Plant Surfaces and Interfaces in Plants: Lignin, Suberin and Cutin

As plants evolved from aquatic to terrestrial environment complex phenolic compounds (lignin) and natural polyesters (cutin, suberin) have played a major role by building hydrophobic surfaces and interfaces. The deposition of lignin as an additional interface between the cellulose microfibrils and hemicelluloses has been essential for the functioning of the developed water conducting elements (xylem) and for mechanical strength to reach considerable heights. The outermost surfaces had to be adapted to regulate water loss, which has been achieved by suberisation (stem, root) and cutinisation (leaf). Although general principles on the three compounds are known, they are the least understood plant polymers due to their high variability in amount and structure and still unresolved questions regarding polymerisation. Adaptation to survive in the great variety of habitats has resulted in quite diverse body plans that require highly specialized tissues with distinct properties and functions. These are achieved by changing composition and structure at the different hierarchical levels (mm-m-nm) and there is still a huge knowledge gap on the microchemistry and nanostructure of the native cell walls and its interfaces and surfaces. We will bridge this gap by application of non-destructive Raman imaging and Atomic force microscopy (AFM). Raman imaging gives access to the chemical composition on the micro level and AFM complements by elucidating nanostructure and-mechanics. Every Raman image is based on thousands of spectra, each a position resolved (0.3m) molecular fingerprint of the cell wall. Currently only a part of the chemical and structural information in the Raman signature is extracted and we aim at maximising the knowledge gain by applying new multivariate data analysis approaches. To drive the chemical information below the diffraction limit of light for probing tiny plant interfaces and surfaces we will explore the challenging tip-enhanced Raman spectroscopy (TERS) technique. With these sophisticated in-situ approaches we will 1) follow the lignification spatially resolved within the native cell wall to tackle unresolved questions around lignin polymerisation, 2) reveal the chemistry and structure of tracheid and vessel walls on the micro and nano level (including vessel surface and pit membranes) and its implications on hydraulic and mechanical properties 3) investigate the microchemistry and nanostructure of suberized and cutinized layers and its relation to the barrier properties, 4) additionally assess the impact of water stress as drought is becoming more and more a limiting factor for tree growth. New insights into the micro and nano distribution and composition of the plant polymers and as affected by drought stress will be gained and important structure-function relationships revealed. This will break new scientific grounds in the field of plant physiology (plant hydraulics, lipophilic barriers) and wood science (lignification, material properties) and have an impact on optimised plant feedstock development (forestry, agriculture, breeding, genetic engineering) and utilisation (biorefinery, pulp and paper, biopolymers) and probably inspire new biomimetic approaches.

As plants evolved from aquatic to terrestrial environment three complex polymers, lignin, suberin, and cutin, have played a major role. These hydrophobic components impregnate plant surfaces and interfaces to ensure plant survival in a great variety of terrestrial habitats. Plants build tissues with distinct properties and functions by changing composition and structure at the different hierarchical levels (mm-m-nm). Cellulose microfibrils and other carbohydrates build the basis and then aromatic and lipidic components modify plant surfaces and interfaces. These "modifiers" are the least understood plant polymers due to their high variability in amount and structure and unknown distribution and interactions on the micro- and nano-level. In this project we tackled microchemistry of native plant tissues by Raman microscopy. This spectroscopic imaging approach is based on the interaction of laser light with the plants molecules, resulting in the energy of the laser photons being shifted up or down. The energy shift (Raman spectrum) gives information about the vibrational modes and thus molecular structure at any pixel (0.3 x 0.3 m). In each scan, thousands of Raman spectra were acquired and used to calculate chemical images based on univariate and multivariate analysis methods. Especially, unmixing and mixture analysis resulted in the most informative chemical images and purest component spectra. The purest component and multicomponent spectra were compared or fit with spectra of our unique database, which now comprises about 300 plant relevant reference spectra. This enabled us to interpret multicomponent spectra and visualize all components, carbohydrates, aromatics and lipids, at once within the native plant tissues and surfaces on the microscale. Our methodological advances paved the way for new insights into lignification of plant cell walls in different species and mutants and to reveal the impact of lignin on mechanical properties as well as the influence of drought. In plant surfaces, we imaged for the first time phenolic acids as well as flavonoids within the cuticle and at the interface to the epidermal layers, which resulted in new models and a discussion of the role of aromatics in the cuticle. To enlarge the view on the nanoscale, we combined Raman and Atomic force microscopy and with the nanoscale tip "feeling" the surface we succeeded to image nanochemistry in compressed wood based on adhesion values. The results suggested a new more "loose" cell wall model based on flexible lignin nanodomains and advanced our knowledge of the molecular reorganization during wood deformation. Our research opens new scientific grounds in the field of plant physiology and wood science, can have an impact on optimised plant feedstock development (forestry, agriculture, breeding, genetic engineering) as well as utilisation (biorefinery, pulp and paper, biopolymers) and has potential to inspire biomimetic nature based solutions.