Chen Fang, Zhuo Chunliu, Xiao Xirong, Pendergast Thomas H, Devos Katrien M
BioDiscovery Institute and Department of Biological Sciences, University of North Texas, 1155 Union Circle #311428, Denton, TX, 76203, USA.
Center for Bioenergy Innovation (CBI), Oak Ridge National Laboratory, Oak Ridge, TN, 37831, USA.
Biotechnol Biofuels. 2021 Jan 11;14(1):18. doi: 10.1186/s13068-020-01865-y.
Biomass composition varies from plant to plant and greatly affects biomass utilization. Lignin is a heterogeneous phenolic polymer derived mainly from p-coumaryl, coniferyl, and sinapyl alcohols and makes up to 10-25% of lignocellulosic biomass. Recently, tricin, an O-methylated flavone, was identified as a lignin monomer in many grass species. Tricin may function as a nucleation site for lignification and is advocated as a novel target for lignin engineering to reduce lignin content and improve biomass digestibility in grasses. Thioacidolysis is an analytical method that can be adapted to analyze both lignin monomeric composition and tricin content in the lignin polymer. However, the original thioacidolysis procedure is complex, laborious, and time consuming, making it difficult to be adopted for large-scale screening in biomass research. In this study, a modified, rapid higher throughput thioacidolysis method was developed.
In combination with gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS), the modified thioacidolysis method can be used to simultaneously characterize the lignin composition and tricin content using 2-5 mg of dry samples. The modified method eliminates the solvent extraction and drastically improves the throughput; 80 samples can be processed in one day per person. Our results indicate that there is no significant difference in the determination of lignin S/G ratio and tricin content between the original and modified methods.
A modified thioacidolysis protocol was established. The results demonstrate that the modified method can be used for rapid, high-throughput, and reliable lignin composition and tricin content analyses for screening transgenic plants for cell wall modifications or in large-scale genome-wide association studies (GWAS).
生物质组成因植物种类而异，并极大地影响生物质的利用。木质素是一种主要由对香豆醇、松柏醇和芥子醇衍生而来的异质酚类聚合物，占木质纤维素生物质的10%-25%。最近，三甲氧基黄酮（tricetin）这种O-甲基化黄酮，在许多禾本科植物中被鉴定为木质素单体。三甲氧基黄酮可能作为木质化的成核位点，被倡导作为木质素工程的一个新靶点，以降低木质素含量并提高禾本科植物生物质的消化率。硫代酸解是一种分析方法，可用于分析木质素聚合物中的木质素单体组成和三甲氧基黄酮含量。然而，原始的硫代酸解方法复杂、费力且耗时，难以应用于生物质研究中的大规模筛选。在本研究中，开发了一种改进的、快速的高通量硫代酸解方法。
结合气相色谱-质谱联用（GC-MS）和液相色谱-质谱联用（LC-MS），改进后的硫代酸解方法可使用2-5毫克干燥样品同时表征木质素组成和三甲氧基黄酮含量。改进后的方法省去了溶剂萃取步骤，大幅提高了通量；每人每天可处理80个样品。我们的结果表明，原始方法和改进方法在木质素S/G比和三甲氧基黄酮含量的测定上没有显著差异。
建立了一种改进的硫代酸解方案。结果表明，改进后的方法可用于快速、高通量且可靠地分析木质素组成和三甲氧基黄酮含量，用于筛选细胞壁修饰的转基因植物或大规模全基因组关联研究（GWAS）。
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