dr. RA (Ruud) de Maagd

dr. RA (Ruud) de Maagd

senior researcher

Interested in doing a BSc or MSc thesis, or doing an internship with us? This is possible through the chair group Molecular Biology (Prof. G.C. Angenent). Contact me or check out the pages of our PhD students Rufang Wang, Vera Veltkamp,  and Ellen Slaman

I also collaborate with colleagues from Bioscience in a part of the EU project "CHIC", where we use CRISPR-mutagenesis of genes encoding fructan-degrading enzymes to increase the inulin content of Chicory. Students interested in an internship in that project may contact  Ingrid van der Meer.

 

 

 

REGULATORY NETWORKS IN TOMATO FRUIT DEVELOPMENT AND RIPENING

Introduction

Fruits and their products are an indispensable part of the human diet; they provide us with calories, as well as with many essential nutrients, vitamins and other health-sustaining compounds and, importantly, with many pleasant taste sensations. Although not always recognized by the casual observer, all flowering plants produce fruits in some form or another, but it is particularly the juicy, fleshy fruits that are cultivated for human consumption.

Since the beginning of agriculture some 10,000 years ago, farmers have domesticated, selected, and improved fruit-producing plant species for higher production and better taste. In the past century, this task has been gradually taken over by professional breeders. For wild plants the fruits are the organs that protect the developing seeds and, when mature, promote the dispersal of mature seeds. Fleshy fruits often enhance seed dispersal through their attractiveness to the animals that eat them. During their development, a fleshy fruit grows, through cell division and cell expansion, often to a final size many times that of the ovary that gave rise to it. Additionally, many fleshy fruits undergo ripening at the end of their development, when seeds are mature and ready for dispersal. Ripening comprises a large number of visible and not-so visible changes, such as changes in color, taste and firmness. In our group we are studying the regulation of fruit growth and ripening at the molecular level. We focus particularly on transcription factors, which are proteins that regulate the activity of genes, through the activation or inhibition of their expression. Many of these transcription factors work in concert or have opposing activities, and often regulate each others activities in either a positive or a negative fashion. This forms a regulatory network of considerable complexity. Studying how this network functions and its variations might contribute to obtaining higher yields or better quality of cultivated fruits. We use tomato as the main model in most of our studies, but are interested in studying other fruits as well.

Transcription factors

Tomato is a model plant for research in fleshy fruit development and ripening. Ripening in the cultivated tomato comprises a series of biochemical and physiological events, including softening, pigment change, development of flavor components, autocatalytic ethylene production, and climacteric respiratory behavior, which together result in ripe fruits. Several naturally occurring ripening mutants have been characterized. These include, for example, colorless non-ripening (cnr), ripening-inhibitor (rin), and never-ripe (nr), all of which have been identified by positional cloning or by genetic mapping. Both the rin and cnr loci encode transcription factors, providing the first insights into fruit-specific transcriptional control of ripening. Another transcription factor that has been shown to be involved in tomato fruit ripening is SlAP2a, which was characterized in our group (Karlova et al. 2011, Plant Cell 23: 923-941). SlAP2a was shown to regulate different aspects of ripening in seemingly opposing ways, being required for both the proper progression of ripening, as well as simultaneous inhibition of ethylene production. Two other transcription factors, SlFUL1 and SlFUL2, were shown by our group to play more subtle and redundant roles in tomato fruit ripening (Bemer et al. 2012, Plant Cell 24: 4437-4451). Curiously all these transcription factor genes are orthologs (derived from a common ancestral gene) of genes that have been shown to be involved in flowering and floral architecture in the model plant Arabidopsis thaliana (arabidopsis; thale cress or mouse-eared cress). However, arabidopsis does not have fleshy fruit and lacks most of the developmental changes that occur during tomato ripening, indicating that these similar genes have been used for different purposes depending on the evolutionary path they followed. The relative position of these proteins in the fruit ripening regulatory network, and the way their functions interact, remains largely unknown and is the subject of our research.

CRISPR/Cas-mutagenesis and gene editing

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Novel site-specific nucleases such as the highly versatile CRISPR/Cas9 nucleases offer a wealth of possibilities for precise genome modification, either by introduction of mutations due to erroneous repair of double strand DNA-breaks (non-homologous end joining, NHEJ) or by genome editing through homology directed repair (HDR). In their most elegant form, targeted mutations or deletions introduced by these nucleases are indistinguishable from variations that can be found in nature or can be introduced after well-established chemical or radiation-induced mutagenesis. This fuels the public debate as to whether organisms with precise mutations made via the novel nucleases would need to be considered as genetically modified organisms (GMOs), while those obtained by crossings or after treatment with mutagens are not. While this discussion is ongoing, novel methods using nucleases with properties that are complementary to Cas9 (such as Cpf1), improving Cas9, or using the targeting capacity of Cas9 for additional modifications (such as regulating gene expression by epigenetic modifications) have been developed in animal systems and are finding their way to crop plants. Gene editing, gene replacement, or knock-in using homologous recombination around a nuclease-induced DSB is possible in plants, but much less effective than in animal cells or yeast, and therefore not yet practically applicable. However, the rewards in terms of fundamental and applied plant research makes it likely that it will be developed as a practical tool in the near future.

We are developing applications of Cas9 and Cpf1 to answer fundamental questions in our research on regulation of fruit development and ripening, and for practical applications in improved fruit quality or yield. We have been successfully using Cas9 for targeted knock-out of regulatory genes and for the creation of promoter deletion-mutants in tomato for over four years. We have recently started to test the properties of Cpf1 for similar applications. Additionally we have just started a project addressing questions of efficacy and specificity of different CRISPR technologies in tomato in order to, among others, be better able to predict the occurrence of and mitigate unwanted effects of these technologies (if any). This involves testing and optimizing Cas9 and Cpf1 activity as well as different delivery methods in plants, and single protoplasts for short term, multiplexed, and high throughput mutagenesis assays. Furthermore we are aiming at harnassing homology-directed repair and other methods of allele replacement for improvement of tomato.

microRNAs

In plants, many transcription factors are also regulated at the post-transcriptional level by microRNAs. microRNAs negatively regulate protein levels by either slicing the messenger RNAs (protein precursors) that they bind to, or by blocking their translation to a protein. Both CNR as well as SlAP2a are members of two separate, conserved miRNA-target gene families, and miRNA activity on CNR and AP2a mRNA has been demonstrated. Many other tomato targets of miRNAs are transcription factors, and a yet unknown number of them might be involved in fruit development or ripening (Karlova et al. 2013, J. Exp. Bot. 64: 1863-1878). This allows for a new layer of regulatory interactions during fruit ripening, including the regulation of miRNA expression itself. We have identified the miRNA genes that are active during ripening and are modifying their expression to see what role they play.

 

 

Natural variation

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Tomato and its wild relatives, which can be crossed with each other, comprise a stunning variety of fruit colors, sizes and shapes, tastes, shelf life, metabolite profiles, disease resistances and other traits, which are the starting material for further improvement of tomato varieties by breeding. The availability of a large number of sequenced genomes and, advances in genome sequencing technology and throughput allows us to study in detail the molecular mechanisms underlying the variety of tomato fruit traits. We are studying a collection of cultivated tomatoes and wild relatives (Aflitos et al. 2014, Plant J. 80:136-148) by phenotyping them for yield and quality traits, and correlating these traits to DNA sequence variation in genes known to be involved in these traits.