Research objectives: (1) how the cell wall is assembled into coherent yet dynamic network, (2) what are the key changes in cell wall architecture and chemistry during growth, (3) understand the fast signaling by which cells perceive and coordinate wall expansion and remodeling, and (4) build a dynamic hybrid model to explain how plants coordinate wall expansion, (5) develop novel methods in nanoimaging, biosensing, optogenetics, multiplexed imaging to achieve goals 1-2.
Develop in silico models to explain how wall expansion is governed by pH-dependent effects, enzymatic activities, and interactions of systems components. The model will explain how sustained growth oscillations arise and what are the underlying differences between tip- and diffuse growth, e.g. different time-delays. Our objective is to integrate the three growth mechanisms and combine these with rapid regulatory processes to build a dynamic hybrid model explaining how plants coordinate wall expansion. This includes: (i) building dynamic models based on pectin remodelling and cell wall loosening including mechanical (stress/strain), physicochemical (e.g. pH, Ca2+ concentration) and biochemical (e.g. expansin, cell wall modifying enzyme activity; interactions) regulation; (ii) build hybrid models that will integrate growth geometry (FEM for pavement cells, mathematical description of a growing root hair) and molecular interactions (ordinary/partial differential equations, ODE/PDE). We will investigate the parallels between polar growth in tip-growing cells and lobe formation in diffusely growing cells.
Toolbox for high spatiotemporal precision observations to simultaneously monitor the downstream effects with high spatiotemporal resolution permitting to establish the causality between observed phenomena. Multiplexed optogenetics and FRET sensing Photoactivable activators (optogenetics) will elicit a fast (~s-min) spatially delineated (~µm2) perturbation of the physico- and biochemical aspects of wall expansion; a set of multicolor biosensors will monitor, in parallel, the downstream effects. Optogenetic activators. This includes photoactivable ion channels (H+, Ca2+) to manipulate cell wall pH and Ca+. Photoactivable (pa) exocytosis (paEXO), paRALF, photoactivable enzymes: paPMEI, paPME, paPectolyase (paPL). Biochemical read-outs We develop cell wall directed FRET biosensors for pH and Ca2+, PME/RALF maturation by subtilisin 1 protease (S1P).
Develop ultrahigh precision nanoscopy techniques to create an atlas of wall structures and unravel wall changes during growth. Toolbox for Ultrahigh Precision Nanoscopy: We are in the process of developing single-chain antibodies (nanobodies) targeting key cell wall elements, including differentially methylated HG, expansins, extensins, and RALF-LRX. This initiative aims to enhance resolution and improve cell wall penetrability, enabling high-density epitope mapping. The small probe size (3-5 nm) will facilitate the development of in vivo nanoimaging protocols for plant polysaccharides. Using dSTORM super-resolution microscopy (Haas et al., 2020) we will characterize the nanoarchitecture of the key cell wall epitopes cellulose, xyloglucan, pectins (HG, RG), arabinogalactan proteins, extensins, expansins and LRX1-RALF. We will determine how cell wall nano- and mesoarchitecture changes during cell wall expansion.
Decode the rapid mechanisms coordinating cell wall expansion. The goal is to determine temporal dynamics of growth and associated variables in diffusely growing hypocotyl cells or tip-growing root hairs and observe their behavior upon photoperturbation. Since it is not yet known if the growth of hypocotyl epidermis cells is periodic, the root hair is needed, because periodic processes are more prone to reveal underlying phenomena. We are now developing (see above) in vivo optogenetic tools to rapidly, locally, and reversibly perturb biochemical and cellular events involved in cell wall expansion and simultaneously monitor the downstream effects with high spatiotemporal resolution using biosensors and reporter dyes. The key variables we are observing is intra- and extracellular pH, Ca2+, and ROS, exocytosis, RALF localization, enzymatic activity, and cell wall hydration. Meanwhile we extensively use microfluidics to deliver pharmacological treatment or change ionic strength of the growing media.
Develop in silico models to explain how wall expansion is governed by pH-dependent effects, enzymatic activities, and interactions of systems components. The model will explain how sustained growth oscillations arise and what are the underlying differences between tip- and diffuse growth, e.g. different time-delays. Our objective is to integrate the three growth mechanisms and combine these with rapid regulatory processes to build a dynamic hybrid model explaining how plants coordinate wall expansion. This includes: (i) building dynamic models based on pectin remodelling and cell wall loosening including mechanical (stress/strain), physicochemical (e.g. pH, Ca2+ concentration) and biochemical (e.g. expansin, cell wall modifying enzyme activity; interactions) regulation; (ii) build hybrid models that will integrate growth geometry (FEM for pavement cells, mathematical description of a growing root hair) and molecular interactions (ordinary/partial differential equations, ODE/PDE). We will investigate the parallels between polar growth in tip-growing cells and lobe formation in diffusely growing cells.
Toolbox for high spatiotemporal precision observations to simultaneously monitor the downstream effects with high spatiotemporal resolution permitting to establish the causality between observed phenomena. Multiplexed optogenetics and FRET sensing Photoactivable activators (optogenetics) will elicit a fast (~s-min) spatially delineated (~µm2) perturbation of the physico- and biochemical aspects of wall expansion; a set of multicolor biosensors will monitor, in parallel, the downstream effects. Optogenetic activators. This includes photoactivable ion channels (H+, Ca2+) to manipulate cell wall pH and Ca+. Photoactivable (pa) exocytosis (paEXO), paRALF, photoactivable enzymes: paPMEI, paPME, paPectolyase (paPL). Biochemical read-outs We develop cell wall directed FRET biosensors for pH and Ca2+, PME/RALF maturation by subtilisin 1 protease (S1P).
Develop ultrahigh precision nanoscopy techniques to create an atlas of wall structures and unravel wall changes during growth. Toolbox for Ultrahigh Precision Nanoscopy: We are in the process of developing single-chain antibodies (nanobodies) targeting key cell wall elements, including differentially methylated HG, expansins, extensins, and RALF-LRX. This initiative aims to enhance resolution and improve cell wall penetrability, enabling high-density epitope mapping. The small probe size (3-5 nm) will facilitate the development of in vivo nanoimaging protocols for plant polysaccharides. Using dSTORM super-resolution microscopy (Haas et al., 2020) we will characterize the nanoarchitecture of the key cell wall epitopes cellulose, xyloglucan, pectins (HG, RG), arabinogalactan proteins, extensins, expansins and LRX1-RALF. We will determine how cell wall nano- and mesoarchitecture changes during cell wall expansion.
Decode the rapid mechanisms coordinating cell wall expansion. The goal is to determine temporal dynamics of growth and associated variables in diffusely growing hypocotyl cells or tip-growing root hairs and observe their behavior upon photoperturbation. Since it is not yet known if the growth of hypocotyl epidermis cells is periodic, the root hair is needed, because periodic processes are more prone to reveal underlying phenomena. We are now developing (see above) in vivo optogenetic tools to rapidly, locally, and reversibly perturb biochemical and cellular events involved in cell wall expansion and simultaneously monitor the downstream effects with high spatiotemporal resolution using biosensors and reporter dyes. The key variables we are observing is intra- and extracellular pH, Ca2+, and ROS, exocytosis, RALF localization, enzymatic activity, and cell wall hydration. Meanwhile we extensively use microfluidics to deliver pharmacological treatment or change ionic strength of the growing media.
