Simple preparation of plant epidermal tissue for laser microdissection and downstream quantitative proteome and carbohydrate analysis

Abstract

The outwardly directed cell wall and associated plasma membrane of epidermal cells represent the first layers of plant defense against intruding pathogens. Cell wall modifications and the formation of defense structures at sites of attempted pathogen penetration are decisive for plant defense. A precise isolation of these stress-induced structures would allow a specific analysis of regulatory mechanism and cell wall adaption. However, methods for large-scale epidermal tissue preparation from the model plant Arabidopsis thaliana, which would allow proteome and cell wall analysis of complete, laser-microdissected epidermal defense structures, have not been provided. We developed the adhesive tape – liquid cover glass technique (ACT) for simple leaf epidermis preparation from A. thaliana, which is also applicable on grass leaves. This method is compatible with subsequent staining techniques to visualize stress-related cell wall structures, which were precisely isolated from the epidermal tissue layer by laser microdissection (LM) coupled to laser pressure catapulting. We successfully demonstrated that these specific epidermal tissue samples could be used for quantitative downstream proteome and cell wall analysis. The development of the ACT for simple leaf epidermis preparation and the compatibility to LM and downstream quantitative analysis opens new possibilities in the precise examination of stress- and pathogen-related cell wall structures in epidermal cells. Because the developed tissue processing is also applicable on A. thaliana, well-established, model pathosystems that include the interaction with powdery mildews can be studied to determine principal regulatory mechanisms in plant–microbe interaction with their potential outreach into crop breeding.

Induced ligno‐suberin vascular coating and tyramine‐derived hydroxycinnamic acid amides restrict Ralstonia solanacearum colonization in resistant tomato

“…In natural environments plants are constantly exposed to diverse microbiota, including pathogenic organisms. In addition to pre‐existing structural cell barriers that act as a first line of defense (Serrano et al ., 2014; Falter et al ., 2015), pathogen perception results in activation of a complex, multi‐layered immune system in plants (Jones & Dangl, 2006). As part of the suite of inducible defenses, de novo formation of physico‐chemical barriers prevents pathogen colonization and spread inside the plant.…”

Variation in genome size, cell and nucleus volume, chromosome number and rDNA loci among duckweeds

“…Due to the small frond size, a single epidermis layer is difficult to obtain especially for species of the genus Wolffia (frond diameter ~1 mm). Therefore, we modified the epidermis preparation methods described 40–42 , by using domestic adhesive tape. Because stomata are located on the upper surface in floating plants 9,18 , duckweed fronds were placed with their upper side on the adhesive tape.…”

Protein Extraction Methods Shape Much of the Extracted Proteomes

“…The protein extracts were analyzed by nanoUPLC separation combined with ESI-Q-TOF MS, which successfully identified 137 and 44 proteins in nucellar projection and endosperm transfer cells, respectively (Kaspar et al, 2010 ). In addition, a method of mechanical separation of leaf epidermal, vascular, and mesophyll tissues has been developed in Arabidopsis (Falter et al, 2015 ), tomato, and cassava (Svozil et al, 2016 ), and the separated tissue samples can be used for quantitative LCM-assisted microproteomic analysis.…”