Multilevel Modeling of Lignin Biosynthesis in Brachypodium distachyon

Despite the potential of lignin as raw material for conversion into renewable bioproducts, our current understanding of its biosynthesis is still incomplete, especially in the economically important Poaceae family of grasses. Here, we applied a combined proteomics and isotope labeling approach to study lignification in a set of knockdown lines for several lignin biosynthetic genes using the model grass Brachypodium distachyon. We identified the lignin protein family members in mature stems, and found that PTAL, the first enzyme of the lignin pathway in grasses, was the fourth most abundant protein among the +11,000 proteins identified. Accordingly, p-coumarate, the product of the PTAL reaction, was the most abundant lignin pathway intermediate. Overall, our ¹³C-labeling data, metabolic flux analysis and in situ hybridization experiments support a metabolic separation of the carbon flux from L-phenylalanine and L-tyrosine into different lignin subunits and flavonoid classes associated with distinct cellular localization. Notably, co-downregulation of pairs of monolignol biosynthetic genes did not result in additive impacts on plant growth or lignin deposition, but instead induced pronounced proteome changes associated with oxidative stress and vitamin E synthesis. These results highlight the pivotal role of PTAL and the flexibility of the monolignol biosynthetic pathway in grasses.