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Protein phosphorylation is instrumental to early signaling events. Studying system-wide phosphorylation in relation to processes under investigation requires a quantitative proteomics approach. In Arabidopsis, auxin application can induce pericycle cell divisions and lateral root formation. Initiation of lateral root formation requires transcriptional reprogramming following auxin-mediated degradation of transcriptional repressors. The immediate early signaling events prior to this derepression are virtually uncharacterized. To identify the signal molecules responding to auxin application, we used a lateral root-inducible system that was previously developed to trigger synchronous division of pericycle cells. To identify and quantify the early signaling events following this induction, we combined (15)N-based metabolic labeling and phosphopeptide enrichment and applied a mass spectrometry-based approach. In total, 3068 phosphopeptides were identified from auxin-treated root tissue. This root proteome dataset contains largely phosphopeptides not previously reported and represents one of the largest quantitative phosphoprotein datasets from Arabidopsis to date. Key proteins responding to auxin treatment included the multidrug resistance-like and PIN2 auxin carriers, auxin response factor2 (ARF2), suppressor of auxin resistance 3 (SAR3), and sorting nexin1 (SNX1). Mutational analysis of serine 16 of SNX1 showed that overexpression of the mutated forms of SNX1 led to retarded growth and reduction of lateral root formation due to the reduced outgrowth of the primordium, showing proof of principle for our approach.

In Arabidopsis thaliana, lateral roots (LRs) initiate acropetally and their dynamic development forms the basis on which root system architecture is elaborated. This thesis work focuses on revealing the underlying molecular network of how redundant PLETHORA (PLT) genes, encode APETALA2 (AP2)-domain transcription factors, jointly control rhizotaxis and LR outgrowth. Rhizotaxis represents the spatial distribution of lateral root primordia (LRP) and LRs along primary roots. LR outgrowth, which is staged according to the number of radial cell layers, is defined as a ‘primordium-intrinsic’ establishment of new organ tissues and meristem in an orthogonal axis to the parental roots. PLT3, PLT5 and PLT7 prevent the formation of LRP close to one another, because, in their absence, successive LRP are frequently grouped in close clusters. In addition, LR outgrowth is disrupted in plt3plt5plt7 LRP from the Stage I to II transition phase, when central cells fail to induce formative cell divisions to define the stage-specific layers and the expression of key root tissue-specific and meristematic genes is mis-regulated. Re-introduction of any PLT proteins in plt3plt5plt7 LRP from Stage I onward completely restores LR morphology, tissue specificity and meristem. Thus, PLT3, PLT5 and PLT7 genes are ‘bottleneck’ genes to activate the orthogonal program of de novo LR tissue pattern and meristem activity after LR initiation. Moreover, we have identified three LATERAL ORGAN BOUNDARIES DOMAIN (LBD) family members, LBD14, LBD18 and LBD29 as direct PLT promoter binding proteins that are associated with rhizotaxis and LR outgrowth.

Plant roots are colonized by an immense number of microbes, referred to as the root microbiome. Selected strains of beneficial soil-borne bacteria can protect against abiotic stress and prime the plant immune system against a broad range of pathogens. Pseudomonas spp. rhizobacteria represent one of the most abundant genera of the root microbiome. Here, by employing a germ-free experimental system, we demonstrate the ability of selected Pseudomonas spp. strains to promote plant growth and drive developmental plasticity in the roots of Arabidopsis (Arabidopsis thaliana) by inhibiting primary root elongation and promoting lateral root and root hair formation. By studying cell type-specific developmental markers and employing genetic and pharmacological approaches, we demonstrate the crucial role of auxin signaling and transport in rhizobacteria-stimulated changes in the root system architecture of Arabidopsis. We further show that Pseudomonas spp.-elicited alterations in root morphology and rhizobacteria-mediated systemic immunity are mediated by distinct signaling pathways. This study sheds new light on the ability of soil-borne beneficial bacteria to interfere with postembryonic root developmental programs.

Plants and animal cells use intricate signaling pathways to respond to a diverse array of stimuli. These stimuli include signals from the environment, such as biotic and abiotic stress signals, as well as cell-to-cell signaling required for pattern formation during development. The transduction of the signal often relies on protein phosphorylation, which in eukaryotic cells occurs mainly on serine (Ser), threonine (Thr) and tyrosine (Tyr) residues. In animal systems Tyr phosphorylation plays a prominent role in signaling, however the relative contribution of Tyr phosphorylation to plant signal transduction has remained an open question. We implemented an approach to selectively measure and quantify Tyr phosphorylation in plant cells, which can also be applied to whole plants. We reproducibly quantified 23 pTyr peptides in two inversely labeled biological replicates, identifying 11 differentially phosphorylated proteins. These include a set of 3 well-characterized flagellin responsive mitogen-activated protein kinases (MAPKs) and 4 novel MAPKs. In plant signal transduction MAPKs are important in transducing signals from the upstream receptor to the downstream targets and they are pivotal signaling modules in pathogen-associated molecular patterns (PAMP)-triggered immunity (PTI). In Arabidopsis thaliana (Arabidopsis), the MEK4/5-MPK3/6 module is an important part of a major MAPK cascade in immune responses. We report the functional analysis of a Arabidopsis MAPK kinase kinase (MAPKKK) as a negative regulator of PTI signaling and basal immunity. We show that this function requires phosphorylation of this protein on specific Ser residues. Based on genetic evidence we observed, that this MAPKKK is also involved in root meristem maintenance and meristem cell proliferation, which are required for root growth. Our results show that through phosphorylation of this MAPKKK, signaling pathways controlling growth and immune response are antagonistically regulated.

Multicellular organisms develop their large variety of cell types from just one single cell, the zygote. Both plants and animals use asymmetric cell division to establish a multicellular body plan How different cell and tissue types are determined, how patterns are created and maintained, and which players are involved, are fundamental questions in developmental biology. Plants grow from their meristems, groups of dividing cells residing at foci of development. Within a meristem, stem cell niches are vital for maintaining the balance between stem cell self-renewal and differentiation. Stem cell divisions in Arabidopsis roots are asymmetric. Each root stem cell division gives rise to a new stem cell and a daughter cell that embarks on a trajectory towards differentiation, even though a limited number of cell divisions may still occur. Often the model plant Arabidopsis thaliana is used to illustrate the implementation of asymmetric cell division to create pattern and diversity. The heat shock transcription factor SCHIZORIZA (SCZ) is relevant for specification of root ground tissueand segregation of surrounding QC/columella and epidermal/lateral root cap fates (Mylona et al. 2002, ten Hove et al. 2010, Pernas, Ryan & Dolan 2010). This makes it an intriguing factor for studying asymmetric cell division and fate determination in Arabidopsis roots. The molecular foundation to SCZs mode of action is still unclear. This study aims to contribute a deeper understanding of asymmetric cell division and fate segregation. The SCZ target genes found endorse the view on the role of SCZ in cortex specification and outer tissue fate segregation. Three transcription factor genes, formerly unknown to pattern the rot meristem; ANTHOCYANINLESS2 (ANL2), TUBBY LIKE PROTEIN 8 (TLP8) and a C2H2 type Zinc Finger, ZN7571 are described. SCZ genetic interaction with the well characterized root patterning transcription factor genes SHORTROOT (SHR) and SCARECROW (SCR) is established. And it was shown that both SCZ and SHR/SCR are needed to establish the root stem cell niche during embryogenesis. Proteins that are able to physically interact with SCZ are identified.

Genetic variation in natural populations of Arabidopsis thaliana has allowed the species to adapt to a wide range of environmental conditions. The identification of natural alleles affecting important traits can be used to enhance our understanding of the molecular mechanisms deployed by Arabidopsis to respond to the environment. These responses are generally controlled by multiple genes contributing quantitatively to the observed phenotypic traits. The molecular dissection of quantitative traits has been aided by the availability of RILs and genetic maps allowing the linkage of traits to genotypes by QTL analysis. The concept of genetical genomics was based on the idea that natural variation not only affects phenotypic traits, but also the variation in gene expression that can be measured on a genome-wide scale. In this thesis it was shown that the quantitative analyses of genome-wide gene expression in the Landsberg erecta (Ler)/Cape Verde Islands (Cvi) RIL population revealed heritable expression patterns for which thousands of eQTLs could be mapped. Local regulation was as prevalent as distant regulation. Loci were detected though, that harbor distant eQTLs for a large number of genes, which possibly point to master regulators. The possibility to dissect gene regulatory networks using eQTL data is based on the assumption that the expression of target genes correlates with the expression of the regulator gene. We successfully applied this principle to construct regulatory networks for genes known to be involved in flowering time and to genes downstream of the master regulator ERECTA. The two genetical genomics experiments described in this thesis allowed an initial comparison which gave an impression of plasticity in gene expression regulation. About 1000, predominantly locally regulated, linkages were identified in both experiments. The majority of eQTLs, however, was specific for each experiment, and enriched for distant eQTLs. We explored the added value of the genetical genomics in the analysis of the response of Arabidopsis to the virulent bacterial strain Pseudomonas syringae pv tomato (Pst) and to treatment with low light in the Ler/Cvi RILs respectively, in search for new factors in the regulation of these stress responses. The higher susceptibility of Cvi compared to Ler could be mapped to several disease susceptibility QTLs in the Ler/Cvi RILs. The major QTL had large impact on the transcriptional response to jasmonic acid during infection. The genetical genomics data allowed the identification of NUDT8, a Nudix hydrolase co-expressed with JA biosynthesis genes. Further studies are needed to analyze the role of NUDT8 in susceptibility of Arabidopsis to Pst infection. The low light treatment is known to induce the shade avoidance syndrome for which Ler and Cvi showed considerable natural variation. A major HTR affected the expression of components of the circadian clock, a ROS-activated signaling cascade and auxin- and sterol homeostasis. A common feature linking these processes could be the regulation of protein ubiquitination. The local regulation of CSN5A by the HTR could indicate a role for the CSN in the HTR-mediated processes. These results demonstrate the added value of genetical genomics in the dissection of quantitative traits.

Mutant genotypes are essential to uncover the role of any gene of interest (GOI), however, the absence of a wt (normal) allele in the entire organism might be obstructive for taking such approaches. If a GOI is essential in early developmental stages (e.g., embryogenesis), homozygous knockout individuals do not reach adulthood and mutant alleles cannot be studied in later stages. Together with advances in molecular biological techniques that are designed to address more specific and fine-tuned questions, cell/tissue specific knockout (or knockdown) methods are continuously developed. Several methods make use of two-component systems such as the Cre/lox mechanism to excise a genomic sequence, which can subsequently lead to elimination and/or activation of a GOI. One of our focuses was the cell specific role of the RETINOBLASTOMA-RELATED (RBR) gene in the Arabidopsis root stem cell (SC) niche. In animals, the homologous gene, RETINOBLASTOMA (Rb), was the first identified tumor suppressor, leading to 2.5 decades of research that disclosed Rb as one of the chief regulators of cell cycle, cell differentiation and cell death. Previous studies in plants suggested that RBR functions in a variety of developmental processes, amongst which, the maintenance of shoot and root meristem. We sought to create cell specific RBR loss of function using the Cre/lox system to study its distinct (and/or overlapping) activity in several types of root meristem cells. This kind of analysis, also known as clonal deletion, relies on loss of heterozygosity, where null and wt sectors are compared to study the local effect of a knockout genotype. However, these methods are not applicable for RBR since the protein is essential in the parental gametophytes. Therefore, hemizygous genotypes are rare and practically not available for research. To circumvent this obstacle, we design a new recombination system, Brother Of Brainbow (BOB) and also made use of tissue specific, inducible Cre recombinase to create and visually identify mutant clones. We found that RBR is required cell-autonomously in the QC and columella SCs to suppress unscheduled proliferation. Using the BOB system as well as artificial microRNA based silencing method (amiGO), we showed that the QC serves as a reservoir for columella SCs and that RBR interaction with SCR is necessary to keep its quiescent state as well as to protect it from genotoxic stress. Further examination of hypomorphic RBR genotypes revealed that reduced RBR levels lead to cell death. This phenotype partially requires the ATM/ATR machinery, whereas several mechanisms that induce differentiation including RBR over-expression partially reduced the cell death. Last, we investigated transcriptional regulation by the root specific transcription factor, PLETHORA2. We identified two targets, HAN and ARF10 that could either mimic PLT2 over-expression phenotype or complement plt1;plt2 mutant, respectively. Over-expression of either PLT2 or HAN was especially intriguing since it was sufficient to revert differentiating cells back to proliferation state. These findings together with the BOB and amiGO tools can set the basis for further studies on the mechanisms that underlie proliferation and differentiation in the root SC niche and the interaction between the two pathways

SummaryIn plants, where cells cannot migrate, asymmetric cell divisions (ACDs) must be confined to the appropriate spatial context. We investigate tissue-generating asymmetric divisions in a stem cell daughter within the Arabidopsis root. Spatial restriction of these divisions requires physical binding of the stem cell regulator SCARECROW (SCR) by the RETINOBLASTOMA-RELATED (RBR) protein. In the stem cell niche, SCR activity is counteracted by phosphorylation of RBR through a cyclinD6;1-CDK complex. This cyclin is itself under transcriptional control of SCR and its partner SHORT ROOT (SHR), creating a robust bistable circuit with either high or low SHR-SCR complex activity. Auxin biases this circuit by promoting CYCD6;1 transcription. Mathematical modeling shows that ACDs are only switched on after integration of radial and longitudinal information, determined by SHR and auxin distribution, respectively. Coupling of cell-cycle progression to protein degradation resets the circuit, resulting in a “flip flop” that constrains asymmetric cell division to the stem cell region.

We report a surface morphology study on single-shot submicron features fabricated on silicon on insulator by tightly focused femtosecond laser pulses. In the regime just below single-shot ablation threshold nano-tips are formed, whereas in the regime just above single-shot ablation threshold, a saturation in the ablation depth is found. We attribute this saturation by secondary laser absorption in the laser-induced plasma. In this regime, we find excellent agreement between the measured depths and a simple numerical model. When the laser fluence is further increased, a sharp increase in ablation depth is observed accompanied by a roughening of the ablated hole.

The mature Arabidopsis embryo contains two stem cell niches that upon germination bring forth and maintain the root meristem (RM) and shoot apical meristem (SAM). During seedling growth, the RM enables indeterminate elongation of the primary root, while the SAM produces true leaves. The resulting seedling further increases in complexity by continuous de novo organ formation, which involves positioning, formation and patterning of organ primordia. These primordia can produce leaves, flowers or the new growth axes of lateral roots or inflorescence stems. Cells within meristems are required to divide, elongate and differentiate without consuming the meristem to facilitate organ growth. Plant growth is plastic, but displays profound regularities that are thought to result from the interplay between endogenously determined developmental cues and environmental factors. In this thesis we have attempted to understand better the endogenous factors and mechanisms that control Arabidopsis development and architecture. We show that embryonic root formation requires the redundant activity of four PLETHORA (PLT) genes (PLT1-4), which function in a gene-dosage-dependent manner. These PLT proteins accumulate in gradients throughout the RM, with the highest levels required for stem cell niche maintenance, intermediate levels promoting mitotic cell division and low levels required for cell elongation. Lateral root initiation involves the formation of local auxin activity maxima. We have combined experiments and computational modelling to study the mechanism that establish these maxima and thus determine root system architecture. We found that root bending induces lateral root initiation on the outside of the bend. We performed computational modelling and found that cell size changes due to bending perturb auxin transport in such a manner that it positions higher auxin levels at the positions where lateral roots normally form. We show that such differences can be amplified by auxin influx carriers, which allows the precise definition of a new lateral root initiation site. Lateral organ formation in roots and shoots appears to be very different, but auxin has been shown to regulate both. The pattern of root and shoot branching thus might relate to similar mechanisms that position and restrict auxin activity maxima. So far, no transcription factors have been found to regulate branching of both roots and shoots, which would indicate that these branching systems evolved separately. However, we demonstrate that both branching mechanisms depend on the activity of three PLT genes, PLT3, 5 and 7. These genes are expressed through auxin accumulation at the sites of lateral root formation and their absence results in the clustering of root initiation sites. In the shoot, they regulate accumulation patterns of the PIN1 auxin transport protein to position primordia. Strikingly, plants that fail to express these PLT genes switch their pattern of leaf initiation from spiral to other regular patterns that are often observed within other plant species.

Asymmetric cell division is an essential and universal mechanism for generating diversity and pattern in multicellular organisms. Divisions generating daughter cells different in size, shape, identity and function are fundamental to many developmental processes including fate specification, tissue patterning and self-renewal. It is hypothesized that the angiosperm root meristem has evolved from the shoot apical meristem. Presumably, this is the outcome of the plants adaptation to changing environmental conditions. Accordingly, key gene network motifs present in the shoot have been found to be important in the development and regulation of the root meristem, such as peptide ligands and their receptors. In this thesis Arabidopsis root patterning and meristem maintenance has been investigated using forward and reverse genetics approaches. I describe the identification and analysis of SCHIZORIZA (SCZ). SCZ encodes a member of the family of heat shock transcription factors, albeit one that appears to be recruited for development instead of stress signaling. The pleiotropic effects of the scz mutation define the existence of a novel mechanism for patterning cell identity in the Arabidopsis root. SCZ acts in a parallel pathway with SHR/SCR to specify the root stem cell niche in the early embryo. Overexpression of CLE family peptides restricts the size of both the shoot and root meristem, suggesting that signaling pathways involved in shoot and root meristem maintenance are conserved. An activation tagging screen was performed on transgenic plants ectopically expressing CLE19 in the root meristem, aimed to identify new components of a root CLE signaling pathway. A recessive mutant, sol3, was isolated, which suppresses the CLE19 overexpression phenotype. SOL3 has a dual role in the root controlling growth and formative cell divisions. Using a reverse genetics approach I established and analyzed a collection of homozygous T-DNA insertion lines for 69 leucine-rich repeat receptor-like kinases (LRR RLKs) for developmental and conditional phenotypes. Possibly due to genetic redundancy the functional loss-of-function studies revealed developmental phenotypes for only one mutant line, rlk902. T-DNA insertion mutants assayed for their response after exposure to environmental, hormonal/chemical and abiotic stress, reveal several novel conditional functions for a number of RLK genes. rlk902 mutants show both reduced root growth and resistance to the oomycete pathogen Hyaloperonospora arabidopsidis. These phenotypes are not caused by RLK902 inactivation but are linked to the T-DNA insertion. Microarray analysis revealed downregulated gene expression over an 84 kb region upstream of and including RLK902 in rlk902, putatively encompassing the causal gene(s). The identification of SCZ as factor involved in tissue specification and cell fate segregation provides a basis for future research into mechanisms of asymmetric division. Work on CLE ligand signaling identified SOL3 as a factor controlling root growth and formative divisions and the LRR RLK reverse genetics indicated the existence of extensive cross talk and signal integration among different RLK signaling pathways in the Arabidopsis root. A challenge for the future will be to integrate these results with signaling networks for root patterning and growth.

Nephronophthisis (NPHP) is the major cause of pediatric renal failure, yet the disease remains poorly understood, partly due to the lack of appropriate animal models. Joubert syndrome (JBTS) is an inherited ciliopathy giving rise to NPHP with cerebellar vermis aplasia and retinal degeneration. Among patients with JBTS and a cerebello-oculo-renal phenotype, mutations in CEP290 (NPHP6) are the most common genetic lesion. We present a Cep290 gene trap mouse model of JBTS that displays the kidney, eye, and brain abnormalities that define the syndrome. Mutant mice present with cystic kidney disease as neonates. Newborn kidneys contain normal amounts of lymphoid enhancer-binding factor 1 (Lef1) and transcription factor 1 (Tcf1) protein, indicating normal function of the Wnt signaling pathway; however, an increase in the protein Gli3 repressor reveals abnormal Hedgehog (Hh) signaling evident in newborn kidneys. Collecting duct cells from mutant mice have abnormal primary cilia and are unable to form spheroid structures in vitro. Treatment of mutant cells with the Hh agonist purmorphamine restored normal spheroid formation. Renal epithelial cells from a JBTS patient with CEP290 mutations showed similar impairments to spheroid formation that could also be partially rescued by exogenous stimulation of Hh signaling. These data implicate abnormal Hh signaling as the cause of NPHP and suggest that Hh agonists may be exploited therapeutically.

Plant roots are colonized by an immense number of microbes, referred to as the root microbiome. Selected strains of beneficial soil-borne bacteria can protect against abiotic stress and prime the plant immune system against a broad range of pathogens. Pseudomonas spp. rhizobacteria represent one of the most abundant genera of the root microbiome. Here, by employing a germ-free experimental system, we demonstrate the ability of selected Pseudomonas spp. strains to promote plant growth and drive developmental plasticity in the roots of Arabidopsis (Arabidopsis thaliana) by inhibiting primary root elongation and promoting lateral root and root hair formation. By studying cell type-specific developmental markers and employing genetic and pharmacological approaches, we demonstrate the crucial role of auxin signaling and transport in rhizobacteria-stimulated changes in the root system architecture of Arabidopsis. We further show that Pseudomonas spp.-elicited alterations in root morphology and rhizobacteria-mediated systemic immunity are mediated by distinct signaling pathways. This study sheds new light on the ability of soil-borne beneficial bacteria to interfere with postembryonic root developmental programs.

Plants have many polarized cell types, but relatively little is known about the mechanisms that establish polarity. The orc mutant was identified originally by defects in root patterning, and positional cloning revealed that the affected gene encodes STEROL METHYLTRANSFERASE1, which is required for the appropriate synthesis and composition of major membrane sterols. smt1orc mutants displayed several conspicuous cell polarity defects. Columella root cap cells revealed perturbed polar positioning of different organelles, and in the smt1orc root epidermis, polar initiation of root hairs was more randomized. Polar auxin transport and expression of the auxin reporter DR5--glucuronidase were aberrant in smt1orc. Patterning defects in smt1orc resembled those observed in mutants of the PIN gene family of putative auxin efflux transporters. Consistently, the membrane localization of the PIN1 and PIN3 proteins was disturbed in smt1orc, whereas polar positioning of the influx carrier AUX1 appeared normal. Our results suggest that balanced sterol composition is a major requirement for cell polarity and auxin efflux in Arabidopsis.