Osteoblast iron genes: real time PCR and microarray hybridization approach under hyperoxia

Objectives Iron is essential for cell growth, differentiation, electron transfer, and oxygen transport. Hyperoxia may increase the turnover of bone matrix components with a net effect of accelerated bone growth. Although hyperoxia was claimed could increase osteoblast activity, but expression level in possible genes which play role in proliferation is still unclear. This research aims to prove the differences of expression level of transferrin receptor gene and iron regulated transporter and other genes of 7F2 under 24 h normoxia, 24 h hyperoxia, and 48 h hyperoxia and the effect of hyperoxia by using osteoblast cell culture 7F2. Methods Reverse transcriptase, real time Polymerase Chain Reaction (PCR), and microarray is used to qualitatively detect gene expression. The computer softwares such as National Center for Biotechnology Information (NCBI) data base, Software Affymetrix, DNA Strider program, Genomatix – DiAlign program, Oligo 5.0 program (Software primer design) from Wojciech & Piotr Rychlik, and Genetyx-Mac version 8.0 have been used to analyze the PCR result. Results Under 24 h hyperoxia, there were 3,884 copies of transferrin receptor mRNA per 1,000,000 copies of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA. After 24 h hyperoxia, 8,325 copies of transferrin receptor mRNA per 1,000,000 GAPDH mRNA copies were found showing 2.1-fold up regulation. After 48 h hyperoxia, there was no significant increase at the level of expression of transferrin receptor mRNA, 8,079 mRNA copies per 1,000,000 copies of mRNA were found (2.0-fold up regulation compared with 24 h normoxia). Conclusions It can be concluded that hyperoxia might have an effect on upregulating the expression of some osteoblast genes which might have an impact on osteoblast activity.

Aedes aegypti dyspepsia encodes a novel member of the SLC16 family of transporters and is critical for reproductive fitness

As a key vector for major arthropod-borne viruses (arboviruses) such as dengue, Zika and chikungunya, control of Aedes aegypti represents a major challenge in public health. Bloodmeal acquisition is necessary for the reproduction of vector mosquitoes and pathogen transmission. Blood contains potentially toxic amounts of iron while it provides nutrients for mosquito offspring; disruption of iron homeostasis in the mosquito may therefore lead to novel control strategies. We previously described a potential iron exporter in Ae. aegypti after a targeted functional screen of ZIP (zinc-regulated transporter/Iron-regulated transporter-like) and ZnT (zinc transporter) family genes. In this study, we performed an RNAseq-based screen in an Ae. aegypti cell line cultured under iron-deficient and iron-excess conditions. A subset of differentially expressed genes were analyzed via a cytosolic iron-sensitive dual-luciferase reporter assay with several gene candidates potentially involved in iron transport. In vivo gene silencing resulted in significant reduction of fecundity (egg number) and fertility (hatch rate) for one gene, termed dyspepsia. Silencing of dyspepsia reduced the induction of ferritin expression in the midgut and also resulted in delayed/impaired excretion and digestion. Further characterization of this gene, including a more direct confirmation of its substrate (iron or otherwise), could inform vector control strategies as well as to contribute to the field of metal biology.

Enhanced Cadmium Accumulation and Tolerance in Transgenic Hairy Roots of Solanum nigrum L. Expressing Iron-Regulated Transporter Gene IRT1

Solanum nigrum L., a hyperaccumulator of cadmium (Cd), is regarded as a promising candidate for phytoremediation of heavy metal pollution. In the present study, the hairy roots of Solanum nigrum L. were selected as a model plant system to study the potential application of Iron-regulated Transporter Gene (IRT1) for the efficient phytoremediation of Cd pollution. The transgenic hairy roots of Solanum nigrum L. expressing the IRT1 gene from Arabidopsis thaliana were successfully obtained via the Agrobacterium tumegaciens-mediated method. Expression of IRT1 reduced Cd stress-induced phytotoxic effects. Significantly superior root growth, increased antioxidant enzyme activities, decreased reactive oxygen species (ROS) levels, and less cell apoptosis were observed in the transgenic hairy roots of Solanum nigrum L. compared to the wild-type lines under Cd stress. Enhanced Cd accumulation was also carried out in the transgenic hairy roots compared to the control (886.8 μg/g vs. 745.0 μg/g). These results provide an important understanding of the Cd tolerance mechanism of transgenic IRT1 hairy roots of Solanum nigrum L., and are of particular importance to the development of a transgenic candidate for efficient phytoremediation process.

The Interplay between Toxic and Essential Metals for Their Uptake and Translocation Is Likely Governed by DNA Methylation and Histone Deacetylation in Maize

The persistent nature of lead (Pb) and cadmium (Cd) in the environment severely affects plant growth and yield. Conversely, plants acquire zinc (Zn) from the soil for their vital physiological and biochemical functions. However, the interplay and coordination between essential and toxic metals for their uptake and translocation and the putative underlying epigenetic mechanisms have not yet been investigated in maize. Here, we report that the presence of Zn facilitates the accumulation and transport of Pb and Cd in the aerial parts of the maize plants. Moreover, the Zn, Pb, and Cd interplay specifically interferes with the uptake and translocation of other divalent metals, such as calcium and magnesium. Zn, Pb, and Cd, individually and in combinations, differentially regulate the expression of DNA methyltransferases, thus alter the DNA methylation levels at the promoter of Zinc-regulated transporters, Iron-regulated transporter-like Protein (ZIP) genes to regulate their expression. Furthermore, the expression of histone deacetylases (HDACs) varies greatly in response to individual and combined metals, and HDACs expression showed a negative correlation with ZIP transporters. Our study highlights the implication of DNA methylation and histone acetylation in regulating the metal stress tolerance dynamics through Zn transporters and warns against the excessive use of Zn fertilizers in metal contaminated soils.

Transporters and ascorbate–glutathione metabolism for differential cadmium accumulation and tolerance in two contrasting willow genotypes

Salix matsudana Koidz is a low cadmium (Cd)-accumulating willow, whereas its cultivated variety, Salix matsudana var. matsudana f. umbraculifera Rehd., is a high Cd-accumulating and tolerant willow (HCW). The physiological and molecular mechanisms underlying differential Cd accumulation and tolerance in the two Salix species are poorly understood. Here, we confirmed that the differential Cd translocation capacity from roots to the shoots leads to the differential Cd accumulation in their aboveground parts between these two willow genotypes. Cadmium accumulation happens preferentially in the transport pathway, and Cd is mainly located in the vacuolar, cell wall and intercellular space in HCW bark by cadmium location analysis at tissue and subcellular levels. Comparative transcriptome analysis revealed that higher expressions of several metal transporter genes (ATP-binding cassette transporters, K+ transporters/channels, yellow stripe-like proteins, zinc-regulated transporter/iron-regulated transporter-like proteins, etc.) are involved in root uptake and translocation capacity in HCW; meanwhile, ascorbate–glutathione metabolic pathways play essential roles in Cd detoxification and higher tolerance of the Cd-accumulator HCW. These results lay the foundation for further understanding the molecular mechanisms of Cd accumulation in woody plants and provide new insights into molecular-assisted-screening woody plant varieties for phytoremediation.

Improving Zinc and Iron Accumulation in Maize Grains Using the Zinc and Iron Transporter ZmZIP5

Zinc (Zn) and iron (Fe) are essential micronutrients for plant growth. Thus, it is important to understand the mechanisms of uptake, transport and accumulation of these micronutrients in maize to improve crop nutritional quality. Members of the zinc-regulated transporters, iron-regulated transporter-like protein (ZIP) family are responsible for the uptake and transport of divalent metal ions in plant. Previously, we showed that ZmZIP5 functionally complemented the Zn uptake double mutant zrt1zrt2, Fe-uptake double mutant fet3fet4 in yeast. In our β-glucuronidase (GUS) assay, the germinated seeds, young sheaths, and stems of ZmZIP5-promoter-GUS transgenic plants were stained. We generated and compared two maize lines for this study: Ubi-ZmZIP5, in which ZmZIP5 was constitutively overexpressed, and ZmZIP5i, a RNAi line. At the seedling stage, high levels of Zn and Fe were found in the roots and shoots of Ubi-ZmZIP5 plants, whereas low levels were found in the ZmZIP5i plants. Zn and Fe contents decreased in the seeds of Ubi-ZmZIP5 plants and remained unchanged in the seeds of ZmZIP5i plants. The seeds of Leg-ZmZIP5 plants, in which ZmZIP5 overexpression is specific to the endosperm, had higher levels of Zn and Fe. Our results imply that ZmZIP5 may play a role in Zn and Fe uptake and root-to-shoot translocation. Endosperm-specific ZmZIP5 overexpression could be useful for Zn and Fe biofortification of cereal grains.