Single-run HPLC Quantification of Plant Cell Wall Monosaccharides

Background

Understanding plant cell wall structure and composition have been at the forefront of both academic and industrial plant research for many years. Polysaccharide and cell wall protein interactions provide structure to plant cells, while also actively playing important biological roles during growth and adaptation to diverse external conditions (Cosgrove, 2005 and 2016; Kesten et al., 2017). The plant cell wall acts as an important physical barrier during biotic or abiotic interactions, but can also provide an endogenous source of signaling molecules released during stress response (Cosgrove, 2005 and 2016; Kesten et al., 2017 and 2019). These cell wall-derived molecules can activate signaling cascades to alert the plant host of an invading pathogen or a need to redirect growth resources. Cell wall polysaccharides are also important for industrial uses–pectins are widely used in the food and cosmetics industries, while cellulose is important for the food, paper, and textile industries (Pettolino et al., 2012). As such, efficient and informative methods for characterizing plant cell wall polysaccharides are vital in both academia and industry to further understand the biological role of the plant cell wall and improve ease of polymer extraction while reducing waste during industrial applications.

Experiments from the 1940s conducted in wood established the use of dilute acid hydrolysis at high temperature to study sugar decomposition into monomers (Saeman, 1945). Such studies have provided a foundation for many current protocols, including the one presented here. Streamlining this analysis has allowed us to improve both the accuracy and efficiency of our measurements. Other recently described protocols have established the combination of the widely-used trifluoroacetic acid (TFA) hydrolysis approach with a single HPLC run, or a dual sulfuric acid-based h ydrolysis method, referred to as a “one-step two-step sulfuric acid hydrolysis” approach, with multiple HPAEC-PAD runs (Zhang et al., 2012; Voiniciuc et al., 2016; Yeats et al., 2016a and 2016b). This “one-step two-step” approach makes use of a Saeman hydrolysis procedure followed by what has been previously referred to as a “matrix hydrolysis” (Yeats et al., 2016a). Saeman hydrolysis, the “two-step” portion of this approach, is essentially the addition of concentrated sulfuric acid hydrolysis (72% sulfuric acid) directly to plant material in order to swell the sample. After one hour at room temperature, water is added to the sample to dilute the sulfuric acid concentration to 4%. This portion of the hydrolysis is then performed at 121 °C for one hour. The use of a dilute acid hydrolysis immediately following a strong acid swelling hydrolysis allows the release of glucose from all possible sources, including heavily cross-linked and rigidly packed cell wall components, such as crystalline cellulose. The “one-step” portion of the hydrolysis, also called the “matrix hydrolysis”, refers to the hydrolysis of samples only at 121 °C with 4% sulfuric acid. By simply treating samples with a “matrix hydrolysis”, only monosaccharides from easily hydrolysable sources will be released. Therefore, the difference between these two hydrolyses allows the quantification of crystalline cellulose in what is referred to as a “one-step two-step” hydrolysis approach. In our protocol, we combine the efficient “one-step two-step” sulfuric acid hydrolysis described by Yeats et al. (2016a) with the inclusion of an optional enzymatic starch degradation and the use of alternate internal standards to elute all cell wall sugars and quantify glucose derived from crystalline cellulose in a single HPAEC-PAD run. Our additions to the previously mentioned protocols contribute significant improvements in both ease and accuracy of monosaccharide measurement that will greatly benefit the field of plant cell wall research.