https://www.dbt.univr.it/documenti/Avviso/all/all023820.pdf

Fluorescent protein FRET: the good, the bad and the ugly

Members of the MADS-box transcription factor family play essential roles in almost every developmental process in plants. Many MADS-box genes have conserved functions across the flowering plants, but some have acquired novel functions in specific species during evolution. The analyses of MADS-domain protein interactions and target genes have provided new insights into their molecular functions. Here, we review recent findings on MADS-box gene functions in Arabidopsis and discuss the evolutionary history and functional diversification of this gene family in plants. We also discuss possible mechanisms of action of MADS-domain proteins based on their interactions with chromatin-associated factors and other transcriptional regulators.

Developmental and evolutionary diversity of plant MADS-domain factors: insights from recent studies

Floral organs are specified by the combinatorial action of MADS-domain transcription factors, yet the mechanisms by which MADS-domain proteins activate or repress the expression of their target genes and the nature of their cofactors are still largely unknown. Here, we show using affinity purification and mass spectrometry that five major floral homeotic MADS-domain proteins (AP1, AP3, PI, AG, and SEP3) interact in floral tissues as proposed in the "floral quartet" model. In vitro studies confirmed a flexible composition of MADS-domain protein complexes depending on relative protein concentrations and DNA sequence. In situ bimolecular fluorescent complementation assays demonstrate that MADS-domain proteins interact during meristematic stages of flower development. By applying a targeted proteomics approach we were able to establish a MADS-domain protein interactome that strongly supports a mechanistic link between MADS-domain proteins and chromatin remodeling factors. Furthermore, members of other transcription factor families were identified as interaction partners of floral MADS-domain proteins suggesting various specific combinatorial modes of action.

https://www.pnas.org/content/pnas/109/5/1560.full.pdf

Characterization of MADS-domain transcription factor complexes in Arabidopsis flower development

The molecular mechanisms by which floral homeotic genes act as major developmental switches to specify the identity of floral organs are still largely unknown. Floral homeotic genes encode transcription factors of the MADS-box family, which are supposed to assemble in a combinatorial fashion into organ-specific multimeric protein complexes. Major mediators of protein interactions are MADS-domain proteins of the SEPALLATA subfamily, which play a crucial role in the development of all types of floral organs. In order to characterize the roles of the SEPALLATA3 transcription factor complexes at the molecular level, we analyzed genome-wide the direct targets of SEPALLATA3. We used chromatin immunoprecipitation followed by ultrahigh-throughput sequencing or hybridization to whole-genome tiling arrays to obtain genome-wide DNA-binding patterns of SEPALLATA3. The results demonstrate that SEPALLATA3 binds to thousands of sites in the genome. Most potential target sites that were strongly bound in wild-type inflorescences are also bound in the floral homeotic agamous mutant, which displays only the perianth organs, sepals, and petals. Characterization of the target genes shows that SEPALLATA3 integrates and modulates different growth-related and hormonal pathways in a combinatorial fashion with other MADS-box proteins and possibly with non-MADS transcription factors. In particular, the results suggest multiple links between SEPALLATA3 and auxin signaling pathways. Our gene expression analyses link the genomic binding site data with the phenotype of plants expressing a dominant repressor version of SEPALLATA3, suggesting that it modulates auxin response to facilitate floral organ outgrowth and morphogenesis. Furthermore, the binding of the SEPALLATA3 protein to cis-regulatory elements of other MADS-box genes and expression analyses reveal that this protein is a key component in the regulatory transcriptional network underlying the formation of floral organs.

/pdf/target-genes-of-the-mads-transcription-factor-sepallata3-25ash392r9.pdf

Target Genes of the MADS Transcription Factor SEPALLATA3: Integration of Developmental and Hormonal Pathways in the Arabidopsis Flower

The study of biological systems relies to a large extent on DNA cloning technologies enabling the analysis of recombinant genes through transgenic research. In this context, the advent of recombinational cloning methods was a significant progress because DNA fragments can now be assembled regardless

https://biblio.ugent.be/publication/409524/file/3067793.pdf

Recombinational Cloning with Plant Gateway Vectors

Plant MADS box proteins play important roles in a plethora of developmental processes. In order to regulate specific sets of target genes, MADS box proteins dimerize and are thought to assemble into multimeric complexes. In this study a large-scale yeast three-hybrid screen is utilized to provide insight into the higher-order complex formation capacity of the Arabidopsis MADS box family. SEPALLATA3 (SEP3) has been shown to mediate complex formation and, therefore, special attention is paid to this factor in this study. In total, 106 multimeric complexes were identified; in more than half of these at least one SEP protein was present. Besides the known complexes involved in determining floral organ identity, various complexes consisting of combinations of proteins known to play a role in floral organ identity specification, and flowering time determination were discovered. The capacity to form this latter type of complex suggests that homeotic factors play essential roles in down-regulation of the MADS box genes involved in floral timing in the flower via negative auto-regulatory loops. Furthermore, various novel complexes were identified that may be important for the direct regulation of the floral transition process. A subsequent detailed analysis of the APETALA3, PISTILLATA, and SEP3 proteins in living plant cells suggests the formation of a multimeric complex in vivo. Overall, these results provide strong indications that higher-order complex formation is a general and essential molecular mechanism for plant MADS box protein functioning and attribute a pivotal role to the SEP3 'glue' protein in mediating multimerization.

/pdf/sepallata3-the-glue-for-mads-box-transcription-factor-57h74pho9f.pdf

SEPALLATA3: the 'glue' for MADS box transcription factor complex formation

MADS-box transcription factors are major regulators of development in flowering plants. The factors act in a combinatorial manner, either as homo- or heterodimers, and they control floral organ formation and identity and many other developmental processes through a complex network of protein-protein and protein-DNA interactions. Despite the fact that many studies have been carried out to elucidate MADS-box protein dimerization by yeast systems, very little information is available on the behaviour of these molecules in planta. Here, evidence for specific interactions between the petunia MADS-box proteins FBP2, FBP11, and FBP24 is provided in vivo. The dimers identified in yeast for the ovule-specific FBP24 protein have been confirmed in living plant cells by means of fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy and, in addition, some of the most likely, less stable homo- and heterodimers were identified. This in vivo approach revealed that particular dimers could only be detected in specific sub-nuclear domains. In addition, evidence for the in planta assembly of these ovule-specific MADS-box transcription factors into higher-order complexes is provided.

In vivo imaging of MADS-box transcription factor interactions

Since its introduction in plants 10 years ago, the bimolecular fluorescence complementation (BiFC) method, or split-YFP (yellow fluorescent protein), has gained popularity within the plant biology field as a method to study protein-protein interactions. BiFC is based on the restoration of fluorescence after the two non-fluorescent halves of a fluorescent protein are brought together by a protein-protein interaction event. The major drawback of BiFC is that the fluorescent protein halves are prone to self-assembly independent of a protein-protein interaction event. To circumvent this problem, several modifications of the technique have been suggested, but these modifications have not lead to improvements in plant BiFC protocols. Therefore, it remains crucial to include appropriate internal controls. Our literature survey of recent BiFC studies in plants shows that most studies use inappropriate controls, and a qualitative rather than quantitative read-out of fluorescence. Therefore, we provide a cautionary note and beginner’s guideline for the setup of BiFC experiments, discussing each step of the protocol, including vector choice, plant expression systems, negative controls, and signal detection. In addition, we present our experience with BiFC with respect to self-assembly, peptide linkers, and incubation temperature. With this note, we aim to provide a guideline that will improve the quality of plant BiFC experiments.

A Cautionary Note on the Use of Split-YFP/BiFC in Plant Protein-Protein Interaction Studies

Regulation of flowering time is best understood in the dicot model species Arabidopsis thaliana. Molecular analyses revealed that genes belonging to the MADS box transcription factor family play pivotal regulatory roles in both the vernalization- and photoperiod-regulated flowering pathways. Here the analysis of three APETALA1 (AP1)-like MADS box proteins (LpMADS1– 3) and a SHORT VEGETATIVE PHASE (SVP)-like MADS box protein (LpMADS10) from the monocot perennial grass species Lolium perenne is reported. Features of these MADS box proteins were studied by yeast twohybrid assays. Protein–protein interactions among the Lolium proteins and with members of the Arabidopsis MADS box family have been studied. The expression pattern for LpMADS1 and the protein properties suggest that not the Arabidopsis AP1 gene, but the SUPPRESSOR OF CONSTANS1 (SOC1) gene, is the functional equivalent of LpMADS1. To obtain insight into the molecular mechanism underlying the regulation of LpMADS1 gene expression in vernalizationsensitive and -insensitive Lolium accessions, the upstream sequences of this gene from a winter and spring growth habit variety were compared with respect to MADS box protein binding. In both promoter elements, a putative MADS box transcription factorbinding site (CArG-box) is present; however, the putative spring promoter has a short deletion adjacent to this DNA motif. Experiments using yeast one-hybrid and gel retardation assays demonstrated that the promoter element is bound by an LpMADS1–LpMADS10 higher order protein complex and, furthermore, that this complex binds efficiently to the promoter element from the winter variety only. This strongly supports the model that LpMADS1 together with LpMADS10 controls the vernalization-dependent regulation of the LpMADS1 gene, which is part of the vernalizationinduced flowering process in Lolium.

Isabella A. Nougalli Tonaco

Papers

SEPALLATA3: the 'glue' for MADS box transcription factor complex formation

In vivo imaging of MADS-box transcription factor interactions

A Cautionary Note on the Use of Split-YFP/BiFC in Plant Protein-Protein Interaction Studies

Protein interactions of MADS box transcription factors involved in flowering in Lolium perenne