A Robust Method to Quantify Cell Wall Bound Phenolics in Plant Suspension Culture Cells Using Pyrolysis-Gas Chromatography/Mass Spectrometry

Lindsey M Kline; Priya Voothuluru; Scott C Lenaghan; Jason N Burris; Mikhael Soliman; Laurene Tetard; C Neal Stewart Jr; Timothy G Rials; Nicole Labbé

doi:10.3389/fpls.2020.574016

A Robust Method to Quantify Cell Wall Bound Phenolics in Plant Suspension Culture Cells Using Pyrolysis-Gas Chromatography/Mass Spectrometry

Front Plant Sci. 2020 Sep 9:11:574016. doi: 10.3389/fpls.2020.574016. eCollection 2020.

Authors

Lindsey M Kline¹, Priya Voothuluru¹, Scott C Lenaghan^{1

2

3}, Jason N Burris^{2

3

4}, Mikhael Soliman⁵, Laurene Tetard⁵, C Neal Stewart Jr^{3

4}, Timothy G Rials¹, Nicole Labbé¹

Affiliations

¹ Center for Renewable Carbon, University of Tennessee, Knoxville, TN, United States.
² Department of Food Science, University of Tennessee, Knoxville, TN, United States.
³ Center for Agricultural Synthetic Biology, University of Tennessee Institute of Agriculture, Knoxville, TN, United States.
⁴ Department of Plant Sciences, University of Tennessee, Knoxville, TN, United States.
⁵ Nanoscience Technology Center, Department of Physics, University of Central Florida, Orlando, FL, United States.

Abstract

The wide-scale production of renewable fuels from lignocellulosic feedstocks continues to be hampered by the natural recalcitrance of biomass. Therefore, there is a need to develop robust and reliable methods to characterize and quantify components that contribute to this recalcitrance. In this study, we utilized a method that incorporates pyrolysis with successive gas chromatography and mass spectrometry (Py-GC/MS) to assess lignification in cell suspension cultures. This method was compared with other standard techniques such as acid-catalyzed hydrolysis, acetyl bromide lignin determination, and nitrobenzene oxidation for quantification of cell wall bound phenolic compounds. We found that Py-GC/MS can be conducted with about 250 µg of tissue sample and provides biologically relevant data, which constitutes a substantial advantage when compared to the 50-300 mg of tissue needed for the other methods. We show that when combined with multivariate statistical analyses, Py-GC/MS can distinguish cell wall components of switchgrass (Panicum virgatum) suspension cultures before and after inducing lignification. The deposition of lignin precursors on uninduced cell walls included predominantly guaiacyl-based units, 71% ferulic acid, and 5.3% p-coumaric acid. Formation of the primary and partial secondary cell wall was supported by the respective ~15× and ~1.7× increases in syringyl-based and guaiacyl-based precursors, respectively, in the induced cells. Ferulic acid was decreased by half after induction. These results provide the proof-of-concept for quick and reliable cell wall compositional analyses using Py-GC/MS and could be targeted for either translational genomics or for fundamental studies focused on understanding the molecular and physiological mechanisms regulating plant cell wall production and biomass recalcitrance.

Keywords: cell suspension cultures; chemical composition; gas chromatography/mass spectrometry; lignification; lignin; phenolics; pyrolysis; switchgrass.