Article

Development of Molecular Markers for Iron Metabolism Related Genes in Lentil and Their Expression Analysis under Excess Iron Stress

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Abstract

Iron uptake and translocation in plants from soil is regulated by multiple genes and transcription factors. This study was designed to develop iron metabolism related molecular markers for Ferritin-1, BHLH-1 (Basic helix loop helix) or FER-like transcription factor protein and IRT-1 (Iron related transporter) genes using genome synteny with barrel medic (Medicago truncatula) to facilitate allele mining for these genes in lentil germplasms. The another objective of this study was to analyze differential gene expression under excess iron over time (2h, 8h, 24h). Specific molecular markers were developed for iron metabolism related genes (Ferritin-1, BHLH-1, IRT-1) and validated in lentil. Gene specific markers for Ferritin-1 and IRT-1 were used for quantitative PCR (qPCR) studies based on their amplification efficiency. Significant differential expression of Ferritin-1 and IRT-1 was observed under excess iron conditions. Future studies should involve field conditions under external iron supply.

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... Each of these processes is likely governed by a set of genes, many of which are still unknown. Several studies have discovered genes involved in uptake and mobilization of Fe (Palmer and Guerinot, 2009;Xiong et al., 2012;Sen Gupta et al., 2017) and Zn (Palmgren et al., 2008;Urwat et al., 2021) from different parts of plant to the seeds. ...
... A plasma membrane-localized transporter protein, YS1, is a crucial protein for acquiring Fe via uptake of Fe(III)-phytosiderophores (Curie et al., 2001). Expression analysis has revealed several genes that encode Fe transporter protein families in several legume species such as in Arachis hypogaea (AhIRT1, Xiong et al., 2012), Medicago truncatula-natural resistance-associated macrophage protein 1 (MtNRAMP1, Tejada-Jiménez et al., 2015;MtZIP3, MtZIP5, and MtZIP6, Lopez-Millan et al., 2004), Glycine max (NRAMP, Qin et al., 2017), Lens culinaris [Ferritin-1, basic helix-loop-helix-1 (bHLH-1), and FER-like transcription factor protein and IRT1, Sen Gupta et al., 2017] and in Cicer arietinum (CaFer1, Parveen et al., 2016). Fe sensing mechanisms are still unknown, despite the discovery of distinct transcriptional networks that regulate absorption and intracellular distribution (Thomine and Vert, 2013). ...
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Global food security, both in terms of quantity and quality remains as a challenge with the increasing population. In parallel, micronutrient deficiency in the human diet leads to malnutrition and several health-related problems collectively known as “hidden hunger” more prominent in developing countries around the globe. Biofortification is a potential tool to fortify grain legumes with micronutrients to mitigate the food and nutritional security of the ever-increasing population. Anti-nutritional factors like phytates, raffinose (RFO’s), oxalates, tannin, etc. have adverse effects on human health upon consumption. Reduction of the anti-nutritional factors or preventing their accumulation offers opportunity for enhancing the intake of legumes in diet besides increasing the bioavailability of micronutrients. Integrated breeding methods are routinely being used to exploit the available genetic variability for micronutrients through modern “omic” technologies such as genomics, transcriptomics, ionomics, and metabolomics for developing biofortified grain legumes. Molecular mechanism of Fe/Zn uptake, phytate, and raffinose family oligosaccharides (RFOs) biosynthesis pathways have been elucidated. Transgenic, microRNAs and genome editing tools hold great promise for designing nutrient-dense and anti-nutrient-free grain legumes. In this review, we present the recent efforts toward manipulation of genes/QTLs regulating biofortification and Anti-nutrient accumulation in legumes using genetics-, genomics-, microRNA-, and genome editing-based approaches. We also discuss the success stories in legumes enrichment and recent advances in development of low Anti-nutrient lines. We hope that these emerging tools and techniques will expedite the efforts to develop micronutrient dense legume crop varieties devoid of Anti-nutritional factors that will serve to address the challenges like malnutrition and hidden hunger.
... Nutrient availability and minerals, such as boron and iron imbalances, are of local importance, causing substantial yield loss. Variations in response to iron and boron deficiency and boron toxicity have been found among lentil genotypes under Fe-deficiency or excess (Gupta et al. 2017), B-deficiency, and B-toxicity (Yau and Erskine 2000;Hobson et al. 2006). This variability is necessary for developing genotypes with high yield potential in soils with mineral imbalances (Kumar et al. 2016a). ...
... Lentil reduces ferric iron at the rhizosphere which is then absorbed as ferrous iron by the root. Recently, specific DNA markers have been developed for iron metabolism related genes (Ferritin-1, BHLH-1, IRT-1) in lentil, with significant differential expression for Ferritin-1 and IRT-1 genes at the transcriptional level (Gupta et al. 2017). Transcriptome-based SSR and SNP markers have been used to develop a genetic map for elucidating the segregation of boron tolerance in lentil leading to the identification of two SNP loci associated with B-tolerance and three candidate orthologous genes with B transport-associated functions (Kaur et al. 2014). ...
Chapter
Lentil is an ancient legume crop cultivated thousands of years for its nutritious seeds, its ability to improve soil colonized by nitrogen fixing symbiotic bacteria, and providing income to local farmers at semiarid areas. During the centuries, numerous landraces and traditional varieties have been developed, providing a wealth of genetic material for lentil cultivation and use by local communities worldwide. However, current improved lentil varieties suffer from many biotic and abiotic challenges, and breeding new cultivars should exploit the breadth of genetic potential reserved within the Lens gene pool. Landraces and wild relatives are more tolerant to adverse environmental conditions and can provide valuable genes to develop improved varieties in modern agriculture, adapted to environmental abiotic and biotic stresses, suitable as well for other industrial non-food uses, such as biomass production and use as energy crop. Molecular tools to assist breeding efforts in lentil are less well developed in comparison with other crops, although progress has been made in germplasm characterization using molecular markers. Genomic research is delayed by the large (4.3 GB) lentil genome size, and progress towards the release of the complete lentil genome sequence is expected to accelerate breeding efforts. In this chapter we review current knowledge on lentil domestication and landrace distribution, cultivar improvement and breeding, efforts to characterize abiotic and biotic stress tolerance, the research strategies and major advancements made by modern molecular technologies for identification and utilization of important markers/QTLs in lentil breeding, and future prospects for this important legume crop.
... Nutrient availability and minerals, such as boron and iron imbalances, are of local importance, causing substantial yield loss. Variations in response to iron and boron deficiency and boron toxicity have been found among lentil genotypes under Fe-deficiency or excess (Gupta et al. 2017), B-deficiency, and B-toxicity (Yau and Erskine 2000;Hobson et al. 2006). This variability is necessary for developing genotypes with high yield potential in soils with mineral imbalances (Kumar et al. 2016a). ...
... Lentil reduces ferric iron at the rhizosphere which is then absorbed as ferrous iron by the root. Recently, specific DNA markers have been developed for iron metabolism related genes (Ferritin-1, BHLH-1, IRT-1) in lentil, with significant differential expression for Ferritin-1 and IRT-1 genes at the transcriptional level (Gupta et al. 2017). Transcriptome-based SSR and SNP markers have been used to develop a genetic map for elucidating the segregation of boron tolerance in lentil leading to the identification of two SNP loci associated with B-tolerance and three candidate orthologous genes with B transport-associated functions (Kaur et al. 2014). ...
Chapter
Lentil is an ancient legume crop cultivated thousands of years for its nutritious seeds, its ability to improve soil colonized by nitrogen fixing symbiotic bacteria, and providing income to local farmers at semiarid areas. During the centuries, numerous landraces and traditional varieties have been developed, providing a wealth of genetic material for lentil cultivation and use by local communities worldwide. However, current improved lentil varieties suffer from many biotic and abiotic challenges, and breeding new cultivars should exploit the breadth of genetic potential reserved within the Lens gene pool. Landraces and wild relatives are more tolerant to adverse environmental conditions and can provide valuable genes to develop improved varieties in modern agriculture, adapted to environmental abiotic and biotic stresses, suitable as well for other industrial non-food uses, such as biomass production and use as energy crop. Molecular tools to assist breeding efforts in lentil are less well developed in comparison with other crops, although progress has been made in germplasm characterization using molecular markers. Genomic research is delayed by the large (4.3 GB) lentil genome size, and progress towards the release of the complete lentil genome sequence is expected to accelerate breeding efforts. In this chapter we review current knowledge on lentil domestication and landrace distribution, cultivar improvement and breeding, efforts to characterize abiotic and biotic stress tolerance, the research strategies and major advancements made by modern molecular technologies for identification and utilization of important markers/QTLs in lentil breeding, and future prospects for this important legume crop.
... About 80% of the stored Fe in plants is found in the chloroplast, that accounts for the maintenance of structural and functional integrity of the thylakoid membranes (Adamski et al., 2011;Rout and Sahoo, 2015). The optimum concentration of soil Fe 2+ ion ranges from 5.6 to 300 ppm for most of the plants, including rice throughout their life cycle (R€ omheld and Schaaf, 2004;Sen Gupta et al., 2017). Above 300 ppm soil Fe 2+ ion concentration creates toxicity in rice, whereas below 60 ppm is considered as deficient (Dobermann and Fairhurst, 2000;Vose, 1982), making it a difficult nutrient disorder to manage. ...
Chapter
Rice is the third largest crop produced worldwide, the majority of which is flood irrigated and accounts for about one-third of the global irrigated area. Relative to other crops, rice requires a large amount of scarce water input. Research has shown that irrigated rice water use can be substantially reduced by decreasing the period of permanent flooding through water saving irrigation techniques. For regions where irrigation water can be controlled, increased labour demand, weed and pest infestation and risk of water stress are major inhibiting factors for widescale adoption of water saving rice. Automation of irrigation has the potential to resolve some of these issues. Automated irrigation has long been commercially practised in pressurized systems and proven to save significant labor; however, it has not been adopted in gravity surface irrigation systems. The purpose of this chapter is to review the current status of automated gravity surface irrigation in rice, identify potential technical adoption-limiting factors of the few previously developed systems and outline additional functionality required of an automated irrigation system for water savings in commercial scale rice systems during both ponded and non-ponded periods. To harness the full economic, social and environmental benefits that automation could provide, research is required to determine the most appropriate parameters to schedule irrigation during non-ponded periods and regionally and varietal specific thresholds. Resolution of the research and technical gaps outlined in this chapter could enable widescale adoption of irrigation practice that significantly reduce irrigation water input whilst simultaneously reducing paddy GHG emissions.
... Fe-induced genomic alterations in Lens culinaris were investigated and an up-regulation of Fe metabolism genes (Ferritin-1, BHLH-1 and IRT-1) were identified. Under Fe excess conditions, these genes maintain a balance between absorption and translocation of Fe [122]. In the leaves of the Japonica variety of rice, genes involved in hormonal control, senescence, absorption, transport, and stress oxidant were examined under Fe stress and were extensively characterized. ...
Article
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Micronutrients are essential for plants. Their growth, productivity and reproduction are directly influenced by the supply of micronutrients. Currently, there are eight trace elements considered to be essential for higher plants: Fe, Zn, Mn, Cu, Ni, B, Mo, and Cl. Possibly, other essential elements could be discovered because of recent advances in nutrient solution culture techniques and in the commercial availability of highly sensitive analytical instrumentation for elemental analysis. Much remains to be learned about the physiology of micronutrient absorption, translocation and deposition in plants, and about the functions they perform in plant growth and development. With the recent advancements in the proteomic and molecular biology tools, researchers have attempted to explore and address some of these questions. In this review, we summarize the current knowledge of micronutrients in plants and the proteomic/genomic approaches used to study plant nutrient deficiency and toxicity.
... To cope up with iron deficiency in the soil, foliar spray of 1% FeSO 4 solution was found to be effective against yield loss in chickpea and lentil. Ferritin-1 and IRT-1 genes were found to be the key regulatory genes during Fe deficiency as well as toxicity sen Gupta et al. 2017). Twenty-one QTLs regulating Fe content have been identified in a lentil population segregating for seed iron concentration using genotyping by sequencing (GBS) (Aldemir et al. 2017). ...
Chapter
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Lentil (Lens culinaris Medik.) is a cool-season grain legume crop that is mainly cultivated across the semi-arid regions of Australia, South Asia, Africa, and North America. The crop is highly valued for its nutritional attributes such as dietary proteins (22–35%), carbohydrates, minerals, and fiber that play a significant role in alleviating malnutrition and micronutrient deficiencies across populations in developing countries. The last five decades have seen an upward trend in global production of lentils from 0.85 to 5.73 Mt. suggesting its increasing demand and utilization. However, various abiotic stresses such as drought, heat, cold, salinity, and nutrient deficiency impose severe threats to the global lentil yield and productivity. The current book chapter is an attempt to comprehend the morpho-physiological and biochemical changes occurring during these stresses and the developmental plasticity shown by the plant to counteract them. Furthermore, the current status of research focusing on the development of novel molecular and functional markers/tags, identification of candidate genes/QTLs responsible for abiotic stress tolerance, the intervention of high throughput genotyping and phenotyping platforms, development of populations and linkage maps, and omics studies have been discussed. Some tolerant germplasm and varieties developed through conventional and next-generation breeding approaches are also enlisted making the book chapter a concise platform for reports of abiotic stress tolerance in lentils.KeywordsLentilLens culinaris MedikAbiotic stressClimate changeOmics approaches
... As a legume species, the roots of lentils can induce the reduction of Fe and Mn by the ferric reductase enzyme expressed on the epidermal cells of roots, which can greatly help the root system to absorb cation micronutrients [23][24][25]. Furthermore, because lentils can fix N 2 in symbiosis with rhizobia, there is an unbalanced uptake of cations that results in pumping H + to the rhizosphere to maintain the membrane potential in normal metabolic ranges, triggering the acidification of the rhizosphere [26,27]. ...
Article
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Iron (Fe) and manganese (Mn) toxicity is a widespread problem in lentil production in the coastal dryland of Chile. Increasing the soil pH by liming with CaCO3 or incrementing grain yields through nitrogen fertilization can help the plants to reduce metal concentration. Thus, the main objective of this work was to evaluate two different fertilization strategies (lime (CaCO3) and nitrogen (N) additions) to reduce Fe and Mn toxicities in lentils. Lentils grown under field conditions with the highest Fe and Mn concentrations showed toxicity symptoms, but without grain yield reductions. In a pot experiment using the same soil as in the field with toxicity symptoms, the dry matter (DM) produced at the end of the trial was higher in the plants that received N while the lowest DM production was recorded in those plants treated with lime. In particular, higher root DM sustained the growth of the N-fertilized shoots, which also positively affected the grain yields being 33% higher than the control treatment (no fertilization addition). In the plants fertilized with N, the Fe and Mn levels in the shoots were lower than the control plants and those grown in soils treated with lime, but showed higher concentrations of Fe and Mn in roots. In parallel, roots exhibited high concentrations of Fe and Mn that were 13- and 9-fold higher than in the shoots. Additionally, a significant decrease of 29% in Mn concentration in the grains of plants treated with N was reported. Overall, our results suggest that an increase in DM of lentils by the addition of N can reduce the Mn concentration on leaves to a level that is likely under the threshold that causes toxicity in plant tissues. Finally, we conclude that the increase of Fe and Mn in the roots may be connected to the reduction of these metals on leaves.
... The genes involved in phosphorylation and dephosphorylation related genes have both the up and down expression quality under Mn excess indicates the balancing nature in between phosphorylation and dephosphorylation caused by Mn-toxicity in C. grandis (Zhou et al. 2013) oxidative stress Fe uptake and genomic changes was examined in Lens culinaris by considering Fe metabolism genes (Ferritin-1, BHLH-1 and IRT-1). A result signifies that the up-regulation of Ferritin-1 and IRT-1 under excess Fe conditions to maintain a homeostasis in between Fe uptake and translocation (Gupta et al. 2017). The genes having functions of Fe uptake, transport, hormonal regulation, stress oxydant, senescence and stress was studied and well described in leaves of Japonica rice. ...
Article
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Minerals or trace elements in small amount are essential nutrients for every plant, but when the internal concentration exceeds the threshold, these essential elements do create phytotoxicity. Plant responses to elemental stresses are very common due to different anthropogenic activities; however it is a complex phenomenon with individual characteristics for various species. To cope up with the situation, a plant produces a group of strategies both in proteomic and genomic level to overcome it. Controlling the metal stress is known to activate a multigene response resulting in the changes in various proteins, which directly affects almost all biological processes in a living cell. Therefore, proteomic and genomic approaches can be useful for elucidating the molecular responses under metal stress. For this, it is tried to provide the latest knowledge and techniques used in proteomic and genomic study during nutritional stress and is represented here in review form.
... In wild and cultivated wheat, variability in grain for Fe and Zn contents have been found related to allelic variation at a chromosomal locus involved in remobilization of protein, Fe, Zn and Mn from senescing leaves to seeds [21]. Recently, Gupta et al. [22] developed markers for some iron metabolism related genes (Ferritin-1, BHLH-1, IRT-1) in lentil. Thus, the intra-as well as interspecific variability observed for seed elemental composition in the present study also highlights the hidden role of tight genetic control besides influence of plant morphology. ...
Article
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Lentil, generally known as poor man’s’ meat due to its high protein value is also a good source of dietary fiber, antioxidants and vitamins along with fast cooking characteristics. It could be used globally as a staple food crop to eradicate hidden hunger, if this nutritionally rich crop is further enriched with essential minerals. This requires identification of essential mineral rich germplasm. So, in the present study, a core set of 96 wild accessions extracted from 405 global wild annual collections comprising different species was analyzed to determine its bio-fortification potential. Impressive variation (mg/100 g) was observed for different minerals including Na (30–318), K (138.29–1578), P (37.50–593.75), Ca (4.74–188.75), Mg (15–159), Fe (2.82–14.12), Zn (1.29–12.62), Cu (0.5–7.12), Mn (1.22–9.99), Mo (1.02–11.89), Ni (0.16–3.49), Pb (0.01–0.58), Cd (0–0.03), Co (0–0.63) and As (0–0.02). Hierarchical clustering revealed high intra- and inter-specific variability. Further, correlation study showed positive significant association among minerals and between minerals including agro-morphological traits. Accessions representation from Turkey and Syria had maximum variability for different minerals. Diversity analysis exhibited wide geographical variations across gene-pool in core set. Potential use of the identified trait-specific genetic resources could be initial genetic material, for genetic base broadening and biofortification of cultivated lentil.
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Two procedures were developed for removing DNA from agarose after electrophoretic separation of DNA fragments according to size. Both involve dissolving the DNA-containing agarose in NaI. The preparative technique uses binding of DNA to glass in the presence of NaI. The method is rapid and convenient, and DNA of all molecular weight ranges can be recovered in high yield and without degradation. The DNA is free of agarose and remains susceptible to digestion by restriction enzymes. The analytical technique uses selective precipitation of DNA with acetone and has been adapted to molecular hybridization scans of sequences in agarose gels. The sequence-monitoring system is quantitative, directly measuring the proportion of the probe complementary to a given DNA fragment and vice versa. It is especially suitable for analyzing restriction enzyme digests of DNA in mapping experiments.
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An algorithm is presented for the multiple alignment of sequences, either proteins or nucleic acids, that is both accurate and easy to use on microcomputers. The approach is based on the conventional dynamic-programming method of pairwise alignment. Initially, a hierarchical clustering of the sequences is performed using the matrix of the pairwise alignment scores. The closest sequences are aligned creating groups of aligned sequences. Then close groups are aligned until all sequences are aligned in one group. The pairwise alignments included in the multiple alignment form a new matrix that is used to produce a hierarchical clustering. If it is different from the first one, iteration of the process can be performed. The method is illustrated by an example : a global alignment of 39 sequences of cytochrome c.
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Iron cores from native pea seed (Pisum sativum) ferritin have been analysed by electron microscopy and Mössbauer spectroscopy and shown to be amorphous. This correlates with their relatively high phosphate content (Fe: P = 2.83; 1800 Fe, 640 P atoms/molecule). Reconstituted cores obtained by adding iron (2000 Fe atoms/molecule) in the absence of phosphate to pea seed apoferritin were crystalline ferrihydrite. In vitro rates of formation of pea-seed ferritin iron cores were intermediate between those of recombinant human H-chain and horse spleen apoferritin and this may reflect the amino-acid residues of its ferroxidase and putative nucleation centres. The high phosphate content of pea-seed ferritin suggests that this molecule could be involved in both phosphorus and iron storage. The high phosphate concentration found within plastids, from which the molecules were isolated, is a possible source of the ferritin phosphate.
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Iron deficiency afflicts more than three billion people worldwide, and plants are the principal source of iron in most diets. Low availability of iron often limits plant growth because iron forms insoluble ferric oxides, leaving only a small, organically complexed fraction in soil solutions. The enzyme ferric-chelate reductase is required for most plants to acquire soluble iron. Here we report the isolation of the FRO2 gene, which is expressed in iron-deficient roots of Arabidopsis. FRO2 belongs to a superfamily of flavocytochromes that transport electrons across membranes. It possesses intramembranous binding sites for haem and cytoplasmic binding sites for nucleotide cofactors that donate and transfer electrons. We show that FRO2 is allelic to the frd1 mutations that impair the activity of ferric-chelate reductase. There is a nonsense mutation within the first exon of FRO2 in frd1-1 and a missense mutation within FRO2 in frd1-3. Introduction of functional FRO2 complements the frd1-1 phenotype in transgenic plants. The isolation of FRO2 has implications for the generation of crops with improved nutritional quality and increased growth in iron-deficient soils.
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Use of the real-time polymerase chain reaction (PCR) to amplify cDNA products reverse transcribed from mRNA is on the way to becoming a routine tool in molecular biology to study low abundance gene expression. Real-time PCR is easy to perform, provides the necessary accuracy and produces reliable as well as rapid quantification results. But accurate quantification of nucleic acids requires a reproducible methodology and an adequate mathematical model for data analysis. This study enters into the particular topics of the relative quantification in real-time RT–PCR of a target gene transcript in comparison to a reference gene transcript. Therefore, a new mathematical model is presented. The relative expression ratio is calculated only from the real-time PCR efficiencies and the crossing point deviation of an unknown sample versus a control. This model needs no calibration curve. Control levels were included in the model to standardise each reaction run with respect to RNA integrity, sample loading and inter-PCR variations. High accuracy and reproducibility (<2.5% variation) were reached in LightCycler PCR using the established mathematical model.
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The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data.
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Iron mobilization responses are induced by low iron supply at transcriptional level. In tomato, the basic helix-loop-helix gene FER is required for induction of iron mobilization. Using molecular-genetic techniques, we analyzed the function of BHLH029, named FRU (FER-like regulator of iron uptake), the Arabidopsis thaliana homolog of the tomato FER gene. The FRU gene was mainly expressed in roots in a cell-specific pattern and induced by iron deficiency. FRU mutant plants were chlorotic, and the FRU gene was found necessary for induction of the essential iron mobilization genes FRO2 (ferric chelate reductase gene) and IRT1 (iron-regulated transporter gene). Overexpression of FRU resulted in an increase of iron mobilization responses at low iron supply. Thus, the FRU gene is a mediator in induction of iron mobilization responses in Arabidopsis, indicating that regulation of iron uptake is conserved in dicot species.
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Compared to other organisms, plants have expanded families of transporters that are involved in the uptake and efflux of metals. Fortunately, in many cases, the examination of double mutants has been sufficient to overcome the challenge of studying functionally redundant gene families. Plants that lack two heavy-metal-transporting P-type ATPase family members (HMA2 and HMA4) reveal a function for these transporters in Zn translocation from roots to shoots. Likewise, the phenotype of plants that lack two natural resistance associated macrophage protein (NRAMP) homologs (NRAMP3 and NRAMP4) implicate these metal uptake proteins in the mobilization of vacuolar Fe stores during seed germination. Most families of metal transporters are ubiquitous but the Yellow Stripe1-Like (YSL) family is plant specific and YSL family members have been implicated in the transport of metals that are complexed with a plant specific chelator called nicotianamine (NA).
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The first predominantly gene-based genetic linkage map of lentil (Lens culinaris ssp. culinaris) was constructed using an F5 population developed from a cross between the cultivars Digger (ILL5722) and Northfield (ILL5588) using 79 intron-targeted amplified polymorphic (ITAP) and 18 genomic simple sequence repeat (SSR) markers. Linkage analysis revealed seven linkage groups (LGs) comprised of 5-25 markers that varied in length from 80.2 to 274.6 cM. The genome map spanned a total length of 928.4 cM. Clear evidence of a simple and direct macrosyntenic relationship between lentil and Medicago truncatula was observed. Sixty-six out of the 71 gene-based markers, which were previously assigned to M. truncatula genetic and physical maps, were found in regions syntenic between the Lens c. ssp. culinaris and M. truncatula genomes. However, there was evidence of moderate chromosomal rearrangements which may account for the difference in chromosome numbers between these two legume species. Eighteen common SSR markers were used to connect the current map with the most comprehensive and recent map that exists for lentil, providing the syntenic context of four important domestication traits. The composite map presented, anchored with orthologous markers mapped in M. truncatula, provides a strong foundation for the future use of genomic and genetic information in lentil genetic analysis and breeding.
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Biofortification of staple foods with iron (Fe) in the form of ferritin (Ft) is now possible, both by conventional plant breeding methods and transgenic approaches. Ft-Fe from plants and animals is absorbed well (25-30%) by human subjects, but little is known about dietary factors affecting its absorption. We used human intestinal Caco-2 cells and compared Fe absorption from animal Ft and FeSO4 to determine the effects of inhibitors and enhancers, such as phytic acid, ascorbic acid, tannic acid, calcium and heme. When postconfluent cells were coincubated with 59Fe-labeled (1 microM) FeSO4 and dietary factors, at different molar ratios of dietary factor to Fe (phytic acid:Fe, 10:1; ascorbic acid:Fe, 50:1; tannic acid:Fe, 50:1; calcium:Fe, 10:1 and hemin:Fe, 10:1), all inhibited uptake from FeSO4, except ascorbate, confirming earlier studies. In contrast, these dietary factors had little or no effect on Fe uptake from undigested Ft or Ft digested in vitro at pH 4, except tannins. However, results after in vitro digestion of Ft at pH 2 were similar to those obtained for FeSO4. These results suggest that Fe uptake occurs from both undigested as well as digested Ft but, possibly, via different mechanisms. The Fe-Ft stability shown here could minimize Fe-induced oxidation of Fe-supplemented food products.
A new mathematical model for relative quantification in real-time RT–PCR Extensive macrosynteny between Medicago truncatula and ssp. culinaris
  • M W H T Pfaffl
  • S R Ellwood
  • J K Hane
  • R Ford
  • M Materne
  • R P Oliver
Pfaffl, M. W. (2001). A new mathematical model for relative quantification in real-time RT–PCR. Nucleic Acids Res. 29:e45. doi: 10.1093/nar/29.9.e45 Phan, H. T., Ellwood, S. R., Hane, J. K., Ford, R., Materne, M., and Oliver, R. P. (2007). Extensive macrosynteny between Medicago truncatula and ssp. culinaris. Theor. Appl. Genet. 114, 549–558. doi: 10.1007/s00122-006-0455-3