Bypassing Iron Storage in Endodermal Vacuoles Rescues the Iron Mobilization Defect in the natural resistance associated-macrophage protein3natural resistance associated-macrophage protein4 Double Mutant.

To improve seed iron (Fe) content and bioavailability, it is crucial to decipher the mechanisms that control Fe storage during seed development. In Arabidopsis (Arabidopsis thaliana) seeds, most Fe is concentrated in insoluble precipitates, with phytate in the vacuoles of cells surrounding the vasculature of the embryo. NATURAL RESISTANCE ASSOCIATED-MACROPHAGE PROTEIN3 (AtNRAMP3) and AtNRAMP4 function redundantly in Fe retrieval from vacuoles during germination. When germinated under Fe-deficient conditions, development of the nramp3nramp4 double mutant is arrested as a consequence of impaired Fe mobilization. To identify novel genes involved in seed Fe homeostasis, we screened an ethyl methanesulfonate-mutagenized population of nramp3nramp4 seedlings for mutations suppressing their phenotypes on low Fe. Here, we report that, among the suppressors, two independent mutations in the VACUOLAR IRON TRANSPORTER1 (AtVIT1) gene caused the suppressor phenotype. The AtVIT1 transporter is involved in Fe influx into vacuoles of endodermal and bundle sheath cells. This result establishes a functional link between Fe loading in vacuoles by AtVIT1 and its remobilization by AtNRAMP3 and AtNRAMP4. Moreover, analysis of subcellular Fe localization indicates that simultaneous disruption of AtVIT1, AtNRAMP3, and AtNRAMP4 limits Fe accumulation in vacuolar globoids.

The Arabidopsis MTP8 transporter determines the localization of manganese and iron in seeds.

Understanding how seeds obtain and store nutrients is key to developing crops with higher agronomic and nutritional value. We have uncovered unique patterns of micronutrient localization in seeds using synchrotron X-ray fluorescence (SXRF). Although all four members of the Arabidopsis thaliana Mn-CDF family can transport Mn, here we show that only mtp8-2 has an altered Mn distribution pattern in seeds. In an mtp8-2 mutant, Mn no longer accumulates in hypocotyl cortex cells and sub-epidermal cells of the embryonic cotyledons, but rather accumulates with Fe in the cells surrounding the vasculature, a pattern previously shown to be determined by the vacuolar transporter VIT1. We also show that MTP8, unlike the other three Mn-CDF family members, can transport Fe and is responsible for localization of Fe to the same cells that store Mn. When both the VIT1 and MTP8 transporters are non-functional, there is no accumulation of Fe or Mn in specific cell types; rather these elements are distributed amongst all cell types in the seed. Disruption of the putative Fe binding sites in MTP8 resulted in loss of ability to transport Fe but did not affect the ability to transport Mn.

Mn-euvering manganese: the role of transporter gene family members in manganese uptake and mobilization in plants.

Manganese (Mn), an essential trace element, is important for plant health. In plants, Mn serves as a cofactor in essential processes such as photosynthesis, lipid biosynthesis and oxidative stress. Mn deficient plants exhibit decreased growth and yield and are more susceptible to pathogens and damage at freezing temperatures. Mn deficiency is most prominent on alkaline soils with approximately one third of the world's soils being too alkaline for optimal crop production. Despite the importance of Mn in plant development, relatively little is known about how it traffics between plant tissues and into and out of organelles. Several gene transporter families have been implicated in Mn transport in plants. These transporter families include NRAMP (natural resistance associated macrophage protein), YSL (yellow stripe-like), ZIP (zinc regulated transporter/iron-regulated transporter [ZRT/IRT1]-related protein), CAX (cation exchanger), CCX (calcium cation exchangers), CDF/MTP (cation diffusion facilitator/metal tolerance protein), P-type ATPases and VIT (vacuolar iron transporter). A combination of techniques including mutant analysis and Synchrotron X-ray Fluorescence Spectroscopy can assist in identifying essential transporters of Mn. Such knowledge would vastly improve our understanding of plant Mn homeostasis.

Methodological approaches for using synchrotron X-ray fluorescence (SXRF) imaging as a tool in ionomics: examples from Arabidopsis thaliana.

Here we present approaches for using multi-elemental imaging (specifically synchrotron X-ray fluorescence microscopy, SXRF) in ionomics, with examples using the model plant Arabidopsis thaliana. The complexity of each approach depends on the amount of a priori information available for the gene and/or phenotype being studied. Three approaches are outlined, which apply to experimental situations where a gene of interest has been identified but has an unknown phenotype (phenotyping), an unidentified gene is associated with a known phenotype (gene cloning) and finally, a screening approach, where both gene and phenotype are unknown. These approaches make use of open-access, online databases with which plant molecular genetics researchers working in the model plant Arabidopsis will be familiar, in particular the Ionomics Hub and online transcriptomic databases such as the Arabidopsis eFP browser. The approaches and examples we describe are based on the assumption that altering the expression of ion transporters can result in changes in elemental distribution. We provide methodological details on using elemental imaging to aid or accelerate gene functional characterization by narrowing down the search for candidate genes to the tissues in which elemental distributions are altered. We use synchrotron X-ray microprobes as a technique of choice, which can now be used to image all parts of an Arabidopsis plant in a hydrated state. We present elemental images of leaves, stem, root, siliques and germinating hypocotyls.

An erythropoietin autocrine/paracrine axis modulates the growth and survival of human prostate cancer cells.

Erythropoietin receptors have been identified on a variety of cancer-derived cell lines and primary cancer cells, including those of prostate cancer. The functional status of these extrahematopoietic erythropoietin receptors remains a matter of some dispute. The publication of several important clinical trials suggesting a direct effect of erythropoietin on the growth and survival of primary tumors adds further importance to the question of whether erythropoietin receptors on cancer cells are functional. We have reported previously that human prostate cancer cell lines and primary prostate cancer cells express functional erythropoietin receptors that respond to exogenous erythropoietin by increased cell proliferation and STAT5 phosphorylation. We now show that prostate cancer cell lines express both the EPO gene and the biologically active erythropoietin. The coexpression of functional receptor and biologically active ligand in the cells has led us to hypothesize an autocrine/paracrine mechanism, driven by endogenous erythropoietin, which may modulate the growth and progression of prostate cancer. To test our hypothesis, we have knocked down, independently, erythropoietin receptor and erythropoietin on prostate cancer cells by transfection with short hairpin RNAs. Erythropoietin receptor knockdown cells grow significantly more slowly than their erythropoietin receptor-bearing counterparts in monolayer culture, produce fewer, smaller colonies in soft agar, and do not exhibit erythropoietin-induced signaling. Erythropoietin knockdown cells exhibit dramatically slower rates of growth, which could be restored by transfecting the cells with a murine erythropoietin gene. Taken together, our data suggest that the coordinated regulation of a functional erythropoietin/erythropoietin receptor axis in prostate cancer cells may be integral to the growth and progression of prostate cancer.