Understanding Temporal Dynamics of Plant Specialized Metabolites in Response to Simulated Browsing in Winter
Authors: Bright Bryanna Hope 
Published: September 2023

Plants are continually defending themselves from the herbivores that consume them, often using an array of plant specialized metabolites (PSMs).
Volatile organic compounds, including monoterpenes, are one such type of PSMs that can be emitted and induced by plants in response to mechanical
damage and herbivory. These volatiles serve as direct defenses against herbivores and can alert neighboring plants about potential threats,
resulting in protection against future attacks. However, how these chemicals change over time in response to browsing by vertebrates in the
winter has received limited attention and is crucial to interpreting how monoterpenes defend plants against vertebrate herbivores. To assess
induced defenses of plants in the winter, we investigated temporal changes of monoterpenes in Wyoming big
sagebrush (Artemisia tridentate subsp. wyomingensis) naturally occurring in the Wyoming landscape
following either a single event of mechanical damage (acute damage) or repeated damage (chronic damage) to leaves that simulated “bites” by the
avian herbivore, Greater Sage-grouse (Centrocercus urophasianus). We hypothesized that plants would exhibit changes in specific
monoterpenes through the process of emission which releases volatiles into the air in response to damage and then through induction which
involves biochemical synthesis. We also hypothesized that the intensity of damage would influence monoterpene profiles. Based on these
hypotheses, we predicted an initial decrease in the concentration of monoterpenes in leaves soon after damage due to emission which would
be followed by increases in monoterpenes in leaves as the time course progressed associated with induction. We also predicted that
treatment plants would be less variable in monoterpene profiles compared to control plants and that higher levels of damage would
result in greater changes in monoterpenes. Monoterpene profiles and concentrations were analyzed using gas chromatography. Multiple
Principal Component Analyses (PCA) using Euclidean distance, ANOVAs, correlation matrices, and nonparametric Kruskal-Wallis tests were
used to compare changes in monoterpenes over time within treatments, between treatment groups, and between levels of collection intensity
after an initial simulated browsing event. We identified fourteen potential compounds of interest with the largest vector loadings from
the PCAs which were reduced to six compounds of interest based on strong correlation with other compounds. There was no evidence that any
compound of interest changed over time after simulated browsing within individual treatment groups. We found no evidence that browsing type
or browsing intensity changed monoterpene profiles or concentrations of compound of interest. Results suggest that damage to sagebrush in
the winter results in only minor changes in monoterpenes over a six-day time course. However, review of correlation among clusters of
compounds following browsing indicate that chronic browsing results in more negative correlations between compounds suggesting conversion
of monoterpenes into other metabolites. Lack of induced defenses associated with winter browsing is inconsistent with observed induced
defenses in sagebrush damaged in the summer and suggests stable winter chemistry following leaf damage that may explain the relatively
high winter foraging fidelity observed in sage-grouse. This research demonstrates the value of a multivariate approach to detect
chemicals that might normally be ignored due to their relatively low concentration and rarity in a plant-herbivore system. The
multivariate approach could be used to assess the relative plasticity of chemical defenses as a consequence of gene by environment
interactions. Metrics of chemical stability relative to seasonality, climate, or herbivory could be used to inform management decisions
of what plant genotypes to select for restoration of disturbed areas and predict how chemical responses will cascade up to influence
species of conservation concern and entire communities.