Photoprotection during iron deficiency is mediated by the bHLH transcription factors PYE and ILR3.

Iron (Fe) is an essential micronutrient whose availability is limiting in many soils. During Fe deficiency, plants alter the expression of many genes to increase Fe uptake, distribution, and utilization. In a genetic screen for suppressors of Fe sensitivity in the E3 ligase mutant bts-3, we isolated an allele of the bHLH transcription factor (TF) ILR3, ilr3-4 We identified a striking leaf bleaching phenotype in ilr3 mutants that was suppressed by limiting light intensity, indicating that ILR3 is required for phototolerance during Fe deficiency. Among its paralogs that are thought to be partially redundant, only ILR3 was required for phototolerance as well as repression of genes under Fe deficiency. A mutation in the gene-encoding PYE, a known transcriptional repressor under Fe deficiency, also caused leaf bleaching. We identified singlet oxygen as the accumulating reactive oxygen species (ROS) in ilr3-4 and pye, suggesting photosensitivity is due to a PSII defect resulting in ROS production. During Fe deficiency, ilr3-4 and pye chloroplasts retain normal ultrastructure and, unlike wild type (WT), contain stacked grana similar to Fe-sufficient plants. Additionally, we found that the D1 subunit of PSII is destabilized in WT during Fe deficiency but not in ilr3-4 and pye, suggesting that PSII repair is accelerated during Fe deficiency in an ILR3- and PYE-dependent manner. Collectively, our results indicate that ILR3 and PYE confer photoprotection during Fe deficiency to prevent the accumulation of singlet oxygen, potentially by promoting reduction of grana stacking to limit excitation and facilitate repair of the photosynthetic machinery.

Redundant roles of four ZIP family members in zinc homeostasis and seed development in Arabidopsis thaliana.

Zinc (Zn) is essential for normal plant growth and development. The Zn-regulated transporter, iron-regulated transporter (IRT)-like protein (ZIP) family members are involved in Zn transport and cellular Zn homeostasis throughout the domains of life. In this study, we have characterized four ZIP transporters from Arabidopsis thaliana (IRT3, ZIP4, ZIP6, and ZIP9) to better understand their functional roles. The four ZIP proteins can restore the growth defect of a yeast Zn uptake mutant and are upregulated under Zn deficiency. Single and double mutants show no phenotypes under Zn-sufficient or Zn-limited growth conditions. In contrast, triple and quadruple mutants show impaired growth irrespective of external Zn supply due to reduced Zn translocation from root to shoot. All four ZIP genes are highly expressed during seed development, and siliques from all single and higher-order mutants exhibited an increased number of abnormal seeds and decreased Zn levels in mature seeds relative to wild type. The seed phenotypes could be reversed by supplementing the soil with Zn. Our data demonstrate that IRT3, ZIP4, ZIP6, and ZIP9 function redundantly in maintaining Zn homeostasis and seed development in A. thaliana.

A transporter for delivering zinc to the developing tiller bud and panicle in rice.

Tiller number is one of the most important agronomic traits that determine rice (Oryza sativa) yield. Active growth of tiller bud (TB) requires high amount of mineral nutrients; however, the mechanism underlying the distribution of mineral nutrients to TB with low transpiration is unknown. Here, we found that the distribution of Zn to TB is mediated by OsZIP4, one of the ZIP (ZRT, IRT-like protein) family members. The expression of OsZIP4 was highly detected in TB and nodes, and was induced by Zn deficiency. Immunostaining analysis revealed that OsZIP4 was mainly expressed in phloem of diffuse vascular bundles in the nodes and the axillary meristem. The mutation of OsZIP4 did not affect the total Zn uptake, but altered Zn distribution; less Zn was delivered to TB and new leaf, but more Zn was retained in the basal stems at the vegetative growth stage. Bioimaging analysis showed that the mutant aberrantly accumulated Zn in enlarged and transit vascular bundles of the basal node, whereas in wild-type high accumulation of Zn was observed in the meristem part. At the reproductive stage, mutation of OsZIP4 resulted in delayed panicle development, which is associated with decreased Zn distribution to the panicles. Collectively, OsZIP4 is involved in transporting Zn to the phloem of diffuse vascular bundles in the nodes for subsequent distribution to TBs and other developing tissues. It also plays a role in transporting Zn to meristem cells in the TBs.

The iron deficiency response in Arabidopsis thaliana requires the phosphorylated transcription factor URI.

Iron is an essential nutrient for plants, but excess iron is toxic due to its catalytic role in the formation of hydroxyl radicals. Thus, iron uptake is highly regulated and induced only under iron deficiency. The mechanisms of iron uptake in roots are well characterized, but less is known about how plants perceive iron deficiency. We show that a basic helix-loop-helix (bHLH) transcription factor Upstream Regulator of IRT1 (URI) acts as an essential part of the iron deficiency signaling pathway in Arabidopsis thaliana The uri mutant is defective in inducing Iron-Regulated Transporter1 (IRT1) and Ferric Reduction Oxidase2 (FRO2) and their transcriptional regulators FER-like iron deficiency-induced transcription factor (FIT) and bHLH38/39/100/101 in response to iron deficiency. Chromatin immunoprecipitation followed by sequencing (ChIP-seq) reveals direct binding of URI to promoters of many iron-regulated genes, including bHLH38/39/100/101 but not FIT While URI transcript and protein are expressed regardless of iron status, a phosphorylated form of URI only accumulates under iron deficiency. Phosphorylated URI is subject to proteasome-dependent degradation during iron resupply, and turnover of phosphorylated URI is dependent on the E3 ligase BTS. The subgroup IVc bHLH transcription factors, which have previously been shown to regulate bHLH38/39/100/101, coimmunoprecipitate with URI mainly under Fe-deficient conditions, suggesting that it is the phosphorylated form of URI that is capable of forming heterodimers in vivo. We propose that the phosphorylated form of URI accumulates under Fe deficiency, forms heterodimers with subgroup IVc proteins, and induces transcription of bHLH38/39/100/101 These transcription factors in turn heterodimerize with FIT and drive the transcription of IRT1 and FRO2 to increase Fe uptake.

All together now: regulation of the iron deficiency response.

Iron (Fe) is one of the essential micronutrients required by both plants and animals. In humans, Fe deficiency causes anemia, the most prevalent nutritional disorder. Most people rely on plant-based foods as their major Fe source, but plants are a poor source of dietary Fe. Therefore, there is a critical need to better understand the mechanisms involved in the uptake and trafficking of Fe and how plants adapt to Fe deficiency. Fe participates in key cellular functions such as photosynthesis and respiration. Perturbations of Fe uptake, transport, or storage affect plant growth as well as crop yield and plant product quality. Excess Fe has toxic effects due to its high redox activity. Plants, therefore, tightly regulate Fe uptake, distribution, and allocation. Here, we review the regulatory mechanisms involved at the transcriptional and post-translational levels that are critical to prevent Fe uptake except when plants experience Fe deficiency. We discuss the key regulatory network of basic helix-loop-helix (bHLH) transcription factors, including FIT, subgroup Ib, subgroup IVc, and URI (bHLH121), crucial for regulating Fe uptake in Arabidopsis thaliana. Furthermore, we describe the regulators of these transcription factors that either activate or inhibit their function, ensuring optimal Fe uptake that is essential for plant growth.

Targeted expression of the arsenate reductase HAC1 identifies cell type specificity of arsenic metabolism and transport in plant roots.

High Arsenic Concentration 1 (HAC1), an Arabidopsis thaliana arsenate reductase, plays a key role in arsenate [As(V)] tolerance. Through conversion of As(V) to arsenite [As(III)], HAC1 enables As(III) export from roots, and restricts translocation of As(V) to shoots. To probe the ability of different root tissues to detoxify As(III) produced by HAC1, we generated A. thaliana lines expressing HAC1 in different cell types. We investigated the As(V) tolerance phenotypes: root growth, As(III) efflux, As translocation, and As chemical speciation. We showed that HAC1 can function in the outer tissues of the root (epidermis, cortex, and endodermis) to confer As(V) tolerance, As(III) efflux, and limit As accumulation in shoots. HAC1 is less effective in the stele at conferring As(V) tolerance phenotypes. The exception is HAC1 activity in the protoxylem, which we found to be sufficient to restrict As translocation, but not to confer As(V) tolerance. In conclusion, we describe cell type-specific functions of HAC1 that spatially separate the control of As(V) tolerance and As translocation. Further, we identify a key function of protoxylem cells in As(V) translocation, consistent with the model where endodermal passage cells, above protoxylem pericycle cells, form a 'funnel' loading nutrients and potentially toxic elements into the vasculature.

Genome-wide association mapping for grain manganese in rice (Oryza sativa L.) using a multi-experiment approach.

Manganese (Mn) is an essential trace element for plants and commonly contributes to human health; however, the understanding of the genes controlling natural variation in Mn in crop plants is limited. Here, the integration of two of genome-wide association study approaches was used to increase the identification of valuable quantitative trait loci (QTL) and candidate genes responsible for the concentration of grain Mn across 389 diverse rice cultivars grown in Arkansas and Texas, USA, in multiple years. Single-trait analysis was initially performed using three different SNP datasets. As a result, significant loci could be detected using the high-density SNP dataset. Based on the 5.2 M SNP dataset, major QTLs were located on chromosomes 3 and 7 for Mn containing six candidate genes. In addition, the phenotypic data of grain Mn concentration were combined from three flooded-field experiments from the two sites and 3 years using multi-experiment analysis based on the 5.2 M SNP dataset. Two previous QTLs on chromosome 3 were identified across experiments, whereas new Mn QTLs were identified that were not found in individual experiments, on chromosomes 3, 4, 9 and 11. OsMTP8.1 was identified in both approaches and is a good candidate gene that could be controlling grain Mn concentration. This work demonstrates the utilisation of multi-experiment analysis to identify constitutive QTLs and candidate genes associated with the grain Mn concentration. Hence, the approach should be advantageous to facilitate genomic breeding programmes in rice and other crops considering QTLs and genes associated with complex traits in natural populations.

Natural variation in a molybdate transporter controls grain molybdenum concentration in rice.

Molybdenum (Mo) is an essential micronutrient for most living organisms, including humans. Cereals such as rice (Oryza sativa) are the major dietary source of Mo. However, little is known about the genetic basis of the variation in Mo content in rice grain. We mapped a quantitative trait locus (QTL) qGMo8 that controls Mo accumulation in rice grain by using a recombinant inbred line population and a backcross introgression line population. We identified a molybdate transporter, OsMOT1;1, as the causal gene for this QTL. OsMOT1;1 exhibits transport activity for molybdate, but not sulfate, when heterogeneously expressed in yeast cells. OsMOT1;1 is mainly expressed in roots and is involved in the uptake and translocation of molybdate under molybdate-limited condition. Knockdown of OsMOT1;1 results in less Mo being translocated to shoots, lower Mo concentration in grains and higher sensitivity to Mo deficiency. We reveal that the natural variation of Mo concentration in rice grains is attributed to the variable expression of OsMOT1;1 due to sequence variation in its promoter. Identification of natural allelic variation in OsMOT1;1 may facilitate the development of rice varieties with Mo-enriched grain for dietary needs and improve Mo nutrition of rice on Mo-deficient soils.

Using membrane transporters to improve crops for sustainable food production.

With the global population predicted to grow by at least 25 per cent by 2050, the need for sustainable production of nutritious foods is critical for human and environmental health. Recent advances show that specialized plant membrane transporters can be used to enhance yields of staple crops, increase nutrient content and increase resistance to key stresses, including salinity, pathogens and aluminium toxicity, which in turn could expand available arable land.

OPT3 Is a Phloem-Specific Iron Transporter That Is Essential for Systemic Iron Signaling and Redistribution of Iron and Cadmium in Arabidopsis.

Iron is essential for both plant growth and human health and nutrition. Knowledge of the signaling mechanisms that communicate iron demand from shoots to roots to regulate iron uptake as well as the transport systems mediating iron partitioning into edible plant tissues is critical for the development of crop biofortification strategies. Here, we report that OPT3, previously classified as an oligopeptide transporter, is a plasma membrane transporter capable of transporting transition ions in vitro. Studies in Arabidopsis thaliana show that OPT3 loads iron into the phloem, facilitates iron recirculation from the xylem to the phloem, and regulates both shoot-to-root iron signaling and iron redistribution from mature to developing tissues. We also uncovered an aspect of crosstalk between iron homeostasis and cadmium partitioning that is mediated by OPT3. Together, these discoveries provide promising avenues for targeted strategies directed at increasing iron while decreasing cadmium density in the edible portions of crops and improving agricultural productivity in iron deficient soils.

Bypassing Iron Storage in Endodermal Vacuoles Rescues the Iron Mobilization Defect in the natural resistance associated-macrophage protein3natural resistance associated-macrophage protein4 Double Mutant.

To improve seed iron (Fe) content and bioavailability, it is crucial to decipher the mechanisms that control Fe storage during seed development. In Arabidopsis (Arabidopsis thaliana) seeds, most Fe is concentrated in insoluble precipitates, with phytate in the vacuoles of cells surrounding the vasculature of the embryo. NATURAL RESISTANCE ASSOCIATED-MACROPHAGE PROTEIN3 (AtNRAMP3) and AtNRAMP4 function redundantly in Fe retrieval from vacuoles during germination. When germinated under Fe-deficient conditions, development of the nramp3nramp4 double mutant is arrested as a consequence of impaired Fe mobilization. To identify novel genes involved in seed Fe homeostasis, we screened an ethyl methanesulfonate-mutagenized population of nramp3nramp4 seedlings for mutations suppressing their phenotypes on low Fe. Here, we report that, among the suppressors, two independent mutations in the VACUOLAR IRON TRANSPORTER1 (AtVIT1) gene caused the suppressor phenotype. The AtVIT1 transporter is involved in Fe influx into vacuoles of endodermal and bundle sheath cells. This result establishes a functional link between Fe loading in vacuoles by AtVIT1 and its remobilization by AtNRAMP3 and AtNRAMP4. Moreover, analysis of subcellular Fe localization indicates that simultaneous disruption of AtVIT1, AtNRAMP3, and AtNRAMP4 limits Fe accumulation in vacuolar globoids.

MYB10 and MYB72 are required for growth under iron-limiting conditions.

Iron is essential for photosynthesis and is often a limiting nutrient for plant productivity. Plants respond to conditions of iron deficiency by increasing transcript abundance of key genes involved in iron homeostasis, but only a few regulators of these genes have been identified. Using genome-wide expression analysis, we searched for transcription factors that are induced within 24 hours after transferring plants to iron-deficient growth conditions. Out of nearly 100 transcription factors shown to be up-regulated, we identified MYB10 and MYB72 as the most highly induced transcription factors. Here, we show that MYB10 and MYB72 are functionally redundant and are required for plant survival in alkaline soil where iron availability is greatly restricted. myb10myb72 double mutants fail to induce transcript accumulation of the nicotianamine synthase gene NAS4. Both myb10myb72 mutants and nas4-1 mutants have reduced iron concentrations, chlorophyll levels, and shoot mass under iron-limiting conditions, indicating that these genes are essential for proper plant growth. The double myb10myb72 mutant also showed nickel and zinc sensitivity, similar to the nas4 mutant. Ectopic expression of NAS4 rescues myb10myb72 plants, suggesting that loss of NAS4 is the primary defect in these plants and emphasizes the importance of nicotianamine, an iron chelator, in iron homeostasis. Overall, our results provide evidence that MYB10 and MYB72 act early in the iron-deficiency regulatory cascade to drive gene expression of NAS4 and are essential for plant survival under iron deficiency.

Dirigent domain-containing protein is part of the machinery required for formation of the lignin-based Casparian strip in the root.

The endodermis acts as a "second skin" in plant roots by providing the cellular control necessary for the selective entry of water and solutes into the vascular system. To enable such control, Casparian strips span the cell wall of adjacent endodermal cells to form a tight junction that blocks extracellular diffusion across the endodermis. This junction is composed of lignin that is polymerized by oxidative coupling of monolignols through the action of a NADPH oxidase and peroxidases. Casparian strip domain proteins (CASPs) correctly position this biosynthetic machinery by forming a protein scaffold in the plasma membrane at the site where the Casparian strip forms. Here, we show that the dirigent-domain containing protein, enhanced suberin1 (ESB1), is part of this machinery, playing an essential role in the correct formation of Casparian strips. ESB1 is localized to Casparian strips in a CASP-dependent manner, and in the absence of ESB1, disordered and defective Casparian strips are formed. In addition, loss of ESB1 disrupts the localization of the CASP1 protein at the casparian strip domain, suggesting a reciprocal requirement for both ESB1 and CASPs in forming the casparian strip domain.

Reciprocal interaction of the circadian clock with the iron homeostasis network in Arabidopsis.

In plants, iron (Fe) uptake and homeostasis are critical for survival, and these processes are tightly regulated at the transcriptional and posttranscriptional levels. Circadian clocks are endogenous oscillating mechanisms that allow an organism to anticipate environmental changes to coordinate biological processes both with one another and with the environmental day/night cycle. The plant circadian clock controls many physiological processes through rhythmic expression of transcripts. In this study, we examined the expression of three Fe homeostasis genes (IRON REGULATED TRANSPORTER1 [IRT1], BASIC HELIX LOOP HELIX39, and FERRITIN1) in Arabidopsis (Arabidopsis thaliana) using promoter:LUCIFERASE transgenic lines. Each of these promoters showed circadian regulation of transcription. The circadian clock monitors a number of clock outputs and uses these outputs as inputs to modulate clock function. We show that this is also true for Fe status. Fe deficiency results in a lengthened circadian period. We interrogated mutants impaired in the Fe homeostasis response, including irt1-1, which lacks the major high-affinity Fe transporter, and fit-2, which lacks Fe deficiency-induced TRANSCRIPTION FACTOR1, a basic helix-loop-helix transcription factor necessary for induction of the Fe deficiency response. Both mutants exhibit symptoms of Fe deficiency, including lengthened circadian period. To determine which components are involved in this cross talk between the circadian and Fe homeostasis networks, we tested clock- or Fe homeostasis-related mutants. Mutants defective in specific clock gene components were resistant to the change in period length under different Fe conditions observed in the wild type, suggesting that these mutants are impaired in cross talk between Fe homeostasis and the circadian clock.

Sphingolipids in the root play an important role in regulating the leaf ionome in Arabidopsis thaliana.

Sphingolipid synthesis is initiated by condensation of Ser with palmitoyl-CoA producing 3-ketodihydrosphinganine (3-KDS), which is reduced by a 3-KDS reductase to dihydrosphinganine. Ser palmitoyltransferase is essential for plant viability. Arabidopsis thaliana contains two genes (At3g06060/TSC10A and At5g19200/TSC10B) encoding proteins with significant similarity to the yeast 3-KDS reductase, Tsc10p. Heterologous expression in yeast of either Arabidopsis gene restored 3-KDS reductase activity to the yeast tsc10Δ mutant, confirming both as bona fide 3-KDS reductase genes. Consistent with sphingolipids having essential functions in plants, double mutant progeny lacking both genes were not recovered from crosses of single tsc10A and tsc10B mutants. Although the 3-KDS reductase genes are functionally redundant and ubiquitously expressed in Arabidopsis, 3-KDS reductase activity was reduced to 10% of wild-type levels in the loss-of-function tsc10a mutant, leading to an altered sphingolipid profile. This perturbation of sphingolipid biosynthesis in the Arabidopsis tsc10a mutant leads an altered leaf ionome, including increases in Na, K, and Rb and decreases in Mg, Ca, Fe, and Mo. Reciprocal grafting revealed that these changes in the leaf ionome are driven by the root and are associated with increases in root suberin and alterations in Fe homeostasis.

Mapping and validation of quantitative trait loci associated with concentrations of 16 elements in unmilled rice grain.

KEY MESSAGE: QTLs controlling the concentrations elements in rice grain were identified in two mapping populations. The QTLs were clustered such that most genomic regions were associated with more than one element. In this study, quantitative trait loci (QTLs) affecting the concentrations of 16 elements in whole, unmilled rice (Oryza sativa L.) grain were identified. Two rice mapping populations, the 'Lemont' × 'TeQing' recombinant inbred lines (LT-RILs), and the TeQing-into-Lemont backcross introgression lines (TILs) were used. To increase opportunity to detect and characterize QTLs, the TILs were grown under two contrasting field conditions, flooded and irrigated-but-unflooded. Correlations between the individual elements and between each element with grain shape, plant height, and time of heading were also studied. Transgressive segregation was observed among the LT-RILs for all elements. The 134 QTLs identified as associated with the grain concentrations of individual elements were found clustered into 39 genomic regions, 34 of which were found associated with grain element concentration in more than one population and/or flooding treatment. More QTLs were found significant among flooded TILs (92) than among unflooded TILs (47) or among flooded LT-RILs (40). Twenty-seven of the 40 QTLs identified among the LT-RILs were associated with the same element among the TILs. At least one QTL per element was validated in two or more population/environments. Nearly all of the grain element loci were linked to QTLs affecting additional elements, supporting the concept of element networks within plants. Several of the grain element QTLs co-located with QTLs for grain shape, plant height, and days to heading; but did not always differ for grain elemental concentration as predicted by those traits alone. A number of interesting patterns were found, including a strong Mg­P­K complex.

A chloroplast localized heavy metal-associated domain containing protein regulates grain calcium accumulation in rice.

Calcium (Ca) is an essential mineral nutrient and plays a crucial signaling role in all living organisms. Increasing Ca content in staple foods such as rice is vital for improving Ca nutrition of humans. Here we map a quantitative trait locus that controls Ca concentration in rice grains and identify the causal gene as GCSC1 (Grain Ca and Sr Concentrations 1), which encodes a chloroplast vesicle localized homo-oligomeric protein. GCSC1 exhibits Ca2+ transport activity in heterologous assays in yeast and Xenopus laevis oocytes and is involved in the efflux of Ca2+ from the chloroplast to the cytosol. Knockout of GCSC1 results in increased chloroplast Ca concentration, lower stomatal conductance in leaves and enhanced Ca allocation to grains. Natural variation in grain Ca concentration is attributed to the variable expression of GCSC1 resulting from its promoter sequence variation. Our study identifies a chloroplast localized heavy metal-associated domain containing protein that regulates chloroplast Ca2+ efflux and provides a way to biofortify Ca in rice to benefit human nutrition.

Getting a sense for signals: regulation of the plant iron deficiency response.

Understanding the Fe deficiency response in plants is necessary for improving both plant health and the human diet, which relies on Fe from plant sources. In this review we focus on the regulation of the two major strategies for iron acquisition in plants, exemplified by the model plants Arabidopsis and rice. Critical to our knowledge of Fe homeostasis in plants is determining how Fe is sensed and how this signal is transmitted and integrated into a response. We will explore the evidence for an Fe sensor in plants and summarize the recent findings on hormones and signaling molecules which contribute to the Fe deficiency response. This article is part of a Special Issue entitled: Cell Biology of Metals.

The role of CAX1 and CAX3 in elemental distribution and abundance in Arabidopsis seed.

The ability to alter nutrient partitioning within plants cells is poorly understood. In Arabidopsis (Arabidopsis thaliana), a family of endomembrane cation exchangers (CAXs) transports Ca(2+) and other cations. However, experiments have not focused on how the distribution and partitioning of calcium (Ca) and other elements within seeds are altered by perturbed CAX activity. Here, we investigate Ca distribution and abundance in Arabidopsis seed from cax1 and cax3 loss-of-function lines and lines expressing deregulated CAX1 using synchrotron x-ray fluorescence microscopy. We conducted 7- to 10-µm resolution in vivo x-ray microtomography on dry mature seed and 0.2-µm resolution x-ray microscopy on embryos from lines overexpressing deregulated CAX1 (35S-sCAX1) and cax1cax3 double mutants only. Tomograms showed an increased concentration of Ca in both the seed coat and the embryo in cax1, cax3, and cax1cax3 lines compared with the wild type. High-resolution elemental images of the mutants showed that perturbed CAX activity altered Ca partitioning within cells, reducing Ca partitioning into organelles and/or increasing Ca in the cytosol and abolishing tissue-level Ca gradients. In comparison with traditional volume-averaged metal analysis, which confirmed subtle changes in seed elemental composition, the collection of spatially resolved data at varying resolutions provides insight into the impact of altered CAX activity on seed metal distribution and indicates a cell type-specific function of CAX1 and CAX3 in partitioning Ca into organelles. This work highlights a powerful technology for inferring transport function and quantifying nutrient changes.