Phytoplasmas are obligate plant pathogens that include species causing severe developmental abnormalities in diverse plant families. Phytoplasma infections may result in dwarfism, witches' broom, virescence and phyllody, the conversion of reproductive organs into leaf-like structures. Phyllody is due to the activity of phytoplasma effector proteins, called phyllogens, that interact with the host MADS transcription factor (MTF) network. This interaction was believed to be specific for members of the AP1/SEP/AGL6 superclade of tetrameric MTFs, key regulators of floral meristem identity and organogenesis. Here, we determined the molecular and atomic basis for the interaction between the phyllogen​ from Onions Yellow phytoplasma (PHYLOY) and the floral MTFs, APETALA1, SEPALLATA and AGAMOUS using biochemical, structural and genetic experiments. The DNA-binding patterns of homo and heteromeric MTFs with PHYLOY are shown using electrophoretic mobility shift assays, revealing broader interaction patterns between PHYLOY and MTFs that depend on highly conserved hydrophobic residues. Point mutations of these residues allowed gain or loss of interaction between MTFs and PHYLOY. Expressing PHYLOY under the control of the SEP3 and AP1 promoter in Arabidopsis further demonstrated whorl specific phenotypes, recapitulating the in vitro interaction and DNA-binding patterns. These findings reveal how PHYLOY targets specific MTFs through structural mimicry, resulting in impaired MTF tetramer formation and homeotic conversion of floral organs due to loss of active MTF tetramers.

MADS genes encode transcription factors that act as master regulators of plant reproduction and flower development. The SEPALLATA (SEP) subfamily is required for the development of floral organs and plays roles in inflorescence architecture and development of the floral meristem. The SEPALLTAs act as organizers of MADS complexes, forming both heterodimers and heterotetramers in vitro . To date, the MADS complexes characterized in angiosperm floral organ development contain at least one SEPALLATA protein. Whether DNA-binding by SEPALLATA-containing dimeric MADS complexes are sufficient for launching floral organ identity programs, however, is not clear as only defects in floral meristem determinacy were observed in tetramerization impaired SEPALLATA mutants. Here, we used a combination of genome-wide binding studies, high resolution structural studies of the SEP3/AGAMOUS tetramerization domain, structure-based mutagenesis and complementation experiments in sep1 sep2 sep3 and sep1 sep2 sep3 ag-4 plants transformed with versions of SEP3 encoding tetramerization mutants. We demonstrate that while SEP3 heterodimers are able to bind DNA both in vitro and in vivo and recognize the majority of SEP3 wild type binding sites genome-wide, tetramerization is not only required for floral meristem determinacy, but also absolutely required for floral organ identity in the second, third and fourth whorls.

Floral homeotic MADS-box transcription factors ensure the correct morphogenesis of floral organs, which are organized in different cell layers deriving from distinct meristematic layers. How cells from these distinct layers acquire their respective identities and coordinate their growth to ensure normal floral organ morphogenesis is unresolved. Here, we studied petunia (Petunia x hybrida) petals that form a limb and tube through congenital fusion. We identified petunia mutants (periclinal chimeras) expressing the B-class MADS-box gene DEFICIENS in the petal epidermis or in the petal mesophyll, called wico and star, respectively. Strikingly, wico flowers form a strongly reduced tube while their limbs are almost normal, while star flowers form a normal tube but greatly reduced and unpigmented limbs, showing that petunia petal morphogenesis is highly modular. These mutants highlight the layer-specific roles of PhDEF during petal development. We explored the link between PhDEF and petal pigmentation, a well-characterized limb epidermal trait. The anthocyanin biosynthesis pathway was strongly downregulated in star petals, including its major regulator ANTHOCYANIN2 (AN2). We established that PhDEF directly binds to the AN2 terminator in vitro and in vivo, suggesting that PhDEF might regulate AN2 expression and therefore petal epidermis pigmentation. Altogether, we show that cell layer-specific homeotic activity in petunia petals differently impacts tube and limb development, revealing the relative importance of the different cell layers in the modular architecture of petunia petals.

The MADS transcription factors (TF) are an ancient protein family with a high degree of sequence identity that bind almost identical DNA sequences across all eukaryotic kingdoms of life, yet fulfill dramatically different physiological roles. In plants, the family is divided into two main lineages, type I and II, based on sequence conservation of the DNA-binding MADS-box domain (M domain) with yeast and animal M domains. Here, we demonstrate that DNA binding in both lineages absolutely requires a short amino acid sequence C-terminal to the M domain called the Intervening domain (I domain) in type II MADS. Structural elucidation of the MI domains from the floral regulator, SEPALLATA3 (SEP3), shows a highly conserved MADS-box fold with the I domain forming an alpha helix and acting to stabilize the M domain. Based on secondary structure prediction, sequences fulfilling the same function as the SEP3 I domain can be found in both lineages of plant MADS TFs, suggesting the I domain is a conserved and required part of the DNA-binding domain. Using the floral organ identity MADS TFs, SEP3, APETALA1 (AP1) and AGAMOUS (AG), domain swapping demonstrate that the I domain alters DNA-binding specificity based on seq-DAP-seq experiments. Yeast 2-hybrid experiments further revealed the role of the I domain in dimerization specificity. Surprisingly, introducing AG carrying the I domain of AP1 in the Arabidopsis ap1 mutant, resulted in a high degree of complementation and restoration of first and second whorl organs. Taken together, these data demonstrate that the I domain acts both as an integral part of the DNA-binding domain and strongly contributes to the functional identity of the MADS TF.

Background The effect of global warming on dormancy and flowering patterns of crop trees threatens world-wide fruit production and food security. In Rosaceous tree species, it is believed that a group of genes encoding MADS transcription factors (TFs) controls temperature-mediated dormancy cycle. These genes are similar to SHORT VEGETATIVE PHASE ( SVP ) from Arabidopsis thaliana and referred as DORMANCY-ASSOCIATED MADS-BOX ( DAM ) genes. Results By making use of apple tree ( Malus x domestica ) as a model for Rosaceous species, we have investigated the function of MADS TFs during the dormancy cycle. We found that MdDAM and other dormancy related MADS TFs form multimeric complexes with MdSVPa, and that MdSVPa is essential for the transcriptional complex activity. Then, for the first time in non-model plant species, we performed sequential DNA Affinity Purification sequencing (seq-DAP-seq) to define the genome-wide binding sites of these MADS TF complexes. Target genes associated with the binding sites were identified by combining seq-DAP-seq data with transcriptomics datasets obtained by the inducible glucocorticoid receptor expression system, and reanalyzing preexisting data related to dormancy cycle in apple trees. Conclusion We have determined a gene regulatory network formed by MdSVPa-containing complexes that regulate the dormancy cycle in apple trees in response to environmental cues. Key genes identified with our genomic approach and the elucidated regulatory relationships provide leads for breeding fruit trees better adapted to changing climate conditions. Moreover, we provide novel molecular evidence on the evolutionary functional segregation between DAM and SVP proteins in the Rosaceae family.

The MADS transcription factors (TF), SEPALLATA3 (SEP3) and AGAMOUS (AG) are required for floral organ identity and floral meristem determinacy. While dimerization is obligatory for DNA binding, SEP3 and SEP3-AG also form tetrameric complexes. How homo and hetero-dimerization and tetramerization of MADS TFs affect genome-wide DNA-binding and gene regulation is not known. Using sequential DNA affinity purification sequencing (seq-DAP-seq), we determined genome-wide binding of SEP3 homomeric and SEP3-AG heteromeric complexes, including SEP3Δtet-AG, a complex with a SEP3 splice variant, SEP3Δtet, which is largely dimeric and SEP3-AG tetramer. SEP3 and SEP3-AG share numerous bound regions, however each complex bound unique sites, demonstrating that protein identity plays a role in DNA-binding. SEP3-AG and SEP3Δtet-AG share a similar genome-wide binding pattern; however the tetrameric form could access new sites and demonstrated a global increase in DNA-binding affinity. Tetramerization exhibited significant cooperative binding with preferential distances between two sites, allowing efficient binding to regions that are poorly recognized by dimeric SEP3Δtet-AG. By intersecting seq-DAP-seq with ChIP-seq and expression data, we identified unique target genes bound either in SEP3-AG seq-DAP-seq or in SEP3/AG ChIP-seq. Seq-DAP-seq is a versatile genome-wide technique and complements in vivo methods to identify putative direct regulatory targets.

The Evening Complex (EC), composed of the DNA-binding protein LUX ARRHYTHMO (LUX) and two additional proteins, EARLY FLOWERING 3 (ELF3) and ELF4, is a transcriptional repressor complex and a core component of the plant circadian clock. In addition to maintaining oscillations in clock gene expression, the EC also participates in temperature and light entrainment and regulates important clock output genes such as PHYTOCHROME INTERACTING FACTOR 4 ( PIF4 ), a key transcription factor involved in temperature dependent plant growth. These properties make the EC an attractive target for altering plant development through targeted mutations to the complex. However, the molecular basis for EC function was not known. Here we show that binding of the EC requires all three proteins and that ELF3 decreases the ability of LUX to bind DNA whereas the presence of ELF4 restores interaction with DNA. To be able to manipulate this complex, we solved the structure of the DNA-binding domain of LUX bound to DNA. Using structure-based design, a LUX variant was constructed that showed decreased in vitro binding affinity but retained specificity for its cognate sequences. This designed LUX allele modulates hypocotyl elongation and flowering. These results demonstrate that modifying the DNA-binding affinity of LUX can be used to titrate the repressive activity of the entire EC, tuning growth and development in a predictable manner. Significance Statement Circadian gene expression oscillates over a 24 hr. period and regulates many genes critical for growth and development. In plants, the Evening Complex (EC), a three-protein repressive complex made up of LUX ARRYTHMO, EARLY FLOWERING 3 and EARLY FLOWERING 4, acts as a key component of the circadian clock and is a regulator of thermomorphogenic growth. However, the molecular mechanisms of complex formation and DNA-binding have not been identified. Here we determine the roles of each protein in the complex and present the structure of the LUX DNA-binding domain in complex with DNA. Based on these data, we used structure-based protein engineering to produce a version of the EC with altered in vitro and in vivo activity. These results demonstrate that the EC can be modified to alter plant growth and development at different temperatures in a predictable manner.

MADS transcription factors (TFs) are DNA binding proteins found in almost all eukaryotes that play essential roles in diverse biological processes. While present in animals and fungi as a small TF family, the family has dramatically expanded in plants over the course of evolution, with the model flowering plant, Arabidopsis thaliana, possessing over 100 type I and type II MADS TFs. All MADS TFs contain a core and highly conserved DNA binding domain called the MADS or M domain. Plant MADS TFs have diversified this domain with plant-specific auxiliary domains. Plant type I MADS TFs have a highly diverse and largely unstructured Carboxy-terminal (C domain), whereas type II MADS have added oligomerization domains, called Intervening (I domain) and Keratin-like (K domain), in addition to the C domain. In this mini review, we describe the overall structure of the type II "MIKC" type MADS TFs in plants, with a focus on the K domain, a critical oligomerization module. We summarize the determining factors for oligomerization and provide mechanistic insights on how secondary structural elements are required for oligomerization capability and specificity. Using MADS TFs that are involved in flower organ specification as an example, we provide case studies and homology modeling of MADS TFs complex formation. Finally, we highlight outstanding questions in the field.

MADS family transcription factors are crucial during plant reproductive development, and have evolved a complex protein- protein interaction (PPI) network. Proteins of the SEPALLATA (SEP) clade are required for tetramer formation and can act as critical ` hubs' in the network. Rumpler et al. (2018) have now provided quantitative measures of the contribution of individual amino acids to cooperative DNA binding, laying a foundation for predicting MADS tetramer formation based on primary sequence. It is an important step forward in understanding how cooperativity affects processes from flowering time to floral organ identity.

The MADS transcription factors (TF) constitute an ancient family of TF found in all eukaryotes that bind DNA as obligate dimers. Plants have dramatically expanded the functional diversity of the MADS family during evolution by adding protein-protein interaction domains to the core DNA-binding domain, allowing the formation of heterotetrameric complexes. Tetramerization of plant MADS TFs is believed to play a central role in the evolution of higher plants by acting as one of the main determinants of flower formation and floral organ specification. The MADS TF, SEPALLATA3 (SEP3), functions as a central protein-protein interaction hub, driving tetramerization with other MADS TFs. Here, we use a SEP3 splice variant, SEP3(Delta tet), which has dramatically abrogated tetramerization capacity to decouple SEP3 tetramerization and DNA-binding activities. We unexpectedly demonstrate that SEP3 heterotetramer formation is required for correct termination of the floral meristem, but plays a lesser role in floral organogenesis. The heterotetramer formed by SEP3 and the MADS protein, AGAMOUS, is necessary to activate two target genes, KNUCKLES and CRABSCLAW, which are required for meristem determinacy. These studies reveal unique and highly specific roles of tetramerization in flower development and suggest tetramerization may be required to activate only a subset of target genes in closed chromatin regions.

Unlike most transcription factors (TF), pioneer TFs have a specialized role in binding closed regions of chromatin and initiating the subsequent opening of these regions. Thus, pioneer TFs are key factors in gene regulation with critical roles in developmental transitions, including organ biogenesis, tissue development, and cellular differentiation. These developmental events involve some major reprogramming of gene expression patterns, specifically the opening and closing of distinct chromatin regions. Here, we discuss how pioneer TFs are identified using biochemical and genome-wide techniques. What is known about pioneer TFs from animals and plants is reviewed, with a focus on the strategies used by pioneer factors in different organisms. Finally, the different molecular mechanisms pioneer factors used are discussed, highlighting the roles that tertiary and quaternary structures play in nucleosome-compatible DNA-binding.

Circular RNAs (circRNAs) are a diverse and abundant class of hyper-stable, non-canonical RNAs that arise through a form of alternative splicing (AS) called back-splicing. These single-stranded, covalently-closed circRNA molecules have been identified in all eukaryotic kingdoms of life1, yet their functions have remained elusive. Here, we report that circRNAs can be used as bona fide biomarkers of functional, exon-skipped AS variants in Arabidopsis, including in the homeotic MADS-box transcription factor family. Furthermore, we demonstrate that circRNAs derived from exon 6 of the SEPALLATA3 (SEP3) gene increase abundance of the cognate exon-skipped AS variant (SEP3.3 which lacks exon 6), in turn driving floral homeotic phenotypes. Toward demonstrating the underlying mechanism, we show that the SEP3 exon 6 circRNA can bind strongly to its cognate DNA locus, forming an RNA:DNA hybrid, or R-loop, whereas the linear RNA equivalent bound significantly more weakly to DNA. R-loop formation results in transcriptional pausing, which has been shown to coincide with splicing factor recruitment and AS2-4. This report presents a novel mechanistic insight for how at least a subset of circRNAs probably contribute to increased splicing efficiency of their cognate exon-skipped messenger RNA and provides the first evidence of an organismal-level phenotype mediated by circRNA manipulation.

Understanding the evolutionary leap from non-flowering (gymnosperms) to flowering (angiosperms) plants and the origin and vast diversification of the floral form has been one of the focuses of plant evolutionary developmental biology. The evolving diversity and increasing complexity of organisms is often due to relatively small changes in genes that direct development. These "developmental control genes" and the transcription factors (TFs) they encode, are at the origin of most morphological changes. TFs such as LEAFY (LFY) and the MADS-domain TFs act as central regulators in key developmental processes of plant reproduction including the floral transition in angiosperms and the specification of the male and female organs in both gymnosperms and angiosperms. In addition to advances in genome wide profiling and forward and reverse genetic screening, structural techniques are becoming important tools in unraveling TF function by providing atomic and molecular level information that was lacking in purely genetic approaches. Here, we summarize previous structural work and present additional biophysical and biochemical studies of the key master regulators of plant reproduction - LEAFY and the MADS-domain TFs SEPALLATA3 and AGAMOUS. We discuss the impact of structural biology on our understanding of the complex evolutionary process leading to the development of the bisexual flower.

The function of Selenium Binding Protein 1 (SBP1), present in almost all organisms, has not yet been established. In mammals, SBP1 is known to bind the essential element selenium but the binding site has not been identified. In addition, the SBP family has numerous potential metal binding sites that may play a role in detoxification pathways in plants. In Arabidopsis thaliana, AtSBP1 over-expression increases tolerance to two toxic compounds for plants, selenium and cadmium, often found as soil pollutants. For a better understanding of AtSBP1 function in detoxification mechanisms, we investigated the chelating properties of the protein towards different ligands with a focus on selenium using biochemical and biophysical techniques. Thermal shift assays together with inductively coupled plasma mass spectrometry revealed that AtSBP1 binds selenium after incubation with selenite (SeO3) with a ligand to protein molar ratio of 1:1. Isothermal titration calorimetry confirmed the stoichiometry 1:1 and revealed an unexpectedly large value of binding enthalpy suggesting a covalent bond between selenium http://www.jbc.org/cgi/doi/10.1074/jbc.M114.571208 The latest version is at JBC Papers in Press. Published on October 1, 2014 as Manuscript M114.571208 Copyright 2014 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on M arch 2, 2020 hp://w w w .jb.org/ D ow nladed from Identification of the Selenium binding site in A. thaliana SBP1 2 and AtSBP1. Titration of reduced Cys residues and comparative mass spectrometry on AtSBP1 and the purified selenium-AtSBP1 complex identified Cys21 and Cys22 as being responsible for the binding of one selenium. These results were validated by site directed mutagenesis. Selenium K–edge X-ray absorption near edge spectroscopy performed on the selenium-AtSBP1 complex demonstrated that AtSBP1 reduced SeO3 to form a R-S-Se-S-R-type complex. The capacity of AtSBP1 to bind different metals and selenium is discussed with respect to the potential function of AtSBP1 in detoxification mechanisms and selenium metabolism. _____________________________________ INTRODUCTION At low concentration, selenium (Se) is an essential nutrient to many organisms including some archaea, bacteria, protozoan, green algae and nearly all animals but it is non-essential in land plants (1-4). The daily Se requirement in human adults is 60 to 70 μg and the main source of dietary Se is plants (3,5). Se dietary consumption has been associated with a reduced risk of many diseases such as cardiovascular diseases, diabetes and cancer. In addition, a lack of Se can lead to KeshinBeck and Keshan disease that can be treated by Se supplementation (3,4). In organisms where Se is an essential nutrient, it is required for the biosynthesis of the selenoaminoacid Se-Cys, used for the translation of 25 selenoproteins, that are involved in critical functions such as redox reactions, free radical scavenging and hormone regulation (1,3,4). In addition to its role as a micronutrient, Se can have toxic effects. Se toxicity (selenosis) can occur in some areas where exploitation of seleniferous soils or fossil fuels leads to toxic accumulation of Se in the environment and in plants. In mammals, excess selenium targets the cardiovascular, gastrointestinal, neurological and hematopoietic systems (3,5-7). As the line between selenium deficiency and toxicity is very narrow, both Se deficiency and Se toxicity are common problems worldwide. Se concentration in soils ranges from 0.01 to 2 mg/kg and can be >10mg/kg in seleniferous soils (3,5,8). When present in soil, Se is absorbed and accumulated in plants and is subsequently disseminated along the whole food chain. In this context, plants may help to alleviate both Se deficiency and toxicity problems. A better understanding of the mechanisms involved in the plant response to Se including accumulation, protection and sequestration can be considered as one of the most important challenges in the coming decades. Engineering plants with nutrientenriched content for biofortification, using plants to remove toxic Se for phytoremediation and selecting for Se resistant plants are critical goals (5,8). The impact of Se on plant physiology has been extensively studied. Se is not essential to land plants, unlike in mammals. Plant homologues to selenoproteins from mammals and bacteria have a Cys residue in their sequence instead of selenocysteine. However, low concentrations of Se can have a positive effect on plant growth and be beneficial in facing biotic and abiotic stress (9,10) notably by protecting plants against oxidative stress. At higher concentration, Se is generally highly toxic to plants. The major forms of inorganic Se in soils are Se(VI) (SeO4) and Se(IV) (SeO3) which are taken up by roots via sulfate and phosphate transporters, respectively (6,11). Once inside the cells, Se toxicity results from its chemical similarity with sulfur (S) that leads to nonspecific replacement of sulphur containing amino acids with their seleno derivatives (7,12,13). In addition, at high concentrations, Se triggers oxidative stress by reducing the pool of glutathione (GSH) (12). The main mechanisms of Se tolerance in plants are the conversion of SeMet and SeCys into their methylated forms, which are non-incorporable into proteins, and volatilization (5,8). In addition to its incorporation into selenoproteins, Se can be bound to proteins belonging to the Selenium Binding Protein family (SBP). Many of the beneficial impacts of Se on mammalian health have been attributed to its role as a critical constituent of selenoproteins and to its binding to SBP1. Although the function of selenoproteins is well established, the activity of SBP1 proteins is still unclear and the link between Se binding and SBP1 function has not yet been determined. Mammalian SBP1 was first identified in mouse liver (14) in experiments designed to find new selenoproteins. Two homologues are present in humans. Today, SBP genes have been identified in many organisms including plants (14-18) and additional sequences of SBP homologues are by gest on M arch 2, 2020 hp://w w w .jb.org/ D ow nladed from Identification of the Selenium binding site in A. thaliana SBP1 3 available in public databases from many additional organisms. In humans, down regulation of SBP1 expression has been correlated with rapid tumor development in many organs (19-27) and SBP1 expression is considered to be a predictor of clinical outcome. Recently, interaction of SBP1 and the selenoprotein glutathione peroxidase GPx1 was observed, revealing a cross talk between members of distinct families of Se containing proteins (19,21,28). SBP1 was also characterized as a biomarker for schizophrenia as up regulation of SBP1 is observed in the brains of patients with the disease (29,30). Other functions, such as intra-Golgi protein transport have been assigned to mammalian SBP1 (31). Its SBP2 homologue was described as playing a protective role as a scavenger of toxic electrophiles or oxidant species (32-34). In the A. thaliana genome, 3 genes encoding SBP are present (16). AtSBP1 is the isoform that is the most highly expressed (35). One of the first functions that was assigned to SBP1 in plants was a putative role in Se tolerance (15). Indeed, plants over-expressing AtSBP1 have increased resistance to selenite (SeO3) while reducing AtSBP1 and AtSBP2 expression increased plant sensitivity to the toxic compound (15). AtSBP1 could therefore be involved in Se metabolism but no reports were available in the literature on the ability of AtSBP1 to bind Se like its mammalian homologues. In addition to its putative role in Se tolerance, AtSBP1 has been identified as a protein accumulating in response to the heavy metal Cd in A. thaliana cultured cells using differential proteomic analysis (36). Cd is toxic to most organisms and is one of the most toxic pollutants in the world. One of the main mechanisms that plants use to face Cd toxicity is the synthesis of polymers of glutathione (GSH), called phytochelatines (PC), that chelate Cd and are then transferred into the vacuoles (37). AtSBP1 shows the ability to bind 3 Cd in vitro and AtSBP1 over-expression in A. thaliana seedlings led to enhanced tolerance to Cd (35). This phenotype is more important in GSHand PCdeficient Arabidopsis seedlings (35). Therefore, AtSBP1 may have chelating properties in vivo towards Cd and may represent a new detoxification mechanism that plants use to face heavy metal toxicity, possibly throught direct binding to the metal (35). In addition to Cd, AtSBP1 overexpressing A. thaliana plants showed increased tolerance to stress such as Se and H2O2 that also require GSH for detoxification (38). Overexpression of OsSBP1 in rice enhanced tolerance to various pathogens (17) and the importance of GSH in plant defence to biotic stress has been recently reviewed (39). These results suggest that SBP1 may share similar functions with GSH in response to stress. In line with these results, stresses inducing SBP1 expression were also inducers of PRH43, which encodes 5' adenylylphosphosulfate reductase 2, a key enzyme of the sulfur assimilation pathway and GSH biosynthesis (38). As an example, AtSBP1 and AtPRH43 are induced by Cd, SeO4, H202 and sulfur starvation. An internal sulfur demand of the cell could be a signal that triggers SBP1 expression in response to the different stresses (37) and this correlates well with potential functional redundancy between SBP1 and GSH. To date, all the data accumulated on SBP1 function in plants points to the importance of SBP1 in response to stress. The fact that AtSBP1 shows the ability to bind Cd, a toxic metal, in vitro, and that the mammalian homologue can bind Se in vivo, another toxic compounds for plants, raises the question of whether AtSBP1 may have some chelating properties toward Se as well as other metals in addition to Cd. Because of the importance of Se in huma

Uranium is a natural element which is mainly redistributed in the environment due to human activity, including accidents and spillages. Plants may be useful in cleaning up after incidents, although little is yet known about the relationship between metal speciation and plant response. Here, J-Chess modeling was used to predict U speciation and exposure conditions affecting U bioavailability for plants. The model was confirmed by exposing Arabidopsis thaliana plants to U under hydroponic conditions. The early root response was characterized using complete Arabidopsis transcriptome microarrays (CATMA). Expression of 111 genes was modified at the three timepoints studied. The associated biological processes were further examined by real-time quantitative RT-PCR. Annotation revealed that oxidative stress, cell wall and hormone biosynthesis, and signaling pathways (including phosphate signaling) were affected by U exposure. The main actors in iron uptake and signaling (IRT1, FRO2, AHA2, AHA7 and FIT1) were strongly down-regulated upon exposure to uranyl. A network calculated using IRT1, FRO2 and FIT1 as bait revealed a set of genes whose expression levels change under U stress. Hypotheses are presented to explain how U perturbs the iron uptake and signaling response. These results give preliminary insights into the pathways affected by uranyl uptake, which will be of interest for engineering plants to help clean areas contaminated with U.

Background: The selenium-binding site in selenium-binding protein (SBP) homologues was not identified. Results: The Arabidopsis thaliana SBP1 selenium-binding site was characterized as a R-S-Se(II)-S-R-type complex involving Cys(21) and Cys(22). Conclusion: This is the first identification of the selenium-binding site in any SBP. Significance: It is an important step toward a better understanding of the link between selenium binding and function of SBP.The function of selenium-binding protein 1 (SBP1), present in almost all organisms, has not yet been established. In mammals, SBP1 is known to bind the essential element selenium but the binding site has not been identified. In addition, the SBP family has numerous potential metal-binding sites that may play a role in detoxification pathways in plants. In Arabidopsis thaliana, AtSBP1 over-expression increases tolerance to two toxic compounds for plants, selenium and cadmium, often found as soil pollutants. For a better understanding of AtSBP1 function in detoxification mechanisms, we investigated the chelating properties of the protein toward different ligands with a focus on selenium using biochemical and biophysical techniques. Thermal shift assays together with inductively coupled plasma mass spectrometry revealed that AtSBP1 binds selenium after incubation with selenite (SeO32-) with a ligand to protein molar ratio of 1:1. Isothermal titration calorimetry confirmed the 1:1 stoichiometry and revealed an unexpectedly large value of binding enthalpy suggesting a covalent bond between selenium and AtSBP1. Titration of reduced Cys residues and comparative mass spectrometry on AtSBP1 and the purified selenium-AtSBP1 complex identified Cys(21) and Cys(22) as being responsible for the binding of one selenium. These results were validated by site-directed mutagenesis. Selenium K-edge x-ray absorption near edge spectroscopy performed on the selenium-AtSBP1 complex demonstrated that AtSBP1 reduced SeO32- to form a R-S-Se(II)-S-R-type complex. The capacity of AtSBP1 to bind different metals and selenium is discussed with respect to the potential function of AtSBP1 in detoxification mechanisms and selenium metabolism.

Monitoring molecular dynamics of an organism upon stress is probably the best approach to decipher physiological mechanisms involved in the stress response. Quantitative analysis of proteins and metabolites is able to provide accurate information about molecular changes allowing the establishment of a range of more or less specific mechanisms, leading to the identification of major players in the considered pathways. Such tools have been successfully used to analyze the plant response to cadmium (Cd), a major pollutant capable of causing severe health issues as it accumulates in the food chain. We present a summary of proteomics and metabolomics works that contributed to a better understanding of the molecular aspects involved in the plant response to Cd. This work allowed us to provide a finer picture of general signaling, regulatory and metabolic pathways that appeared to be affected upon Cd stress. In particular, we conclude on the advantage of employing different approaches of global proteome- and metabolome-wide techniques, combined with more targeted analysis to answer molecular questions and unravel biological networks. Finally, we propose possible directions and methodologies for future prospectives in this field, as many aspects of the plant-Cd interaction remain to be discovered.