Direct Depolymerization Coupled to Liquid Extraction Surface Analysis-High-Resolution Mass Spectrometry for the Characterization of the Surface of Plant Tissues.

Chiara Giorio,Edwige Moyroud,Beverley J Glover,Markus Kalberer

doi:10.1021/acs.analchem.9b01094

Abstract

The cuticle, the outermost layer covering the epidermis of most aerial organs of land plants, can have a heterogeneous composition even on the surface of the same organ. The main cuticle component is the polymer cutin which, depending on its chemical composition and structure, can have different biophysical properties. In this study, we introduce a new on-surface depolymerization method coupled to liquid extraction surface analysis (LESA) high-resolution mass spectrometry (HRMS) for a fast and spatially resolved chemical characterization of the cuticle of plant tissues. The method is composed of an on-surface saponification, followed by extraction with LESA using a chloroform–acetonitrile–water (49:49:2) mixture and direct HRMS detection. The method is also compared with LESA-HRMS without prior depolymerization for the analysis of the surface of the petals of Hibiscus richardsonii flowers, which have a ridged cuticle in the proximal region and a smooth cuticle in the distal region. We found that on-surface saponification is effective enough to depolymerize the cutin into its monomeric constituents thus allowing detection of compounds that were not otherwise accessible without a depolymerization step. The effect of the depolymerization procedure was more pronounced for the ridged/proximal cuticle, which is thicker and richer in epicuticular waxes compared with the cuticle in the smooth/distal region of the petal.

Highlights

Previous studies showed a heterogeneous composition of the cuticle even on the same organ.[2,9−11] there is a need to perform a spatially resolved characterization of the cuticle chemistry on the surface of the same organ
We introduce a new on-surface depolymerization method coupled to liquid extraction surface analysis (LESA) high-resolution mass spectrometry (HRMS) for a fast and spatially resolved chemical characterization of the cuticle of plant tissues
The new cuticle characterization method developed here was adapted from Mendez-Millan et al.[22] to translate a bulk saponification procedure into a direct/on surface saponification of the cutin prior to LESA-HRMS analysis

Summary

Introduction

Previous studies showed a heterogeneous composition of the cuticle even on the same organ.[2,9−11] there is a need to perform a spatially resolved characterization of the cuticle chemistry on the surface of the same organ. In order to characterize the cutin polymer with mass spectrometry, it is necessary to depolymerize it to break down the macromolecules into their monomeric constituents This is done by extracting and depolymerizing bulk samples of cutin, losing any spatial resolution on the same tissue and risking contaminations from compounds coming from the bulk of the sample rather than the surface only.[14−20] Another option is to mechanically strip off[21] the cuticle before extraction and depolymerization. Depolymerization was done by adapting a method proposed by Mendez-Millan et al.[22] for bulk samples which was modified here into a fast and direct approach that provides spatially resolved characterization on the surface of the same organ This method was successfully applied to the characterization of the cuticle of the petals of Hibiscus richardsonii,[23,24] a flower characterized by a ridged/proximal and a smooth/distal portion (Figure 1). Chemical composition of the different portions of the petals are here compared and discussed to gain insights concerning the compounds that may play a role in the formation of cuticular ridges on the surface of the petals

Methods

Results

Conclusion