Characterization of plant cell wall polysaccharides with surface enhanced Raman spectroscopic imaging

Abstract

<p>The polysaccharides in plant cell walls provide a potentially huge amount of fermentable sugars which could be the source of renewable biofuels. They are an important part of dietary fibers in cereal grains which have the potentials to reduce the risks of human disease. And the plant cell wall is the first barrier to protect the plant from pathogenic infections. A better understanding of plant cell wall polysaccharides organization, function, and interactions can provide a knowledge foundation to further improving crop yields, nutritional values, and disease resistance. Raman spectroscopy is a noninvasive optical molecular spectroscopic imaging technique that has been explored to study the plant cell wall. Raman spectroscopy provides chemical structural information at the molecular level with micrometer scale resolution.</p> <p>Pectin is the most complex family of polysaccharides in nature. Its functions and structures are versatile but still lack understanding. A better understanding of plant cell wall pectin organization, function, and interactions with other polysaccharides is needed. However, the overlapping of plant cell wall polysaccharides Raman peaks makes the accurate identification and quantification of different polysaccharides difficult to be achieved. The weak Raman scattering from the plant cell wall requires a longer spectrum collection time. And the Raman imaging resolution is limited by the diffraction limit of the microscope. Methods that have the potential to improve the specificity, sensitivity and spatial resolution of (surface enhancement Raman spectroscopy) SERS-enabled chemical imaging for plant cell wall polysaccharides in the plant cell wall were explored. To improve the specificity, a pectin enzyme was employed to specifically hydrolyze the pectin inside the plant cell wall, and the resulting Raman signal changes were correlated to the localization and distribution of pectin in the plant cell wall. The combination of Raman labeling and Shell-isolated nanoparticles (SHIN) enabled SERS was shown to improve the sensitivity of pectin characterization. The silica nanosphere induced near-field focusing was implemented to improve the spatial resolution and sensitivity.</p> <p>A Raman label based SERS image technique was applied to reveal the distribution of pectin and its co-localization xyloglucan inside onion epidermal cell wall. The Raman label 4-Aminothiophenol (4ATP) and silver nanoparticles (AgNP) were uniformly self-assembled on the polysaccharides in onion epidermal cell walls. This technique significantly decreased the required spectrum collection time by a factor of more than 15 times and increased the signal-to-noise (S/N) ratio. The resulted Raman images based on 4ATP signature peak intensity successfully reflected the pectin distribution of onion epidermal cell walls. The pectin distribution, its coexistence with xyloglucan and their interactions with endo-polygalacturonase (EPG) and xyloglucan-specific endo-b-1,4-glucanase (XEG) enzymes were further studied through the Raman images collected before and after the enzymatic treatments.</p> <p>Confocal Raman Microscopy (CRM) is a powerful tool that has the potential to provide the details about chemical distribution and quantity in the plant cell wall with micrometer resolution. However, the low S/N ratio Raman spectrum of polysaccharides in the plant cell wall and low throughput of Raman imaging limited its potential in plant cell wall study. We improved the Raman images collected from the plant cell wall and studied the plant cell wall polysaccharides interaction with the enzyme by introducing the principal component analysis (PCA) and hierarchical clustering analysis (HCA). The endo-polygalacturonase (EPG) was introduced to specifically hydrolyze the pectin in the onion epidermal plant cell (OEC) wall. The Raman images were collected before and after EPG hydrolysis at high scanning speed. The obtained low S/N ratio Raman spectra were processed with PCA, the Raman spectra with signal features were then recovered from the dataset. The resulted changes in the Raman signal reflected the changes in the polysaccharides contents caused specifically by the enzyme. The further analysis of principal components (PCs) revealed the pectin distribution and its coexistence with other polysaccharides.</p>