Iron Stress Response Research Articles

Role of HFE in Hepatic Iron Overload: Novel Findings Demonstrate Direct Effects of Mutant C282Y on Hepcidin and Calreticulin mRNA Expression.

Studies done in non-hepatic cell lines, focusing on the interaction between HFE with TFR1 and β-2M proved insufficient to explain the discrepancies found in the clinical penetrance of hemochromatosis in subjects carrying the C282Y mutation. Our first goal was to investigate the role of HFE wild type (wt) and mutant proteins (C282Y and H63D) in a human hepatic cell line, focusing on the cellular localization and interaction of HFE with the expression of other iron related proteins. HFE mutant C282Y was found to be retained in the endoplasmic reticulum (ER). Thus, in addition, we investigated the effect of HFE wt and mutant proteins on Calreticulin, which is a chaperon protein that responds to ER stress and has a protective effect on oxidative damage in some cell lines. Here we report setting up a stable transfection of wt- and mutant-HFE in a hepatic cell line (HepG2) and examine the intracellular distribution of wt- and HFE mutants, their effect on iron intake independently of TFR1 and on the expression of other iron and ER stress response genes, namely Hepcidin and Calreticulin. In addition, we validated some of the novel effects of HFE on Calreticulin using peripheral blood mononuclear cells from HFE patients. The localization of the HFE variants was analyzed using KDEL and Golgin-97 as ER and the Golgi complex markers, respectively. HFE C282Y shows a high degree of overlap with the ER markers, confirming a retention of this variant in this organelle. Over-expression of the HFE wt impaired the intake of 55Fe relatively to transfected control cells (P<0.008) independently of TFR1, as demonstrated by RNAi silencing. Hamp RNA expression was decreased in cells over expressing C282Y in comparison to HFE wt cells (P<0.011). Finally over-expression of HFE wt decreases Calreticulin mRNA, whereas the C282Y had an opposite effect, compared to the control cell line. A similar result was observed in peripheral blood mononuclear cells (PMBC) of C282Y homozygous HFE patients, compared to wild type blood donors (P<0.006). Interestingly, this data suggest that synthesis of the HFE mutant C282Y triggers a protective effect on oxidative damage mediated by Calreticulin. In fact, HepG2 cells over-expressing C282Y showed lower levels of ROS than HFE wt (P<0.004). This observation might contribute to explain some of the discrepancies seen in the clinical penetrance of the disease in C282Y carrying subjects. The direct effect of the mutant HFE C282Y on mRNA expression of hepcidin also demonstrated here for the first time corroborates and provides a molecular basis for earlier reports of low hepcidin levels in HH patients and in Hfe-KO mice.

Suppression of the Lethality of High Light to a Quadruple HLI Mutant by the Inactivation of the Regulatory Protein PfsR in Synechocystis PCC 6803

A regulatory gene, designated pfsR (photosynthesis, Fe homeostasis and stress-response regulator), was discovered by a genetic screen in Synechocystis PCC 6803. Deletion of the gene from a high light-sensitive strain lacking four hli genes (4Xhli) restored viability to the parental strain under high light conditions. The quintuple mutant pfsR-/4Xhli retained photosystem-II and oxygen evolution capacity at levels similar to the wild-type levels under high light conditions. The transcripts of the two bfr genes (encoding bacterioferritin) were found to be constitutively up-regulated, whereas the transcripts of ho1 gene (encoding a heme oxygenase) were greatly down-regulated in high light upon deletion of pfsR. Under intermediate high intensity light, the pfsR deletion strains accumulated carotenoids and chlorophyll a to a significantly higher level than their corresponding parental strains. An exacerbated, transient increase in oxygen evolution during the early hours of high light acclimation and a somewhat increased steady-state level of photosystem-II-mediated oxygen evolution observed in the 4Xhli strain were brought back to the wild-type levels upon deletion of pfsR from the strain. The pfsR deletion mutants were found to be less sensitive to iron limitation under low light conditions and to suffer less lipid peroxidation following exposure to high light. Therefore, inactivation of PfsR resulted in tighter control of iron availability, which in turn reduced oxidative stress during photosynthesis in high light. These studies have revealed a critical role of PfsR in regulation of iron homeostasis and stress response.

Proteomic analysis of iron overload in human hepatoma cells

Iron-mediated organ damage is common in patients with iron overload diseases, namely, hereditary hemochromatosis. Massive iron deposition in parenchymal organs, particularly in the liver, causes organ dysfunction, fibrosis, cirrhosis, and also hepatocellular carcinoma. To obtain deeper insight into the poorly understood and complex cellular response to iron overload and consequent oxidative stress, we studied iron overload in liver-derived HepG2 cells. Human hepatoma HepG2 cells were exposed to a high concentration of iron for 3 days, and protein expression changes initiated by the iron overload were studied by two-dimensional electrophoresis and mass spectrometry. From a total of 1,060 spots observed, 21 spots were differentially expressed by iron overload. We identified 19 of them; 11 identified proteins were upregulated, whereas 8 identified proteins showed a decline in response to iron overload. The differentially expressed proteins are involved in iron storage, stress response and protection against oxidative stress, protein folding, energy metabolism, gene expression, cell cycle regulation, and other processes. Many of these molecules have not been previously suggested to be involved in the response to iron overload and the consequent oxidative stress.

The Saccharomyces cerevisiae Ca 2+ channel Cch1pMid1p is essential for tolerance to cold stress and iron toxicity

Cch1p and Mid1p are components of a high-affinity Ca 2+-permeable channel in the yeast plasma membrane. Here, we show that growth of mutants in the Cch1pMid1p channel is markedly hypersensitive to low temperature and to high iron concentration in the medium. Both phenotypes were suppressed by high Ca 2+ concentration. Iron stress elicited an increased Ca 2+ influx into both wild type and cch1Δ mid1Δ yeast. Inhibition of calcineurin strongly depressed growth of iron-stressed wild type yeast, indicating that calcineurin is a downstream element of the iron stress response. Iron hypersensitivity of the cch1Δ mid1Δ mutant was not associated with an increased iron uptake. An involvement of oxidative stress in the iron-hypersensitive phenotype was indicated by the findings that the antioxidants tocopheryl acetate and (ethyl)glutathione improved growth and viability of the iron-stressed mutant. Further, the degree of glutathione oxidation was increased in the presence of iron. The results indicate that iron stress leads to an increased oxidative poise and that Cch1pMid1p is essential to tolerate this condition.

Quantitative traits associated with adaptation of three barley (Hordeum vulgare L) cultivars to suboptimal iron supply

Iron stress responses were examined for 3 barley cultivars (Hordeum vulgare L. cvs. Steptoe, Morex and Dicktoo) grown at 5 iron levels (pFe 16.5, 17, 17.5, 18, 19) in chelator-buffered hydroponic solutions. Among the 3 cultivars, Steptoe produced the highest biomass and maintained the highest leaf chlorophyll content at each incremental decrease in Fe supply, followed by Dicktoo and Morex. All 3 cultivars responded to Fe limitation by early release of phytosiderophore prior to onset of Fe-deficiency chlorosis sp., but differed in the length of time prior to maximum phytosiderophore release. With Steptoe, near maximum release [42 μmol g−1 root (2 h)−1] occurred at day 6 for plants grown at pFe 18, and the plants maintained the same relative growth rate as Fe-sufficient plants (pFe 16.5) until day 15. In contrast, maximum rates of phytosiderophore release in Dicktoo and Morex peaked higher at 67 and 56 μmol g−1 root (2 hr)−1, but not until day 12 and day 18, respectively. By day 10, both Dicktoo and Morex were chlorotic and had a ∼50% decrease in relative growth rate. Under moderate Fe stress, all 3 cultivars decreased their shoot:root ratios by 25–30%, but differed with respect to specific root length and numbers of root tips per unit root length. The results suggest that differences in plant adaptation to Fe limitation cannot always be attributed to differences in quantities of phytosiderophore release, but also reflect several additive responses including timing of phytosiderophore release, root morphological adaptations and efficiency in Fe metabolism at low tissue Fe concentrations.

Hormonal regulation of iron-stress response in sunflower roots: a morphological and cytological investigation

Sunflowers are known to respond to Fe deficiency (-Fe) with a typical root tip swelling and the formation of root hairs and transfer cells in the rhizodermis. The possible regulation of this process was examined by a comparative study of root morphology and cytology of intact seedlings (Helianthus annuus L. cv. Giganteus) under -Fe and hormonal treatment in nutrient solution. Longitudinal sections of -Fe roots showed root tip swelling is due to cessation of cell elongation and isodiarnetric volume increase of the cortical cells. Enhanced cell division in the pericycle leads to the formation of lateral root primordia in the swollen zone. Xylem vessel differentiation is markedly accelerated and accompanied by early differentiation of the casparian band in the endodermis. Exogenous application of IAA (10−8-10−7 M) via the nutrient solution to Fe sufficient plants causes symptoms which closely mimick the characteristics of Fe deficiency including root hair development. Moreover, rhizodermal cells produce peripheral protuberances reminiscent of -Fe transfer cells. Ethylene-releasing ethephon (10−4M) also causes subapical swelling and root hair formation. However, wall protuberance development is less pronounced. ABA (10−5 M) leads to similar root thickening and root hair formation but without any comparable transfer cell differentiation. From the striking similarities between -Fe and IAA treatment it is concluded that this hormone (possibly in cooperation with ethylene) is involved in the Fe stress response of sunflower roots. The importance of a continuous polar IAA transport for this process is discussed.

Transcriptional regulation of the iron-responsive sigma factor gene pbrA.

In response to the intracellular iron concentration Pseudomonas fluorescens M114 coordinately regulates the production of pseudobactin M114, its cognate receptor PbuA, and a casein protease. Transcriptional initiation of this coordinate iron-stress response requires the sigma factor PbrA. PbrA is a member of the ECF (Extracytoplasmic function) subgroup of the sigma 70 family of eubacterial RNA polymerase sigma factors. Regulatory studies of the pbrA gene utilising promoter-lacZ transcriptional fusions demonstrate that expression of pbrA dictates the cellular response to iron. pbrA is transcribed in all phases of iron-limited growth but maximally at late-logarithmic to stationary phase. pbrA expression is independent of autoregulatory control but is strictly repressed in iron-rich conditions in a Fur-dependent fashion. Constitutive expression of pbrA from an inducible tac promoter permits the induction of PbrA-dependent transcription and pseudobactin M114 biosynthesis in high-iron conditions. A PbrA consensus sequences was derived from significant DNA sequence homologies observed within the "-25 bp" and "-16 bp" regions conserved among all PbrA-dependent promoters. The predicted PbrA target promoter consensus is homologous for the promoter recognition sites for other environmentally responsive ECF sigma factors.

Iron stress response of three peach rootstock cultivars: Ferric‐iron reduction capacity1

Abstract Three peach rootstocks were evaluated for their capacity to reduce ferric‐iron (Fe3+) after iron‐stress treatment. Neither of the rootstocks with tolerance to iron (Fe) chlorosis [Titan1 x Nemaguard (Prunus dulcis (all.) D. A. Webb x Prunus persica (Batsch) L.] nor ‘Montclar’ (P. persica) showed consistently higher Fe‐stress induced Fe3+ reduction/g root fresh weight (RFW) compared to the less tolerant rootstock Nemaguard [(P. persica)]. However, non‐stressed ‘Titan’ x Nemaguard (TNG) showed higher leaf chlorophyll content than both ‘Montclar’ (MC) and Nemaguard (NG) and higher mean Fe3+ reduction by root released reductants than Nemaguard. The reduction rate by root released reductants was approximately 22–34% for NG, 15% for MC, and 29% by TNG. ‘Titan’ x Nemaguard and Montclar maintained better root growth (i.e., had less root necrosis) under Fe‐stress than NG which resulted in greater Fe3+ reduction per plant. This indicates that the maintenance of a greater root surface area during Fe‐stress ...

Iron Chelation in Plants and Soil Microorganisms

Part 1 Production and characteristics of metal chelator: classes of microbial siderophores, M. Hofte biochemical and genetic analysis of siderophores produced by plant-associated Pseudomonas spp. and Erwinia spp, C.A. Ischimaru and J.E. Loper growth conditions for the demonstration of siderophores and iron repressible outer membrane proteins in soil bacteria, with an emphasis on free-living diazotrophs, W.J. Page production of phytosiderophores, S.-I Takagi plant and microbial ferritins, E.C. Theil and T. Hase glutathione-derived metal-binding polypeptides and metallothioneins, P.J. Jackson and C.R. Kuske. Part 2 Enzymes and interactive systems: overview of bacterial iron transport and siderophore systems in Rhizobia, J.B. Neilands iron and the nodule, M.L. Guerinot kinetics, energetics, and mechanisms of siderophore-iron transport in fungi, G. Winkelmann enzymatic reduction of iron in siderophores, J.S. Lodge the role of iron in fungal phytopathologies, I, Barash, et al the role of iron in the suppression of bacterial-plant pathogens by fluorescent Pseudomonas, A.H.M. Bakker and B. Schippers ferrochelatase and related enzymes, L.L. Barton three genetically distinct nitrogenase systems in Asotobacter vinelandii, P.E. Bishop. Part 3 Physiological and chemical characteristics of the iron stress response: iron and plant pigments, J. Abadia and A. Abadia plant iron uptake physiology by non-siderophore systems, G.W. Welkie and G.W. Miller selected physiological responses associated with Fe(III) and Fe(II) metabolism, G.V. Johnson and L.L. Barton a case study with soy-beans - iron-efficiency evaluations in field tests versus controlled conditions, S. Rodriquez de Cianzio assays for microbial siderophores, F.A. Fekete Mossbauer spectroscopy, E.V. Mielczarek sample preparation and determination of iron in biological material, Q. Wallace and J.B. Jones, Jr evaluation of iron in soil samples, R.C. Hartwig and R.H. Loeppert.

The role of active Bradyrhizobium japonicum in iron stress response of soybeans

The role of active Bradyrhizobium japonicum in iron stress response of soybeans

Influences of ultra‐violet (UV)‐blue light radiation on the growth of cotton. I. Effect on iron nutrition and iron stress response

Abstract Cool white fluorescent (CWF) light reduces Fe3+ to Fe2+ while low pressure sodium (LPS) light does not. Cotton plants grown under CWF light are green, while those yrown under LPS light develop a chlorosis very similar to the chlorosis that develops when the plants are deficient in iron (Fe). It could be that CWF light (which has ultra violet) makes iron more available for plant use by maintaining more Fe2+ in the plant. Two of the factors commonly induced by Fe‐stress in dicotyledonous plants‐‐hydroyen ions and reductants released by the roots‐‐were measured as indicators of the Fe‐deficiency stress response mechanism in M8 cotton. The plants were grown under LPS and CWF light in nutrient solutions containing either NO3‐N or NH4‐N as the source of nitrogen, and also in a fertilized alkaline soil. Leaf chlorophyll concentration varied significantly in plants grown under the two light sources as follows: CWF+Fe > LPS+Fe > CWF‐Fe ≥ LPS‐Fe. The leaf nitrate and root Fe concentrations were significant...

Increased Fe-efficiency in soybeans through plant breeding related to increased response to Fe-deficiency stress. I. Iron stress response

Abstract A7, a new soybean [Glvcine max (L.) Merr.], developed through plant breeding is considered to be more Fe‐efficient than cultivars currently available. The objective of this investigation was to determine if recognized factors in the Fe‐stress response mechanism may have been altered in A7 soybean to make it more Fe‐efficient than the A2, Hawkeye (HA) and Bragg (Fe‐efficlent) and the Pride B216 (Pride), T203, and Anoka cultivars (Fe‐inef‐ficient). The release of hydrogen ions and ‘reductants’ by the roots were the two parameters of the Fe‐stress response studied in this investigation. The effect of plant number per liter of solution was also considered by using 20 and 28 plants per 14 liters of nutrient solution. Compared to A2, HA, Bragg, Pride, Anoka and T203 soybean cultivars, A7 released more hydrogen ions and ‘reductant’ from its roots in the zero Fe treatment and also maintained higher Fe and chlorophyll concentrations. This could explain why A7 soybean grows better than the other soybeans o...

The relationships among iron-stress response, iron-efficiency and iron uptake of plants

Abstract The relationships among iron‐stress response, iron‐efficiency and iron uptake in plants is discussed. The literature concerning the relationships among iron‐stress response and iron‐efficiency or chlorosis resistance in plants is confusing. Not all plants which exhibit a classical iron‐stress response in nutrient solution culture are more resistant to chlorosis than plants which do not. The term “iron‐efficiency”; has different meanings for different researchers. For example, there are statements in the literature that monocots are “iron‐inefficient”; based on results of laboratory experiments which show a lack of iron‐stress response. Incongruently, separate studies are done using “iron‐efficient”; varieties of sorghum, oats or other monocots. We suggest that the term iron‐deficiency chlorosis (IDC) resistance be used for observations regarding field‐grown plants. There is still a debate about whether or not iron must be reduced before being transported across the root cell plasmalemma. There is much evidence to support the hypothesis of “obligatory reduction”; of Fe‐III to Fe‐II during the enhanced rate of iron uptake by iron‐stressed plants. However, there is also evidence supporting the contention that iron can be absorbed as a metal complex in toto by plants which are not iron‐stressed or which do not exhibit an enhanced rate of iron uptake in response to iron‐stress. Further research is needed to resolve this debate.

Effect of paclobutrazol on the development of iron chlorosis in soybeans

Abstract Iron‐efficient (A7) and ‐inefficient (T203) soybean cultivars were grown in nutrient solution culture both with and without the plant growth regulator paclobutrazol at low levels of Fe. Paclobutrazol affected root morphology, greatly reduced shoot growth, and delayed the onset of chlorosis in the penultimate leaf of both cultivars by several days at all Fe levels as compared to untreated controls. The chemical delayed, but did not hinder, the iron stress response mechanism (proton and reductant extrusion). Paclobutrazol did not delay the onset of chlorosis by mobilizing Fe to the leaves because foliar Fe content in paclobutrazol treated plants was similar to that of untreated ones. These results suggest that paclobutrazol may be a useful tool in studying the interactions between growth, chlorophyll synthesis, the development of Fe chlorosis, and the Fe stress response mechanism.

A critical evaluation of iron mobilization mechanisms with special reference to the contact effect phenomenon

Abstract The mechanism whereby plants growing on aerated and neutral to basic soils obtain adequate amounts of iron is, in spite of several experimentally observed iron stress responses, not yet clear. We have evaluated the currently held models of iron mobilization, and have discussed whether they could, as they presently stand, explain a very old observation, namely the contact phenomenon according to which mere physical contact between plant roots and iron‐containing solid particles enhances the uptake of iron by plant roots. It seems that in aerated well‐buffered soils, the iron mobilization mechanisms such as H+ efflux, reduction of iron, or chelation by organic ligands can be operational to an effective and significant extent only if these chemical processes take place in domains isolated from the chemical reactions taking place in the soil milieu. It is shown that roots of corn attach themselves firmly to sand particles and build extensive root hairs at the contact sites. In the two‐phase experiments in which the nutrient phase lacked iron, corn plants with roots in contact with quartz sand containing traces of iron impurities grew better and absorbed more Fe, Cu, and Mn than plants with roots suspended in nutrient solution. When iron was present in the nutrient phase, however, the roots’ contact with quartz sand had no effect on the plant's growth and the content of the above nutrients. It is postulated that protected micropockets, formed at the root and solid interfaces and sealed off from the rest of the soil milieu by mucigel plugs, shelter a chemical environment favorable to nutrient mobilization. The nutrients thus mobilized diffuse to the root cell membrane but cannot leak out and are not accessible to other roots.

Iron stress response in tomato affected by potassium and renewing nutrient solutions

Abstract Iron‐efficient T3238FER tomatoes (Lycopersicon esculentum Mill.) did not respond to Fe‐deficiency stress by releasing hydrogen ions and reductants from their roots when the plants were grown in a K‐deficient nutrient solution with or without sodium. When increments of K were added to the nutrient solution, the plants responded proportionally to Fe‐deficiency stress, Fe was transported to plant tops and the chlorophyll concentration in plant tops increased. As the leaf Fe concentration was increasing, root K concentration was increasing and root Mn concentration was decreasing. The K and Mn in tops did not show the marked differences observed in roots. In the presence of adequate K, renewing the solutions each time the pH was lowered to near 4 (days 7 and 11) caused an increased concentration of most elements in the plant, especially Mn in both tops and roots. These plants had the same Fe concentration as plants grown in unchanged solutions but they contained much less chlorophyll. Balance of nutrient elements to some degree seems required in order for iron to be made available to function properly in the plant.

A model chapter on iron for a textbook on plant nutrition

Abstract New ideas are being developed on iron relationships in plants. Older textbooks on plant nutrition are obsolete in respect to iron and some newer textbooks are somewhat obsolete. It is very important that students be kept near the front of this changing field to insure that progress continues. The chapter by Mengel and Kirkby (1982) is excellent although it does not go into siderophores and their possible relationships with iron uptake and to microbial and crop ecology, or deeply into redox in relationship with uptake of iron from synthetic chelating agents, or into iron uptake by plants from organic iron amendments, or into the riboflavin aspect of the iron response mechanism, or into hormonal control of iron transport, or into the role of iron chelates on the flowering of Lemna, or into the role of iron on thylakoid synthesis or much into the diagnosis of iron status of plants and soils as examples, or much into the significance of iron interactions in ecological problems related to lime soils. The chapter does a good work of putting together the iron stress response mechanism and its characteristics although it could expand more into its genetic control.

Excretion of riboflavin by iron deficient plants

Abstract Roots of mild iron deficient plants have frequently been observed to excrete riboflavin. There is some evidence that the riboflavin contributes to iron efficiency. Previous papers of this symposium have described several iron response mechanisms. Another possible iron stress response is that of excretion of riboflavin which has been demonstrated with iron deficiency in many plant species (Frolich and Wallace, 1966; Nagarajah and Ulrich, 1966; Wallace et al., 1968, 1971a,b; and Welkie and Miller, 1960). After a short period of iron deficiency, roots of tobacco, soybeans, sugar beets. Ambrosia, and others excrete riboflavin. Enhanced iron transport in the xylem exudate of tobacco under iron deficiency was associated with riboflavin in the exudate (Wallace, 1971a). The same workers found improved greening of iron deficient tobacco plants after the riboflavin color had disappeared from the nutrient solution (Wallace, 1969). The role of riboflavin has never been fully evaluated even though riboflavin production has been frequently observed as an iron stress response mechanism.

Application of high performance liquid chromatography in the characterization of iron stress response

Abstract When Fe‐efficient plants become Fe deficient, physiological responses occur which may increase the availability of Fe in the root environment. Iron‐stress responses of the sunflower (Helianthus annus L.) include a lowering of the medium pH, an increase in the reducing capacity of the root, and a release of riboflavin into the root medium. In this investigation, compounds released into the root medium during Fe‐stress were characterized using high performance liquid chrbmatography (HPLC). Samples of nutrient solution from hydroponically grown sunflowers (cultivar Large Russian) were analyzed by HPLC using an acetonitrile : water gradient.. Absorbance at 254 nm was recorded as compounds were separated. Negligible concentrations of compounds absorbing at 254 nm were found to be released by non‐stressed sunflower plants; however, nutrient solutions from Fe‐stressed plants contained riboflavin and at least two other compounds not present in control nutrient solutions. The concentration of these compou...

Differences in iron stress response and iron uptake in some sorghum varieties

Abstract Six sorghum (Sorghum vulqaris Pers.,) varieties representing male and female lines and hybrids were examined for their response to Fe‐stress, and their ability to absorb and transport Fe in the absence and presence of phosphate or CaCO,. It was found that the variety 2077‐A (male sterile female line) and the variety CSH‐5 (hybrid of 2077‐A r CS‐3541) showed distinct mechanism to tolerate and recover completely from chlorosis induced by Fe‐stress in 10 days. Furthermore, these 2 varieties were able to reduce the pH from 6.4 to 3.8 in about 9 days, and this was increased to about 6.2 in another 6 days, which period corresponded with the onset of chlorosis and subsequent recovery. The variety 2219‐A (male‐sterile female line) was also able to withstand Fe‐stress, although the chlorosis appeared only mild throughout. This variety also reduced pH from 6.4 to 4.5, but only after 14 days in the presence of CaCO3 (8 mg/1), and this time corresponded to the period of complete recovery from chlorosis. Ther...