生物化学

Epichloë species form agriculturally important symbioses with many cool season grasses. To study these symbioses, such as the interaction of Epichloë festucae with perennial ryegrass (Lolium perenne), host plants can be infected by artificial inoculation of etiolated seedlings. This inoculation is performed by placing mycelium into an incision in the meristem, as previously described by Latch and Christensen (1985). In recent years, this method has been broadly used to study this interaction at the molecular level using different Epichloë festucae mutants that can cause incompatible interactions. We have developed and adapted methods to study four of the most important host plant responses to infection, including cell death, callose deposition, lignin production, and hydrogen peroxide (H₂O₂) production, which are useful in defining the host response to infection at a very early time point.

Lignin is the second most abundant biopolymer on Earth, providing plants with mechanical support in secondary cell walls and defense against abiotic and biotic stresses. However, lignin also acts as a barrier to biomass saccharification for biofuel generation (Carroll and Somerville, 2009; Zhao and Dixon, 2011; Wang et al., 2013). For these reasons, studying the properties of lignin is of great interest to researchers in agriculture and bioenergy fields. This protocol describes the acetyl bromide method of total lignin extraction and quantification, which is favored among other methods for its high recovery, consistency, and insensitivity to different tissue types (Johnson et al., 1961; Chang et al., 2008; Moreira-Vilar et al., 2014; Kapp et al., 2015). In brief, acetyl bromide digestion causes the formation of acetyl derivatives on free hydroxyl groups and bromide substitution of α-carbon hydroxyl groups on the lignin backbone to cause a complete solubilization of lignin, which can be quantified using known extinction coefficients and absorbance at 280 nm (Moreira-Vilar et al., 2014).