bHLH121 and clade IVc bHLH transcription factors synergistically function to regulate iron homeostasis in Arabidopsis thaliana.

Iron is an essential micronutrient for plant growth and development. In Arabidopsis thaliana, an intricate regulatory network involving several bHLH transcription factors (TFs) controls the homeostasis of iron. Among these TFs, bHLH121 plays a crucial role. bHLH121 interacts in vivo with clade IVc bHLH TFs and activates the expression of FIT and clade Ib bHLH TFs to stimulate the uptake of iron. How bHLH121 and clade IVc bHLH TFs function collectively and efficiently to maintain iron homeostasis is still unclear. Herein, we found that double loss-of-function mutants involving bhlh121 and one of the clade IVc bHLH displayed more severe iron deficiency associated growth defects than each of the single ones. We also found that among the four clade IVc bHLH TFs, solely bHLH34 and bHLH105 could partially complement the iron-associated growth defects of bhlh121 when overexpressed. These data, together with protein localization analysis, support that bHLH121 and clade IVc bHLH TFs act synergistically to regulate iron homeostasis and that different bHLH121/clade IVc and clade IVc/clade IVc protein complexes are involved in this process.

The arabidopsis bHLH transcription factor family.

Basic helix-loop-helices (bHLHs) are present in all eukaryotes and form one of the largest families of transcription factors (TFs) found in plants. bHLHs function as transcriptional activators and/or repressors of genes involved in key processes involved in plant growth and development in interaction with the environment (e.g., stomata and root hair development, iron homeostasis, and response to heat and shade). Recent studies have improved our understanding of the functioning of bHLH TFs in complex regulatory networks where a series of post-translational modifications (PTMs) have critical roles in regulating their subcellular localization, DNA-binding capacity, transcriptional activity, and/or stability (e.g., protein-protein interactions, phosphorylation, ubiquitination, and sumoylation). Further elucidating the function and regulation of bHLHs will help further understanding of the biology of plants in general and for the development of new tools for crop improvement.

The Impact of Elevated Atmospheric Carbon Dioxide Exposure on Magic Tomatoes' Nutrition-Health Properties.

The release of carbon dioxide (CO2) into the atmosphere has accelerated during the last two decades. Elevated atmospheric CO2 concentration (eCO2) is known as an agent that improves plant photosynthesis. However, eCO2 was also correlated with alterations in the macronutrient and micronutrient compositions of various dietary crops. In order to explore the effect of eCO2 on the nutritional and health properties of tomatoes, three parental lines of the Magic population, which includes a large part of the genetic diversity present in large fruit varieties, were used as models. The plants were grown in growth chambers under ambient (400 ppm) or eCO2 (900 ppm) conditions. The macronutrient and micronutrient contents were measured. The anti-oxidant and anti-inflammatory bioactivities were assessed in vitro on activated macrophages. These analyses highlighted that the carbohydrate content was not affected by the eCO2, whereas the protein, carotenoid, lycopene, and mineral contents decreased. Regarding the anti-oxidant properties, no influence of eCO2 exposure was observed. Similarly, the anti-inflammatory properties were not affected by the eCO2. These data are in contrast with previous studies conducted on different plant species or accessions, indicating that the effect of eCO2 on crops' nutrition and health properties is based on complex mechanisms in which growth conditions and genetic backgrounds play a central role.

Reduction in PLANT DEFENSIN 1 expression in Arabidopsis thaliana results in increased resistance to pathogens and zinc toxicity.

Ectopic expression of defensins in plants correlates with their increased capacity to withstand abiotic and biotic stresses. This applies to Arabidopsis thaliana, where some of the seven members of the PLANT DEFENSIN 1 family (AtPDF1) are recognised to improve plant responses to necrotrophic pathogens and increase seedling tolerance to excess zinc (Zn). However, few studies have explored the effects of decreased endogenous defensin expression on these stress responses. Here, we carried out an extensive physiological and biochemical comparative characterization of (i) novel artificial microRNA (amiRNA) lines silenced for the five most similar AtPDF1s, and (ii) a double null mutant for the two most distant AtPDF1s. Silencing of five AtPDF1 genes was specifically associated with increased aboveground dry mass production in mature plants under excess Zn conditions, and with increased plant tolerance to different pathogens - a fungus, an oomycete and a bacterium, while the double mutant behaved similarly to the wild type. These unexpected results challenge the current paradigm describing the role of PDFs in plant stress responses. Additional roles of endogenous plant defensins are discussed, opening new perspectives for their functions.

The Use of Spectral Imaging to Follow the Iron and pH-Dependent Accumulation of Fluorescent Coumarins.

Plants challenged with iron deficiency produce in their roots and secrete into the rhizosphere several small molecules named coumarins that derive from the phenylpropanoid pathway. Coumarins are biosynthesized in different root cell types and transported to the root epidermis prior to their secretion in the surrounding media. Taking advantage of the natural fluorescence of most coumarins glycosides when exposed to UV light, we developed a method to uncover their individual cellular localization and accumulation. This approach couples spectral imaging acquisition and linear unmixing analysis. In this protocol, we describe guidelines, experimental setup, and conditions for the analysis of coumarins localization and accumulation in Arabidopsis thaliana root seedlings grown in control and iron deficiency conditions, at both acidic and alkaline pH.

Chromatin Immunoprecipitation (ChIP) to Study the Transcriptional Regulatory Network that Controls Iron Homeostasis in Arabidopsis thaliana.

In plants, gene expression is orchestrated by thousands of transcription factors (TFs). For instance, a large set of bHLH TFs are involved in the regulation of iron homeostasis in Arabidopsis thaliana. The identification of the direct target genes of TFs through uncovering the interaction between the TFs and cis-regulatory elements has become an essential step toward a comprehensive understanding of the iron homeostasis transcriptional regulatory network in Arabidopsis. Chromatin immunoprecipitation (ChIP) followed by qRT-PCR (ChIP-qPCR), sequencing (ChIP-seq), or chip hybridization (ChIP-chip) is a robust tool to investigate protein-DNA interactions in plants in a physiological context. The procedure generally includes six steps: DNA-protein crosslink, isolation of nuclei, shearing of chromatin, immunoprecipitation, DNA purification, and qRT-PCR analyses. In this protocol, we describe guidelines, experimental setup, and conditions for ChIP experiment in Arabidopsis. This protocol focuses on seedlings grown in control and iron deficiency conditions, but can readily be adapted for use with other Arabidopsis tissues or samples. In addition, the protocol could also be applied to perform ChIP-chip or ChIP-seq experiments.

A gene regulatory network in Arabidopsis roots reveals features and regulators of the plant response to elevated CO2.

The elevation of CO2 in the atmosphere increases plant biomass but decreases their mineral content. The genetic and molecular bases of these effects remain mostly unknown, in particular in the root system, which is responsible for plant nutrient uptake. To gain knowledge about the effect of elevated CO2 on plant growth and physiology, and to identify its regulatory in the roots, we analyzed genome expression in Arabidopsis roots through a combinatorial design with contrasted levels of CO2 , nitrate, and iron. We demonstrated that elevated CO2 has a modest effect on root genome expression under nutrient sufficiency, but by contrast leads to massive expression changes under nitrate or iron deficiencies. We demonstrated that elevated CO2 negatively targets nitrate and iron starvation modules at the transcriptional level, associated with a reduction in high-affinity nitrate uptake. Finally, we inferred a gene regulatory network governing the root response to elevated CO2 . This network allowed us to identify candidate transcription factors including MYB15, WOX11, and EDF3 which we experimentally validated for their role in the stimulation of growth by elevated CO2 . Our approach identified key features and regulators of the plant response to elevated CO2 , with the objective of developing crops resilient to climate change.

Iron Nutrition in Plants: Towards a New Paradigm?

Iron (Fe) is an essential micronutrient for plant growth and development. Fe availability affects crops' productivity and the quality of their derived products and thus human nutrition. Fe is poorly available for plant use since it is mostly present in soils in the form of insoluble oxides/hydroxides, especially at neutral to alkaline pH. How plants cope with low-Fe conditions and acquire Fe from soil has been investigated for decades. Pioneering work highlighted that plants have evolved two different strategies to mine Fe from soils, the so-called Strategy I (Fe reduction strategy) and Strategy II (Fe chelation strategy). Strategy I is employed by non-grass species whereas graminaceous plants utilize Strategy II. Recently, it has emerged that these two strategies are not fully exclusive and that the mechanism used by plants for Fe uptake is directly shaped by the characteristics of the soil on which they grow (e.g., pH, oxygen concentration). In this review, recent findings on plant Fe uptake and the regulation of this process will be summarized and their impact on our understanding of plant Fe nutrition will be discussed.

Effect of Elevated Carbon Dioxide Exposure on Nutrition-Health Properties of Micro-Tom Tomatoes.

(1) Background: The anthropogenically induced rise in atmospheric carbon dioxide (CO2) and associated climate change are considered a potential threat to human nutrition. Indeed, an elevated CO2 concentration was associated with significant alterations in macronutrient and micronutrient content in various dietary crops. (2) Method: In order to explore the impact of elevated CO2 on the nutritional-health properties of tomato, we used the dwarf tomato variety Micro-Tom plant model. Micro-Toms were grown in culture chambers under 400 ppm (ambient) or 900 ppm (elevated) carbon dioxide. Macronutrients, carotenoids, and mineral contents were analyzed. Biological anti-oxidant and anti-inflammatory bioactivities were assessed in vitro on activated macrophages. (3) Results: Micro-Tom exposure to 900 ppm carbon dioxide was associated with an increased carbohydrate content whereas protein, minerals, and total carotenoids content were decreased. These modifications of composition were associated with an altered bioactivity profile. Indeed, antioxidant anti-inflammatory potential were altered by 900 ppm CO2 exposure. (4) Conclusions: Taken together, our results suggest that (i) the Micro-Tom is a laboratory model of interest to study elevated CO2 effects on crops and (ii) exposure to 900 ppm CO2 led to the decrease of nutritional potential and an increase of health beneficial properties of tomatoes for human health.

B3 Transcription Factors Determine Iron Distribution and FERRITIN Gene Expression in Embryo but Do Not Control Total Seed Iron Content.

Iron is an essential micronutrient for humans and other organisms. Its deficiency is one of the leading causes of anemia worldwide. The world health organization has proposed that an alternative to increasing iron content in food is through crop biofortification. One of the most consumed part of crops is the seed, however, little is known about how iron accumulation in seed occurs and how it is regulated. B3 transcription factors play a critical role in the accumulation of storage compounds such as proteins and lipids. Their role in seed maturation has been well characterized. However, their relevance in accumulation and distribution of micronutrients like iron remains unknown. In Arabidopsis thaliana and other plant models, three master regulators belonging to the B3 transcription factors family have been identified: FUSCA3 (FUS3), LEAFY COTYLEDON2 (LEC2), and ABSCISIC ACID INSENSITIVE 3 (ABI3). In this work, we studied how seed iron homeostasis is affected in B3 transcription factors mutants using histological and molecular approaches. We determined that iron distribution is modified in abi3, lec2, and fus3 embryo mutants. For abi3-6 and fus3-3 mutant embryos, iron was less accumulated in vacuoles of cells surrounding provasculature compared with wild type embryos. lec2-1 embryos showed no difference in the pattern of iron distribution in hypocotyl, but a dramatic decrease of iron was observed in cotyledons. Interestingly, for the three mutant genotypes, total iron content in dry mutant seeds showed no difference compared to wild type. At the molecular level, we showed that genes encoding the iron storage ferritins proteins are misregulated in mutant seeds. Altogether our results support a role of the B3 transcription factors ABI3, LEC2, and FUS3 in maintaining iron homeostasis in Arabidopsis embryos.

2000 years of agriculture in the Atacama desert lead to changes in the distribution and concentration of iron in maize.

We performed a histological and quantitative study of iron in archaeological maize seeds from prehispanic times recovered from Tarapacá, Atacama Desert. Also, we examined iron distribution changes at the cell level in embryos from ancient versus new varieties of maize. Our results show a progressive decrease in iron concentration from the oldest maize to modern specimens. We interpret the results as an effect of prehispanic agriculture over the micronutrient composition of maize.

Transcriptional integration of plant responses to iron availability.

Iron is one of the most important micronutrients for plant growth and development. It functions as the enzyme cofactor or component of electron transport chains in various vital metabolic processes, including photosynthesis, respiration, and amino acid biosynthesis. To maintain iron homeostasis, and therefore prevent any deficiency or excess that could be detrimental, plants have evolved complex transcriptional regulatory networks to tightly control iron uptake, translocation, assimilation, and storage. These regulatory networks are composed of various transcription factors; among them, members of the basic helix-loop-helix (bHLH) family play an essential role. Here, we first review recent advances in understanding the roles of bHLH transcription factors involved in the regulatory cascade controlling iron homeostasis in the model plant Arabidopsis, and extend this understanding to rice and other plant species. The importance of other classes of transcription factors will also be discussed. Second, we elaborate on the post-translational mechanisms involved in the regulation of these regulatory networks. Finally, we provide some perspectives on future research that should be conducted in order to precisely understand how plants control the homeostasis of this micronutrient.

The Coumarins: Secondary Metabolites Playing a Primary Role in Plant Nutrition and Health.

Although abundant in soils, iron (Fe) is poorly bioavailable for plants. Improving Fe uptake in crops, enabling them to grow in Fe-depleted soils, has become a major focal interest. The secretion of Fe-mobilizing coumarins by plant roots recently emerged as an important factor allowing nongrass species to cope with low Fe bioavailability. The main molecular actors involved in the biosynthesis and secretion of coumarins have been identified, but the precise regulatory mechanisms that tune their production remain poorly understood. Here, we review the recent progress in coumarin synthesis and transport in plants and future research directions to gain knowledge of these mechanisms, which will offer novel opportunities for improving plant growth and health and for generating Fe-fortified crops.

Coumarin accumulation and trafficking in Arabidopsis thaliana: a complex and dynamic process.

Iron (Fe) is a major micronutrient and is required for plant growth and development. Nongrass species have evolved a reduction-based strategy to solubilize and take up Fe. The secretion of Fe-mobilizing coumarins (e.g. fraxetin, esculetin and sideretin) by plant roots plays an important role in this process. Although the biochemical mechanisms leading to their biosynthesis have been well described, very little is known about their cellular and subcellular localization or their mobility within plant tissues. Spectral imaging was used to monitor, in Arabidopsis thaliana, the in planta localization of Fe-mobilizing coumarins and scopolin. Molecular, genetic and biochemical approaches were also used to investigate the dynamics of coumarin accumulation in roots. These approaches showed that root hairs play a major role in scopoletin secretion, whereas fraxetin and esculetin secretion occurs through all epidermis cells. The findings of this study also showed that the transport of coumarins from the cortex to the rhizosphere relies on the PDR9 transporter under Fe-deficient conditions. Additional experiments support the idea that coumarins move throughout the plant body via the xylem sap and that several plant species can take up coumarins present in the surrounding media. Altogether, the data presented here demonstrate that coumarin storage and accumulation in roots is a highly complex and dynamic process.

A Global Proteomic Approach Sheds New Light on Potential Iron-Sulfur Client Proteins of the Chloroplastic Maturation Factor NFU3.

Iron-sulfur (Fe-S) proteins play critical functions in plants. Most Fe-S proteins are synthetized in the cytosol as apo-proteins and the subsequent Fe-S cluster incorporation relies on specific protein assembly machineries. They are notably formed by a scaffold complex, which serves for the de novo Fe-S cluster synthesis, and by transfer proteins that insure cluster delivery to apo-targets. However, scarce information is available about the maturation pathways of most plastidial Fe-S proteins and their specificities towards transfer proteins of the associated SUF machinery. To gain more insights into these steps, the expression and protein localization of the NFU1, NFU2, and NFU3 transfer proteins were analyzed in various Arabidopsis thaliana organs and tissues showing quite similar expression patterns. In addition, quantitative proteomic analysis of an nfu3 loss-of-function mutant allowed to propose novel potential client proteins for NFU3 and to show that the protein accumulation profiles and thus metabolic adjustments differ substantially from those established in the nfu2 mutant. By clarifying the respective roles of the three plastidial NFU paralogs, these data allow better delineating the maturation process of plastidial Fe-S proteins.

Sulphur availability modulates Arabidopsis thaliana responses to iron deficiency.

Among the mineral nutrients that are required for plant metabolism, iron (Fe) and sulphur (S) play a central role as both elements are needed for the activity of several proteins involved in essential cellular processes. A combination of physiological, biochemical and molecular approaches was employed to investigate how S availability influences plant response to Fe deficiency, using the model plant Arabidopsis thaliana. We first observed that chlorosis symptom induced by Fe deficiency was less pronounced when S availability was scarce. We thus found that S deficiency inhibited the Fe deficiency induced expression of several genes associated with the maintenance of Fe homeostasis. This includes structural genes involved in Fe uptake (i.e. IRT1, FRO2, PDR9, NRAMP1) and transport (i.e. FRD3, NAS4) as well as a subset of their upstream regulators, namely BTS, PYE and the four clade Ib bHLH. Last, we found that the over accumulation of manganese (Mn) in response to Fe shortage was reduced under combined Fe and S deficiencies. These data suggest that S deficiency inhibits the Fe deficiency dependent induction of the Fe uptake machinery. This in turn limits the transport into the root and the plant body of potentially toxic divalent cations such as Mn and Zn, thus limiting the deleterious effect of Fe deprivation.

Further insights into the role of bHLH121 in the regulation of iron homeostasis in Arabidopsis thaliana.

Iron (Fe) is an important micronutrient for plant growth and development but any excess of Fe is toxic because of the Fe-dependent generation of reactive oxygen species (ROS). Thus, Fe homeostasis must be tightly regulated. In Arabidopsis thaliana, a cascade of transcription factors has been identified as involved in the regulation of this process by modulating the expression of genes related to Fe uptake, transport, and storage. Recently, it was demonstrated that in response to Fe deficiency, bHLH121/URI (UPSTREAM REGULATOR OF IRT1) directly activates the expression of several genes involved in this regulatory network. It was also shown that bHLH121 interacts with ILR3 (bHLH105) and its homologs. Herein it is shown that bHLH121 is necessary for the expression of the main markers of the plant responses to Fe excess, the ferritin genes (i.e. FER1, FER3, and FER4). bHLH121 regulates ferritin genes expression by directly binding to their promoters, at the same locus than the ILR3-PYE repressive complex. Therefore, this study highlight that bHLH121, PYE, and ILR3 form a chain of antagonistic switches that regulate the expression of ferritin genes. The implication of this finding is discussed.