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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.

Despite widespread evidence that biological invasion influences the biotic and abiotic soil environments, the extent to which each of these pathways underpins the effects of invasion on native plant traits and performance is unknown. Leveraging a long-term (14-yr) manipulative field experiment, we show that an allelochemical-producing invader, Alliaria petiolata, affects native plants through biotic mechanisms, altering the soil fungal community composition, with no apparent shifts in soil nutrient availability. These changes in belowground soil fungal communities resulted in a high cost of resource uptake for native forest perennial herbs and a shift in functional traits linked to their carbon and nutrient economies. Furthermore, we illustrate that some species in the invaded community compensate for high nutrient costs by reducing nutrient uptake and maintaining photosynthesis by expending more water. This demonstrates a trade-off in trait investment that increases nutrient use efficiency as nutrient costs increase. Our results show that invasion-induced disruptions in the soil fungal community belowground can cascade to affect aboveground plant communities via shifts in physiological traits needed to maintain plant water and nutrient economies. These complex above-belowground linkages suggest that plant invasions should be evaluated at the system-level to fully understand and predict their impact on native plants and communities.

Despite widespread evidence that biological invasion influences the biotic and abiotic soil environments, the extent to which each of these pathways underpins the effects of invasion on native plant traits and performance is unknown. Leveraging a long-term (14-yr) manipulative field experiment, we show that an allelochemical-producing invader, Alliaria petiolata, affects native plants through biotic mechanisms, altering the soil fungal community composition, with no apparent shifts in soil nutrient availability. These changes in belowground soil fungal communities resulted in a high cost of resource uptake for native forest perennial herbs and a shift in functional traits linked to their carbon and nutrient economies. Furthermore, we illustrate that some species in the invaded community compensate for high nutrient costs by reducing nutrient uptake and maintaining photosynthesis by expending more water. This demonstrates a trade-off in trait investment that increases nutrient use efficiency as nutrient costs increase. Our results show that invasion-induced disruptions in the soil fungal community belowground can cascade to affect aboveground plant communities via shifts in physiological traits needed to maintain plant water and nutrient economies. These complex above-belowground linkages suggest that plant invasions should be evaluated at the system-level to fully understand and predict their impact on native plants and communities.