Understanding the differences in molecular conformation of carbohydrate and protein in endosperm tissues of grains with different biodegradation kinetics using advanced synchrotron technology

Abstract

Conventional "wet" chemical analyses rely heavily on the use of harsh chemicals and derivatization, thereby altering native seed structures leaving them unable to detect any original inherent structures within an intact tissue sample. A synchrotron is a giant particle accelerator that turns electrons into light (million times brighter than sunlight) which can be used to study the structure of materials at the molecular level. Synchrotron radiation-based Fourier transform IR microspectroscopy (SR-FTIRM) has been developed as a rapid, direct, non-destructive and bioanalytical technique. This technique, taking advantage of the brightness of synchrotron light and a small effective source size, is capable of exploring the molecular chemistry within the microstructures of a biological tissue without the destruction of inherent structures at ultraspatial resolutions within cellular dimensions. This is in contrast to traditional 'wet' chemical methods, which, during processing for analysis, often result in the destruction of the intrinsic structures of feeds. To date there has been very little application of this technique to the study of plant seed tissue in relation to nutrient utilization. The objective of this study was to use novel synchrotron radiation-based technology (SR-FTIRM) to identify the differences in the molecular chemistry and conformation of carbohydrate and protein in various plant seed endosperms within intact tissues at cellular and subcellular level from grains with different biodegradation kinetics. Barley grain (cv. Harrington) with a high rate (31.3%/h) and extent (78%), corn grain (cv. Pioneer) with a low rate (9.6%/h) and extent of (57%), and wheat grain (cv. AC Barrie) with an intermediate rate (23%/h) and extent (72%) of ruminal DM degradation were selected for evaluation. SR-FTIRM evaluations were performed at the National Synchrotron Light Source at the Brookhaven National Laboratory (Brookhaven, NY). The molecular structure spectral analysis involved the fingerprint regions of ca. 1720-1485 cm(-1) (attributed to protein amide I C=O and C-N stretching; amide II N-H bending and C-N stretching), ca. 1650-950 cm(-1) (non-structural CHO starch in endosperms), and ca. 1185-800 cm(-1) (attributed to total CHO C-O stretching vibrations) together with agglomerative hierarchical cluster and principal component analyses. Analyses involving the protein amide I features consistently identified differences between all three grains. Other analyses involving carbohydrate features were able to differentiate between wheat and barley but failed however to differentiate between wheat and corn. These results suggest that SR-FTIRM plus the multivariate analyses can be used to identify spectral features associated with the molecular structure of endosperm from grains with different biodegradation kinetics, especially in relation to protein structure. The Novel synchrotron radiation-based bioanalytical technique provides a new approach for plant seed structural molecular studies at ultraspatial resolution and within intact tissue in relation to nutrient availability.

Fig. 1

Synchrotron-based FTIR spectrum in plant seed endosperm tissue (wheat) (pixel size: 10 μm × 10 μm): (a) whole region: ca. 4000–800 cm⁻¹; (b) fingerprint region: ca. 1800–800 cm⁻¹; (c) protein amide I region: 1720–1575 cm⁻¹; (d) protein amide II region: 1575–1485 cm⁻¹; (e) total carbohydrate region: ca. 1185–800 cm⁻¹; (f) non-structural carbohydrate (starch) region: ca.1065–950 cm⁻¹.

Fig. 2

Multivariate spectral analyses of protein internal structures in the endosperms: compared AC Barrie wheat with Pioneer corn and AC Barrie wheat with Harrington barley. I: cluster analysis (1) select spectral region: amides I and II 1720–1485 cm⁻¹; (2) distance method: Euclidean; (3) cluster method: Ward’s algorithm]; II: principal component analysis: Scatter plots of the 1st principal components (PC1) vs. the 2nd principal components (PC2).

Fig. 3

Multivariate spectral analyses of carbohydrates internal structures in the endosperms: compared AC Barrie wheat with Pioneer corn and AC Barrie wheat with Harrington barley. I: cluster analysis (1) select spectral region: ca. 1185–800 cm⁻¹; (2) distance method: Euclidean; (3) cluster method: Ward’s algorithm]; II: principal component analysis: Scatter plots of the 1st principal components (PC1) vs. the 2nd principal components (PC2).

Fig. 4

Multivariate spectral analyses of the non-structural carbohydrate (NSC) structures in the endosperms: compared AC Barrie wheat with Pioneer corn and AC Barrie wheat with Harrington barley. I: cluster analysis (1) select spectral region: ca. 1065–950 cm⁻¹; (2) distance method: Euclidean; (3) cluster method: Ward’s algorithm]; II: principal component analysis: scatter plots of the 1st principal components (PC1) vs. the 2nd principal components (PC2).