The Arabidopsis root meristem consists of concentrically arranged tissues that surround the central vasculature. While the outer cell layers, the epidermis and ground tissue (GT), contribute to the radial symmetry of the entire root, the internal vasculature displays bilateral symmetry. These spatial patterns are established by the activity of stem cells located at defined positions within the stem cell niche. Daughter cells undergo cell expansion and differentiation to form shootward files along the longitudinal axis of the root. In the first part of this thesis, I describe GT specification and maturation. GT comprises the endodermis and cortex, and their lineages bifurcate following the asymmetric cell division (ACD) of the cortex-endodermis initial. The ACD requires the function of SHORT-ROOT (SHR) and its downstream regulator SCARECROW (SCR), which both belong to the GRAS transcription factor (TF) family. GA in the endodermis modulates the timing of GT maturation by promoting a second periclinal cell division which gives rise to an additional cortical layer between the endodermis and cortex. This additional cortex layer is named the middle cortex, and this process also requires SHR/SCR activity. Here, we have genetically and molecularly demonstrated that another GRAS member, SCARECROW-LIKE 3 (SCL3), is a direct downstream target of SHR/SCR, and its expression is also under the direct control of REPRESSOR OF ga1-3 (RGA), one of the DELLA proteins that are Gibberellin (GA) signaling inhibitors. SCL3 is expressed exclusively in the endodermis, where it integrates the SHR/SCR-mediated developmental signal and the GA-mediated hormonal signal to fine-tune the timing of middle cortex formation. The second part of my thesis investigates phloem sieve element (SE) development. SEs form a conductive tissue in the phloem that mediates the long-distance transport of sucrose. In Arabidopsis roots, protophloem SEs (PSE) comprise 20-25 cells from the stem cell to the enucleating cell. This cell file can serve as a model system to study tissue morphogenesis at single-cell resolution. Using confocal time lapse imaging technology, we monitored single-cell behaviors along the PSE lineage and defined five discernible stages: quiescent (stem cell), active division (transit amplifying cells), transition, differentiation and nuclear degradation (enucleation). We then focused on PSE differentiation, which involves dynamic cellular rearrangement ending with enucleation. We discovered a regulatory cascade in which ALTERED PHLOEM DEVELOPMENT (APL) and two closely related NAC-domain containing TFs – NAC45 and NAC86 – play major roles. We also identified NAC45/86-DEPENDENT EXONUCLEASE-DOMAIN PROTEINs (NENs) that is involved in nuclear degradation downstream of the APL-NAC pathway. In order to comprehensively understand PSE development, we carried out phloem-specific transcriptome profiling by performing fluorescence activated cell sorting of various phloem reporter lines followed by RNA-seq. By analyzing the transcriptome data, we identified 925 phloem-abundant genes. We focused on a cluster of genes which initiate their expression early in the protophloem sieve element (PSE) lineage. Interestingly, this cluster includes six DOF transcription factor family members. Knocking out all six genes resulted in a very narrow vasculature and impaired phloem transport, indicating that these DOFs are required for phloem differentiation as well as specification. We also determined that these DOFs promote PSE differentiation by directly activating the APL pathway. As part of the root meristem, the PSE must differentiate in coordination with the surrounding tissues. In fact, defects in vascular tissue development often lead to systemic growth inhibition and even seedling lethality in extreme cases. In the final part of this thesis, I demonstrate that the global root meristem regulator PLETHORA (PLT) overrides DOF function in the early stages of phloem development by suppressing the expression of APL, thereby preventing premature PSE differentiation.

Flowers and their number and arrangement within inflorescences are essential for the reproductive fitness and adaptive success of plants as well as for human sustenance. Comparative studies using species representing various plant lineages are a prerequisite for a comprehensive understanding of the development of diverse flower forms during the evolution of plants. In addition to a few classical model plants, new models, including core eudicots Asteraceae and basal eudicots Papaveraceae, have emerged to enhance our current understanding of the genetic networks in the regulation of plant growth and development from an evolutionary developmental biology perspective. This thesis seeks to understand the evolutionary origin of the Asteraceae capitulum and the regulatory networks of flower type specification as well as to illustrate the ancestral functions of CYCLOIDEA/TEOSINTE BRANCHED1 (CYC/TB1)-like (CYL) genes in basal eudicots. The Asteraceae inflorescence (capitulum, or flower head) superficially resembles a solitary flower, but it is a tightly packed structure composed of different types of flowers, including marginal ray flowers and central disc flowers. Such evolutionary innovations, as studied in Gerbera hybrida here, are owing to the novel functions of the gerbera orthologues of flower meristem identity (FMI) genes LEAFY (GhLFY) and UNUSUAL FLORAL ORGANS (GhUFO) as well as the regulatory networks involving MADS-box and TCP transcription factors (TFs). The expression domain of GhLFY at the inflorescence meristem mimics that of a single flower meristem, suggesting that the Asteraceae capitulum resembles a solitary flower not only morphologically but also at the molecular level. This expression pattern defines the capitulum as a determinate structure that can display floral fate upon ectopic GhUFO expression. Additionally, suppression of GhLFY and GhUFO led to a loss of FMI and suppressed MADS-box floral organ identity genes in a flower type-dependent manner. In particular, GhLFY regulates the early ontogeny of ray flowers, providing the first molecular evidence for how this structure has evolved. We speculate that the differentiation of flower types in Asteraceae is associated with their independent evolutionary origins from separate branching systems. Furthermore, the establishment of the regulatory networks amongst TCP and MADS-box gene family members in flower type specification provides additional support for distinct genetic origins of flower types. As trans-acting regulators of GhCYC3, the strongest CYC-like TCP gene in specifying ray flower identity, the CINCINNATA (CIN)-like TCP GhCIN1 co-localises with GhCYC3 in ray primordia and specifies the development of ray ligules. Moreover, we discovered that the whorl-specific MADS-box TFs control GhCYC3 expression during flower organ development: SEPALLATA3-like GERBERA REGULATOR OF CAPITULUM DEVELOPMENT5 (GRCD5) specifies ray ligule elongation, and C-class GERBERA AGAMOUS-LIKE1 (GAGA1) controls staminode development in ray flowers. Papaveraceae, belonging to a lineage basal to all other eudicots, represents a phylogenetically important family to understand the origin, evolution, and diversification of CYL genes in eudicots. Comparative studies between Eschscholzia californica and Cysticapnos vesicaria revealed their conserved functional roles in shoot branching, petal size, flower symmetry, and stamen growth, although in a species-specific manner. Nonetheless, our studies revealed a novel function in perianth development for CyveCYLs that were shown to specify the distinction of sepal and petal identity by suppressing B-class genes expression in C. vesicaria. These data indicate that instead of recruiting completely new regulators, the evolution of divergent morphologies employs conserved regulatory genes that show varying spatial/temporal expression patterns to perform their functions within modified regulatory networks, thereby contributing to the morphological diversification and origin of evolutionary innovations.

Plants possess the rare capability to shape the own architecture according to biotic and abiotic stimuli received from the environment. Spatially defined groups of cells, called meristems, contribute to the division and differentiation processes continuously occurring inside the organism. Meristems can be classified as primary meristems, if they are specified during embryogenesis, or secondary meristems, if they form from undifferentiated, quiescent cells outside the primary meristems. Primary meristems, like the Root Apical Meristem (RAM) and the Shoot Apical Meristem (SAM), coordinate the apical growth of the plant in opposite directions, while secondary meristems shape the radial architecture, regulating the thickness and branching of the primary root and shoot. Cambium is a secondary meristem which produces the vascular tissues xylem and phloem. Xylem transports water and minerals from the root to the photosynthetic tissues; it comprises lignified dead conducting cells called tracheary elements, living parenchyma cells, and lignified dead cells, called fibres, which confer mechanical support and strength. Phloem distributes glucose, RNA, viruses, and proteins from the photosynthetic sources to the sink cells; it consists of empty living sieve elements, supporting companion cells, and parenchyma cells. In order to investigate the regulation of primary and secondary growth, we developed a new chemically inducible system to control the timing and location of the induction of an effector or gene of interest. This enables us to avoid deleterious effects such as seed lethality or sterility when studying the role of a gene in a particular cell type. For example, the meristem cambium is difficult to access through normal techniques, since mutations affecting cambial cell divisions often inhibit the primary growth, too. We developed the inducible system by combining the Multi-Site Gateway cloning technology with the already extant XVE inducible system. This system was used to perform part of the research presented in the thesis. Phytohormones are involved in virtually every aspect of plant life, from development to stress response. They are small molecules which act cellautonomously or non-cell-autonomously to mediate the majority of developmental and environmental responses and, consequently, the activity of the meristems throughout the plant life cycle. Auxin and cytokinins, which were among the first phytohormones discovered, regulate almost every aspect of plant life, such as the division and differentiation processes occurring continuously in the RAM and SAM. The two phytohormones have long been known to interact, and recent studies have uncovered significant crosstalk on the level of biosynthesis, transport, signalling and degradation. We investigated the dynamic role of auxin in maintaining the balance between division, elongation, differentiation in the RAM of the model organism Arabidopsis thaliana. Our results confirm that an optimal level of auxin response is required for division and elongation, while differentiation mechanisms require just a minimal concentration of auxin to proceed normally. We discovered that auxin and cytokinin responses interact synergistically to specify the stem cells and to regulate the timing of divisions in the cambium of Arabidopsis thaliana. The auxin and cytokinin signalling pathways both have a positive role in triggering secondary growth, but the hierarchy of the crosstalk between them is still unclear. Finally, auxin transported via the AUX1/LAX auxin influx carriers regulates the differentiation of vessel elements in the later stages of root cambium development. In summary, we confirm that auxin and cytokinins behave as master regulators of meristematic activities throughout the root, as the signalling pathways associated with both phytohormones heavily influence primary and secondary growth.