Plant Developmental Systems (PDS, Angenent Group)

Flower development is one of the best understood and economically most important developmental processes in plants. It serves as a model system to understand organ specification and cellular differentiation, starting from pools of undifferentiated ‘stem cells’ residing in meristems. Flower development is controlled by transcription factors of the MADS-box family. These proteins act as major developmental switches to specify the identity of floral meristems and floral organs. They supposedly act in larger protein complexes that bind to the promoters of target genes and regulate them in an organ-specific fashion.

We want to understand how and why MADS protein complexes control the expression of their target genes in different floral organs and at different stages during flower development. The goal of this project is to identify MADS-box target genes that are dynamically regulated during development based on the results of genome-wide DNA-binding and expression studies. The expression dynamics of these target genes will be studied using fluorescent reporter gene studies (e.g. GFP). Binding of MADS-domain proteins and protein complexes will be studied by chromatin immunoprecipitation (ChIP) in planta and/or by Electrophoretic Mobility Shift Assays (EMSAs) in vitro.

Methods 

Gateway cloning, plant transformation, Confocal microscopy (CLSM), chromatin immunoprecipitation, Realtime quantitative PCR, Electrophoretic Mobility Shift Assays (EMSAs).

Flower development in Arabidopsis is controlled by transcription factors of the MADS-box family. These proteins act as major developmental switches to specify the identity of floral meristems and floral organs. According to the current model of flower development, MADS-box transcription factors interact in a combinatorial fashion to specify the different types of floral organs, and to control organ differentiation and growth. MADS proteins are expressed in a highly cell-type specific and temporally dynamic fashion in developing flowers.

The goal of this project is to characterize and quantify MADS protein expression levels during flower development at the cellular level using fluorescent reporter gene analysis. These data will be used for modelling approaches to understand regulatory interactions within the MADS-box transcription factor network that are essential for flower formation.

Methods

Handling Arabidopsis - genotyping, making crosses, phenotypic analysis. Confocal microscopy (CLSM), CLSM data analysis. 

In vitro embryogenesis

Plants are developmentally-plastic organisms. Not only do they continually differentiate new organs from the stem cell niche throughout their lifespan, but they also regenerate organs and even embryos during in vitro culture. We study different in vitro embryogenesis systems to gain a deeper understanding of how differentiated cells switch developmental pathways to form embryos in vitro. These include somatic embryogenesis, induced from vegetative plant cells by the synthetic auxin 2,4-D or by the BABY BOOM (BBM) and LEAFY COTYLEDON1 (LEC1) transcription factors, and embryogenesis induced from immature pollen grains or egg cells by stress, by inhibition of chromatin regulatory proteins or by BBM/LEC1. Our model systems include arabidopsis, Brassica napus and tomato. Identification of cis-regulatory elements We aim to identify cis-regulatory elements that control BBM and LEC1 expression in planta. We identify highly conserved promoter regions by phylogenetic footprinting and then mutate them using CRISPR-Cas9 mutagenesis. Reporter analysis, yeast one-hybrid analysis, mutant analysis and transient expression techniques are then used to correlate conserved promoter motifs with regulatory functions and specific DNA binding proteins.  

Molecular control of haploid embryogenesis 

Haploid cells like pollen grains can be converted into embryos in vitro. We aim to understand this process by identifying the changes that take place at the chromatin and gene expression levels as cells are induced toward embryogenesis. We study how tissue culture-induced changes in chromatin architecture and histone modifications activate or repress specific developmental pathways at the gene expression level. Much of this work is done on cell-type specific populations collected by flow sorting.  

Techniques

ChIP-seq, mRNA-seq, ATAC-seq, FACS, qRT-PCR, yeast n-hybrid screens, mutant analysis, CRISPR mutagenesis, Gateway and Golden Gate cloning, reporter analysis, confocal/DIC microscopy, chemical genomics, tissue culture, transformation. 

Plants are very special because they can continually differentiate new organs from the stem cell niche throughout their lifespan, but they are also capable of regenerating new cell types and organs after wounding or from explants in tissue culture. 

Microspore embryogenesis is a unique process that beautifully illustrates this developmental plasticity of plant cells. In this process, the immature male gametophyte (microspore) is induced by a simple and short heat stress treatment to form haploid embryos in culture. The haploid embryos that arise can be germinated and converted to homozygous doubled haploids (DHs). Microspore embryogenesis is therefore widely used as a means to generate homozygous plants in a single generation, allowing plant breeders to accelerate their breeding programs. However, despite the practical advances of this process, the (epi)genetic and molecular mechanisms underlying this process remain poorly understood. 

The phytohormone auxin, which is essential for correct tissue patterning and organ formation, seems to play an early and important role in microspore embryogenesis: we observed the presence of an auxin response shortly after the heat stress treatment and this auxin response was specifically present in those cells that will eventually form haploid embryos in culture. Based on our current knowledge, we hypothesize that microspore embryogenesis is a two-step process comprising i) a stress‐related event needed for the switch from pollen development to embryo development, followed by ii) an endogenous auxin‐related event required for cell proliferation. 

This project aims to examine the role of stress and early auxin signaling during microspore embryogenesis. To this end, we will use different fluorescent auxin reporters in combination with transcriptome analysis to identify the stress- and auxin-related events during microspore embryogenesis. Additionally, we use novel and existing auxin compounds to identify the specific role of this hormone in microspore embryo induction. The overall focus is to improve our understanding of the molecular basis of microspore embryogenesis.

Requirements

Good theoretical and practical basis in (plant) molecular biology.

Fruits provide humans with essential nutrients for health benefits, such as vitamins, dietary fiber and minerals. Tomato is the model species for studying fleshy fruit development and ripening. 

In my group, the transcriptional regulation of tomato fruit growth and ripening is the main subject of study. We are particularly interested in the role of transcription factors (TFs) and their interaction with the genes they regulate, with the gaseous hormone ethylene, and each other. Some TF's are known to underlie spontaneous mutations that completely block ripening. However, using CRISPR mutagenesis, we and others have shown that these mutants contain gain-of-function mutations that do not represent their actual function. How these and other TFs we have discovered work together to control color, flavor, and texture during fruit ripening by regulating many common downstream effector genes is poorly understood. We are also studying some of these TFs' roles in early fruit growth and the differentiation of fruit tissues. 

We are experienced in editing the tomato genome using CRISPR/Cas9 or Cas12a. We are continuing to develop new strategies for mutagenesis and "real" editing, such as homology-directed repair and prime editing. Null (knockout) mutants in many genes encoding major transcription factors and downstream genes have been obtained, and promoter mutagenesis experiments by multiple guide RNAs are ongoing. Fruit ripening is also epigenetically regulated. We recently started a project on epigenetic modification using Cas9 fused to epigenetic modifiers to study the effects on gene expression.

Methods & skills

mRNA-seq, ATAC-seq, qRT-PCR, yeast n-hybrid screens, mutant analysis, CRISPR mutagenesis and editing, Gateway and Golden Gate cloning, reporter analysis, EMSA, tissue culture, transformation

Requirements

Good theoretical and practical basis in (plant) molecular biology.

Transcription factors (TFs) act together in complexes to regulate the expression of downstream target genes. MADS domain TFs are key regulators of developmental processes such as flowering time control and flower organ development, and form specific dimers and higher-order protein complexes. We are investigating how interaction specificity is laid down in the primary protein sequence and aim to identify short protein motifs and key amino-acid residues determining protein-protein interaction specificity. We follow a wet-lab based experimental approach but also collaborate with Bioinformaticians, who develop and apply Machine Learning (ML)-based tools to predict which amino-acids and short protein motifs determine interaction specificity. Prediction of protein structure and how this affects the biochemical characteristics of the MADS TFs is part of the approach. Ultimately, we check and confirm our findings and predictions by the generation of specific mutations in MADS TFs in planta, followed by detailed phenotyping of developmental effects.

Methods

Protein interaction studies with yeast reverse-, 2-, or 3-hybrid assays, BIFC or FRET-FLIM to confirm interactions, CRISPR/CAS9-based mutagenesis and mutant characterization and phenotyping, plant transformation and tissue culture, molecular cloning and sequencing, applying Bioinformatics tools.