Cadmium (Cd) is a highly toxic heavy metal for plants, but several unique Cd-hyperaccumulating plant species are able to accumulate this metal to extraordinary concentrations in the aboveground tissues without showing any toxic symptoms. However, the molecular mechanisms underlying this hypertolerance to Cd are poorly understood. Here we have isolated and functionally characterized an allelic gene, TcHMA3 (heavy metal ATPase 3) from two ecotypes (Ganges and Prayon) of Thlaspi caerulescens contrasting in Cd accumulation and tolerance. The TcHMA3 alleles from the higher (Ganges) and lower Cd-accumulating ecotype (Prayon) share 97.8% identity, and encode a P(1B)-type ATPase. There were no differences in the expression pattern, cell-specificity of protein localization and transport substrate-specificity of TcHMA3 between the two ecotypes. Both alleles were characterized by constitutive expression in the shoot and root, a tonoplast localization of the protein in all leaf cells and specific transport activity for Cd. The only difference between the two ecotypes was the expression level of TcHMA3: Ganges showed a sevenfold higher expression than Prayon, partly caused by a higher copy number. Furthermore, the expression level and localization of TcHMA3 were different from AtHMA3 expression in Arabidopsis. Overexpression of TcHMA3 in Arabidopsis significantly enhanced tolerance to Cd and slightly increased tolerance to Zn, but did not change Co or Pb tolerance. These results indicate that TcHMA3 is a tonoplast-localized transporter highly specific for Cd, which is responsible for sequestration of Cd into the leaf vacuoles, and that a higher expression of this gene is required for Cd hypertolerance in the Cd-hyperaccumulating ecotype of T. caerulescens.

SUMMARYThe Zn/Cd hyperaccumulator, Noccaea caerulescens, has been studied extensively for its ability to accumulate high levels of Zn and Cd in its leaves. Previous studies have indicated that the Zn and Cd hyperaccumulation trait exhibited by this species involves different transport and tolerance mechanisms. It has also been well documented that certain ecotypes of N. caerulescens are much better Cd hyperaccumulators than others. However, there does not seem to be much ecotypic variation for Zn hyperaccumulation in N. caerulescens. In this study we employed a comparative transcriptomics approach to look at root and shoot gene expression in Ganges and Prayon plants in response to Cd stress to identify transporter genes that were more highly expressed in either the roots or shoots of the superior Cd accumulator, Ganges. Comparison of the transcriptomes from the two ecotypes of Noccaea caerulescens identified a number of genes that encoded metal transporters that were more highly expressed in the Ganges ecotype in response to Cd stress. Characterization of one of these transporters, NcNramp1, showed that it is involved in the influx of Cd across the endodermal plasma membrane and thus may play a key role in Cd flux into the stele and root-to-shoot Cd transport. NcNramp1 may be one of the main transporters involved in Cd hyperaccumulation in N. caerulescens and copy number variation appears to be the main reason for high NcNramp1 gene expression underlying the increased Cd accumulation in the Ganges ecotype.

Borrelia burgdorferi binds strongly to the extracellular matrix and cells of the connective tissue, a binding apparently mediated by specific proteins and proteoglycans. We investigated the interactions between B. burgdorferi cells and intact type I collagen using hydrated lattices that reproduce features of in vivo collagen matrices. B. burgdorferi cells of several strains adhered avidly to these acellular matrices by a mechanism that was not mediated by decorin or other proteoglycans. Moreover, following adhesion to these matrices, B. burgdorferi grew and formed microcolonies. The collagen used in these studies was confirmed to lack decorin by immunoblot analysis; B. burgdorferi cells lacking the decorin adhesin bound readily to intact collagen matrices. B. burgdorferi also bound to collagen lattices that incorporated enzymes that degraded glycosaminoglycan chains in any residual proteoglycans. Binding of the bacteria to intact collagen was nonetheless specific, as bacteria did not bind agar and showed only minimal binding to bovine serum albumin, gelatin, pepsinized type I collagen, and intact collagen that had been misassembled under nonphysiological pH and ionic-strength conditions. Proteinase K treatment of B. burgdorferi cells decreased the binding, as did a lack of flagella, suggesting that surface-exposed proteins and motility may be involved in the ability of B. burgdorferi to interact with intact collagen matrices. The high efficiency of binding of B. burgdorferi strains to intact collagen matrices permits replacement of the commonly used isotopic binding assay with visual fluorescent microscopic assays and will facilitate future studies of these interactions.Borrelia burgdorferi, the etiologic agent of Lyme disease, is transmitted to animals and humans mainly by nymphae of the tick Ixodes scapularis, which during a blood meal deposit a small number of microorganisms into the skin (8,43,51). The inoculation of the bacteria by the tick bite results after a few days in a characteristic rash, erythema migrans, which may be accompanied by systemic symptoms, including malaise, fatigue, fever, headache, neck stiffness, arthralgias, or myalgias (30,51,58). Adhesion, colonization, and proliferation within the skin and other host organs and tissues by B. burgdorferi necessitates interaction between the spirochete and cells of the connective tissue, including macrophages, dendritic cells, fibroblasts (24,48), and the associated extracellular matrix (ECM) (24).B. burgdorferi expresses cell surface proteins that interact specifically with different components of the ECM of the host organism and of mammalian cells in culture (8,23,26,30). These B. burgdorferi surface proteins include the fibronectin receptor encoded by BBK32 (42); proteins that bind directly to glycosaminoglycans (GAGs), such as Bgp (39-41); and membrane lipoproteins, such as DbpA and DbpB, which bind to decorin (4,16,(20)(21)(22). The B. burgdorferi outer membrane protein p66 binds to the beta3 integrin chain of host ECM receptors (9-12). The var...

Summary• Differential sorption and transport characteristics of the leaf mesophyll layer of the Prayon and Ganges ecotypes of the hyperaccumulator Thlaspi caerulescens were examined.• 109 Cd influx and efflux experiments were conducted with leaf sections, and X-ray absorption near edge structure (XANES) data were collected from leaves as a general comparison of in vivo cadmium (Cd) coordination.• There were modest differences in cell wall sorption of Cd between ecotypes. There were obvious differences in time-and concentration-dependent Cd influx, including a greater V MAX for Prayon but a lower K M for Ganges for concentration-dependent Cd uptake and a notably greater Cd uptake by Ganges leaf sections at 1000 µM Cd. Leaf sections of Prayon had a greater Cd efflux than Ganges. The XANES spectra from the two ecotypes suggested differences in Cd coordination.• The fundamental differences observed between the two ecotypes may reflect differential activity and/or expression of plasma membrane and tonoplast transporters. More detailed study of these transporters and the in vivo coordination of Cd are needed to determine the contribution of these processes to metal homeostasis and tolerance.

Selenium (Se) biofortification has been practiced in Se-deficient regions throughout the world primarily by adding inorganic sources of Se to the soil. Considering the use of adding organic sources of Se could be useful as an alternative Se amendment for the production of Se-biofortified food crops. In this multi-year micro-plot study, we investigate growing carrots and broccoli in soils that had been previously amended with Se-enriched Stanleya pinnata Pursh (Britton) three and 4 years prior to planting one and two, respectively. Results showed that total and extractable Se concentrations in soils (0–30 cm) were 1.65 mg kg-1 and 88 μg L-1, and 0.92 mg kg-1 and 48.6 μg L-1 at the beginning of the growing season for planting one and two, respectively. After each respective growing season, total Se concentrations in the broccoli florets and carrots ranged from 6.99 to 7.83 mg kg-1 and 3.15 to 6.25 mg kg-1 in planting one and two, respectively. In broccoli and carrot plant tissues, SeMet (selenomethionine) was the predominant selenoamino acid identified in Se aqueous extracts. In postharvest soils from planting one, phospholipid fatty acid (PLFA) analyses showed that amending the soil with S. pinnata exerted no effect on the microbial biomass, AMF (arbuscular mycorrhizal fungi), actinomycetes and Gram-positive and bacterial PLFA at both 0–5 and 0–30 cm, respectively, 3 years later. Successfully producing Se-enriched broccoli and carrots 3 and 4 years later after amending soil with Se-enriched S. pinnata clearly demonstrates its potential source as an organic Se enriched fertilizer for Se-deficient regions.