and above 100 microM Al reduced the concentrations of calcium (Ca) and magnesium (Mg) in roots and leaves, whereas Al at or above 50 microM reduced root and leaf phosphorous (P) concentrations. The majority of Al taken up was retained in the root system. Even with the induction of OA exudation and accumulation, P. tremuloides and P. trichocarpa remained sensitive to Al, as evidenced by elevated antioxidative enzyme activities, which may reflect inhibition of Ca or P uptake and destabilization of cell homeostasis in these poplar species. Although plants exhibited reductions in growth and evidence of oxidative and nutritional stress, total C rhizodeposition rates for both species increased with increasing Al exposure concentration. Estimated C deposition rates of 16 mg C plant(-1) day(-1) were four-times larger than previously reported values for forest tree species, indicating that edaphic stress plays an important role in C flux to the rhizosphere.

Carbon allocation and partitioning in Populus tremuloides are modulated by ectomycorrhizal fungi under phosphorus limitation.

The fate of carbon (C) captured by forest trees during photosynthesis is influenced by the supply of other resources. Fixed C may be partitioned among biomolecules within the leaf and/or allocated throughout the tree to growth, storage and maintenance activities. Phosphorus (P) availability often limits tree productivity due to its high biological demand and strong interactions with soil minerals. As ectomycorrhizal (ECM) fungi play critical roles in enhancing phosphate (Pi) acquisition by their hosts, these symbioses will influence the fate of C within trees and forested ecosystems. Using Populus tremuloides Michx. (trembling aspen) in symbiosis with Laccaria bicolor (Marie) P.D. Orton or Paxillus involutus (Batsch) Fr., we assessed C acquisition, allocation and partitioning under Pi limitation, specifically focusing on primary and secondary C compounds. Both ECM fungi moderated the effects of low P on photosynthesis and C partitioning among carbohydrates and secondary metabolites by sustaining Pi uptake and translocation in P. tremuloides under Pi limitation. As leaf P declined, reductions in photosynthesis were accompanied by significant shifts in C partitioning from nonstructural carbohydrates (NSCs) to phenolic glycosides and tannins. Carbon partitioning in roots exhibited more complex patterns, with distinct increases in NSCs in nonmycorrhizal (NM) plants under Pi limitation that were not evident in plants colonized by either ECM symbiont. In general, aspen colonized by L. bicolor exhibited C partitioning patterns intermediate between those of NM and P. involutus aspen. The C cost of symbiosis was pronounced for plants supporting P. involutus, where ECM plants exhibited maintenance of photosynthesis yet reduced biomass in comparison with NM and L. bicolor aspen under Pi replete conditions. Our results indicate that the ECM symbiosis affects the disposition of C in forest trees in part by altering the acquisition of other limiting resources from soils, but also through ECM species-specific influences on host physiology. This modulation of C partitioning will have broad implications for forest ecosystem C capture, storage and cycling where nutrient resources may be limited.

Variations in organic acid exudation and aluminum resistance among arbuscular mycorrhizal species colonizing Liriodendron tulipifera.

Aluminum (Al) in acidic soils is toxic to plants, affecting growth, water uptake and nutrient assimilation. Aluminum resistance in some plant species and genotypes has been ascribed to organic acid exudation from roots and arbuscular mycorrhizal (AM) fungal symbiosis. We investigated variation among several AM species in altering Al resistance of Liriodendron tulipifera L. and evaluated AM influence on organic acid production as a potential Al resistance mechanism. Growth, nutritional responses and rhizosphere organic acid profiles were assessed for seedlings in association with Acaulospora morrowiae Spain & Schenck, Glomus claroideum Schenck & Smith, G. clarum Nicol. & Schenck or Paraglomus brasilianum (Spain & Miranda) Morton & Redecker and non-mycorrhizal seedlings exposed to 0, 50 or 200 microM Al. Plants colonized by G. clarum had the greatest biomass, least Al and most phosphorus (P) in leaf tissues and exuded malate and citrate into the rhizosphere at rates that complexed 99% of delivered Al in all treatments. Other AM fungi did not confer significant Al resistance on L. tulipifera and did not maintain citrate and malate exudation in response to Al exposure. This study illustrates functional diversity among AM fungal species in conferred Al resistance to plants and highlights the potential importance of fungal diversity in ecosystem responses to environmental stresses.

Pseudomonas fluorescens Transportome Is Linked to Strain-Specific Plant Growth Promotion in Aspen Seedlings under Nutrient Stress.

Diverse communities of bacteria colonize plant roots and the rhizosphere. Many of these rhizobacteria are symbionts and provide plant growth promotion (PGP) services, protecting the plant from biotic and abiotic stresses and increasing plant productivity by providing access to nutrients that would otherwise be unavailable to roots. In return, these symbiotic bacteria receive photosynthetically-derived carbon (C), in the form of sugars and organic acids, from plant root exudates. PGP activities have been characterized for a variety of forest tree species and are important in C cycling and sequestration in terrestrial ecosystems. The molecular mechanisms of these PGP activities, however, are less well-known. In a previous analysis of Pseudomonas genomes, we found that the bacterial transportome, the aggregate activity of a bacteria's transmembrane transporters, was most predictive for the ecological niche of Pseudomonads in the rhizosphere. Here, we used Populus tremuloides Michx. (trembling aspen) seedlings inoculated with one of three Pseudomonas fluorescens strains (Pf0-1, SBW25, and WH6) and one Pseudomonas protegens (Pf-5) as a laboratory model to further investigate the relationships between the predicted transportomic capacity of a bacterial strain and its observed PGP effects in laboratory cultures. Conditions of low nitrogen (N) or low phosphorus (P) availability and the corresponding replete media conditions were investigated. We measured phenotypic and biochemical parameters of P. tremuloides seedlings and correlated P. fluorescens strain-specific transportomic capacities with P. tremuloides seedling phenotype to predict the strain and nutrient environment-specific transporter functions that lead to experimentally observed, strain, and media-specific PGP activities and the capacity to protect plants against nutrient stress. These predicted transportomic functions fall in three groups: (i) transport of compounds that modulate aspen seedling root architecture, (ii) transport of compounds that help to mobilize nutrients for aspen roots, and (iii) transporters that enable bacterial acquisition of C sources from seedling root exudates. These predictions point to specific molecular mechanisms of PGP activities that can be directly tested through future, hypothesis-driven biological experiments.

Proteomic and genetic analysis of the response of S. cerevisiae to soluble copper leads to improvement of the antimicrobial function of cellulosic copper nanoparticles.

Copper (Cu) was used in antiquity to prevent waterborne and food diseases because, as a broad-spectrum antimicrobial agent, it generates reactive oxygen species, ROS. New technologies incorporating Cu into low-cost biodegradable nanomaterials built on cellulose, known as cellulosic cupric nanoparticles or c-CuNPs, present novel approaches to deliver Cu in a controlled manner to control microbial growth. We challenged strains of Saccharomyces cerevisiae with soluble Cu and c-CuNPs to evaluate the potential of c-CuNPs as antifungal agents. Cells exposed to c-CuNPs demonstrated greater sensitivity to Cu than cells exposed to soluble Cu, although Cu-resistant strains were more tolerant than Cu-sensitive strains of c-CuNP exposure. At the same level of growth inhibition, 157 µM c-CuNPs led to the same internal Cu levels as did 400 µM CuSO4, offering evidence for alternative mechanisms of toxicity, perhaps through ß-arrestin dependent endocytosis, which was supported by flow cytometry and fluorescence microscopy of c-CuNPs distributed both on the cell surface and within the cytoplasm. Genes responsible for genetic variation in response to copper were mapped to the ZRT2 and the CUP1 loci. Through proteomic analyses, we found that the expression of other zinc (Zn) transporters increased in Cu-tolerant yeast compared to Cu-sensitive strains. Further, the addition of Zn at low levels increased the potency of c-CuNPs to inhibit even the most Cu-tolerant yeast. Through unbiased systems biological approaches, we identified Zn as a critical component of the yeast response to Cu and the addition of Zn increased the potency of the c-CuNPs.

Activation of metallothioneins and alpha-crystallin/sHSPs in human lens epithelial cells by specific metals and the metal content of aging clear human lenses.

PURPOSE: To identify those metallothionein and alpha-crystallin/small heat-shock genes induced by toxic metals in human lens cells and to evaluate the levels of these metals between young and aged human lenses. METHODS: Human SRA01/04 and primary human lens epithelial cells were cultured and exposed to Cd(2+), Cu(2+), and Zn(2+). The levels of lens metallothioneins (Ig, If, Ih, Ie, and IIa) and alpha-crystallin/small heat-shock (alphaA-crystallin, alphaB-crystallin, and HSP27) genes were analyzed by semiquantitative and quantitative competitive RT-PCR. The content of aluminum, cadmium, calcium, chromium, copper, iron, lead, magnesium, manganese, nickel, potassium, sodium, and zinc in young (mean, 32.8 years), middle-aged (mean, 52.3 years), and old (mean, 70.5 years) human lenses was analyzed by inductively coupled plasma-emission spectroscopy. RESULTS: Lens metallothioneins (Ig, If, Ih, Ie, and IIa) and alpha-crystallin/small heat-shock genes (alphaA-crystallin, alphaB-crystallin, and HSP27) were differentially induced by specific metals in SRA01/04 human lens epithelial cells. Cd(2+) and Zn(2+), but not Cu(2+), induced the metallothioneins, whereas Cd(2+) and Cu(2+), but not Zn(2+), induced alphaB-crystallin and HSP27. alphaA-crystallin was induced by Cu(2+) only. Similar responses of the metallothionein IIa gene were detected in identically treated primary human lens epithelial cells. Cd(2+) and Zn(2+) induced metallothionein IIa to five times higher levels than metallothionein Ig. Of 13 different metals, only iron was altered, exhibiting an 81% decrease in old versus young lenses. CONCLUSIONS: Induction of metallothioneins and alpha-crystallin/small heat shock proteins by different metals indicates the presence of metal-specific lens regulatory pathways that are likely to be involved in protection against metal-associated stresses.

Phosphate-limitation physiology in ectomycorrhizal pitch pine (Pinus rigida) seedlings.

Foliar and root P concentrations, net H(2)PO(4) (-) (P(i)) uptake rates, and root surface acid phosphatase (APase) rates were assessed in pitch pine (Pinus rigida Mill.) seedlings inoculated with the ectomycorrhizal fungi Laccaria bicolor (Maire) Pat., Paxillus involutus (Batsch.) Fr., or Pisolithus tinctorius (Pers.) Coker and Couch, and grown at 10 or 100 micro M P(i) in sand culture. Following a 6-week period of acclimation to the P(i) regimes, seedlings grown at 100 micro M P(i) had greater foliar and root P concentrations than seedlings grown at 10 micro M P(i). Mycorrhizal colonization increased the concentration of P in roots and, under P(i)-limiting conditions, this retention was at the expense of P translocation to foliage. There were no differences in P(i) uptake rates between non-mycorrhizal and mycorrhizal roots grown at 100 micro M P(i). However, mycorrhizal colonization enhanced P(i) uptake in seedlings grown at 10 micro M P(i), with rates 1.3-, 2.6-, and 3.3-fold greater in roots colonized with L. bicolor, P. involutus, and P. tinctorius, respectively, than in non-mycorrhizal roots. Root acid phosphatase (APase) activity was greater in non-mycorrhizal roots than in roots colonized with any of the three mycorrhizal fungi, and increases in activity in response to P(i) limitation occurred only in non-mycorrhizal roots. These results highlight the importance of seedling acclimation to prevailing P(i) availability and the role of mycorrhizal fungi in altering the allocation of P between roots and shoots. The activities of the APase systems of the mycorrhizal species tested do not support the hypothesis that this enzyme system plays an important role in P(i) acquisition under P-limiting conditions.

Arbuscular mycorrhizal fungi enhance aluminium resistance of broomsedge (Andropogon virginicus L.).

In the eastern United States, broomsedge (Andropogon virginicus L.) is found growing on abandoned coal-mined lands that have extremely acidic soils with high residual aluminium (Al) concentrations. Broomsedge may be inherently metal-resistant and nutrient-efficient or may rely on the arbuscular mycorrhizal (AM) fungal association to overcome limitations on such sites. Broomsedge plants were grown with and without an acidic ecotype AM fungal consortium and exposed to controlled levels of Al in two experiments. The AM fungal consortium conferred Al resistance to broomsedge. Arbuscular mycorrhizal fungi reduced Al uptake and translocation in host plants, potentially reflecting measured reductions in inorganic Al availability in the rhizosphere of mycorrhizal plants. Mycorrhizal plants exhibited lower shoot P concentrations, higher phosphorus use efficiency, and lower root acid phosphatase rates than non-mycorrhizal plants. Aluminium significantly reduced calcium (Ca) and magnesium (Mg) tissue concentrations in both mycorrhizal and non-mycorrhizal plants. However, plant response to any change in nutrient acquisition was substantially less pronounced in mycorrhizal plants. The exclusion of Al and greater stability of tissue biomass accretion-tissue nutrient relationships in mycorrhizal broomsedge plants exposed to Al may be important mechanisms that allow broomsedge to grow on unfavourable acidic soils.