New aspects of iron-copper crosstalk uncovered by transcriptomic characterization of Col-0 and the copper uptake mutant spl7 in Arabidopsis thaliana.

Iron (Fe) and copper (Cu) are essential micronutrients for energy metabolism and reactive oxygen species (ROS) scavenging. Some Cu-containing proteins can be substituted with Fe-containing proteins, and vice versa, while several Arabidopsis genes are regulated by both metals. Few details of how plants coordinate Fe-Cu crosstalk are known. Gene expression was measured in the roots and rosettes of Fe, Cu, and simultaneously Fe and Cu deficient WT plants and a mutant of the Cu-uptake transcription factor SPL7. The spl7 mutant accumulated excess Fe under normal conditions, and lower Fe supply rescued the growth phenotype and normalized the Fe : Cu ratios. Most Fe regulated genes were expressed similarly in the WT and spl7 mutant, although at higher fold-change levels in spl7 mutants. Expression patterns indicated that both SPL7 and the FIT Fe uptake transcription factor influenced the expression of many key Fe uptake genes. Most notably, the newly discovered IMA/FEP genes and the subgroup Ib bHLH genes, which are upstream of Fe uptake responses, were repressed in the WT under Cu deficiency. Several AP2/ethylene response factor (AP2/ERF) genes and other redox homeostasis network genes were derepressed in spl7 mutants. Together, we present new information about Fe-Cu crosstalk in plants that could be applied for developing abiotic stress tolerant crops.

Genome-Wide Association Study Reveals Novel Genomic Regions Associated with 10 Grain Minerals in Synthetic Hexaploid Wheat.

Synthetic hexaploid wheat (SHW; Triticum durum L. × Aegilops tauschii Coss.) is a means of introducing novel genes/genomic regions into bread wheat (T. aestivum L.) and a potential genetic resource for improving grain mineral concentrations. We quantified 10 grain minerals (Ca, Cd, Cu, Co, Fe, Li, Mg, Mn, Ni, and Zn) using an inductively coupled mass spectrometer in 123 SHWs for a genome-wide association study (GWAS). A GWAS with 35,648 single nucleotide polymorphism (SNP) markers identified 92 marker-trait associations (MTAs), of which 60 were novel and 40 were within genes, and the genes underlying 20 MTAs had annotations suggesting a potential role in grain mineral concentration. Twenty-four MTAs on the D-genome were novel and showed the potential of Ae. tauschii for improving grain mineral concentrations such as Ca, Co, Cu, Li, Mg, Mn, and Ni. Interestingly, the large number of novel MTAs (36) identified on the AB genome of these SHWs indicated that there is a lot of variation yet to be explored and to be used in the A and B genome along with the D-genome. Regression analysis identified a positive correlation between a cumulative number of favorable alleles at MTA loci in a genotype and grain mineral concentration. Additionally, we identified multi-traits and stable MTAs and recommended 13 top 10% SHWs with a higher concentration of beneficial grain minerals (Cu, Fe, Mg, Mn, Ni, and Zn), a large number of favorable alleles compared to low ranking genotypes and checks that could be utilized in the breeding program for the genetic biofortification. This study will further enhance our understanding of the genetic architecture of grain minerals in wheat and related cereals.

Gene Expression Profiling of Iron Deficiency Chlorosis Sensitive and Tolerant Soybean Indicates Key Roles for Phenylpropanoids under Alkalinity Stress.

Alkaline soils comprise 30% of the earth and have low plant-available iron (Fe) concentration, and can cause iron deficiency chlorosis (IDC). IDC causes soybean yield losses of $260 million annually. However, it is not known whether molecular responses to IDC are equivalent to responses to low iron supply. IDC tolerant and sensitive soybean lines provide a contrast to identify specific factors associated with IDC. We used RNA-seq to compare gene expression under combinations of normal pH (5.7) or alkaline pH (7.7, imposed by 2.5 mM bicarbonate, or pH 8.2 imposed by 5 mM bicarbonate) and normal (25 µM) or low (1 µM) iron conditions from roots of these lines. Thus, we were able to treat pH and Fe supply as separate variables. We also noted differential gene expression between IDC sensitive and tolerant genotypes in each condition. Classical iron uptake genes, including ferric-chelate reductase (FCR) and ferrous transporters, were upregulated by both Fe deficiency and alkaline stress, however, their gene products did not function well at alkaline pH. In addition, genes in the phenylpropanoid synthesis pathway were upregulated in both alkaline and low Fe conditions. These genes lead to the production of fluorescent root exudate (FluRE) compounds, such as coumarins. Fluorescence of nutrient solution increased with alkaline treatment, and was higher in the IDC tolerant line. Some of these genes also localized to previously identified QTL regions associated with IDC. We hypothesize that FluRE become essential at alkaline pH where the classical iron uptake system does not function well. This work could result in new strategies to screen for IDC tolerance, and provide breeding targets to improve crop alkaline stress tolerance.

Mapping and Characterization of the fefe Gene That Controls Iron Uptake in Melon (Cucumis melo L.).

Iron (Fe) deficiency in plants limits crop growth and productivity. Molecular mechanisms that plants use to sense and respond to Fe deficiency by coordinated expression of Fe-uptake genes are not fully understood. The C940-fe chlorotic melon (Cucumis melo) mutant known as fefe is unable to upregulate Fe-uptake genes, however, the FeFe gene had not been identified. In this study, we used two F2 mapping populations to map and identify the FeFe gene as bHLH38, a homolog of subgroup Ib bHLH genes from Arabidopsis thaliana that are involved in transcriptional regulation of Fe-uptake genes in partnership with the FIT gene. A Ty1-copia type retrotransposon insertion of 5.056 kb within bHLH38 is responsible for the defect in bHLH38 in fefe, based on sequencing and expression analysis. This retrotransposon insertion results in multiple non-functional transcripts expected to result in an altered and truncated protein sequence. Hairy root transformation of fefe plants using wild-type bHLH38 resulted in functional complementation of the chlorotic fefe phenotype. Using a yeast-2-hybrid assay, the transcription factor Fit interacted with the wild-type bHLH38 protein, but did not interact with the fefe bHLH38 protein, suggesting that heterodimer formation of Fit/bHLH38 to regulate Fe-uptake genes does not occur in fefe roots. The second subgroup Ib bHLH gene in the melon genome is not functionally redundant to bHLH38, in contrast to Arabidopsis where four subgroup Ib bHLH genes are functionally redundant. Whereas the Arabidopsis bHLH transcript levels are upregulated by Fe deficiency, melon bHLH38 was not regulated at the transcript level. Thus, the fefe mutant may provide a platform for studying bHLH38 genes and proteins from other plant species.

Alkaline stress and iron deficiency regulate iron uptake and riboflavin synthesis gene expression differently in root and leaf tissue: implications for iron deficiency chlorosis.

Iron (Fe) is an essential mineral that has low solubility in alkaline soils, where its deficiency results in chlorosis. Whether low Fe supply and alkaline pH stress are equivalent is unclear, as they have not been treated as separate variables in molecular physiological studies. Additionally, molecular responses to these stresses have not been studied in leaf and root tissues simultaneously. We tested how plants with the Strategy I Fe uptake system respond to Fe deficiency at mildly acidic and alkaline pH by measuring root ferric chelate reductase (FCR) activity and expression of selected Fe uptake genes and riboflavin synthesis genes. Alkaline pH increased cucumber (Cucumis sativus L.) root FCR activity at full Fe supply, but alkaline stress abolished FCR response to low Fe supply. Alkaline pH or low Fe supply resulted in increased expression of Fe uptake genes, but riboflavin synthesis genes responded to Fe deficiency but not alkalinity. Iron deficiency increased expression of some common genes in roots and leaves, but alkaline stress blocked up-regulation of these genes in Fe-deficient leaves. In roots of the melon (Cucumis melo L.) fefe mutant, in which Fe uptake responses are blocked upstream of Fe uptake genes, alkaline stress or Fe deficiency up-regulation of certain Fe uptake and riboflavin synthesis genes was inhibited, indicating a central role for the FeFe protein. These results suggest a model implicating shoot-to-root signaling of Fe status to induce Fe uptake gene expression in roots.

Metabolomic profiling from leaves and roots of tomato (Solanum lycopersicum L.) plants grown under nitrogen, phosphorus or potassium-deficient condition.

Specific metabolic network responses to mineral deficiencies are not well-defined. Here, we conducted a detailed broad-scale identification of metabolic responses of tomato leaves and roots to N, P or K deficiency. Tomato plants were grown hydroponically under optimal (5mM N, 0.5mM P, or 5mM K) and deficient (0.5mM N, 0.05mM P, or 0.5mM K) conditions and metabolites were measured by LC-MS and GC-MS. Based on these results, deficiency of any of these three minerals affected energy production and amino acid metabolism. N deficiency generally led to decreased amino acids and organic acids, and increased soluble sugars. P deficiency resulted in increased amino acids and organic acids in roots, and decreased soluble sugars. K deficiency caused accumulation of soluble sugars and amino acids in roots, and decreased organic acids and amino acids in leaves. Notable metabolic pathway alterations included; (1) increased levels of α-ketoglutarate and raffinose family oligosaccharides in N, P or K-deficient tomato roots, and (2) increased putrescine in K-deficient roots. These findings provide new knowledge of metabolic changes in response to mineral deficiencies.

Distribution of Cadmium, Iron, and Zinc in Millstreams of Hard Winter Wheat (Triticum aestivum L.).

Hard winter wheat (Triticum aestivum L.) is a major crop in the Great Plains of the United States, and our previous work demonstrated that wheat genotypes vary for grain cadmium accumulation with some exceeding the CODEX standard (0.2 mg kg(-1)). Previous reports of cadmium distribution in flour milling fractions have not included high cadmium grain. This study measured the distribution of cadmium, zinc, and iron in flour and bran streams from high cadmium (0.352 mg kg(-1)) grain on a pilot mill that produced 12 flour and four bran streams. Recovery in flour was substantially greater for cadmium (50%) than for zinc (31%) or iron (22%). Cadmium, zinc, and iron in the lowest mineral concentration flour stream, representing the purest endosperm fraction, were 52, 22, and 11%, respectively, of initial grain concentration. Our results indicate that, relative to zinc and iron, a greater proportion of cadmium is stored in the endosperm, the source of white flour.

The trehalose pathway in maize: conservation and gene regulation in response to the diurnal cycle and extended darkness.

Energy resources in plants are managed in continuously changing environments, such as changes occurring during the day/night cycle. Shading is an environmental disruption that decreases photosynthesis, compromises energy status, and impacts on crop productivity. The trehalose pathway plays a central but not well-defined role in maintaining energy balance. Here, we characterized the maize trehalose pathway genes and deciphered the impacts of the diurnal cycle and disruption of the day/night cycle on trehalose pathway gene expression and sugar metabolism. The maize genome encodes 14 trehalose-6-phosphate synthase (TPS) genes, 11 trehalose-6-phosphate phosphatase (TPP) genes, and one trehalase gene. Transcript abundance of most of these genes was impacted by the day/night cycle and extended dark stress, as were sucrose, hexose sugars, starch, and trehalose-6-phosphate (T6P) levels. After extended darkness, T6P levels inversely followed class II TPS and sucrose non-fermenting-related protein kinase 1 (SnRK1) target gene expression. Most significantly, T6P no longer tracked sucrose levels after extended darkness. These results showed: (i) conservation of the trehalose pathway in maize; (ii) that sucrose, hexose, starch, T6P, and TPS/TPP transcripts respond to the diurnal cycle; and(iii) that extended darkness disrupts the correlation between T6P and sucrose/hexose pools and affects SnRK1 target gene expression. A model for the role of the trehalose pathway in sensing of sucrose and energy status in maize seedlings is proposed.

Transcriptomic and physiological characterization of the fefe mutant of melon (Cucumis melo) reveals new aspects of iron-copper crosstalk.

Iron (Fe) and copper (Cu) homeostasis are tightly linked across biology. In previous work, Fe deficiency interacted with Cu-regulated genes and stimulated Cu accumulation. The C940-fe (fefe) Fe-uptake mutant of melon (Cucumis melo) was characterized, and the fefe mutant was used to test whether Cu deficiency could stimulate Fe uptake. Wild-type and fefe mutant transcriptomes were determined by RNA-seq under Fe and Cu deficiency. FeFe-regulated genes included core Fe uptake, metal homeostasis, and transcription factor genes. Numerous genes were regulated by both Fe and Cu. The fefe mutant was rescued by high Fe or by Cu deficiency, which stimulated ferric-chelate reductase activity, FRO2 expression, and Fe accumulation. Accumulation of Fe in Cu-deficient plants was independent of the normal Fe-uptake system. One of the four FRO genes in the melon and cucumber (Cucumis sativus) genomes was Fe-regulated, and one was Cu-regulated. Simultaneous Fe and Cu deficiency synergistically up-regulated Fe-uptake gene expression. Overlap in Fe and Cu deficiency transcriptomes highlights the importance of Fe-Cu crosstalk in metal homeostasis. The fefe gene is not orthologous to FIT, and thus identification of this gene will provide clues to help understand regulation of Fe uptake in plants.

Global changes in mineral transporters in tetraploid switchgrasses (Panicum virgatum L.).

Switchgrass (Panicum virgatum L) is perennial, C4 grass with great potential as a biofuel crop. An in-depth understanding of the mechanisms that control mineral uptake, distribution and remobilization will benefit sustainable production. Nutrients are mobilized from aerial portions to below-ground crowns and rhizomes as a natural accompaniment to above-ground senescence post seed-set. Mineral uptake and remobilization is dependent on transporters, however, little if any information is available about the specific transporters that are needed and how their relative expression changes over a growing season. Using well-defined classes of mineral transporters, we identified 520 genes belonging to 40 different transporter classes in the tetraploid switchgrass genome. Expression patterns were determined for many of these genes using publically available transcriptomic datasets obtained from both greenhouse and field grown plants. Certain transporters showed strong temporal patterns of expression in distinct developmental stages of the plant. Gene-expression was verified for selected transporters using qRT-PCR. By and large these analyses confirmed the developmental stage-specific expression of these genes. Mineral analyses indicated that K, Fe, Mg, Co, and As had a similar pattern of accumulation with apparent limited remobilization at the end of the growing season. These initial analyses will serve as a foundation for more detailed examination of the nutrient biology of switchgrass.

Optimal copper supply is required for normal plant iron deficiency responses.

Iron (Fe) and copper (Cu) homeostasis are tightly linked across biology. Understanding crosstalk between Fe and Cu nutrition could lead to strategies for improved growth on soils with low or excess metals, with implications for agriculture and phytoremediation. Here, we show that Cu and Fe nutrition interact to increase or decrease Fe and/or Cu accumulation in leaves and Fe uptake processes. Leaf Cu concentration increased under low Fe supply, while high Cu lowered leaf Fe concentration. Ferric reductase activity, an indicator of Fe demand, was inhibited at insufficient or high Cu supply. Surprisingly, plants grown without Fe were more susceptible to Cu toxicity.

Rosette iron deficiency transcript and microRNA profiling reveals links between copper and iron homeostasis in Arabidopsis thaliana.

Iron (Fe) is an essential plant micronutrient, and its deficiency limits plant growth and development on alkaline soils. Under Fe deficiency, plant responses include up-regulation of genes involved in Fe uptake from the soil. However, little is known about shoot responses to Fe deficiency. Using microarrays to probe gene expression in Kas-1 and Tsu-1 ecotypes of Arabidopsis thaliana, and comparison with existing Col-0 data, revealed conserved rosette gene expression responses to Fe deficiency. Fe-regulated genes included known metal homeostasis-related genes, and a number of genes of unknown function. Several genes responded to Fe deficiency in both roots and rosettes. Fe deficiency led to up-regulation of Cu,Zn superoxide dismutase (SOD) genes CSD1 and CSD2, and down-regulation of FeSOD genes FSD1 and FSD2. Eight microRNAs were found to respond to Fe deficiency. Three of these (miR397a, miR398a, and miR398b/c) are known to regulate transcripts of Cu-containing proteins, and were down-regulated by Fe deficiency, suggesting that they could be involved in plant adaptation to Fe limitation. Indeed, Fe deficiency led to accumulation of Cu in rosettes, prior to any detectable decrease in Fe concentration. ccs1 mutants that lack functional Cu,ZnSOD proteins were prone to greater oxidative stress under Fe deficiency, indicating that increased Cu concentration under Fe limitation has an important role in oxidative stress prevention. The present results show that Cu accumulation, microRNA regulation, and associated differential expression of Fe and CuSOD genes are coordinated responses to Fe limitation.

Use of natural variation reveals core genes in the transcriptome of iron-deficient Arabidopsis thaliana roots.

Iron (Fe) is an essential mineral micronutrient for plants and animals. Plants respond to Fe deficiency by increasing root uptake capacity. Identification of gene networks for Fe uptake and homeostasis could result in improved crop growth and nutritional value. Previous studies have used microarrays to identify a large number of genes regulated by Fe deficiency in roots of three Arabidopsis ecotypes. However, a large proportion of these genes may be involved in secondary or genotype-influenced responses rather than in a universal role in Fe uptake or homeostasis. Here we show that a small percentage of the Fe deficiency transcriptome of two contrasting ecotypes, Kas-1 and Tsu-1, was shared with other ecotypes. Kas-1 and Tsu-1 had different timing and magnitude of ferric reductase activity upon Fe withdrawal, and different categories of overrepresented Fe-regulated genes. To gain insights into universal responses of Arabidopsis to Fe deficiency, the Kas-1 and Tsu-1 transcriptomes were compared with those of Col-0, Ler, and C24. In early Fe deficiency (24-48 h), no Fe-downregulated genes and only 10 upregulated genes were found in all ecotypes, and only 20 Fe-downregulated and 58 upregulated genes were found in at least three of the five ecotypes. Supernode gene networks were constructed to visualize conserved Fe homeostasis responses. Contrasting gene expression highlighted different responses to Fe deficiency between ecotypes. This study demonstrates the use of natural variation to identify central Fe-deficiency-regulated genes in plants, and identified genes with potential new roles in signalling during Fe deficiency.

The role of transition metal homeostasis in plant seed development.

For human health, transition metal accumulation in edible seeds like cereal grains is of worldwide importance, since Fe and Zn deficiencies are among the most prevalent human nutritional disorders in the world. There have been many recent developments in our understanding of the patterns in which transition metals accumulate in the seeds, the identity of some specific transporters that are required for efficient seed metal accumulation, and the central role played by the ubiquitous plant metal chelator nicotianamine (NA). These and other recent discoveries will be reviewed here.

Moving micronutrients from the soil to the seeds: genes and physiological processes from a biofortification perspective.

The micronutrients iron (Fe), zinc (Zn), and copper (Cu) are essential for plants and the humans and animals that consume plants. Increasing the micronutrient density of staple crops, or biofortification, will greatly improve human nutrition on a global scale. This review discusses the processes and genes needed to translocate micronutrients through the plant to the developing seeds, and potential strategies for developing biofortified crops.

Wheat (Triticum aestivum) NAM proteins regulate the translocation of iron, zinc, and nitrogen compounds from vegetative tissues to grain.

The NAM-B1 gene is a NAC transcription factor that affects grain nutrient concentrations in wheat (Triticum aestivum). An RNAi line with reduced expression of NAM genes has lower grain protein, iron (Fe), and zinc (Zn) concentrations. To determine whether decreased remobilization, lower plant uptake, or decreased partitioning to grain are responsible for this phenotype, mineral dynamics were quantified in wheat tissues throughout grain development. Control and RNAi wheat were grown in potting mix and hydroponics. Mineral (Ca, Cu, Fe, K, Mg, Mn, P, S, and Zn) and nitrogen (N) contents of organs were determined at regular intervals to quantify the net remobilization from vegetative tissues and the accumulation of nutrients in grain. Total nutrient accumulation was similar between lines, but grain Fe, Zn, and N were at lower concentrations in the NAM knockdown line. In potting mix, net remobilization of N, Fe, and Zn from vegetative tissues was impaired in the RNAi line. In hydroponics with ample nutrients, net remobilization was not observed, but grain Fe and Zn contents and concentrations remained lower in the RNAi line. When Fe or Zn was withheld post-anthesis, both lines demonstrated remobilization. These results suggest that a major effect of the NAM genes is an increased efflux of nutrients from the vegetative tissues and a higher partitioning of nutrients to grain.

Clozapine-induced bowel infarction: a case report.

Resistant schizophrenia is often treated with clozapine after other conventional atypical antipsychotics have failed or have resulted in intolerable side effects. Clozapine is generally reserved for secondary use due to its propensity to cause serious side effects, such as life-threatening agranulocytosis and the necessity for life-long blood monitoring. In this case report we present a 40-year-old female patient who suffered from chronic constipation and bowel infarctions after long-term use with clozapine. Although the use of this medication resulted in multiple abdominal surgeries with subtotal colonic resections, the patient's schizophrenic symptoms only responded to clozapine after several repeat failures with other medications; ultimately, she was continued on this medication.

Quantitative trait locus mapping for seed mineral concentrations in two Arabidopsis thaliana recombinant inbred populations.

Biofortification of foods, achieved by increasing the concentrations of minerals such as iron (Fe) and zinc (Zn), is a goal of plant scientists. Understanding genes that influence seed mineral concentration in a model plant such as Arabidopsis could help in the development of nutritionally enhanced crop cultivars. Quantitative trait locus (QTL) mapping for seed concentrations of calcium (Ca), copper (Cu), Fe, potassium (K), magnesium (Mg), manganese (Mn), phosphorus (P), sulfur (S), and Zn was performed using two recombinant inbred line (RIL) populations, Columbia (Col) x Landsberg erecta (Ler) and Cape Verde Islands (Cvi) x Ler, grown on multiple occasions. QTL mapping was also performed using data from silique hulls and the ratio of seed:hull mineral concentration of the Cvi x Ler population. Over 100 QTLs that affected seed mineral concentration were identified. Twenty-nine seed QTLs were found in more than one experiment, and several QTLs were found for both seed and hull mineral traits. A number of candidate genes affecting seed mineral concentration are discussed. These results indicate that A. thaliana is a suitable and convenient model for discovery of genes that affect seed mineral concentration. Some strong QTLs had no obvious candidate genes, offering the possibility of identifying unknown genes that affect mineral uptake and translocation to seeds.