dr. RA (Ruud) de Maagd

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 and many essential nutrients, vitamins and other health-sustaining compounds and, importantly, with many pleasant taste sensations. Although not consistently recognized by the casual observer, all flowering plants produce fruits in some form or another. However, it is mainly 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 many visible and not-so visible changes, such as changes in colour, 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 other’s activities positively or negatively. This concerted action 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 primary model in most of our studies but are also interested in studying other fruits.

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 link to "Projects" (to the right of this text) for subjects and for the work of our PhD students  Victor Aprilyanto and Xiaowei Wang.

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 flavour components. Driven by autocatalytic ethylene production and climacteric respiratory behaviour, these result in ripe fruits. Several naturally occurring ripening mutants have been characterized. These include, for example, Colorless non-ripening (Cnr), ripening-inhibitor (rin), and non-ripening (nor), all of which have been identified by positional cloning or by genetic mapping. All three loci encode or are near genes for transcription factors, providing the first insights into the fruit-specific transcriptional control of ripening (Wang, R. et al., 2020, Plant Sci. 110436). Another transcription factor that has been shown to be involved in tomato fruit ripening is SlAP2a, which was characterized in our group (Karlova, R. ... de Maagd, R.A. (2011), Plant Cell 923-941). SlAP2a was shown to regulate different aspects of ripening in seemingly opposing ways, being required for both the proper progression of ripening and simultaneous inhibition of ethylene production.

Our group showed two other transcription factors, SlFUL1 and SlFUL2, to play more subtle and redundant roles in tomato fruit ripening (Bemer, M. ... de Maagd, R.A. (2012), Plant Cell 4437-4451). Curiously all these transcription factor genes are orthologs (derived from a common ancestral gene) of genes that were shown to be involved in flowering and floral architecture in the model plant Arabidopsis thaliana. However, arabidopsis lacks fleshy fruits and lacks most developmental changes during tomato ripening, indicating that these similar genes were recruited for different purposes depending on their evolutionary path. The relative position of these proteins in the fruit ripening regulatory network and how their functions interact remains largely unknown and is the subject of our research.

microRNA’s

In plants, expression levels of many transcription factors are also regulated at the post-transcriptional level by microRNAs. microRNAs negatively regulate protein levels by either slicing the messenger RNAs that they bind to or blocking their translation into a protein. In tomatoes, SPL-CNR and AP2a are members of two different, conserved miRNA-targeted gene families, and miRNA activity on their mRNAs has been demonstrated. Many other tomato targets of miRNAs are transcription factors, and a yet unknown number might be involved in fruit development or ripening (Karlova, R. ...de Maagd, R.A.. (2013), J. Exp. Bot. 1863-1878). miRNA action allows for a new layer of regulatory interactions d(uring fruit ripening, including regulating miRNA expression itself. We have identified the active miRNA genes during ripening and are modifying their expression to see what role they play.

Natural variation

Tomato and its wild relatives, which are crossable, comprise a stunning variety of fruit colours, sizes and shapes, tastes, shelf life, metabolite profiles, disease resistance and other traits. These are the starting material for further study on the improvement of tomato varieties by breeding. The availability of many 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. 136-148) by phenotyping them for yield and quality traits and correlating them to DNA sequence variation in genes known to be involved in these traits (Roohanitaziani, R., de Maagd, R.A., et al. (2020). Genes (Basel). 11: 1–22).

CRISPR/Cas-mutagenesis and gene editing

Site-specific nucleases such as the highly versatile CRISPR/Cas9 nucleases offer many possibilities for precise genome modification. This may happen either by introducing mutations due to erroneous repair of double-stranded 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 found in nature or introduced after well-established chemical or radiation-induced mutagenesis. This observation fuels the public debate about whether organisms with precise mutations made via the novel nucleases would need to be considered and regulated 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 Cas12a, and Type I CRISPR systems), improving Cas9, or using the targeting capacity of Cas9 for additional modifications are being studied. These include regulating gene expression by epigenetic modifications, base-editing, and prime editing and have been developed in animal systems. These techniques are currently finding their way to crop plants. Gene editing, gene replacement, or knock-in using homology-driven recombination around a nuclease-induced DSB is possible in plants but much less effective than in animal cells or yeast, and therefore not yet much used. However, the rewards in terms of fundamental insights and applied plant research rewards make them likely to be further developed as practical tools.

Already our use of CRISPR/Cas-mutagenesis for gene function knock-out, as well as the work of others, has wholly overturned our understanding of the function of the transcription factors MADS-RIN, NAC-NOR, and SPL-CNR in tomato ripening regulation (Wang, R., .. de Maagd, R.A. (2019). Sci. Rep. 9: 1696). The natural mutations (rin, nor, Cnr) in these genes were revealed all be dominant-negative or gain-of-function mutations. True knock-out mutations depict a completely different picture of their function (Wang, R., ....de Maagd, R.A. (2020). Trends Plant Sci. 25: 291–301).

We are developing applications of Cas9 and Cas12a to answer fundamental questions in our research on the regulation of fruit development and ripening and for practical applications in improved fruit quality, extended shelf life, or yield. We have been successfully using Cas9 for targeted knock-out of regulatory genes and the creation of promoter deletion mutants in tomatoes for over six years and are currently applying Cas12a for similar applications. Additionally, we have a project addressing questions of efficacy and specificity of different CRISPR technologies in tomatoes to, among others, be better able to predict the occurrence of and mitigate unwanted effects of these technologies (if any). This project involves testing and optimizing Cas9 and Cas12a activity and different delivery methods in plants, and single protoplasts for short-term, multiplexed, and high-throughput mutagenesis assays. Furthermore, we aim to harness homology-directed repair and other allele replacement methods for the improvement of tomatoes.