MADS transcription factors cooperate: complexities of complex formation

International audienceMADS 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. Rümpler 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 identit

The Arabidopsis Putative Selenium-Binding Protein Family: Expression Study and Characterization of SBP1 as a Potential New Player in Cadmium Detoxification Processes

International audienceIn Arabidopsis thaliana, the putative selenium-binding protein gene family is made of 3 members (SBP 1 to 3). RT-PCR analyses showed that SBP1 expression was ubiquitous. SBP2 was expressed at a lower level in flowers and roots whereas SBP3 transcripts were only detected in young seedling tissues. In Cd-treated seedlings, SBP1 level of expression was rapidly increased in roots. In shoots, SBP1 transcripts accumulated later and for higher Cd doses. SBP2 and SBP3 expression showed delayed or no responsiveness to Cd. In addition, luciferase (LUC) activity recorded on Arabidopsis lines expressing the LUC gene under the control of SBP1 promoter further showed dynamic regulation of SBP1 expression during development and in response to Cd stress. Western blot analysis using polyclonal antibodies raised against SBP1 showed that SBP1 protein accumulated in Cd-exposed tissues in correlation with SBP1 transcript amount. The sbp1 null mutant displayed no visible phenotype under normal and stress conditions that was explained by the up regulation of SBP2 expression. SBP1 over-expression enhanced Cd accumulation in roots and reduced sensitivity to Cd in WT and more significantly in Cd-hypersensitive cad mutants that lack phytochelatins. Similarly in Saccharomyces cerevisiae, SBP1 expression led to increased Cd tolerance of the Cd-hypersensitive ycf1 mutant. In vitro experiments showed that SBP1 has the ability to bind Cd. These data highlight the importance of maintaining the adequate SBP protein level under healthy and stress conditions and suggest that during Cd stress, SBP1 accumulation efficiently helps to detoxify Cd potentially through direct binding

Arabidopsis Putative Selenium-Binding Protein1 Expression Is Tightly Linked to Cellular Sulfur Demand and Can Reduce Sensitivity to Stresses Requiring Glutathione for Tolerance1[W]

Selenium-Binding Protein1 (SBP1) gene expression was studied in Arabidopsis (Arabidopsis thaliana) seedlings challenged with several stresses, including cadmium (Cd), selenium {selenate [Se(VI)] and selenite [Se(IV)]}, copper (Cu), zinc (Zn), and hydrogen peroxide (H2O2) using transgenic lines expressing the luciferase (LUC) reporter gene under the control of the SBP1 promoter. In roots and shoots of SBP1∷LUC lines, LUC activity increased in response to Cd, Se(VI), Cu, and H2O2 but not in response to Se(IV) or Zn. The pattern of expression of SBP1 was similar to that of PRH43, which encodes the 5′-Adenylylphosphosulfate Reductase2, a marker for the induction of the sulfur assimilation pathway, suggesting that an enhanced sulfur demand triggers SBP1 up-regulation. Correlated to these results, SBP1 promoter showed enhanced activity in response to sulfur starvation. The sulfur starvation induction of SBP1 was abolished by feeding the plants with glutathione (GSH) and was enhanced when seedlings were treated simultaneously with buthionine sulfoxide, which inhibits GSH synthesis, indicating that GSH level participates in the regulation of SBP1 expression. Changes in total GSH level were observed in seedlings challenged with Cd, Se(VI), and H2O2. Accordingly, cad2-1 seedlings, affected in GSH synthesis, were more sensitive than wild-type plants to these three stresses. Moreover, wild-type and cad2-1 seedlings overexpressing SBP1 showed a significant enhanced tolerance to Se(VI) and H2O2 in addition to the previously described resistance to Cd, highlighting that SBP1 expression decreases sensitivity to stress requiring GSH for tolerance. These results are discussed with regard to the potential regulation and function of SBP1 in plants

A Proteomics Dissection of Arabidopsis thaliana Vacuoles Isolated from Cell Culture * □S Michel Jaquinod‡§¶�**, Florent Villiers�‡‡, Sylvie Kieffer-Jaquinod‡§¶,

To better understand the mechanisms governing cellular traffic, storage of various metabolites, and their ultimate degradation, Arabidopsis thaliana vacuole proteomes were established. To this aim, a procedure was developed to prepare highly purified vacuoles from protoplasts isolated from Arabidopsis cell cultures using Ficoll density gradients. Based on the specific activity of the vacuolar marker �-mannosidase, the enrichment factor of the vacuoles was estimated at �42-fold with an average yield of 2.1%. Absence of significant contamination by other cellular compartments was validated by Western blot using antibodies raised against specific markers of chloroplasts, mitochondria, plasma membrane, and endoplasmic reticulum. Based on these results, vacuole preparations showed the necessary degree of purity for proteomic

Structural Basis for Plant MADS Transcription Factor Oligomerization

International audienceMADS 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

Genome-wide binding of SEPALLATA3 and AGAMOUS complexes determined by sequential DNA-affinity purification sequencing

International audienceThe 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

A Proteomics Approach Highlights a Myriad of Transporters in the Arabidopsis thaliana Vacuolar Membrane

To better understand plant vacuolar functions and identify new transporters present on the tonoplast, a proteomic work was initiated on Arabidopsis thaliana. A procedure was developed to prepare highly purified vacuoles from protoplasts isolated from Arabidopsis cell cultures, and a proteomics approach was designed to identify the protein components present in both the membrane and soluble fractions of the vacuoles. This procedure allowed the identification of 650 proteins, 2/3 of which copurify with the hydrophobic membrane fraction and 1/3 with the soluble fraction. With regard to function, only 20% of the proteins identified were previously known to be associated with vacuolar activities

: Mol Cell Proteomics

International audienceTo better understand the mechanisms governing cellular traffic, storage of various metabolites, and their ultimate degradation, Arabidopsis thaliana vacuole proteomes were established. To this aim, a procedure was developed to prepare highly purified vacuoles from protoplasts isolated from Arabidopsis cell cultures using Ficoll density gradients. Based on the specific activity of the vacuolar marker alpha-mannosidase, the enrichment factor of the vacuoles was estimated at approximately 42-fold with an average yield of 2.1%. Absence of significant contamination by other cellular compartments was validated by Western blot using antibodies raised against specific markers of chloroplasts, mitochondria, plasma membrane, and endoplasmic reticulum. Based on these results, vacuole preparations showed the necessary degree of purity for proteomics study. Therefore, a proteomics approach was developed to identify the protein components present in both the membrane and soluble fractions of the Arabidopsis cell vacuoles. This approach includes the following: (i) a mild oxidation step leading to the transformation of cysteine residues into cysteic acid and methionine to methionine sulfoxide, (ii) an in-solution proteolytic digestion of very hydrophobic proteins, and (iii) a prefractionation of proteins by short migration by SDS-PAGE followed by analysis by liquid chromatography coupled to tandem mass spectrometry. This procedure allowed the identification of more than 650 proteins, two-thirds of which copurify with the membrane hydrophobic fraction and one-third of which copurifies with the soluble fraction. Among the 416 proteins identified from the membrane fraction, 195 were considered integral membrane proteins based on the presence of one or more predicted transmembrane domains, and 110 transporters and related proteins were identified (91 putative transporters and 19 proteins related to the V-ATPase pump). With regard to function, about 20% of the proteins identified were known previously to be associated with vacuolar activities. The proteins identified are involved in ion and metabolite transport (26%), stress response (9%), signal transduction (7%), and metabolism (6%) or have been described to be involved in typical vacuolar activities, such as protein and sugar hydrolysis. The subcellular localization of several putative vacuolar proteins was confirmed by transient expression of green fluorescent protein fusion constructs