Summary
The diets of over two-thirds of the world's population lack one or more essential mineral elements. This can be remedied through dietary diversification, mineral supplementation, food fortification, or increasing the concentrations and/or bioavailability of mineral elements in produce (biofortification). This article reviews aspects of soil science, plant physiology and genetics underpinning crop biofortification strategies, as well as agronomic and genetic approaches currently taken to biofortify food crops with the mineral elements most commonly lacking in human diets: iron (Fe), zinc (Zn), copper (Cu), calcium (Ca), magnesium (Mg), iodine (I) and selenium (Se). Two complementary approaches have been successfully adopted to increase the concentrations of bioavailable mineral elements in food crops. First, agronomic approaches optimizing the application of mineral fertilizers and/or improving the solubilization and mobilization of mineral elements in the soil have been implemented. Secondly, crops have been developed with: increased abilities to acquire mineral elements and accumulate them in edible tissues; increased concentrations of ‘promoter’ substances, such as ascorbate, β-carotene and cysteine-rich polypeptides which stimulate the absorption of essential mineral elements by the gut; and reduced concentrations of ‘antinutrients’, such as oxalate, polyphenolics or phytate, which interfere with their absorption. These approaches are addressing mineral malnutrition in humans globally.

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Biofortification of crops with seven mineral elements often lacking in human diets--iron, zinc, copper, calcium, magnesium, selenium and iodine.

Iron is essential for the survival and proliferation of all plants. Higher plants have developed two distinct strategies to acquire iron, which is only slightly soluble, from the rhizosphere: the reduction strategy of nongraminaceous plants and the chelation strategy of graminaceous plants. Key molecular components—including transporters, enzymes, and chelators—have been clarified for both strategies, and many of these components are now thought to also function inside the plant to facilitate internal iron transport. Transporters for intracellular iron trafficking are also being clarified. A majority of genes encoding these components are transcriptionally regulated in response to iron availability. Recent research has uncovered central transcription factors, cis-acting elements, and molecular mechanisms regulating these genes. Manipulation of these molecular components has produced transgenic crops with enhanced tolerance to iron deficiency or with increased iron content in the edible parts.

https://www.uv.mx/personal/tcarmona/files/2010/08/Kobayashi-Y-Nishizawa-2012.pdf

Iron Uptake, Translocation, and Regulation in Higher Plants

Heavy metal contamination of soil and water causing toxicity/stress has become one important constraint to crop productivity and quality. This situation has further worsened by the increasing population growth and inherent food demand. It have been reported in several studies that counterbalancing toxicity, due to heavy metal requires complex mechanisms at molecular, biochemical, physiological, cellular, tissue and whole plant level, which might manifest in terms of improved crop productivity. Recent advances in various disciplines of biological sciences such as metabolomics, transcriptomics, proteomics etc. have assisted in the characterization of metabolites, transcription factors, stress-inducible proteins involved in heavy metal tolerance, which in turn can be utilized for generating heavy metal tolerant crops. This review summarizes various tolerance strategies of plants under heavy metal toxicity, covering the role of metabolites (metabolomics), trace elements (ionomics), transcription factors (transcriptomics), various stress-inducible proteins (proteomics) as well as the role of plant hormones. We also provide a glance at strategies adopted by metal accumulating plants also known as “metallophytes”.

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Heavy Metal Tolerance in Plants: Role of Transcriptomics, Proteomics, Metabolomics, and Ionomics.

The phytohormone auxin plays critical roles in the regulation of plant growth and development. Indole-3-acetic acid (IAA) has been recognized as the major auxin for more than 70 y. Although several pathways have been proposed, how auxin is synthesized in plants is still unclear. Previous genetic and enzymatic studies demonstrated that both TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA) and YUCCA (YUC) flavin monooxygenase-like proteins are required for biosynthesis of IAA during plant development, but these enzymes were placed in two independent pathways. In this article, we demonstrate that the TAA family produces indole-3-pyruvic acid (IPA) and the YUC family functions in the conversion of IPA to IAA in Arabidopsis (Arabidopsis thaliana) by a quantification method of IPA using liquid chromatography–electrospray ionization–tandem MS. We further show that YUC protein expressed in Escherichia coli directly converts IPA to IAA. Indole-3-acetaldehyde is probably not a precursor of IAA in the IPA pathway. Our results indicate that YUC proteins catalyze a rate-limiting step of the IPA pathway, which is the main IAA biosynthesis pathway in Arabidopsis.

https://www.pnas.org/content/pnas/108/45/18512.full.pdf

The main auxin biosynthesis pathway in Arabidopsis.

Recent advances on the regulation of anthocyanin synthesis in reproductive organs

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 FIT with 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.

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

Requirement and Functional Redundancy of Ib Subgroup bHLH Proteins for Iron Deficiency Responses and Uptake in Arabidopsis thaliana

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.

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

The color of red cabbage (Brassica oleracea var. capitata) is due to anthocyanin accumulation. To investigate the regulatory control of anthocyanin production in red cabbage, the expression of anthocyanin biosynthetic and regulatory genes from eight commercial cultivars was examined. While the four green varieties had negligible amount of anthocyanins under normal growth condition, the four red cultivars contained up to 1.60 mg g(-1) fresh weight. HPLC analysis of the four red cultivars revealed that they produced similar composition of various forms of cyanidin glucosides but at different concentrations. Molecular analysis indicated that all the red cabbage shared common mechanism of regulatory control for anthocyanin biosynthesis. Except CHI which showed similar expression levels between green and red cultivars, the other structural genes, CHS, F3H, F3'H, DFR, LDOX, and GST, were constitutively up-regulated during all stages of vegetative growth in red varieties. The expression of these structural genes was also dramatically increased in green and red cabbage under nutrient stresses. The increased expression of the structural genes coincided with a coordinated increase in transcript levels of a bHLH gene, BoTT8, and a MYB transcription factor, BoMYB2. These results indicate that activation of these two regulatory factors by unknown mechanisms constitutively up-regulates nearly the entire pathway genes for the onset of anthocyanin biosynthesis in red cabbage. Moreover, the amount of total anthocyanins in red cabbage was found to be positively correlated with total antioxidant power, implicating the potential health benefit of red cabbage to human health.

Transcriptional regulation of anthocyanin biosynthesis in red cabbage

Iron chelate reductase is required for iron acquisition from soil and for metabolism in plants. In the genome of Arabidopsis thaliana there are eight genes classified into the iron chelate reductase gene family (AtFROs) based on sequence homology with AtFRO2 (a ferric chelate reductase in Arabidopsis). They are localized on chromosome 1 (three AtFROs) and chromosome 5 (five AtFROs) of Arabidopsis and show a high level of amino acid sequence similarity to each other. An assay for ferric chelate reductase activity revealed that AtFRO2, AtFRO3, AtFRO4, AtFRO5, AtFRO7 and AtFRO8 conferred significantly increased iron reduction activity compared with the control when expressed in yeast cells, indicating that the six AtFROs encode iron chelate reductases functioning in iron homeostasis in Arabidopsis. AtFRO2 displayed the highest iron reduction activity among the AtFROs investigated, further demonstrating that AtFRO2 is a major iron reductase gene in Arabidopsis. AtFRO2 and AtFRO3 were mainly expressed in roots of Arabidopsis, AtFRO5 and AtFRO6 in shoots and flowers, and AtFRO7 in cotyledons and trichomes, whereas the transcription of AtFRO8 was specific for leaf veins. Considering the tissue-specific expression profiles of AtFRO genes, we suggest that AtFRO2 and AtFRO3 are two Fe(III) chelate reductases mainly functioning in iron acquisition and metabolism in Arabidopsis roots, while AtFRO5, AtFRO6, AtFRO7 and AtFRO8 are required for iron homeostasis in different tissues of shoots.

Youxi Yuan

Papers

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

Requirement and Functional Redundancy of Ib Subgroup bHLH Proteins for Iron Deficiency Responses and Uptake in Arabidopsis thaliana

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

Transcriptional regulation of anthocyanin biosynthesis in red cabbage

Molecular and biochemical characterization of the Fe(III) chelate reductase gene family in Arabidopsis thaliana.