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 basic helix-loop-helix (bHLH) transcription factors controls the homeostasis of iron. Among these transcription factors, bHLH121 plays a crucial role. bHLH121 interacts in vivo with clade IVc bHLH transcription factors and activates the expression of FIT and clade Ib bHLH transcription factors to stimulate the uptake of iron. How bHLH121 and clade IVc bHLH transcription factors 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 transcription factors displayed more severe iron deficiency-associated growth defects than each of the single mutants. We also found that among the four clade IVc bHLH transcription factors, only 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 transcription factors act synergistically to regulate iron homeostasis and that different bHLH121/clade IVc and clade IVc/clade IVc protein complexes are involved in this process.

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.

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 Transcription Factor bHLH121 Interacts with bHLH105 (ILR3) and Its Closest Homologs to Regulate Iron Homeostasis in Arabidopsis.

Iron (Fe) is an essential micronutrient for plant growth and development. Any defects in the maintenance of Fe homeostasis will alter plant productivity and the quality of their derived products. In Arabidopsis (Arabidopsis thaliana), the transcription factor ILR3 plays a central role in controlling Fe homeostasis. In this study, we identified bHLH121 as an ILR3-interacting transcription factor. Interaction studies showed that bHLH121 also interacts with the three closest homologs of ILR3 (i.e., basic-helix-loop-helix 34 [bHLH34], bHLH104, and bHLH115). bhlh121 loss-of-function mutants displayed severe defects in Fe homeostasis that could be reverted by exogenous Fe supply. bHLH121 acts as a direct transcriptional activator of key genes involved in the Fe regulatory network, including bHLH38, bHLH39, bHLH100, bHLH101, POPEYE, BRUTUS, and BRUTUS LIKE1, as well as IRONMAN1 and IRONMAN2 In addition, bHLH121 is necessary for activating the expression of transcription factor gene FIT in response to Fe deficiency via an indirect mechanism. bHLH121 is expressed throughout the plant body, and its expression is not affected by Fe availability. By contrast, Fe availability affects the cellular localization of bHLH121 protein in roots. Altogether, these data show that bHLH121 is a regulator of Fe homeostasis that acts upstream of FIT in concert with ILR3 and its closest homologs.

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.

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.

The plastidial Arabidopsis thaliana NFU1 protein binds and delivers [4Fe-4S] clusters to specific client proteins.

Proteins incorporating iron-sulfur (Fe-S) co-factors are required for a plethora of metabolic processes. Their maturation depends on three Fe-S cluster assembly machineries in plants, located in the cytosol, mitochondria, and chloroplasts. After de novo formation on scaffold proteins, transfer proteins load Fe-S clusters onto client proteins. Among the plastidial representatives of these transfer proteins, NFU2 and NFU3 are required for the maturation of the [4Fe-4S] clusters present in photosystem I subunits, acting upstream of the high-chlorophyll fluorescence 101 (HCF101) protein. NFU2 is also required for the maturation of the [2Fe-2S]-containing dihydroxyacid dehydratase, important for branched-chain amino acid synthesis. Here, we report that recombinant Arabidopsis thaliana NFU1 assembles one [4Fe-4S] cluster per homodimer. Performing co-immunoprecipitation experiments and assessing physical interactions of NFU1 with many [4Fe-4S]-containing plastidial proteins in binary yeast two-hybrid assays, we also gained insights into the specificity of NFU1 for the maturation of chloroplastic Fe-S proteins. Using bimolecular fluorescence complementation and in vitro Fe-S cluster transfer experiments, we confirmed interactions with two proteins involved in isoprenoid and thiamine biosynthesis, 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase and 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate synthase, respectively. An additional interaction detected with the scaffold protein SUFD enabled us to build a model in which NFU1 receives its Fe-S cluster from the SUFBC2D scaffold complex and serves in the maturation of specific [4Fe-4S] client proteins. The identification of the NFU1 partner proteins reported here more clearly defines the role of NFU1 in Fe-S client protein maturation in Arabidopsis chloroplasts among other SUF components.

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.

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.

Identification of client iron-sulfur proteins of the chloroplastic NFU2 transfer protein in Arabidopsis thaliana.

Iron-sulfur (Fe-S) proteins have critical functions in plastids, notably participating in photosynthetic electron transfer, sulfur and nitrogen assimilation, chlorophyll metabolism, and vitamin or amino acid biosynthesis. Their maturation relies on the so-called SUF (sulfur mobilization) assembly machinery. Fe-S clusters are synthesized de novo on a scaffold protein complex and then delivered to client proteins via several transfer proteins. However, the maturation pathways of most client proteins and their specificities for transfer proteins are mostly unknown. In order to decipher the proteins interacting with the Fe-S cluster transfer protein NFU2, one of the three plastidial representatives found in Arabidopsis thaliana, we performed a quantitative proteomic analysis of shoots, roots, and seedlings of nfu2 plants, combined with NFU2 co-immunoprecipitation and binary yeast two-hybrid experiments. We identified 14 new targets, among which nine were validated in planta using a binary bimolecular fluorescence complementation assay. These analyses also revealed a possible role for NFU2 in the plant response to desiccation. Altogether, this study better delineates the maturation pathways of many chloroplast Fe-S proteins, considerably extending the number of NFU2 clients. It also helps to clarify the respective roles of the three NFU paralogs NFU1, NFU2, and NFU3.

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.

Transcriptional integration of the responses to iron availability in Arabidopsis by the bHLH factor ILR3.

Iron (Fe) homeostasis is crucial for all living organisms. In mammals, an integrated posttranscriptional mechanism couples the regulation of both Fe deficiency and Fe excess responses. Whether in plants an integrated control mechanism involving common players regulates responses both to deficiency and to excess is still to be determined. In this study, molecular, genetic and biochemical approaches were used to investigate transcriptional responses to both Fe deficiency and excess. A transcriptional activator of responses to Fe shortage in Arabidopsis, called bHLH105/ILR3, was found to also negatively regulate the expression of ferritin genes, which are markers of the plant's response to Fe excess. Further investigations revealed that ILR3 repressed the expression of several structural genes that function in the control of Fe homeostasis. ILR3 interacts directly with the promoter of its target genes, and repressive activity was conferred by its dimerisation with bHLH47/PYE. Last, this study highlighted that important facets of plant growth in response to Fe deficiency or excess rely on ILR3 activity. Altogether, the data presented herein support that ILR3 is at the centre of the transcriptional regulatory network that controls Fe homeostasis in Arabidopsis, in which it acts as both transcriptional activator and repressor.

Iron-sulfur protein NFU2 is required for branched-chain amino acid synthesis in Arabidopsis roots.

Numerous proteins require a metallic co-factor for their function. In plastids, the maturation of iron-sulfur (Fe-S) proteins necessitates a complex assembly machinery. In this study, we focused on Arabidopsis thaliana NFU1, NFU2, and NFU3, which participate in the final steps of the maturation process. According to the strong photosynthetic defects observed in high chlorophyll fluorescence 101 (hcf101), nfu2, and nfu3 plants, we determined that NFU2 and NFU3, but not NFU1, act immediately upstream of HCF101 for the maturation of [Fe4S4]-containing photosystem I subunits. An additional function of NFU2 in the maturation of the [Fe2S2] cluster of a dihydroxyacid dehydratase was obvious from the accumulation of precursors of the branched-chain amino acid synthesis pathway in roots of nfu2 plants and from the rescue of the primary root growth defect by supplying branched-chain amino acids. The absence of NFU3 in roots precluded any compensation. Overall, unlike their eukaryotic and prokaryotic counterparts, which are specific to [Fe4S4] proteins, NFU2 and NFU3 contribute to the maturation of both [Fe2S2] and [Fe4S4] proteins, either as a relay in conjunction with other proteins such as HCF101 or by directly delivering Fe-S clusters to client proteins. Considering the low number of Fe-S cluster transfer proteins relative to final acceptors, additional targets probably await identification.

TransDetect Identifies a New Regulatory Module Controlling Phosphate Accumulation.

Identifying transcription factor (TFs) cooperation controlling target gene expression is still an arduous challenge. The accuracy of current methods at genome scale significantly drops with the increase in number of genes, which limits their applicability to more complex genomes, like animals and plants. Here, we developed an algorithm, TransDetect, able to predict TF combinations controlling the expression level of a given gene. TransDetect was used to identify novel TF modules regulating the expression of Arabidopsis (Arabidopsis thaliana) phosphate transporter PHO1;H3 comprising MYB15, MYB84, bHLH35, and ICE1. These TFs were confirmed to interact between themselves and with the PHO1;H3 promoter. Phenotypic and genetic analyses of TF mutants enable the organization of these four TFs and PHO1;H3 in a new gene regulatory network controlling phosphate accumulation in zinc-dependent manner. This demonstrates the potential of TransDetect to extract directionality in nondynamic transcriptomes and to provide a blueprint to identify gene regulatory network involved in a given biological process.

Integrating bioinformatic resources to predict transcription factors interacting with cis-sequences conserved in co-regulated genes.

BACKGROUND: Using motif detection programs it is fairly straightforward to identify conserved cis-sequences in promoters of co-regulated genes. In contrast, the identification of the transcription factors (TFs) interacting with these cis-sequences is much more elaborate. To facilitate this, we explore the possibility of using several bioinformatic and experimental approaches for TF identification. This starts with the selection of co-regulated gene sets and leads first to the prediction and then to the experimental validation of TFs interacting with cis-sequences conserved in the promoters of these co-regulated genes. RESULTS: Using the PathoPlant database, 32 up-regulated gene groups were identified with microarray data for drought-responsive gene expression from Arabidopsis thaliana. Application of the binding site estimation suite of tools (BEST) discovered 179 conserved sequence motifs within the corresponding promoters. Using the STAMP web-server, 49 sequence motifs were classified into 7 motif families for which similarities with known cis-regulatory sequences were identified. All motifs were subjected to a footprintDB analysis to predict interacting DNA binding domains from plant TF families. Predictions were confirmed by using a yeast-one-hybrid approach to select interacting TFs belonging to the predicted TF families. TF-DNA interactions were further experimentally validated in yeast and with a Physcomitrella patens transient expression system, leading to the discovery of several novel TF-DNA interactions. CONCLUSIONS: The present work demonstrates the successful integration of several bioinformatic resources with experimental approaches to predict and validate TFs interacting with conserved sequence motifs in co-regulated genes.

Complexity and robustness of the flavonoid transcriptional regulatory network revealed by comprehensive analyses of MYB-bHLH-WDR complexes and their targets in Arabidopsis seed.

In Arabidopsis thaliana, proanthocyanidins (PAs) accumulate in the innermost cell layer of the seed coat (i.e. endothelium, chalaza and micropyle). The expression of the biosynthetic genes involved relies on the transcriptional activity of R2R3-MYB and basic helix-loop-helix (bHLH) proteins which form ternary complexes ('MBW') with TRANSPARENT TESTA GLABRA1 (TTG1) (WD repeat protein). The identification of the direct targets and the determination of the nature and spatio-temporal activity of these MBW complexes are essential steps towards a comprehensive understanding of the transcriptional mechanisms that control flavonoid biosynthesis. In this study, various molecular, genetic and biochemical approaches were used. Here, we have demonstrated that, of the 12 studied genes of the pathway, only dihydroflavonol-4-reductase (DFR), leucoanthocyanidin dioxygenase (LDOX), BANYULS (BAN), TRANSPARENT TESTA 19 (TT19), TT12 and H(+) -ATPase isoform 10 (AHA10) are direct targets of the MBW complexes. Interestingly, although the TT2-TT8-TTG1 complex plays the major role in developing seeds, three additional MBW complexes (i.e. MYB5-TT8-TTG1, TT2-EGL3-TTG1 and TT2-GL3-TTG1) were also shown to be involved, in a tissue-specific manner. Finally, a minimal promoter was identified for each of the target genes of the MBW complexes. Altogether, by answering fundamental questions and by demonstrating or invalidating previously made hypotheses, this study provides a new and comprehensive view of the transcriptional regulatory mechanisms controlling PA and anthocyanin biosynthesis in Arabidopsis.

Transcriptional regulation of Arabidopsis LEAFY COTYLEDON2 involves RLE, a cis-element that regulates trimethylation of histone H3 at lysine-27.

LEAFY COTYLEDON2 (LEC2) is a master regulator of seed development in Arabidopsis thaliana. In vegetative organs, LEC2 expression is negatively regulated by Polycomb Repressive Complex2 (PRC2) that catalyzes histone H3 Lys 27 trimethylation (H3K27me3) and plays a crucial role in developmental phase transitions. To characterize the cis-regulatory elements involved in the transcriptional regulation of LEC2, molecular dissections and functional analyses of the promoter region were performed in vitro, both in yeast and in planta. Two cis-activating elements and a cis-repressing element (RLE) that is required for H3K27me3 marking were characterized. Remarkably, insertion of the RLE cis-element into pF3H, an unrelated promoter, is sufficient for repressing its transcriptional activity in different tissues. Besides improving our understanding of LEC2 regulation, this study provides important new insights into the mechanisms underlying H3K27me3 deposition and PRC2 recruitment at a specific locus in plants.

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.

Regulation of flavonoid biosynthesis involves an unexpected complex transcriptional regulation of TT8 expression, in Arabidopsis.

TT8/bHLH042 is a key regulator of anthocyanins and proanthocyanidins (PAs) biosynthesis in Arabidopsis thaliana. TT8 transcriptional activity has been studied extensively, and relies on its ability to form, with several R2R3-MYB and TTG1 (WD-Repeat protein), different MYB-bHLH-WDR (MBW) protein complexes. By contrast, little is known on how TT8 expression is itself regulated. Transcriptional regulation of TT8 expression was studied using molecular, genetic and biochemical approaches. Functional dissection of the TT8 promoter revealed its modular structure. Two modules were found to specifically drive TT8 promoter activity in PA- and anthocyanin-accumulating cells, by differentially integrating the signals issued from different regulators, in a spatio-temporal manner. Interestingly, this regulation involves at least six different MBW complexes, and an unpredicted positive feedback regulatory loop between TT8 and TTG2. Moreover, the results suggest that some putative new regulators remain to be discovered. Finally, specific cis-regulatory elements through which TT8 expression is regulated were identified and characterized. Together, these results provide a molecular model consistent with the specific and highly regulated expression of TT8. They shed new light into the transcriptional regulation of flavonoid biosynthesis and provide new clues and tools for further investigation in Arabidopsis and other plant species.