Sebastian Wolf

At the basis of plant developmental plasticity are continuously active pluripotent stem cells, which fuel the life-long post-embryonic formation of new organs. Since plant cells are encased in cell walls and thus immotile, their individual fate-specification program is dependent on their relative position within the organism. It is assumed that the cell wall and its mechanical properties are under surveillance, linking cell wall state to intracellular gene-regulatory networks. However, the role of these cell wall signalling pathways in plant development and the contribution of cell wall properties to cell behaviour and identity are unclear. Here, we show that control of cell wall properties is essential for the perpetuation of stem cell populations and pattering in the shoot apical meristems. The expression of pectin methylesterases (PMEs), which modify homogalacturonan methylesterification and thereby modulate cell wall mechanics, is maintained at low levels in stem cells by the stem cell specifying transcription factor WUS. Low PME expression is required for stemness, auxin patterning and stem cell-specific mechanical properties. Conversely, WUS depletion reduces wall stiffness in the meristem centre, reinforcing its role in maintaining mechanical homeostasis. Together, our findings show that WUS-mediated control of cell wall-modifying enzymes is essential for sustaining stem cell identity and SAM organization. These results demonstrat that the plant cell wall is not only involved in cell differentiation, but also exerts feedback control on developmental transitions, contributing to our understanding how the immediate physical environment is able to guide cell fate decisions in plants.

Modification of pectin, a component of the plant cell wall, is required to facilitate signaling by a RALF peptide, which is essential for many physiological and developmental processes.

A key question in plant biology is how oriented cell divisions are integrated with patterning mechanisms to generate organs with adequate cell type allocation. In the root vasculature, a gradient of miRNA165/6 controls the abundance of HD-ZIP III transcription factors, which in turn control cell fate and spatially restrict vascular cell proliferation to specific cells. Here, we show that vascular development requires the presence of ARGONAUTE10, which is thought to sequester miRNA165/6 and protect HD-ZIP III transcripts from degradation. Our results suggest that the miR165/6-AGO10-HDZIP III module acts by buffering cytokinin responses and restricting xylem differentiation. Mutants of AGO10 show faster growth rates and strongly enhanced survival under severe drought conditions. However, this superior performance is offset by markedly increased variation and phenotypic plasticity in sub-optimal carbon supply conditions. Thus, AGO10 is required for the control of formative cell division and coordination of robust cell fate specification of the vasculature, while altering its expression provides a means to adjust phenotypic plasticity.

Plant cells survey and modulate their cell wall to control their shape and anisotropic growth. Signaling mediated by the plant steroid hormones brassinosteroids (BR) plays a central role in coordinating cell wall status and cell growth, and alterations in the cell wall BR feedback loop leads to life-threatening defects in tissue and cellular integrity. How the status of the cell wall is relayed to BR signaling remains largely unclear. Increasing evidence shows that RAPID ALKALANIZATION FACTORs (RALFs), a class of secreted peptides, play structural and signaling roles at the cell surface. Here we show that perception of RALF23 promotes the formation and signaling of the main BR receptor complex formed by BRASSINOSTEROID INSENSITIVE 1 (BRI1) and BRI1 BRASSINOSTEROID INSENSITIVE1 BRASSINOSTEROID ASSOCIATED KINASE 1 (BAK1). The loss of the plasma membrane localized RALF receptor complex FERONIA (FER) LORELEI LIKE GPI anchor protein 1 (LLG1) leads to defects in cell expansion and anisotropy, as well as uncontrolled BRI1 BAK1 complex formation and signaling. RALF23 bioactivity relies on pectin status and its perception induces changes in pectin composition and the activity of pectin-modifying enzymes. Our observations suggest a model in which RALF23 functions as a cell wall informed signaling cue initiating a feedback loop that solicits BR signaling, modifies the cell wall, and coordinates cell morphogenesis.

Inducible, tissue-specific gene expression is a potent tool to study gene regulatory networks as it allows spatially and temporally controlled genetic perturbations. To this end, we generated a toolkit that covers many cell types in the three main meristems: the root apical meristem, the shoot apical meristem, and the vascular cambium. The system is based on an extensive set of driver lines expressing a synthetic transcription factor under cell type-specific promoters. Induction leads to nuclear translocation of the transcription factor and expression of response elements under control of a cognate synthetic promoter. In addition, a fluorescent reporter incorporated in driver lines allows to monitor induction. All previously generated driver lines are available from the Nottingham Arabidopsis Stock Center. This protocol describes how users can create their own constructs compatible with the existing set of lines and as well as induction and imaging procedures. Key words Inducible expression Trans-activation Promoter-reporter studies Expression control Mis-expression Functional genetics

Summary: The application of the CRISPR/Cas system as a biotechnological tool for genome editing has revolutionized plant biology. Recently, the repertoire was expanded by CRISPR‐Kill, enabling CRISPR/Cas‐mediated tissue engineering through genome elimination by tissue‐specific expression. Using the Cas9 nuclease from Staphylococcus aureus (SaCas9), CRISPR‐Kill relies on the induction of multiple double‐strand breaks (DSBs) in conserved repetitive genome regions, such as the rDNA, causing cell death of the targeted cells. Here, we show that in addition to spatial control by tissue‐specific expression, temporal control of CRISPR‐mediated cell death is feasible in Arabidopsis thaliana. We established a chemically inducible tissue‐specific CRISPR‐Kill system that allows the simultaneous detection of targeted cells by fluorescence markers. As proof of concept, we were able to eliminate lateral roots and ablate root stem cells. Moreover, using a multi-tissue promoter, we induced targeted cell death at defined time points in different organs at select developmental stages. Thus, using this system makes it possible to gain new insights into the developmental plasticity of certain cell types. In addition to enabling tissue engineering in plants, our system provides an invaluable tool to study the response of developing plant tissue to cell elimination through positional signaling and cell‐to‐cell communication.

Protein-protein interaction studies provide valuable insights into cellular signaling. Brassinosteroid (BR) signaling is initiated by the hormone-binding receptor Brassinosteroid Insensitive 1 (BRI1) and its co-receptor BRI1 Associated Kinase 1 (BAK1). BRI1 and BAK1 were shown to interact independently with the Receptor-Like Protein 44 (RLP44), which is implicated in BRI1/BAK1-dependent cell wall integrity perception. To demonstrate the proposed complex formation of BRI1, BAK1 and RLP44, we established three-fluorophore intensity-based spectral Förster resonance energy transfer (FRET) and FRET-fluorescence lifetime imaging microscopy (FLIM) for living plant cells. Our evidence indicates that RLP44, BRI1 and BAK1 form a ternary complex in a distinct plasma membrane nanodomain. In contrast, although the immune receptor Flagellin Sensing 2 (FLS2) also forms a heteromer with BAK1, the FLS2/BAK1 complexes are localized to other nanodomains. In conclusion, both three-fluorophore FRET approaches provide a feasible basis for studying the in vivo interaction and sub-compartmentalization of proteins in great detail.

Summary: Morphogenesis of multicellular organs requires coordination of cellular growth. In plants, 3D growth is driven by undirected turgor pressure, whereas growth directionality is controlled by cell wall mechanical properties at 2D cell faces. Their shared cell wall also fixes cells in their position, and plants thus have to integrate tissue-scale mechanical stresses arising due to growth in a fixed tissue topology. This implies a need to monitor cell wall mechanical and biochemical status and to adapt growth accordingly. Here, we propose that plant cells use their 1D cell edges to monitor cell wall status. We describe two Receptor-Like Proteins, RLP4 and RLP4-L1, which occupy a unique polarity domain at cell edges established through a targeted secretory transport pathway. We show that at cell edges, RLP4s associate with the cell wall via their extracellular domain, and contribute to directional growth control in Arabidopsis .

Plant architecture fundamentally differs from that of other multicellular organisms in that individual cells serve as osmotic bricks, defined by the equilibrium between the internal turgor pressure and the mechanical resistance of the surrounding cell wall, which constitutes the interface between plant cells and their environment. The state and integrity of the cell wall are constantly monitored by cell wall surveillance pathways, which relay information to the cell interior. A recent surge of discoveries has led to significant advances in both mechanistic and conceptual insights into a multitude of cell wall response pathways that play diverse roles in the development, defense, stress response, and maintenance of structural integrity of the cell. However, these advances have also revealed the complexity of cell wall sensing, and many more questions remain to be answered, for example, regarding the mechanisms of cell wall perception, the molecular players in this process, and how cell wall–related signals are transduced and integrated into cellular behavior. This review provides an overview of the mechanistic and conceptual insights obtained so far and highlights areas for future discoveries in this exciting area of plant biology.