FIT interacts with AtbHLH38 and AtbHLH39 in regulating iron uptake gene expression for iron homeostasis in Arabidopsis

Iron is an essential element for plant growth and development. Iron homeostasis in plants is tightly regulated at both transcriptional and posttranscriptional level. Several bHLH transcription factors involved in iron homeostasis have been identified recently. However, their regulatory mechanisms remain unknown. In this work, we demonstrate that the transcription factor FIT interacted with AtbHLH38 and AtbHLH39 and directly conferred the expression regulation of iron uptake genes for iron homeostasis in Arabidopsis. Yeast two-hybrid analysis and transient expression in Arabidopsis protoplasts showed that AtbHLH38 or AtbHLH39 interacted with FIT, a central transcription factor involved in iron homeostasis in Arabidopsis. Expression of FIT/AtbHLH38 or FIT/AtbHLH39 in yeast cells activated GUS expression driven by ferric chelate reductase （FRO2） and ferrous transporter （IRT1） promoters. Overexpression of FITwith either AtbHLH38 or AtbHLH39 in plants converted the expression of the iron uptake genes FRO2 and IRT1 from induced to constitutive. Further analysis revealed that FRO2 and IRT1 were not regulated at the posttranscriptional level in these plants because IRT1 protein accumulation and high ferric chelate reductase activity were detected in the overexpression plants under both iron deficiency and iron sufficiency. The double overexpression plants accumulated more iron in their shoots than wild type or the plants overexpressing either AtbHLH38, AtbHLH39 or FIT. Our data support that ferric-chelate reductase FRO2 and ferrous-transporter IRT1 are the targets of the three transcription factors and the transcription of FRO2 and IRT1 is directly regulated by a complex of FIT/AtbHLH38 or FIT/AtbHLH39.

Influence of Potassium Nutrition and Exogenous Organic Acids on Iron Uptake by Monocot and Dicot Plants

Iron (Fe) is a vital element for the survival and proliferation of all plants;therefore, Fe-biofortification by the application of chemical and organic fertilizers is being as an effective approach to fight hidden hunger retards the growth and development of crop plants. Two experiments were carried out to investigate the effect of potassium and exogenous organic acids on iron uptake by two different plants: one is monocotyledon, maize (Zea mays L.) and the second is dicotolydon pea (Pisum sativum L.) grown under controlled conditions. The seedlings were grown in sand culture in a greenhouse experiment and irrigated with one-tenth strength modified nutrient solution of Hoagland and Arnon as a base solution (pH 7.5), containing different iron treatments (0, 1, and 5 ppm as FeSO4·7H2O) combined with potassium nutrition (0, 5, 10, and 50 ppm as K2SO4). After 30 days, the best interaction treatment was selected for further experiment including 5.0 ppm Fe as FeSO4.7H2O and 50 ppm K as K2SO4 in combination with 1 × 10-5 mole/liter of one of the following organic acids: Citric acid, Oxalic acid, Formic acid, Acetic acid, Propionic acid, Tartaric acid, Succinic acid, Fumaric acid, Malic acid, Glutamic acid, besides the free organic acid nutrient solution as a control. Results revealed that the interaction between 5.0 ppm Fe and 50 ppm K was the best interaction treatment for increasing biomass production and iron uptake of maize and pea seedlings under applied condition. Furthermore, exogenous application of organic acids improves uptake and translocation of nutrient such as iron, potassium and phosphorus by the maize and pea plants. In conclusion, potassium nutrition and exogenous organic acids have the potential to stimulate Fe-uptake of monocot and dicot plants and mediate iron-biofortified crops.

Four IVa bHLH Transcription Factors Are Novel Interactors of FIT and Mediate JA Inhibition of Iron Uptake in Arabidopsis

Plants have evolved sophisticated genetic networks to regulate iron （Fe） homeostasis for their survival. Several classes of plant hormones including jasmonic acid （JA） have been shown to be involved in regulating the expression of iron uptake and/or deficiency-responsive genes in plants. However, the molecular mechanisms by which JA regulates iron uptake remain unclear. In this study, we found that JA negatively modulates iron uptake by downregulating the expression of FIT （bHLH29）, bHLH38, bHLH39, bHLHIO0, and bHLHI01 and promoting the degradation of FIT protein, a key regulator of iron uptake in Arabidopsis. We further demonstrated that the subgroup IVa bHLH proteins, bHLH18, bHLH19, bHLH20, and bHLH25, are novel interactors of FIT, which promote JA-induced FIT protein degradation. These four IVa bHLHs function redundantly to antagonize the activity of the Ib bHLHs （such as bHLH38） in regulating FIT protein stability under iron deficiency. The four IVa bHLH genes are primarily expressed in roots, and are inducible by JA treatment. Moreover, we found that MYC2 and JAR1, two critical components of the JA signaling pathway, play critical roles in mediating JA suppression of the expression of FIT and Ib bHLH genes, whereas they differentially modulate the expression of bHLH18, bHLH19, bHLH20, and bHLH25 to regulate FIT accumulation under iron deficiency. Taken together, these results indicate that by transcriptionally regulating the expression of different sets of bHLH genes JA signaling promotes FIT degradation, resulting in reduced expression of iron-uptake genes, IRT1 and FRO2, and increased sensitivity to iron deficiency. Our data suggest that there is a multilayered inhibition of iron-deficiency response in the presence JA in Arabidopsis.

Effect of Combined Application of Subsurface Drainage and Mineral Fertilization on Iron-Reducing Bacterial Populations’ Developments and Fe2+ Uptake by Two Rice Varieties in an Iron Toxic Paddy Soil of Burkina Faso (West Africa)

Rice is one of the staple crops in Burkina Faso. However, the local production covers only 47% of the population demands. One of the main reasons of the poor productivity in Burkina Faso is iron toxicity which is related mainly to the activity of Iron Reducing Bacteria in the rice field’s ecosystems. In order to control the harmful effects of Iron Reducing Bacterial populations and to improve rice productivity, a pots experiment was conducted at the experimental site of the University Ouaga I Pr. Joseph KI-ZERBO. An iron toxic soil from Kou Valley (West of Burkina Faso) and two rice varieties, BOUAKE-189 and ROK-5, sensitive and tolerant to iron toxicity, respectively, were used for the experiment. The pots were drained for 14 days (D2) and amended with chemical fertilizers (NPK + Urea and NPK + Urea + Ca + Mg + Zn complexes). Control pots without drainage and fertilization (D0/NF) were prepared similarly. The kinetics of Iron Reducing Bacterial populations and ferrous iron content in soil near rice roots were monitored throughout the cultural cycle using MPN and colorimetric methods, respectively. The total iron content was evaluated in rice plant using a spectrometric method. Data obtained were analyzed in relation to drainage and fertilization mode, rice growth stage and rice yield using the Student’s t-test and XLSTAT 2014 statistical software. The experiment showed that the combined application of subsurface drainage and NPK + Urea + Ca + Mg + Zn fertilization, reduced significantly the number of IRB in the soil near rice roots for both rice varieties (p = 0.050 and p = 0.020) increased the leaf tissue tolerance to excess amounts of Fe, and rice yield.

Cytochrome b5 Reductase 1 Triggers Serial Reactions that Lead to Iron Uptake in Plants

Rhizosphere acidification is essential for iron （Fe） uptake into plant roots. Plasma membrane （PM） H＊-ATPases play key roles in rhizosphere acidification. However, it is not fully understood how PM H＋-ATPase activity is regulated to enhance root Fe uptake under Fe-deficient conditions. Here, we present evidence that cytochrome b5 reductase 1 （CBR1） increases the levels of unsaturated fatty acids, which stimulate PM H＋-ATPase activity and thus lead to rhizosphere acidification. CBRl-overexpressing （CBRI-OX） Arabidopsis thaliana plants had higher levels of unsaturated fatty acids （18：2 and 18：3）, higher PM H＊-ATPase activity, and lower rhizosphere pH than wild-type plants. By contrast, cbrl loss-of-function mutant plants showed lower levels of unsaturated fatty acids and lower PM H＊-ATPase activity but higher rhizosphere pH. Reduced PM H＊-ATPase activity in cbrl could be restored in vitro by addition of unsatu- rated fatty acids. Transcript levels of CBR1, fatty acids desaturase 2 （FAD2）, and fatty acids desaturase 3 （FAD3） were increased under Fe-deficient conditions. We propose that CBR1 has a crucial role in increasing the levels of unsaturated fatty acids, which activate the PM H＊-ATPase and thus reduce rhizosphere pH. This reaction cascade ultimately promotes root Fe uptake.

AtbHLH29 of Arabidopsis thaliana is a functional ortholog of tomato FER involved in controlling iron acquisition in strategy I plants

AtbHLH29 of Arabidopsis, encoding a bHLH protein, reveals a high similarity to the tomato FER which is proposed as a transcriptional regulator involved in controlling the iron deficiency responses and the iron uptake in tomato. For identification of its biological functions, AtbHLH29 was introduced into the genome of the tomato FER mutant T3238fer mediated by Agrobacterium tumefaciencs. Transgenic plants were regenerated and the stable integration of AtbHLH29 into their genomes was confirmed by Southern hybridization. Molecular analysis demonstrated that expression of the exogenous AtbHLH29 of Arabidopsis in roots of the FER mutant T3238fer enabled to complement the defect functions of FER. The transgenic plants regained the ability to activate the whole iron deficiency responses and showed normal growth as the wild type under iron-limiting stress. Our transformation data demonstrate that AtbHLH29 is a functional ortholog of the tomato FER and can completely replace FER in controlling the effective iron acquisition in tomato. Except of iron, FER protein was directly or indirectly involved in manganese homeostasis due to that loss functions of FER in T3238fer resulted in strong reduction of Mn content in leaves and the defect function on Mn accumulation in leaves was complemented by expression of AtbHLH29 in the transgenic plants. Identification of the similar biological functions of FER and AtbHLH29, which isolated from two systematically wide-diverged “strategy I” plants, suggests that FER might be a universal gene presented in all strategy I plants in controlling effective iron acquisition system in roots.

Liver iron transport

The liver plays a central role in iron metabolism. It is the major storage site for iron and also expresses a complex range of molecules which are involved in iron transport and regulation of iron homeostasis. An increasing number of genes associated with hepatic iron transport or regulation have been identified. These include transferrin receptors （TFRI and 2）, a ferrireductase （STEAP3）, the transporters divalent metal transporter-1 （DMT1） and ferroportin （FPN） as well as the haemochromatosis protein, HFE and haemojuvelin （HJV）, which are signalling molecules. Many of these genes also participate in iron regulatory pathways which focus on the hepatic peptide hepcidin. However, we are still only beginning to understand the complex interactions between liver iron transport and iron homeostasis. This review outlines our current knowledge of molecules of iron metabolism and their roles in iron transport and regulation of iron homeostasis.

Transgenic expression of DwMYB2 impairs iron transport from root to shoot in Arabidopsis thaliana

In plant, iron uptake and homeostasis are tightly regulated to ensure its absorption from soil and to avoid excess iron in the cell. Many genes involved in this process have been identified during past several years, but there are many problems remain unsolved in the genetic regulation of whole plant iron trafficking and allocation. MYB transcription factors contain tandem repeats of a -50 amino acid DNA-binding motif （R） and are involved in the regulation of many aspects of plant development, hormone signaling and metabolism. Here, we report that the ectopic expression of orchid R2R3-MYB gene DwMYB2 in Arabidopsis thaliana confers the transgenic plants hypersensitivity to iron deficiency. In DwMYB2 transgenic plants, the iron content in root is two-fold higher compared to that in wild-type root, while the reverse is true in shoot. This imbalance of iron content in root and shoot suggested that the translocation of iron from root to shoot was affected by the expression of DwMYB2 in the transgenic plants. Consistently, gene chip and reverse transcription-polymerase chain reaction analysis revealed that the ferric-chelate reductase gene, AtFRO2, and the iron transporter gene, AtlRT1 and AtlRT2, are up-regulated by DwMYB2 expression, while other potential iron transporters such as AtlREG1, AtFRD3 and NRAMP1 are down-regulated. In addition, the expression of several putative peptide transporters and transcription factors are also altered in the 35S：：DwMYB2 transgenic lines. These data provide us insight into the whole plant translocation of iron and identify candidate genes for iron homeostasis in plants despite the fact that a heterologous gene was expressed.

Effects of Lanthanum Chloride on Activity of Redox System in Plasma Membrane of Rice Seedling Roots

The plasma membrane was isolated and purified by using the method of aqueous two phase partitioning from rice (Oryza sativa) seedling roots. The effect of LaCl 3 on the activity of redox system of plasma membrane has been studied. The reduction rate of Fe(CN) 3- 6 and the oxidation rate of NADH in plasma membrane are stimulated below the concentration of 40 μmol·L -1 , but depressed in pace with the increasing of LaCl 3 over the concentration of 40 μmol·L -1 . The possible effect of LaCl 3 on the uptake of Fe element by rice seedling was also discussed.

Statistical approach for the culture conditions optimization of magnetotactic bacteria for magnetic cells production

The culture of Magnetospirillum magneticum WM-1 depends on several control factors that have great effect on the magnetic cells concentration. Investigation into the optimal culture conditions needs a large number of experiments So it is desirable to minimize the number of experiments and maximize the information gained from them. The orthogonal design of experiments and mathematical statistical method are considered as effective methods to optimize the culture condition of magnetotactic bacteria WMol for high magnetic cells concentration. The effects of the four factors, such as pH value of medium, oxygen concentration of gas phase in the serum bottle, C：C （mtartaric acid： m=succinic acid） ratio and NaNO3 concentration, are simultaneously investigated by only sixteen experiments through the orthogonal design L16（44） method. The optimal culture condition is obtained. At the optimal culture condition （ pH 7.0, an oxygen concentration 4.0%, C：C （mtartaric acid： m=succinic acid） ratio 1：2 and NaNO3 100 mg 1^-1）, the magnetic cells concentration is promoted tO 6.5×10^7 cells ml^-1, approximately 8.3% higher than that under the initial conditions. The pH value of medium is a very important factor for magnetic cells concentration. It can be Proved that the orthogonal design of experiment is of 90% confidence. Ferric iron uptake follows MichaelisoMenten kinetics with a Km of 2.5 pM and a Vmax of 0.83 min^-1.

Fitting into the Harsh Reality： Regulation of Irondeficiency Responses in Dicotyledonous Plants

Iron is an essential element for life on Earth and its shortage, or excess, in the living organism may lead to severe health disorders. Plants serve as the primary source of dietary iron and improving crop iron content is an important step towards a better public health. Our review focuses on the control of iron acquisition in dicotyledonous plants and monocots that apply a reduction-based strategy in order to mobilize and import iron from the rhizosphere. Achieving a balance between shortage and excess of iron requires a tight regulation of the activity of the iron uptake system. A number of studies, ranging from single gene characterization to systems biology analyses, have led to the rapid expansion of our knowledge on iron uptake in recent years. Here, we summarize the novel insights into the regulation of iron ac- quisition and internal mobilization from intracellular stores. We present a detailed view of the main known regulatory networks defined by the Arabidopsis regulators FIT and POPEYE （PYE）. Additionally, we analyze the root and leaf iron- responsive regulatory networks, revealing novel potential gene interactions and reliable iron-deficiency marker genes. We discuss perspectives and open questions with regard to iron sensing and post-translational regulation.

bHLH121 Functions as a Direct Link that Facilitates the Activation of FIT by bHLH IVc Transcription Factors for Maintaining Fe Homeostasis in Arabidopsis

Iron(Fe)deficiency is prevalent in plants grown in neutral or alkaline soil.Plants have evolved sophisticated mechanisms that regulate Fe homeostasis,ensuring survival.In Arabidopsis,FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR(FIT)is a crucial regulator of Fe-deficiency response.FIT is activated indirectly by basic helix-loop-helix(bHLH)IVc transcription factors(TFs)under Fed eficiency;how ever,it remains unclear which protein(s)act as the linker to mediate the activation of FIT by bHLH IVc TFs.In this study,we characterize the functions of bHLH121 and demonstrate that it directly associates with the FIT promoter.We found that loss-of-function mutations of bHLH121 cause severe Fedeficiency symptoms,reduced Feaccum ulation,and disrupted expression of genes associated with Fehomeostasis.Genetic analysis showed that FIT is epistatic to bHLH121 and FIT overexpression partially rescues the bhlh121 mutant.Further investigations revealed that bHLH IVc TFs interact with and promote nuclear accumulation of bHLH121.We demonstrated that bHLH121 has DNA-binding activity and can bind the prom oters of the FIT and bHLHlb genes,but we did not find that it has either direct transcriptional activation or repression activity tow ard these genes.Meanw hile,we found that bHLH121 functions downstream of and is a direct target of bHLH IVc TFs,and its expression is induced by Fe deficiency in a bHLH IV c-dependent manner.Taken together,these results establish that bHLH121 functions together with bHLH IVc TFs to positively regulate the expression of FIT and thus plays a pivotal role in maintaining Fe homeostasis in Arabidopsis.

Binding of Orysata lectin induces an immune response in insect cells

In mammals,plant lectinshave been shown to possess immunomodulatory properties,acting in both the innate and adaptive immune system to modulate the production of mediators of the immune response,ultimately improving host defences.At present,knowledge of immunomodulatory effects of plant lectins in insects is scarce.Treatment of insect cells with the Orysa sativa lectin,Orysata,was previously reported to induce cell aggregation,mimicking the immune process of encapsulation.In this project we investigated the potential immunomodulatory effects of this mannose-binding lectin using Drosophila melanogaster S2 cells.Identification of the Orysata binding partners on the surface of S2 cells through a pull-down assay andproteomic analysis revealed 221 putative interactors,several of which were immunity-related proteins.Subsequent qPCR analysis revealed the upregulation of Toll-and immune deficiency(IMD)-regulated antimicrobial peptides(Drs,Mtk,AttA,and Dpt)and signal transducers(Rel and Hid)belonging to the IMD pathway.In addition,the iron-binding protein Transferrin 3 was identified as a putative interactor for Orysata,and treatment of S2 cells with Orysata was shown to reduce the intracellular iron concentration.All together,we believe these results offer a new perspective on the effects by which plant lectins influence insect cells and contribute to the study of their immunomodulatory properties.