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.

Used skills

• Analysis of GFP reporter lines using Confocal Laser Scanning Microscopy
• Fluorescent activated Cell sorting (FACS) of GFP-labelled cells
• Gene expression analysis (RNA isolation, qRT-PCR)
• Tissue culture (haploid embryo culture, brassica transformation)
• Chemical screens (auxin-related assays)

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. 

We study transcription factors RIPENING INHIBITOR (RIN), NON- RIPENING (NOR) and COLORLESS NON-RIPENING (CNR), which are the main factors controlling ripening, together with gaseous hormone ethylene in tomato fruits. Functions of RIN, NOR and CNR have been known already through the study of naturally occurring mutants, but how they work together in a network to control colour, flavour and texture during fruit ripening by regulating many common downstream effector genes, is poorly understood. We aim to unravel the mechanisms of combinational action of tomato transcription factors on the expression of relevant ripening-related genes through: 

We are experienced in editing genome by using CRISPR/CAS9 and are continuing to develop new strategies in mutagenesis. Null mutants in genes encoding major transcription factors have been obtained and promoter mutagenesis experiments by multiple guide-RNAs are ongoing.

Requirements

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

• Studying the effector genes of combining different gene dosages of the major regulators on fruit ripening using heterozygous progeny of different crossed wild type and mutant parents (using natural mutations as well as our own generated CRISPR/CAS9-mutants)
• Studying downstream gene promoter architecture using targeted deletion with CRISPR/CAS9

Transcription factors (TFs) act together in complexes to regulate the expression of downstream target genes. Developmental processes such as flowering and fruit development are regulated by networks of transcription factor complexes: Gene Regulatory Networks (GRN). MADS-domain TFs play a major role in the GRNs that regulate flowering and fruit development. Within the flowering plants, a huge variety of inflorescences, flowers and fruits has evolved, induced by changes in the underlying GRNs. We are investigating the role that MADS-domain TFs play in the evolution of these traits and also use this for crop research. Currently, we focus on different aspects of flowering, comparing the functions of the MADS-box genes in Arabidopsis and tomato. This involves both in vitro and in planta techniques like yeast two-hybrid, yeast one-hybrid, EMSA, cloning and construct generation, CRISPR/CAS and plant transformation. 

Used skills

• Bioinformatics tools
• Molecular cloning and sequencing
• Protein (-DNA) interaction studies with yeast 1, 2- or 3- hybrid assays
• Gene expression analysis with qRT-PCR
• CRISPR/CAS9 mutagenesis and mutant characterization and phenotyping
• Plant transformation and tissue culture

FRUITFULL (FUL) is a MADS-domain transcription factor that plays a major role in the development of the Arabidopsis fruit, the silique. In ful mutants, the silique remains very small and does not open to disperse the seeds. 

The evolution of plant species largely depends on the evolution of transcription factors. In this project, we are investigating the evolution of the transcription factor FRUITFULL, which is present as a single copy in the model species Arabidopsis, and regulates many important developmental processes such as leaf development, plant architecture, flowering time and fruit development. In tomato, there are four FRUITFULL genes that can each regulate a subset of these functions. 

To understand the evolution of the four tomato FRUITFULL proteins, we are investigating their properties and try to find out to which DNA sequences they can bind, which protein-protein interactions they form, what their functions are in tomato, and to what extent they can rescue the Arabidopsis fruitfull mutant. This information will also help us to dissect the functions of FRUITFULL in Arabidopsis, and to predict what functions FRUITFULL genes from other species will have. 

Practical work 

The following experiments are performed to elucidate the evolution of the tomato FRUITFULL genes:

Seeds are the basis of most crop production systems. Poor seed quality results in poor crop establishment, waste of inputs, lower yields and farmers income. Less well known is that seed vigour also influences the sensitivity to pathogens. We study how we can improve seedling tolerance to pathogens by improving seed vigour, as part of the solutions in reducing or even omitting chemical crop protection. Carrot seeds and Alternaria pathogens are our present model system. 

• Yeast two-hybrid experiments to investigate the protein-protein interactions. Try to identify the amino acids that are responsible for changes in these interactions and perform the experiments with mutated proteins. (Cloning of constructs, PCR, yeast transformation, yeast two-hybrid).
• DIP-Seq experiments to investigate to which DNA-sequence motifs the different FRUITFULL homologs can bind. (Recombinant protein expression, DIP-seq, high-througput sequencing, bioinformatics)
• CRISPR mutagenesis to knock-out the genes in tomato and study the function of the different homologs. (Cloning constructs, tomato transformation, tissue culture, DNA-extraction, PCR, phenotyping)
• Transformation of Arabidopsis fruitfull mutants with the tomato genes to determine to what extent they can rescue the phenotype. (Cloning constructs, Floral dip transformation, DNA-extraction, PCR, phenotyping).
• Expression analysis of the tomato genes. (qPCR, cloning of reporter constructs).