205 research outputs found
MADS on the move : a study on MADS domain protein function and movement during floral development in Arabidopsis thaliana
No full textIn this thesis we investigated the behaviour of fluorescently-tagged MADS domain proteins during floral development in the model plant Arabidopsis thaliana, and explored the importance of intercellular transport via plasmodesmata for MADS domain transcription factor functioning. The MADS domain transcription factor family plays an important regulatory role in the development of flowers, among others by establishing the identities of the different floral organs. Although genetic screens and in vitro and in vivo studies on protein-protein and protein-DNA interactions provide important information on how MADS domain transcription factor complexes are able to regulate downstream target genes, understanding of the behaviour of MADS domain transcription factors in planta is still limited. Also, the extent to which intercellular movement of MADS domain transcription factors via plasmodesmata plays a role in developmental processes is poorly understood. Since the discovery of the GREEN FLUORESCENT PROTEIN (GFP) and the subsequent development of similar fluorescent tags, it has become possible to observe the subcellular localisation and behaviour of fluorescently-tagged proteins in living tissues with confocal laser scanning microscopy. In Chapter 2 of this thesis, different methods of tagging the MADS domain transcription factors AGAMOUS (AG), SEPALLATA3 (SEP3), and FRUITFULL (FUL) for chromatin immunoprecipitation, chromatin affinity purification and in planta imaging are described. This research shows that the addition of a small peptide tag or a fluorescent tag to MADS domain proteins easily leads to transgene silencing and specific loss-of-function mutant phenotypes, especially when the tagged MADS box genes are expressed under the control of the constitutive CaMV35S promoter. Plants that express tagged MADS box genes from genomic fragments that include all or most of the regulatory elements, and therefore mimic the natural expression pattern as much as possible, show lower levels of loss-of-function phenotypes. In addition, these plants are also more useful for investigating biological relevant behaviour of the MADS domain proteins. In Chapter 3, the spatio-temporal localisation patterns of GFP-tagged MADS domain transcription factors AG, SEP3, FUL and APETALA1 (AP1) during floral development are reported. These analyses demonstrate that there are several tissues, often epidermal cell layers, where MADS domain proteins could be detected, while the available literature describes an absence of mRNA in those tissues. This could indicate that there is intercellular transport of MADS domain proteins in meristematic tissues during floral development. The implications of the observed behaviour of the different MADS domain proteins for MADS domain protein functioning are discussed in this chapter. In Chapters 4 and 5 we describe the different methods that were used to investigate whether MADS domain proteins are indeed able to transport between cells during floral development. The difficulties that we encountered in our attempts to investigate intercellular MADS domain protein transport with microinjection techniques and by using the photoconvertible fluorescent mEosFP-tag are discussed. In plants that specifically overexpress GFP-tagged MADS domain transcription factors AG, SEP3, APETALA3 (AP3), or PISTILLATA (PI) in the epidermis, we demonstrated with a photobleaching technique that all tested proteins were able to move within the epidermal cell layer. This mechanism of lateral epidermal movement provides an explanation for most of the unexpected MADS domain protein localisations that we found in the spatio-temporal localisation analyses in Chapter 3. Additionally, we demonstrate that epidermis-expressed GFP-tagged AG is able to move from the epidermis to the subepidermis in the centre of the floral meristem, which provides proof for the suggestions that AG acts non-cell-autonomously in the floral meristem. In these plants we also analyzed the effects of epidermal MADS domain protein expression on the plant phenotype. This showed, among others, that epidermis-expressed AG is able to fully complement its own mutant background, while epidermis-expressed AP3 is not. In Chapter 6, we explore the mechanisms underlying the behaviour of GFP-tagged SEP3 during petal and stamen development that was observed in the spatio-temporal localisation studies described in Chapter 3. Just prior to the initiation of petal and stamen primordia GFP-tagged SEP3 proteins change their subcellular localisation from predominantly nuclear to more cytoplasmic, and at later stages GFP-tagged SEP3 protein seems to disappear in the middle of the primordia without the loss of SEP3 mRNA expression. These two processes could be regulated at a post-transcriptional level by two mechanisms that are discussed, namely 26s proteasome mediated SEP3 protein degradation and epidermal-oriented intercellular transport of SEP3 proteins. Additionally, we demonstrate that there are no clear indications that the observed GFP-tagged SEP3 behaviour is due to the presence of F-box protein UNUSUAL FLORAL ORGANS (UFO), which regulates petal and stamen development. In Chapter 7, this thesis finishes with some concluding remarks on in planta imaging of MADS domain transcription factors and the possible mechanisms of MADS domain protein movement in the floral meristem. Furthermore, we speculate on the importance of MADS domain protein movement in establishing MADS box gene expression patterns and MADS domain protein gradients, and on the need for symplastically isolated domains for proper floral development. <br/
Functional characterization of MADS box transcription factors in Petunia hybrida
No full textContains fulltext :
60667.pdf (Publisher’s version ) (Open Access)Transcription factors play a central role in the regulation and integration of several developmental pathways in all organisms. MADS box proteins are, among transcription factors, key players in the regulation of flower induction, flower architecture and vegetative development and have been isolated and studied in many different plant species. Chapter 2 offers an overview of floral development in several species from which MADS box genes have been analyzed, their function in determining the diversity of floral organs and their role in the evolution of floral morphologies. The rest of the thesis focuses on MADS box transcription factors in Petunia hybrida. The isolation of new genes and their preliminary characterization is described in Chapter 3. Phylogenetic analysis, expression analysis and interaction patterns of all the family members are included in this study. Strategies such as overexpression, cosuppression and knockout via transposon insertion have been adopted for a more detailed functional analysis of a few MADS box genes, as described in Chapter 4. These genes have been selected on the basis of their sequences, expression and interaction patterns which altogether suggested possible roles in floral organ determination, flower induction and vegetative development. A thorough analysis of the genes belonging to the FBP2 subfamily follows in Chapter 5, where it is demonstrated that FBP2 is functionally equivalent to the Arabidopsis SEPALLATA (SEP) proteins on the basis of similarities in sequence, expression, interaction patterns, mutant phenotypes and the functional complementation of the Arabidopsis sep triple mutant with FBP2. In Chapter 6 the function of UNS is studied in detail, by sequence and expression analysis, and overexpression of a full-length and a truncated protein in both petunia and Arabidopsis plants. The function of UNS in petunia is similar to SUPPRESSOR OF OVEREXEPRESSION OF CONSTANS1 (SOC1), a promoter of flowering in Arabidopsis. A possible function of a MADS box complex including C-, D- and E-type proteins in bringing the flower meristem to an end, is proposed in Chapter 7. The hypothesis is supported by the phenotype of transgenic plants simultaneously overexpressing FBP2 and FBP11, which show an early arrest in development and a downregulation of TERMINATOR (TER), the petunia WUS homolog. An upregulation of the petunia C-type gene FBP6 and the D-type FBP7 and FBP11 is also observed. A positive feedback loop among C-, D-, and E- type genes is most likely occurring also within the flower. General considerations about the strategies adopted in this study, the relevance of such a research in the understanding of diversity and conservation in plant development, conclude this thesis.RU Radboud Universiteit Nijmegen, 24 november 2004Promotor : Angenent, G.C.190 p
Continuous-time modeling of cell fate determination in Arabidopsis flowers
No full textBackground The genetic control of floral organ specification is currently being investigated by various approaches, both experimentally and through modeling. Models and simulations have mostly involved boolean or related methods, and so far a quantitative, continuous-time approach has not been explored. Results We propose an ordinary differential equation (ODE) model that describes the gene expression dynamics of a gene regulatory network that controls floral organ formation in the model plant Arabidopsis thaliana. In this model, the dimerization of MADS-box transcription factors is incorporated explicitly. The unknown parameters are estimated from (known) experimental expression data. The model is validated by simulation studies of known mutant plants. Conclusions The proposed model gives realistic predictions with respect to independent mutation data. A simulation study is carried out to predict the effects of a new type of mutation that has so far not been made in Arabidopsis, but that could be used as a severe test of the validity of the model. According to our predictions, the role of dimers is surprisingly important. Moreover, the functional loss of any dimer leads to one or more phenotypic alterations
ChIP-seq Analysis in R (CSAR): An R package for the statistical detection of protein-bound genomic regions
No full textAbstract Background In vivo detection of protein-bound genomic regions can be achieved by combining chromatin-immunoprecipitation with next-generation sequencing technology (ChIP-seq). The large amount of sequence data produced by this method needs to be analyzed in a statistically proper and computationally efficient manner. The generation of high copy numbers of DNA fragments as an artifact of the PCR step in ChIP-seq is an important source of bias of this methodology. Results We present here an R package for the statistical analysis of ChIP-seq experiments. Taking the average size of DNA fragments subjected to sequencing into account, the software calculates single-nucleotide read-enrichment values. After normalization, sample and control are compared using a test based on the ratio test or the Poisson distribution. Test statistic thresholds to control the false discovery rate are obtained through random permutations. Computational efficiency is achieved by implementing the most time-consuming functions in C++ and integrating these in the R package. An analysis of simulated and experimental ChIP-seq data is presented to demonstrate the robustness of our method against PCR-artefacts and its adequate control of the error rate. Conclusions The software ChIP-seq Analysis in R (CSAR) enables fast and accurate detection of protein-bound genomic regions through the analysis of ChIP-seq experiments. Compared to existing methods, we found that our package shows greater robustness against PCR-artefacts and better control of the error rate.</p
Arabidopsis thaliana ambient temperature responsive lncRNAs
No full textBackground: Long non-coding RNAs (lncRNAs) have emerged as new class of regulatory molecules in animals where they regulate gene expression at transcriptional and post-transcriptional level. Recent studies also identified lncRNAs in plant genomes, revealing a new level of transcriptional complexity in plants. Thousands of lncRNAs have been predicted in the Arabidopsis thaliana genome, but only a few have been studied in depth. Results: Here we report the identification of Arabidopsis lncRNAs that are expressed during the vegetative stage of development in either the shoot apical meristem or in leaves. We found that hundreds of lncRNAs are expressed in these tissues, of which 50 show differential expression upon an increase in ambient temperature. One of these lncRNAs, FLINC, is down-regulated at higher ambient temperature and affects ambient temperature-mediated flowering in Arabidopsis. Conclusion: A number of ambient temperature responsive lncRNAs were identified with potential roles in the regulation of temperature-dependent developmental changes, such as the transition from the vegetative to the reproductive (flowering) phase. The challenge for the future is to characterize the biological function and molecular mode of action of the large number of ambient temperature-regulated lncRNAs that have been identified in this study.</p
Use of Petunia to unravel plant meristem functioning
No full textIn the past decade, enormous progress has been made in our understanding of the molecular and genetic control of meristem growth, maintenance and differentiation into plant organs. Several model plants have contributed to our current knowledge of meristem function. Research using Petunia has had a substantial share in this progress. Integration of information obtained from this species gives clues about the common and diverged pathways underlying the formation and functioning of plant meristem
Flower Development of Lilium longiflorum: Characterization of MADS-box transcription factors
No full textLily (Liliumspp.) is among the most traditional and beloved ornamental flowers worldwide. The genus Lilium comprises almost one hundred species, among which is the primary subject of our research, described in this thesis, the species Lilium longiflorum (Thunb.), known as trumpet lily or Easter lily.Despite the great economic importance of ornamental lily species, little is known about its biology at the molecular level so far. In a time when two genomes are fully sequenced, Arabidopsis thaliana and Oryza sativa , only a few genes have been characterized in Lilium spp. yet. Possible reasons for this are discussed throughout this thesis.This work intends to be a contribution to bridging the fundamental research concerning transcription factors involved in development of flower morphology in model species and the applied objectives of molecular breeding for manipulating flower morphology, endeavouring to create new cultivars with specific and novel features, more specifically in Lilium spp.The ABC model for floral development was proposed more than 10 years ago and since then many studies have been performed in model species, such as Arabidopsis thaliana , Antirrhinum majus , petunia and many other species in order to confirm this model. This investigation has led to additional information on flower development and to more complex molecular models.In the first chapter of this thesis, notions of molecular floral development, the difficulties of working with molecular biology of lily, the state-of-the-art in lily transformation are introduced, as well as general overviews of transcription factors, MADS-box genes, the ABCDE model for flower development and functional characterization of genes in heterologous systems. These concepts will guide the reader throughout the work we present here.AGAMOUS( AG ) is the only C type gene found in Arabidopsis and it is responsible for stamen and carpel development as well as floral determinacy. In the second chapter, we describe the isolation of LLAG1 , a putative AG orthologue from lily ( L. longiflorum ) by screening a cDNA library derived from developing floral buds. The deduced amino acid sequence of LLAG1 revealed the MIKC structure and a high homology in the MADS-box among AG and other orthologues. Phylogenetic analysis indicated close relationship between LLAG1 and AG orthologues from monocot species. Spatial expression data showed LLAG1 transcripts exclusively in stamens and carpels, constituting the C domain of the ABC model. Functional analysis was carried out in Arabidopsis by overexpression of LLAG1 driven by the CaMV 35S promoter. Transformed plants showed homeotic changes in the two outer floral whorls with some plants having the second whorl completely converted into stamens. Altogether, these data indicate a functional relationship between LLAG1 and AG .( SEP3 ) is a MADS-box homeotic gene possibly determining the E function in the ABCDE model. This function is essential for proper development of petals, stamens and carpels. In order to gain further information on lily ( Lilium longiflorum ) flower development at the molecular level, the cDNA library constructed from developing floral buds was screened again and our findings are reported in the chapter three. A clone ( LLSEP3 ) was isolated with high similarity to the SEP3 transcription factor from Arabidopsis . LLSEP3 belongs to the AGL2 subfamily of MADS-box genes and shares its closest relationships with DOMADS1 and OM1 , from the orchid species Dendrobium grex and Arandadeborah, respectively. Expression analysis by Northern hybridisation showed that LLSEP3 was expressed throughout lily flower development and in tepals, stamens and carpel tissues of mature flowers, whereas no expression was detected in leaves. Overexpression of LLSEP3 in Arabidopsis under the CaMV35S promoter induced early flowering but did not induce any floral homeotic changes, which is in accordance with the effect of SEP3 overexpression in this species. Altogether, these data are consistent with the putative role of LLSEP3 as an E functional gene in lily flower development.Drawbacks found during our work on functional characterisation of LLAG1 , by means of complementing the agamous mutant of Arabidopsis thaliana are described and critically discussed in chapter four. Such difficulties are, on the one hand, the nature of the AGAMOUS gene, of which the loss of function induces sterility and, on the other hand, the unavailability of the defective ag-1 allele in another Arabidopsis background than the Landsberg erecta ecotype, which is recognisably difficult to transform by the floral dip method. Even though we did not manage to complement the AG function with LLAG1 in a defective ag genotype so far, we could observe clear floral homeotic changes in those Arabidopsis plants ectopically overexpressing LLAG1 , which together with our data on sequence identities and expression profile described in the previous chapter of this thesis, indicated that LLAG1 is a strong candidate to control the C function in L. longiflorum .This work also contributes towards the improvement of lily transformation procedures. In the chapter five we describe a transformation of bulblet slices by particle bombardment using a vector carrying the ArabidopsisSUPERMAN gene driven by the petunia flower-specific FLORAL BINDING PROTEIN 1 promoter and the bialaphos resistance gene phosphinothricin acetyltransferase under the CaMV35S promoter. Our intentions were improving the transformation parameters for lily transformation in order to reach higher efficiency, and creating novel phenotypes in lily flowers using transcription factors originating from dicot plants. We were capable of obtaining transgenic lines expressing in vitro resistance to bialaphos. The transgenic plants were transferred to the greenhouse, grown and monitored for two flowering seasons. Flowers derived from these plants appeared normal and indistinguishable from wild-type flowers and the possible reasons for this are currently under investigation.Homeoticchanges in floral organs of lily ( Lilium spp.) are described in chapter six. Usually, lily flowers show similar organs in their first and second whorls called tepals. They constitute the appealing and colourful features determining flower appearance. Stamens and the pistil appear as the third and fourth whorls, respectively. A double lily flower shows replacement of stamens by tepals and of its carpel by a new flower in a reiterated manner, similar to what is seen in the agamous mutant of Arabidopsis . A novel floral phenotype of lily, denominated festiva here, has never been reported in other species so far and shows a complete homeotic change of stamens into tepals, but keeps the carpel identity. We tried to explain these phenotypes taking into consideration all the evidence on the genetic mechanisms involved in flower development gathered over the last 15 years. This work launches challenges and encouragement for exploiting the molecular mechanisms involved in flower development of lily.Virus-induced gene silencing (VIGS) system has shown to be of great potential in reverse plant genetics. Advantages of VIGS over other approaches, such as T-DNA or transposon tagging, include the circumvention of plant transformation, methodological simplicity and robustness, and speedy results. These features enable VIGS as an alternative instrument in functional genomics, even in a high throughput fashion. The system is already well established in Nicotiana benthamiana , but efforts are being addressed to improve VIGS in other species, including monocots. Current research is focussed on unravelling the silencing mechanisms of post-transcriptional gene silencing (PTGS) and VIGS, as well as finding novel viral vectors in order to broaden the host species spectrum. In chapter seven, we discuss the advantages of using VIGS to assess gene functions in plants. We address the molecular mechanisms involved in the process, present the available methodological elements such as vectors, inoculation procedures, and we show examples in which the system was applied successfully to characterize gene function in plants. Moreover, we analyse the potential application of VIGS in assessing genetic function of floral transcription factors from monocots.Analyses of gene functions involved in lily flower development and generation of useful information on the molecular breeding potential of this species were the main objectives of the work described in this thesis.The field for studying the molecular aspects of lily flower development is now wide open and the future may uncover very interesting aspects that will produce new tools for ornamental breeders as well as reveal particular features of monocots and the Liliaceae plant family
The role of receptor-like proteins in Arabidopsis development
No full textAn intriguing and long-standing question in developmental biology is how plant cells communicate with each other and sense signals from their surrounding environment. Through research over past decades, it became clear that plant cells use membrane-localized receptors to perceive signals from their environment, which subsequently results in the initiation of downstream signaling (Kobe and Kajava, 2001; Torii, 2005). The membrane-associated receptors often form in multimeric complexes that contain receptor-like proteins (RLP) (Song et al., 1997; Jeong et al., 1999; Fritz-Laylin et al., 2005) as well as receptor-like kinase (RLK) proteins (Shiu and Bleecker, 2001a; 2001b). This is the case for the development of the shoot apical meristem (SAM) involving the CLAVATA1 (CLV1) and CLAVATA2 (CLV2) receptor molecules, as well as a small secreted polypeptide CLAVATA3 (CLV3) (Clark et al., 1993; Clark et al., 1995; Clark et al., 1997; Kayes and Clark, 1998; Fletcher et al., 1999; Jeong et al., 1999; Brand et al., 2000; Schoof et al., 2000). The Arabidopsis gene CLV2 encodes an LRR RLP acting in a functional CLV receptor complex that is involved in restricting the size of shoot meristem (Jeong et al., 1999). A total of 57 AtRLPs, which share sequence similarity and domain composition, have been identified in the Arabidopsis genome (Wang et al., 2008). However, the function of most AtRLPs remains elusive despite a genome-wide functional study into the roles of AtRLPs has been carried out recently (Wang et al., 2008). Given that fact that many AtRLPs originated from duplication events (Fritz-Laylin et al., 2005), it is very likely that the lack of identification of biological functions for AtRLP genes may be explained by functional redundancy, a phenomenon that typically obscures studies employing a reverse genetics strategy, as has been described for many RLK gene family members (Cano-Delgado et al., 2004; Shpak et al., 2004; Albrecht et al., 2005; DeYoung et al., 2006; Hord et al., 2006). In the future, RNA interference (RNAi) or artificial microRNA (amiRNA) approaches to target the expression of multiple AtRLP genes simultaneously could be followed to circumvent the functional redundancy (Chuang and Meyerowitz, 2000; Miki and Shimamoto, 2005; Ellendorff et al., 2008; Ossowksi et al., 2008). Alternatively, these multiple mutants of closely related AtRLP genes or potential co-expressed AtRLP genes should be combined to reveal the function of these genes. The clv2 mutant displays weaker, although similar phenotypes as clv1 and clv3 mutants (Kayes and Clark, 1998; Diévart et al., 2003; Chapters 2; 3.1), while loss-of-function mutants of clv3 are phenotypically stronger than clv1 or clv2 null mutants (Fletcher et al., 1999; Kayes and Clark, 1998; Dievart et al., 2003). In addition, CLV2 has a broader expression pattern, which may suggest a wider role for CLV2 in more developmental processes than only meristem development (Kayes and Clarks, 1998; Chapters 2; 3.1). Besides the broader role of CLV2, it is interesting to note that clv2 mutations, similar to clv1 mutations, are significantly affected by natural variation (Diévart et al., 2003; Chapters 3.1; 5), as has also been shown for the strubbelig (sub) and brassinosteroid insensitive1 (bri1) mutants (Chevalier et al., 2005; Cano-Delgado et al., 2004). The phenotype of clv2 in Col-0 (atrlp10) is also significantly enhanced by introduction into Ler background. Although our genetic interaction experiments indicated that the effect does not depend on the ER locus (Chapter 3.1), our observations imply that (a) CLV1/CLV2-modifying factor(s) exist(s) to co-regulate meristem development (Diévart et al., 2003; Chapters 3.1; 5). It would be interesting to identify these co-factor(s) in the future. Several lines of evidence suggest a role for CLV2 in root development. Over-expression of CLV3, CLE19 and CLE40 leads to an arrest of root growth (Casamitjana-Martinez et al., 2003; Hobe et al., 2003; Fiers et al., 2004), while the clv2 mutant can suppress the short-root phenotype caused by ectopic expression of CLE19 (Fiers et al., 2005). In addition, clv2 failed to respond to exogenously supplied synthetic CLE peptide, which corresponds to the conserved CLE motif of the CLV3/ESR gene family (Fiers et al., 2005; Ito et al., 2006; Kondo et al., 2006), indicating that CLV2 is able to perceive the CLE ligands in the root. However, the clv2 mutant exhibits no visible root phenotype under normal growth conditions, suggesting that a redundant protein, most likely another AtRLP gene, compensates for the loss of CLV2 function in the root. We found that only a few AtRLP genes are expressed in the root, although their expression is quite low (Chapters 2; 3.1). Possibly, a root defect only will become apparent in combination of multiple mutants for these genes. This observation argues that a CLV-like pathway also operates in roots (Fiers et al., 2007), but no RLK involved in this process has been found, although some of the RLKs that are expressed in the root (Birnbaum et al., 2003; Nawy et al., 2005). As such, CORYNE/Suppressor of overexpression of LLP-2 (CRN/SOL2) and Barely Any Meristem1-3 (BAM1-3) might be the logical RLK candidates for the redundant role in root development, because of their pronounced expression in roots (DeYoung et al., 2006; Müller et al., 2008; Miwa et al., 2008). Specifically, like clv2 mutants, crn/sol2 mutants did not respond to CLE peptide treatments (Müller et al., 2008; Miwa et al., 2008), suggesting that CRN, like CLV2, is involved in transmitting CLE signals. Therefore, studies with different combinations of mutants of these genes will help to clarify their biological function in root development (Chapter 2; 3.1; 4). Interestingly, WOX5, a homologue of WUS, marks the root Quiescent Centre (QC) identity and is expressed very early in the hypophysial cell (Sarkar et al., 2007), which is strikingly similar to the role of WUS in the shoot meristem (Haecker et al., 2004; Sarkar et al., 2007). Furthermore, it has been shown recently that POL and PLL, in addition to their role in Arabidopsis SAM maintenance (Song and Clark, 2005; Song et al., 2006), also act in the root meristem development through regulating the expression of the WUS homolog WOX5 (Song et al., 2008). These findings strengthen the hypothesis that a CLV-like pathway exists in the root meristem, which might include CLV2, CRN, WOX5, POL and PLL1. Similarly, our results as well as previous reports (Song and Clark, 2005; DeYoung et al., 2006; Müller et al., 2008) suggest that a CLV-related signaling pathway is involved in the regulation of leaf shape/size (Chapter 3.1) and pedicel length (Chapter 5). Interestingly, all these developmental pathways share some conserved factors such as POL and PLL, indicating common regulatory mechanisms exist in the developmental regulation of SAM, root meristem, leaf shape/size and pedicel growth. We provide evidence that two CLV2-related AtRLPs, AtRLP2 and AtRLP12, were capable to rescue clv2 mutants when expressed under the control of the CLV2 promoter (Chapter 4), suggesting that functional specification of these two AtRLPs may reside, at least in part, in their cis-regulatory elements. The importance of variation in expression pattern for the specificity in function of closely-related genes from multi-gene families while the proteins are interchangeable, has been documented for many genes such as the CLV1-like genes (BAM1-3, DeYoung et al., 2006; Hord et al., 2006), ERECTA-family (ER and ERL1-2, Shapk et al., 2004) and BRL-family members (BRI1, BRL1and BRL3; Cano-Delgado et al., 2004). However, the double mutant combinations of atrlp2 and atrlp12 mutants with atrlp10/clv2 did not show additive effects on growth of meristems and other organs when compared to that of the atrlp10/clv2 mutant, suggesting that other AtRLPs, such as AtRLP3 and AtRLP11 that are duplication counterparts of AtRLP2 and AtRLP12 respectively, can also replace the function of CLV2 in the regulation of the meristem development (Chapter 4). Therefore, additional phenotypes and the role of other AtRLP family members will become apparent only in the absence of the entire CLV2 close-related members as has been shown for ER, ERL1 and ERL2 (Shpak et al., 2004). Our studies in Chapter 4 revealed that several members of the AtRLP family can replace each other and are functionally equivalent. However, there are also family members with a similar protein domain organization, but that are clearly distinct from CLV2. This raised the question what determines the specificity in these proteins. We determined the function of the different domains by deletion analysis and generation of hybrid molecules (Chapter 4). CLV2 is still fully functional when the island domain is removed, while the C3-F region can be replaced by a close homologue (Chapter 4). Taken together, this study provided valuable information on the function of CLV2 domains that contribute to functional specificity and conservation. Despite these findings, little is known about the roles and specificity of other CLV2 domains, such as the transmembrane domain which is proposed to be the site for dimerization between CLV2 and CRN/SOL2 (Müller et al., 2008; Miwa et al., 2008). Of particular interest for future investigations are conserved residues flanking the LRR domain, which could be mutated to determine whether they are essential for CLV2 activity (Fritz-Laylin et al., 2005; van der Hoorn et al., 2005; Chapters 1; 3.1; 4). Previous studies proposed that CLV2 dimerizes with CLV1 to form an active receptor complex that binds the CLV3 ligand and initiates the downstream signaling pathway required for the maintenance of the stem cell population in the shoot apical meristem (Jeong et al. 1999; Trotochaud et al., 1999; Rojo et al., 2002; Dievart and Clark, 2004). The CLV2 protein was regarded as a stabilizer for the CLV1 protein based on the observation that CLV1 protein levels were reported to be reduced by over 90% in clv2 loss-of-function mutants (Jeong et al., 1999). In addition to CLV1, the receptor kinase CRN/SOL2 acts closely together with CLV2 to transmit the CLV3 signal independently but in parallel with CLV1 (Müller et al., 2008; Miwa et al., 2008). CRN/SOL2 has a kinase domain which might create a fully functional transmembrane receptor kinase together with CLV2 through dimerization in the transmembrane domains (Müller et al., 2008). This raises the hypothesis that CLV3 could bind CLV2 directly. However, whether CLV3 peptide can directly bind to the extracelluar domain of CLV2 remains to be validated. Nevertheless, the CLV3 signal is probably transduced through two separate receptor complexes, comprising CLV1/CLV2 (or CLV1 alone) and CRN/CLV2 (Müller et al., 2008). Despite these observations, the role of CLV2 in these signaling pathways remains largely unresolved. The CLV pathway is largely built on genetic data whereas direct biochemical evidence for the mode of action of the proteins involved is largely missing (Jeong et al. 1999; Trotochaud et al., 1999; Müller et al., 2008). Only very recently, it has been shown that the CLV3 peptide directly binds to the CLV1 ectodomain (Ogawa et al., 2008). In an effort to understand the physical interactions of the CLV proteins and their localization, we created fluorescently tagged versions of CLV1 and CLV2 (Chapter 5). These fusion proteins appeared to be targeted to the plasma membrane, displaying a common subcellular localization (Chapter 5). The functionality of the fusion proteins was confirmed by complementation of the respective mutants (Chapter 5). It has been postulated that the CLV1/CLV2 receptor complex resides in the plasma membrane to perceive the CLV3 ligand (Jeong et al. 1999; Trotochaud et al., 1999; Diévart et al., 2003). Our localization studies support this scenario. Unfortunately, it was not possible to determine a direct interaction between CLV1 and CLV2 in the framework of this Ph.D study. However, it is obvious that the interaction study can be the immediate next step using the available fluorescently-tagged CLV1 and CLV2 proteins and the stable transgenic lines generated in this study (Chapter 5). Furthermore, the study can be extended to visualize the components of the CLV signaling complex and follow their dynamics during plant growth. Furthermore, the labelled CLV-receptors can also be used for the isolation and identification of unknown components of the CLV receptor complex. For instance, it would be interesting to investigate whether CLV2 and CRN/SOL2 interact directly as proposed (Müller et al., 2008; Miwa et al., 2008). Indeed, a preliminary study using a similar approach supports the interaction of CLV2 and CRN/SOL2 (Y-F. Zhu and C-M. Liu, personal communication). It also intrigues to determine whether the CLV3 or other possible CLE(s) are directly interacting with the CLV2/CRN receptor complex. Undoubtedly, the tools generated in this study will be of great help for future experiments aiming to unravel the CLV signaling pathway. <br/
Systems biology of plant molecular networks: from networks to models
No full textDevelopmental processes are controlled by regulatory networks (GRNs), which are tightly coordinated networks of transcription factors (TFs) that activate and repress gene expression within a spatial and temporal context. In Arabidopsis thaliana, the key components and network structures of the GRNs controlling major plant reproduction processes, such as floral transition and floral organ identity specification, have been comprehensively unveiled. This thanks to advances in ‘omics’ technologies combined with genetic approaches. Yet, because of the multidimensional nature of the data and because of the complexity of the regulatory mechanisms, there is a clear need to analyse these data in such a way that we can understand how TFs control complex traits. The use of mathematical modelling facilitates the representation of the dynamics of a GRN and enables better insight into GRN complexity; while multidimensional data analysis enables the identification of properties that connect different layers from genotype-to-phenotype. Mathematical modelling and multidimensional data analysis are both parts of a systems biology approach, and this thesis presents the application of both types of systems biology approaches to flowering GRNs. Chapter 1 comprehensively reviews advances in understanding of GRNs underlying plant reproduction processes, as well as mathematical models and multidimensional data analysis approaches to study plant systems biology. As discussed in Chapter 1, an important aspect of understanding these GRNs is how perturbations in one part of the network are transmitted to other parts, and ultimately how this results in changes in phenotype. Given the complexity of recent versions of Arabidopsis GRNs - which involves highly-connected, non-linear networks of TFs, microRNAs, movable factors, hormones and chromatin modifying proteins - it is not possible to predict the effect of gene perturbations on e.g. flowering time in an intuitive way by just looking at the network structure. Therefore, mathematical modelling plays an important role in providing a quantitative understanding of GRNs. In addition, aspects of multidimensional data analysis for understanding GRNs underlying plant reproduction are also discussed in the first Chapter. This includes not only the integration of experimental data, e.g. transcriptomics with protein-DNA binding profiling, but also the integration of different types of networks identified by ‘omics’ approaches, e.g. protein-protein interaction networks and gene regulatory networks. Chapter 2 describes a mathematical model for representing the dynamics of key genes in the GRN of flowering time control. We modelled with ordinary differential equations (ODEs) the physical interactions and regulatory relationships of a set of core genes controlling Arabidopsis flowering time in order to quantitatively analyse the relationship between their expression levels and the flowering time response. We considered a core GRN composed of eight TFs: SHORT VEGETATIVE PHASE (SVP), FLOWERING LOCUS C (FLC), AGAMOUS-LIKE 24 (AGL24), SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), APETALA1 (AP1), FLOWERING LOCUS T (FT), LEAFY (LFY) and FD. The connections and interactions amongst these components are justified based on experimental data, and the model is parameterised by fitting the equations to quantitative data on gene expression and flowering time. Then the model is validated with transcript data from a range of mutants. We verify that the model is able to describe some quantitative patterns seen in expression data under genetic perturbations, which supported the credibility of the model and its dynamic properties. The proposed model is able to predict the flowering time by assessing changes in the expression of the orchestrator of floral transition AP1. Overall, the work presents a framework, which allows addressing how different quantitative inputs are combined into a single quantitative output, i.e. the timing of flowering. The model allowed studying the established genetic regulations, and we discuss in Chapter 5 the steps towards using the proposed framework to zoom in and obtain new insides about the molecular mechanisms underlying the regulations. Systems biology does not only involve the use of dynamic modelling but also the development of approaches for multidimensional data analysis that are able to integrate multiple levels of systems organization. In Chapter 3, we aimed at comprehensively identifying and characterizing cis-regulatory mutations that have an effect on the GRN of flowering time control. By using ChIP-seq data and information about known DNA binding motifs of TFs involved in plant reproduction, we identified single-nucleotide polymorphisms (SNPs) that are highly discriminative in the classification of the flowering time phenotypes. Often, SNPs that overlap the position of experimentally determined binding sites (e.g. by ChIP-seq), are considered putative regulatory SNPs. We showed that regulatory SNPs are difficult to pinpoint among the sea of polymorphisms localized within binding sites determined by ChIP-seq studies. To overcome this, we narrowed the resolution by focusing on the subset of SNPs that are located within ChIP-seq peaks but that are also part of known regulatory motifs. These SNPs were used as input in a classification algorithm that could predict flowering time of Arabidopsis accessions relative to Col-0. Our strategy is able to identify SNPs that have a biological link with changes in flowering time. We then surveyed the literature to formulate hypothesis that explain the regulatory mechanism underlying the difference in phenotype conferred by a SNP. Examples include SNPs that disrupt the flowering time gene FT; in which the mutation presumably disrupts the binding region of SVP. In Chapter 5 we discuss the steps towards extending our approach to obtain a more comprehensive survey of variants that have an effect on the flowering time control. In Chapter 4, we propose a method for genome-wide prediction of protein-protein interaction (PPI) sites form the Arabidopsis interactome. Our method, named SLIDERbio, uses features encoded in the sequence of proteins and their interactions to predict PPI sites. More specifically, our method mines PPI networks to find over-represented sequence motifs in pairs of interacting proteins. In addition, the inter-species conservation of these over-represented motifs, as well as their predicted surface accessibility, are take into account to compute the likelihood of these motifs being located in a PPI site. Our results suggested that motifs overrepresented in pairs of interacting proteins that are conserved across orthologs and that have high predicted surface accessibility, are in general good putative interaction sites. We applied our method to obtain interactome-wide predictions for Arabidopsis proteins. The results were explored to formulate testable hypothesis for the molecular mechanisms underlying effects of spontaneous or induced mutagenesis on e.g. ZEITLUPE, CXIP1 and SHY2 (proteins relevant for flowering time). In addition, we showed that the binding sites are under stronger selective pressure than the overall protein sequence, and that this may be used to link sequence variability to functional divergence. Finally, Chapter 5 concludes this thesis and describes future perspectives in systems biology applied to the study of GRNs underlying plant reproduction processes. Two key directions are often followed in systems biology: 1) compiling systems-wide snapshots in which the relationships and interactions between the molecules of a system are comprehensively represented; and 2) generating accurate experimental data that can be used as input for the modelling concepts and techniques or multi-dimensional data analysis. Highlighted in Chapter 5 are the limitations in key steps within the systems biology framework applied to GRN studies. In addition, I discussed improvements and extensions that we envision for our model related to the GRN underlying the control of flowering time. Future steps for multi-dimensional data analysis are also discussed. To sum up, I discussed how to connect the different technologies developed in this thesis towards understanding the interplay between the roles of the genes, developmental stages and environmental conditions.</p
Sequence Motifs in MADS Transcription Factors Responsible for Specificity and Diversification of Protein-Protein Interaction
No full textProtein sequences encompass tertiary structures and contain information about specific molecular interactions, which in turn determine biological functions of proteins. Knowledge about how protein sequences define interaction specificity is largely missing, in particular for paralogous protein families with high sequence similarity, such as the plant MADS domain transcription factor family. In comparison to the situation in mammalian species, this important family of transcription regulators has expanded enormously in plant species and contains over 100 members in the model plant species Arabidopsis thaliana. Here, we provide insight into the mechanisms that determine protein-protein interaction specificity for the Arabidopsis MADS domain transcription factor family, using an integrated computational and experimental approach. Plant MADS proteins have highly similar amino acid sequences, but their dimerization patterns vary substantially. Our computational analysis uncovered small sequence regions that explain observed differences in dimerization patterns with reasonable accuracy. Furthermore, we show the usefulness of the method for prediction of MADS domain transcription factor interaction networks in other plant species. Introduction of mutations in the predicted interaction motifs demonstrated that single amino acid mutations can have a large effect and lead to loss or gain of specific interactions. In addition, various performed bioinformatics analyses shed light on the way evolution has shaped MADS domain transcription factor interaction specificity. Identified protein-protein interaction motifs appeared to be strongly conserved among orthologs, indicating their evolutionary importance. We also provide evidence that mutations in these motifs can be a source for sub- or neo-functionalization. The analyses presented here take us a step forward in understanding protein-protein interactions and the interplay between protein sequences and network evolution
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