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Tomato is economically an important vegetable crop, and its fruit yield is among others determined by the timing of flowering, inflorescence architecture and shoot determinacy. These traits are biological consequences of the activities of reproductive meristems, which are regulated by endogenous conditions, phytohormones and flowering pathway genes. The FUL gene, encoding a pleiotropic MADS domain transcription factor in Arabidopsis, has been implicated in different flowering pathways. Its homologs play conserved roles in flowering and inflorescence development in many other species but had not yet been thoroughly investigated in tomato. The tomato genome carries four FUL-like genes (SlFULs: FUL1, FUL2, MBP10, MBP20), among which only FUL1 and FUL2 are known to be involved in regulation of fruit development. The aim of the studies described in this thesis is to investigate the functions of the SlFUL genes in flowering and inflorescence development, and their redundancy/interaction with MC and SP, as well as their regulation of downstream target genes, such as the SlCKXs. To do so, we employed CRISPR mutagenesis, transcriptome analysis, protein-protein interaction analysis, gene expression analysis, and investigated meristem development in detail. In Chapters 2 and 3, we found that knock-out of both SlFULs and CKXs could lead to additional formation of IMs, but the identity of the specified IMs was not affected. However, previous studies had shown that mutation of the close SlFUL homolog MC did cause inflorescence vegetative reversion after the production of a few flowers. This sparked us to study the unique and overlapping functions of SlFUL and MC in the regulation of meristem phase change and meristem identity specification during reproductive development. In Chapter 4, we show that combined mutations of the AP1/FUL-like genes cause delayed FM maturation that allows additional formation of flanking meristems, which adopt a vegetative fate instead of IM fate, leadi

The timing of flowering and the inflorescence architecture are critical for the reproductive success of tomato (Solanum lycopersicum), but the gene regulatory networks underlying these traits have not been fully explored. Here, we show that the tomato FRUITFULL-like (FUL-like) genes FUL2 and MADS-BOX PROTEIN 20 (MBP20) promote the vegetative-to-reproductive transition and repress inflorescence branching by inducing floral meristem (FM) maturation. FUL1 fulfils a less prominent role and appears to depend on FUL2 and MBP20 for its upregulation in the inflorescence- and floral meristems. MBP10, the fourth tomato FUL-like gene, has probably lost its function. The tomato FUL-like proteins cannot homodimerize in in vitro assays, but heterodimerize with various other MADS-domain proteins, potentially forming distinct complexes in the transition meristem and FM. Transcriptome analysis of the primary shoot meristems revealed various interesting downstream targets, including four repressors of cytokinin signaling that are upregulated during the floral transition in ful1 ful2 mbp10 mbp20 mutants. FUL2 and MBP20 can also bind in vitro to the upstream regions of these genes, thereby probably directly stimulating cell division in the meristem upon the transition to flowering. The control of inflorescence branching does not occur via the cytokinin oxidase/dehydrogenases (CKXs) but may be regulated by repression of transcription factors such as TOMATO MADS-box gene 3 (TM3) and APETALA 2b (AP2b).

Null mutations of tomato FRUITFULL-like genes FUL1, FUL2, MBP10, MBP20 caused delayed flowering and branched inflorescence, so we sequenced mRNA from vegetative meristems (VM), transition meristems (TM), floral meristems (FM), and FM of the first sympodial shoot of tomato mutant ful1/ful2/mbp10/mbp20 (slful) as well as the wild type Moneyberg (WT) to see genome-wide expression changes affected by the mutations. Overall design: Meristems at the same developmental stages were collected from many plants of both mutants and wild types and then pooled together for mRNA extractions and then sequencing library construction. Three biological replicates were analyzed for each stage., Null mutations of tomato FRUITFULL-like genes FUL1, FUL2, MBP10, MBP20 caused delayed flowering and branched inflorescence, so we sequenced mRNA from vegetative meristems (VM), transition meristems (TM), floral meristems (FM), and FM of the first sympodial shoot of tomato mutant ful1/ful2/mbp10/mbp20 (slful) as well as the wild type Moneyberg (WT) to see genome-wide expression changes affected by the mutations. Overall design: Meristems at the same developmental stages were collected from many plants of both mutants and wild types and then pooled together for mRNA extractions and then sequencing library construction. Three biological replicates were analyzed for each stage.

Efficient seed dispersal in flowering plants is enabled by the development of fruits, which can be either dehiscent or indehiscent. Dehiscent fruits open at maturity to shatter the seeds, while indehiscent fruits do not open and the seeds are dispersed in various ways. The diversity in fruit morphology and seed shattering mechanisms is enormous within the flowering plants. How these different fruit types develop and which molecular networks are driving fruit diversification is still largely unknown, despite progress in eudicot model species. The orchid family, known for its astonishing floral diversity, displays a huge variation in fruit dehiscence types, which have been poorly investigated. We undertook a combined approach to understand fruit morphology and dehiscence in different orchid species to get more insight into the molecular network that underlies orchid fruit development. We describe fruit development in detail for the epiphytic orchid species Erycina pusilla and compare it to two terrestrial orchid species: Cynorkis fastigiata and Epipactis helleborine. Our anatomical analysis provides further evidence for the split carpel model, which explains the presence of three fertile and three sterile valves in most orchid species. Interesting differences were observed in the lignification patterns of the dehiscence zones. While C. fastigiata and E. helleborine develop a lignified layer at the valve boundaries, E. pusilla fruits did not lignify at these boundaries, but formed a cuticle-like layer instead. We characterized orthologs of fruit-associated MADS-domain transcription factors and of the Arabidopsis dehiscence-related genes INDEHISCENT (IND)/HECATE 3 (HEC3), REPLUMLESS (RPL) and SPATULA (SPT)/ALCATRAZ (ALC) in E. pusilla, and found that the key players of the eudicot fruit regulatory network appear well-conserved in monocots. Protein-protein interaction studies revealed that MADS-domain complexes comprised of FRUITFULL (FUL), SEPALLATA (SEP) and AGAMOUS (A

The family of small auxin up-regulated RNA (SAUR) genes is a family of auxin-responsive genes with ~60–140 members in most higher plant species. Despite the early discovery of their auxin responsiveness, their function and modeof action remained unknown for a long time. In recent years, the importance of SAUR genes in the regulation of dynamic and adaptive growth, and the molecular mechanisms by which SAUR proteins act are increasingly well understood. SAURs play a central role in auxin-induced acid growth, but can also act independently of auxin, tissue specifically regulated by various other hormone pathways and transcription factors. In this review, we summarize recent advances in the characterization of the SAUR genes in Arabidopsis and other plant species. We particularly elaborate on their capacity to fine-tune growth in response to internal and external signals, and discuss the breakthroughs in understanding the mode of action of SAURs in relation to their complex regulation.

Transmission electron microscopy (TEM) is used to study the fine ultrastructural organization of cells. Delicate specimen preparation is required for results to reflect the "native" ultrastructural organization of subcellular features such as the nucleus. Despite the advent of high-resolution, fluorescent imaging of chromatin components, TEM still provides a unique and complementary level of resolution capturing chromatin organization at the nanoscale level. Here, we describe the workflow, from tissue preparation, TEM image acquisition and image processing, for obtaining a quantitative description of chromatin density distribution in plant cells, informing on local fluctuations and periodicity. Comparative analyses then allow to elucidate the structural changes induced by developmental or environmental cues, or by mutations affecting specific chromatin modifiers at the nanoscale level. We argue that this approach remains affordable and merits a renewed interest by the plant chromatin community.

DNA methylation profiling in the epigenome of Arabidopsis thaliana (Arabidopsis) has provided great insights in the role of this epigenetic mark for the regulation of transcription in plants, and is often based on high-throughput sequencing. The analysis of these data involves a series of steps including quality checks, filtering, alignment, identification of methyl-cytosines, and the identification of differentially methylated regions. This chapter outlines the computational methodology required to profile genome-wide differential methylation patterns based on publicly available Arabidopsis base-resolution bisulfite sequencing data. The methylPipe Bioconductor package is adopted for the identification of the differentially methylated regions, and all the steps from the raw data to the required input are described in detail.

Parent-of-origin effects play important roles in plant reproduction and are often mediated by epigenetic modifications at the histone or DNA level. However, the genetic basis underlying these modifications can be challenging to identify. Here, we describe an approach (Bulk-Seq) that can be used to map loci mediating parent-of-origin-dependent effects using whole-genome sequencing of pools of DNA.

Chromatin organization in eukaryotes is highly dynamic, playing fundamental roles in regulating diverse nuclear processes including DNA replication, transcription, and repair. Thus, the analysis of chromatin organization is of great importance for the elucidation of chromatin-mediated biological processes. Immunostaining coupled with imaging is one of the most powerful tools for chromatin analysis at the cellular level. However, in plants, it is sometimes technically challenging to apply this method due to the inaccessibility of certain cell types and/or poor penetration of the reagents into plant tissues and cells. To circumvent these limitations, we developed a highly efficient protocol enabling the analysis of chromatin modifications and nuclear organization at the single-cell level with high resolution in whole-mount plant tissues. The main procedure consists of five steps: (1) tissue fixation; (2) dissection and embedding; (3) tissue processing; (4) antibody incubation; and (5) imaging. This protocol has been simplified for the processing of multiple samples without the need for laborious tissue sectioning. Additionally, it preserves cellular morphology and chromatin organization, allowing comparative analyses of chromatin organization between different cell types or developmental stages. This protocol was successfully used for various tissues of different plant species, including Arabidopsis thaliana, Oryza sativa (rice), and Zea mays (maize). Importantly, this method is very useful to analyze poorly accessible tissues, such as female meiocytes, gametophytes, and embryos.

Somatic embryogenesis is a form of induced plant cell totipotency where embryos develop from somatic or vegetative cells in the absence of fertilization. Somatic embryogenesis can be induced in vitro by exposing explants to stress or growth regulator treatments. Molecular genetics studies have also shown that ectopic expression of specific embryo- and meristem-expressed transcription factors or loss of certain chromatin-modifying proteins induces spontaneous somatic embryogenesis. We begin this review with a general description of the major developmental events that define plant somatic embryogenesis and then focus on the transcriptional regulation of this process in the model plant Arabidopsis thaliana (arabidopsis). We describe the different somatic embryogenesis systems developed for arabidopsis and discuss the roles of transcription factors and chromatin modifications in this process. We describe how these somatic embryogenesis factors are interconnected and how their pathways converge at the level of hormones. Furthermore, the similarities between the developmental pathways in hormone- and transcription-factor-induced tissue culture systems are reviewed in the light of our recent findings on the somatic embryo-inducing transcription factor BABY BOOM.

Both Transgenic and control Arabidopsis thaliana plants were analysed by whole genome resequencing. Transformation events were analysed in detail showing multiple T-DNA insertions, resulting in deletions, insertions, SNPs, rearrangements and partial insertions., Both Transgenic and control Arabidopsis thaliana plants were analysed by whole genome resequencing. Transformation events were analysed in detail showing multiple T-DNA insertions, resulting in deletions, insertions, SNPs, rearrangements and partial insertions.

[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.

Contains fulltext : 164745.pdf (publisher's version ) (Closed access)

We compared gene expression by microarray analysis between wild type and transgenic sibling progeny from two primary transgenic lines containing a construct for knocking down expression of both tomato fruitfull orthologs, FUL1/TDR4 and FUL2/MBP7.

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Radboud Universiteit Nijmegen, 02 juni 2008, Promotores : Angenent, G.C., Mariani, C., Contains fulltext : 45157_funcanevs.pdf (publisher's version ) (Open Access), MADS-box genes are very important for plant development and especially for the formation of the flower. The MADS box family of transcription factors in plants can be subdivided into two classes: the MIKC-type genes, which play essential roles in flower formation, and the type I genes, which are functionally poorly understood. In this thesis, functional and evolutionary aspects of the type I subfamily are elucidated and a thorough characterization of the Arabidopsis type I MADS box gene DIANA (AGL61) is presented. The research described in this thesis suggests that type I MADS box genes play important roles in female gametophyte development and especially in the process of double fertilization. In addition, the expression of several type I genes in the endosperm suggests that some genes play a role in this tissue as well. The importance of the type I genes for the double fertilization, a unique angiosperm feature, indicates that this subfamily of MADS box genes has been very important for angiosperm evolution

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