MADS specificity : Unravelling the dual function of the MADS domain protein FRUITFULL

Encrypted in the DNA lays most information needed for the development of an organism. The transcription of this information into precise patterns of gene activity results in the development of different cell types, organs, and developmental structures. Moreover, transcriptional regulation enables an organism to respond to changing environmental conditions. Essential for the regulation of transcription are DNA-binding transcription factors (TFs). TFs bind the DNA in a sequence-specific fashion. Upon binding of a TF to its DNA binding site, TFs typically activate or repress the transcription of nearby genes. To better understand transcriptional regulation it is essential to study DNA binding specificity of TFs. In the last decades, technological advances allowed the development of high-throughput methods to study protein-DNA interactions. Traditional in vitro methods study one or a few interactions, while new high-throughput methods can determine TF specificity by measuring relative DNA-binding affinities against a large collection or even all possible binding sites. Several high-throughput techniques to study TF-DNA interactions are discussed in Chapter 1 of this thesis. These new technologies and methods resulted in a fast growing number of studies on DNA binding specificities of TFs, expanding the knowledge about TF specificity. A review on the current knowledge of TF DNA binding specificity is described in Chapter 1. One aspect that influences DNA binding of TFs are differences in ability to form protein-protein interactions. The aim of this thesis was to study the role of protein-protein interactions in determining DNA binding specificity of a developmental regulatory MADS domain TF in Arabidopsis thaliana. While the members of the MADS-box protein family have many, diverse in vivo functions, all members bind in vitro to a 10-bp motif called the CArG-box. Moreover, studies demonstrated that closely related MADS proteins are expressed in the same cells, therefore encountering the same DNA accessibility and DNA methylation patterns, but bind different in vivo targets. Interestingly, MADS domain proteins bind DNA obligatorily as homo- and heterodimers and the interactions between MADS domain proteins are highly protein specific. Hence, MADS domain proteins are a perfect model system to study the influence of intra-family protein interactions on DNA binding specificity. To study the influence of protein-protein interactions on DNA-binding specificity this work focusses on one specific MADS domain protein, FRUITFULL (FUL). FUL is expressed at two stages during flower development and, in both stages FUL has highly diverse functions. In Chapter 2 we demonstrate using RNA-seq that FUL regulates different sets of target genes in the two stages. Moreover, using ChIP-seq we show that FUL genomic DNA binding is partly tissue-specific. These tissue-specifically bound and regulated genes are in line with the known dual functions of FUL during development. Interestingly, using protein complex immunoprecipitation for the two studied tissues/stages we show that the interactions of FUL with other MADS domain proteins are also tissue-specific. To determine whether the tissue-specific in vivo binding pattern are due to differences in DNA binding specificity of the FUL-MADS dimers, we studied the DNA binding specificities of the different protein complexes using SELEX-seq. The SELEX-seq results show that although all tested dimers preferably bind the canonical binding motif of MADS domain proteins, different dimers have different preferences for nucleotides within and surrounding the canonical binding site. Hence, different MADS domain dimers have different in vitro DNA binding specificities. By mapping the SELEX-seq affinities to the genome we were able to compare these results with in vivo tissue-specific ChIP-seq data. This analysis revealed a strong correlation between tissue-specific dimer affinities and tissue-specific genomic binding sites of FUL. Hence, we show that the choice of MADS dimerization partner influences DNA binding specificity, highlighting the role of intra-family protein interactions in defining DNA binding specificity. To allow other researchers to determine genome-wide DNA binding of TFs Chapter 3 provides a step-by-step guide for ChIP-seq experiments and computational analysis. The protocol is designed for wet-lab biologists to perform ChIP-seq experiments and analyse their own ChIP-seq data. Using the genome-wide DNA binding patterns determined by ChIP-seq, Chapter 4 and Chapter 5 take a more detailed look at some of the genes directly bound by FUL. In Chapter 4, we demonstrate a connection between developmentally and environmentally regulated growth programs. We studied a gene directly bound by FUL in pistil tissue, SMALL AUXIN UPREGULATED RNA 10 (SAUR10). SAUR10 expression is regulated by FUL in multiple tissues, among others cauline leaves, stems, and branches. The results show that the expression of SAUR10 at the abaxial side of branches is influenced by a combination of environmental and developmental regulated growth programs: hormones, light conditions, and FUL binding. This spatial regulation possibly affects the angle between the side branches and the main inflorescence stem. Additionally, we discuss several other FUL target genes involved in hormone pathways and light conditions. Chapter 5 focusses on the putative direct targets of FUL in IM tissue. Among the putative direct targets two genes involved in flavonoid synthesis were identified, FLAVONOID SYNTHESE 1 (FLS1) and UDP-GLUCOSYL TRANSFERASE 78D3 (UGT78D3). Interestingly, similar to the ful-7 mutant, the fls1 mutant is late flowering. Moreover, expression data exposed an increased gene expression for both FLS1 and UGT78D3 in developing meristems and showed FLS1 expression to be influenced by light conditions. We report the first link between the MADS domain protein FUL and flavonoid synthesis in Arabidopsis. Moreover, our results indicate a possible link between flavonoids and flowering time. In Chapter 6 I discuss the findings of this thesis and make suggestions for further research. Taken together, the work in this thesis shows that intra-family protein interactions can influence DNA-binding specificity of a protein. Thereby these protein-protein interactions can influence genome-wide binding patterns and, as a result, the function of a protein. Moreover, by studying several putative direct targets of FUL in more detail, we demonstrated a connection between development and environment in growth-regulated programs. Interestingly, the FUL target SAUR10 is repressed by FUL in several tissues, including cauline leaves, inflorescence stems, and branches. However, no influence of FUL on SAUR10 expression could be detected in the pistil. So, despite the binding of FUL to the promotor of SAUR10 in the pistil, this binding does not result in gene regulation. This finding reflects the complex relation between TF occupancy and gene regulation, further research is needed to better understand this relation. Moreover, besides MADS domain protein interactions, we found FUL to interact with several proteins of other families. The role of these cross-family protein interactions in cooperative gene regulation is not fully understood and will be an important research topic in the coming years.</p

Regulation of intercellular TARGET OF MONOPTEROS 7 protein transport in the Arabidopsis root

Intercellular communication coordinates hypophysis establishment in the Arabidopsis embryo. Previously, TARGET OF MONOPTEROS 7 (TMO7) was reported to be transported to the hypophysis, the founder cell of the root cap, and RNA suppression experiments implicated its function in embryonic root development. However, the protein properties and mechanisms mediating TMO7 protein transport, and the role the movement plays in development remained unclear. Here, we report that in the post-embryonic root, TMO7 and its close relatives are transported into the root cap through plasmodesmata in a sequence-dependent manner. We also show that nuclear residence is crucial for TMO7 transport, and postulate that modification, potentially phosphorylation, labels TMO7 for transport. Additionally, three novel CRISPR/Cas9-induced tmo7 alleles confirmed a role in hypophysis division, but suggest complex redundancies with close relatives in root formation. Finally, we demonstrate that TMO7 transport is biologically meaningful, as local expression partially restores hypophysis division in a plasmodesmal protein transport mutant. Our study identifies motifs and amino acids that are pivotal for TMO7 protein transport, and establishes the importance of TMO7 in hypophysis and root development

FRUITFULL controls SAUR10 expression and regulates Arabidopsis growth and architecture

[EN] MADS-domain transcription factors are well known for their roles in plant development and regulate sets of downstream genes that have been uncovered by high-throughput analyses. A considerable number of these targets are predicted to function in hormone responses or responses to environmental stimuli, suggesting that there is a close link between developmental and environmental regulators of plant growth and development. Here, we show that the Arabidopsis MADS-domain factor FRUITFULL (FUL) executes several functions in addition to its noted role in fruit development. Among the direct targets of FUL, we identified SMALL AUXIN UPREGULATED RNA 10 (SAUR10), a growth regulator that is highly induced by a combination of auxin and brassinosteroids and in response to reduced R:FR light. Interestingly, we discovered that SAUR10 is repressed by FUL in stems and inflorescence branches. SAUR10 is specifically expressed at the abaxial side of these branches and this localized activity is influenced by hormones, light conditions and by FUL, which has an effect on branch angle. Furthermore, we identified a number of other genes involved in hormone pathways and light signalling as direct targets of FUL in the stem, demonstrating a connection between developmentally and environmentally regulated growth programs.We thank Arjo Meijering for assistance with the light measurements, Niek Stortenbeker for contributions to the manuscript, and Ueli Grossniklaus (University of Zurich) for financial and technical support. MB was supported by the Dutch Organization for Scientific research (NWO) in the framework of the ERA-NET on Plant Genomics (ERA-PG) program project CISCODE and by an NWO Veni-grant. In part, this work was performed in Ueli Grossniklaus' laboratory at the University of Zurich with support through an EMBO LT Fellowship to MB and a grant from the Swiss National Science Foundation to Ueli Grossniklaus. HM was supported by an NWO Vidi-grant, granted to KK.Bemer, M.; Van Mourik, H.; Muiño, JM.; Ferrandiz Maestre, C.; Kaufmann, K.; Angenent, G. (2017). FRUITFULL controls SAUR10 expression and regulates Arabidopsis growth and architecture. Journal of Experimental Botany. 68(13):3391-3403. https://doi.org/10.1093/jxb/erx184S33913403681

Divergent regulation of Arabidopsis SAUR genes : A focus on the SAUR10-clade

Background: Small Auxin-Upregulated RNA (SAUR) genes encode growth regulators that induce cell elongation. Arabidopsis contains more than 70 SAUR genes, of which the growth-promoting function has been unveiled in seedlings, while their role in other tissues remained largely unknown. Here, we focus on the regulatory regions of Arabidopsis SAUR genes, to predict the processes in which they play a role, and understand the dynamics of plant growth. Results: In this study, we characterized in detail the entire SAUR10-clade: SAUR8, SAUR9, SAUR10, SAUR12, SAUR16, SAUR50, SAUR51 and SAUR54. Overexpression analysis revealed that the different proteins fulfil similar functions, while the SAUR expression patterns were highly diverse, showing expression throughout plant development in a variety of tissues. In addition, the response to application of different hormones largely varied between the different genes. These tissue-specific and hormone-specific responses could be linked to transcription factor binding sites using in silico analyses. These analyses also supported the existence of two groups of SAURs in Arabidopsis: Class I genes can be induced by combinatorial action of ARF-BZR-PIF transcription factors, while Class II genes are not regulated by auxin. Conclusions:SAUR10-clade genes generally induce cell-elongation, but exhibit diverse expression patterns and responses to hormones. Our experimental and in silico analyses suggest that transcription factors involved in plant development determine the tissue specific expression of the different SAUR genes, whereas the amplitude of this expression can often be controlled by hormone response transcription factors. This allows the plant to fine tune growth in a variety of tissues in response to internal and external signals.</p

Divergent regulation of Arabidopsis SAUR genes : A focus on the SAUR10-clade

Background: Small Auxin-Upregulated RNA (SAUR) genes encode growth regulators that induce cell elongation. Arabidopsis contains more than 70 SAUR genes, of which the growth-promoting function has been unveiled in seedlings, while their role in other tissues remained largely unknown. Here, we focus on the regulatory regions of Arabidopsis SAUR genes, to predict the processes in which they play a role, and understand the dynamics of plant growth. Results: In this study, we characterized in detail the entire SAUR10-clade: SAUR8, SAUR9, SAUR10, SAUR12, SAUR16, SAUR50, SAUR51 and SAUR54. Overexpression analysis revealed that the different proteins fulfil similar functions, while the SAUR expression patterns were highly diverse, showing expression throughout plant development in a variety of tissues. In addition, the response to application of different hormones largely varied between the different genes. These tissue-specific and hormone-specific responses could be linked to transcription factor binding sites using in silico analyses. These analyses also supported the existence of two groups of SAURs in Arabidopsis: Class I genes can be induced by combinatorial action of ARF-BZR-PIF transcription factors, while Class II genes are not regulated by auxin. Conclusions:SAUR10-clade genes generally induce cell-elongation, but exhibit diverse expression patterns and responses to hormones. Our experimental and in silico analyses suggest that transcription factors involved in plant development determine the tissue specific expression of the different SAUR genes, whereas the amplitude of this expression can often be controlled by hormone response transcription factors. This allows the plant to fine tune growth in a variety of tissues in response to internal and external signals.</p

Regulation of intercellular target of monopteros 7 protein transport in the arabidopsis root

Intercellular communication coordinates hypophysis establishment in the Arabidopsis embryo. Previously, TARGET OF MONOPTEROS 7 (TMO7) was reported to be transported to the hypophysis, the founder cell of the root cap, and RNA suppression experiments implicated its function in embryonic root development. However, the protein properties and mechanisms mediating TMO7 protein transport, and the role the movement plays in development remained unclear. Here, we report that in the post-embryonic root, TMO7 and its close relatives are transported into the root cap through plasmodesmata in a sequence-dependent manner. We also show that nuclear residence is crucial for TMO7 transport, and postulate that modification, potentially phosphorylation, labels TMO7 for transport. Additionally, three novel CRISPR/Cas9-induced tmo7 alleles confirmed a role in hypophysis division, but suggest complex redundancies with close relatives in root formation. Finally, we demonstrate that TMO7 transport is biologically meaningful, as local expression partially restores hypophysis division in a plasmodesmal protein transport mutant. Our study identifies motifs and amino acids that are pivotal for TMO7 protein transport, and establishes the importance of TMO7 in hypophysis and root development

Data from: The origin of floral organ identity quartets

The origin of flowers has puzzled plant biologists ever since Darwin referred to their sudden appearance in the fossil record as an abominable mystery. Flowers are considered to be an assembly of protective, attractive and reproductive male and female leaf-like organs. Their origin cannot be understood by a morphological comparison to gymnosperms, their closest relatives, which develop separate male or female cones. Despite these morphological differences, gymnosperms and angiosperms possess a similar genetic toolbox consisting of phylogenetically related MADS-domain proteins. Using ancestral MADS-domain protein reconstruction, we trace the evolution of organ identity quartets along the stem lineage of crown angiosperms. We provide evidence that current floral quartets specifying male organ identity, which consist of four types of subunits, evolved from ancestral complexes of two types of subunits through gene duplication and integration of SEPALLATA proteins just before the origin of flowering plants. Our results suggest that protein interaction changes underlying this compositional shift were the result of a gradual and reversible evolutionary trajectory. Modelling shows that such compositional changes may have facilitated the evolution of the perfect, bisexual flower

Supplemental Data Set 2

Multiple alignment of AP3/PI used for estimating time of diversification

Supplemental Data Set 1

List of accessions of SEPALLATA-like, APETALA3- and PISTILLATA-like, AGAMOUS- and SEEDSTICK-like genes used for dating the E-class duplication

Supplemental Data Set 7

Multiple alignment of AG/STK used for ancestral protein reconstruction