Alfalfa MsbHLH115 confers tolerance to cadmium stress through activating the iron deficiency response in Arabidopsis thaliana.

Cadmium (Cd) pollution severely affects plant growth and development, posing risks to human health throughout the food chain. Improved iron (Fe) nutrients could mitigate Cd toxicity in plants, but the regulatory network involving Cd and Fe interplay remains unresolved. Here, a transcription factor gene of alfalfa, MsbHLH115 was verified to respond to iron deficiency and Cd stress. Overexpression of MsbHLH115 enhanced tolerance to Cd stress, showing better growth and less ROS accumulation in Arabidopsis thaliana. Overexpression of MsbHLH115 significantly enhanced Fe and Zn accumulation and did not affect Cd, Mn, and Cu concentration in Arabidopsis. Further investigations revealed that MsbHLH115 up-regulated iron homeostasis regulation genes, ROS-related genes, and metal chelation and detoxification genes, contributing to attenuating Cd toxicity. Y1H, EMSA, and LUC assays confirmed the physical interaction between MsbHLH115 and E-box, which is present in the promoter regions of most of the above-mentioned iron homeostasis regulatory genes. The transient expression experiment showed that MsbHLH115 interacted with MsbHLH121pro. The results suggest that MsbHLH115 may directly regulate the iron-deficiency response system and indirectly regulate the metal detoxification response mechanism, thereby enhancing plant Cd tolerance. In summary, enhancing iron accumulation through transcription factor regulation holds promise for improving plant tolerance to Cd toxicity, and MsbHLH115 is a potential candidate for addressing Cd toxicity issues.

MsYSL6, A Metal Transporter Gene of Alfalfa, Increases Iron Accumulation and Benefits Cadmium Resistance.

Iron (Fe) is necessary for plant growth and development. The mechanism of uptake and translocation in Cadmium (Cd) is similar to iron, which shares iron transporters. Yellow stripe-like transporter (YSL) plays a pivotal role in transporting iron and other metal ions in plants. In this study, MsYSL6 and its promoter were cloned from leguminous forage alfalfa. The transient expression of MsYSL6-GFP indicated that MsYSL6 was localized to the plasma membrane and cytoplasm. The expression of MsYSL6 was induced in alfalfa by iron deficiency and Cd stress, which was further proved by GUS activity driven by the MsYSL6 promoter. To further identify the function of MsYSL6, it was heterologously overexpressed in tobacco. MsYSL6-overexpressed tobacco showed better growth and less oxidative damage than WT under Cd stress. MsYSL6 overexpression elevated Fe and Cd contents and induced a relatively high Fe translocation rate in tobacco under Cd stress. The results suggest that MsYSL6 might have a dual function in the absorption of Fe and Cd, playing a role in the competitive absorption between Fe and Cd. MsYSL6 might be a regulatory factor in plants to counter Cd stress. This study provides a novel gene for application in heavy metal enrichment or phytoremediation and new insights into plant tolerance to toxic metals.

MsNRAMP2 Enhances Tolerance to Iron Excess Stress in Nicotiana tabacum and MsMYB Binds to Its Promoter.

Iron(Fe) is a trace metal element necessary for plant growth, but excess iron is harmful to plants. Natural resistance-associated macrophage proteins (NRAMPs) are important for divalent metal transport in plants. In this study, we isolated the MsNRAMP2 (MN_547960) gene from alfalfa, the perennial legume forage. The expression of MsNRAMP2 is specifically induced by iron excess. Overexpression of MsNRAMP2 conferred transgenic tobacco tolerance to iron excess, while it conferred yeast sensitivity to excess iron. Together with the MsNRAMP2 gene, MsMYB (MN_547959) expression is induced by excess iron. Y1H indicated that the MsMYB protein could bind to the "CTGTTG" cis element of the MsNRAMP2 promoter. The results indicated that MsNRAMP2 has a function in iron transport and its expression might be regulated by MsMYB. The excess iron tolerance ability enhancement of MsNRAMP2 may be involved in iron transport, sequestration, or redistribution.

An RGD-Modified Endostatin Peptide Expressed at E. coli Shows Anti-Tumor Activity In vivo.

Tumor vasculatures express high levels of α(V)ß(3)/α(V)ß(5) and α(5)ß(1) integrins. Peptide containing the RGD (Arg-Gly-Asp) sequence, which is present in ligands of integrins, is effective in targeting therapeutic reagents to tumor vascular endothelium. In this study, we investigated whether the biological activity of endostatin 27 peptides can be enhanced by the addition of an integrin targeting sequence. RGDRGD and GGGRGD sequence were added to the carboxyl terminus of endostatin 27 and 25 peptides, respectively. Modification of endostatin 27 peptides with the RGD motif showed specific and increased binding to endothelial cells and the increased binding is consistent with improved antiangiogenic property. RGD-modified endostatin 27 peptides was more effective than human endostatin and endostatin 27 peptides in inhibiting liver cancer growth in athymic mice. These finding indicates that addition of a vascular targeting sequence can enhance the biological activity of an antiangiogenic peptides molecule.

Insulin-like growth factor-1 fused with thrombopoietin mimetic peptide effectively increase platelets count in vivo.

OBJECTIVE: To design the expression of fusion proteins containing one or 2 thrombopoietin mimetic peptide (TMP). METHODS: This study was conducted at Harbin Pharmaceutical Group Research and Development Center, Harbin, China from June 2009 to January 2010. We designed the protein that was fused to the C-terminus of insulin-like growth factors (IGF-1) by a flexible peptide linker by Dami cell proliferation assay, colony-forming assay, and analysis of platelet in mice to prove our hypothesis. The total number of mice used was 48 in all 4 groups. RESULTS: The fusion proteins were produced in Escherichia coli BL21 (DE3) at up to 26% of the total cell proteins. Subsequent biological activity assays showed that the fusion proteins exhibited higher potency than recombinant human thrombopoietin (TPO). Our results showed that the fusion proteins IGF-1-TMP exhibited higher biological activities than TMP in Dami cell proliferation, human cord blood cell colony-forming assays, and in experiments on acute myeloid radiation sickness mice, which can effectively increase the number of platelets. CONCLUSION: Experiments in mice and biology activity assay, which can effectively increase the number of platelets, indicated that it has a potential role in pharmaceutical applications for the treatment of thrombocytopenia.

Overexpression of AtFRO6 in transgenic tobacco enhances ferric chelate reductase activity in leaves and increases tolerance to iron-deficiency chlorosis.

The Arabidopsis gene FRO6(AtFRO6) encodes ferric chelate reductase and highly expressed in green tissues of plants. We have expressed the gene AtFRO6 under the control of a 35S promoter in transgenic tobacco plants. High-level expression of AtFRO6 in transgenic plants was confirmed by northern blot analysis. Ferric reductase activity in leaves of transgenic plants grown under iron-sufficient or iron-deficient conditions is 2.13 and 1.26 fold higher than in control plants respectively. The enhanced ferric reductase activity led to increased concentrations of ferrous iron and chlorophyll, and reduced the iron deficiency chlorosis in the transgenic plants, compared to the control plants. In roots, the concentration of ferrous iron and ferric reductase activity were not significantly different in the transgenic plants compared to the control plants. These results suggest that FRO6 functions as a ferric chelate reductase for iron uptake by leaf cells, and overexpression of AtFRO6 in transgenic plants can reduce iron deficiency chlorosis.