The Characterisation of Corn and Soya Seed Epicuticular Wax Surfaces

Abstract

All plant surfaces are highly specialised systems that have evolved to survive and thrive in an incredibly
diverse range of environments. Seeds represent the embryonic plant and are an under researched area
of agricultural studies. The multi-functional surfaces of the seed are responsible protection and
information transfer from the environment and play a crucial role in germination and later development.
The surface of the seed is covered in a varying, thin layer of epicuticular wax that functions with the
seed cuticle to protect the seed.
Six seeds were chosen to be characterised: three different Corn seeds and different Soya seeds. The
characterisation of these seeds epicuticular wax showed the differences between not just genetic
varieties but special varieties as well. Through one of the very first applications of Liquid
Chromatography with tandem mass spectrometry on plant lipids, with particular focus on the lipidomics
and molecular confirmation of the surface chemistry the epicuticular waxes of the seeds were quantified.
The following PCA analysis showed clear trends in the surface chemistry of the Soya seeds separate
from the surface chemistry of the Corn seeds. The separation of the Soya seeds shows the similarities
in the surface chemistry within the species, while the separation of each individual Corn seed showed
the varietal differences. From a breakdown of the lipid genres detected in this epicuticular wax
chemistry it was clear one of the main differences was the presence of fatty acids on the surface of the
Corn seeds. The quantity of fatty acids detected for the Yukon, LG32.20 and Nerissa seeds when
compared to the Soya varieties was significantly higher. Fatty acids were also detected in the Soya seeds
but at relatively trace amounts when compared to Corn. This was confirmed with targeted analysis of
the seed surface with ToF-SIMS determining that fatty acids were found in the epicuticular wax.
Further physical characterization was needed to characterise the seed’s topography and morphology
and the role the epicuticular wax had on this system. As part of the chemical analysis, it was shown that
two minutes for the extraction of the surface waxes was able to remove a significant amount of the wax
however an investigation to the extent of the extraction was also needed. It was found that two minutes
is optimum for surface wax elution for the six seeds but then investigated for their surface properties
with and without their epicuticular waxes. Three principal techniques were used for this analysis:
Scanning Electron Microscopy, Atomic Force Microscopy and Optical Profilometry. The Corn seeds
were found to have a striated folding of the cuticle running the length of the seed, and the Soya seeds
exhibit a pitted structure similar to a golf ball. The findings of the epicuticular wax covering and the
tertiary structures of each of the seeds show the need for each seed to be addressed on an individual
basis.
Following the characterisation of the epicuticular waxes chemical and physical compositions it was
clear that the system was incredibly complex. Nerissa Corn seed was chosen for its relative simplicity
of lipid break down and a selection of principal components of its epicuticular waxes were selected to
be contrasted into a model wax replica. 1168 native components were approximated down into four
principal components. Binary investigations followed by ternary investigations were conducted, and it
was found that wax esters functioned as an inhibitor to crystallisation. The interactions of the fatty acids
drove any crystallisation within the ternary and quaternary remodels. The sterol esters as the last
component had complex interactions with the fatty acids and the wax esters.
With the aim of comparing the replica model wax to the Nerissa epicuticular seed wax as closely as
possible, a solid-liquid pseudo-binary replica wax was created from Plant Sterol esters, Fatty acids and
Wax esters found in the Nerissa epicuticular wax. The results of this investigation yield a replica
epicuticular wax model, with clear similarities between it and native waxes observed.

Date of Award	3 Oct 2023
Original language	English
Awarding Institution	University of Bristol
Supervisor	Terence J McMaster (Supervisor) & Neil George (Supervisor)