NEW YORK (GenomeWeb News) – The phenotypic plasticity found in European holly (Ilex aquifolium) leaves may stem from epigenetic marks that vary, in part, based on pressure from herbivores grazing on the plants, according to a study published online this week by a pair of Spanish researchers.
"The results of this study, although correlative in nature, support the emerging three-way link between herbivory, phenotypic plasticity, and epigenetic changes in plants," the Spanish Council for Scientific Research (CSIC)-based team wrote in the Botanical Journal of the Linnean Society paper, "and also contribute to the crystallization of the consensus that epigenetic variation can complement genetic variation as a source of phenotypic variation in natural plant populations."
Using a methylation-sensitive amplified polymorphism method, the research duo compared DNA methylation patterns in prickly and non-prickly European holly leaves from the same plants in an effort to understand if, and how, epigenetic profiles differ in relation to within plant leaf variability, known as heterophylly.
"Heterophylly is often witnessed in holly trees," corresponding author Carlos Herrera, a researcher with the CSIC's Doñana Biological Station, explained in a statement, "where some leaves are prickly, a defense against herbivores, while others are non-prickly, with smooth margins and no defense."
The team's cytosine methylation analyses uncovered a slight dip in methylation frequency at sites tested in the holly genome in the prickly leaves relative to their smoother counterparts from the same plant. And a handful of methylation marks appeared to show promise for distinguishing between the two leaf types.
"The mean per-marker probability of methylation declined significantly from non-prickly to prickly leaves," Herrera and co-author Pilar Bazaga, also from CSIC, noted.
"Methylation differences between leaf types did not occur randomly across the genome," they added, "but affected predominantly certain specific markers."
The researchers saw also saw a jump in prickly leaf production for holly trees growing in places prone to nibbling or "browsing" by herbivores, hinting that the plant may dial methylation up or down in response to herbivory to produce physically distinct leaves that grazing animals are more apt to leave alone in the future.
While the size and age of trees can also influence heterophylly in holly plants, the study authors explained, the plant's prickly and spiny leaves have long been proposed as a mechanism for fending off herbivores.
Ways in which genetically identical leaves within each plant take on such dramatically different physical features is not as well understood, though epigenetic factors were suspected.
To test this theory, Herrera and Bazaga collected hundreds of branchlet samples from 40 holly trees growing in the under story of a forest in southeastern Spain.
The researchers classified these branchlets depending on whether they had spiny, smooth, or a combination (prickly and non-prickly) leaves, noting whether the plants tested showed signs of being snacked on by red deer and wild goats in the area.
Consistent with the apparently high level of browsing in the area, for instance, they deemed 39 of the 40 trees tested to be heterophyllous, while a single representative had exclusively prickly leaves.
Even so, the proportion of prickly leaves on each branchlet varied from just a few percent to 100 percent prickly. As expected, the researchers reported seeing more pronounced leaf prickliness on branches from the nibble-prone zone within reach of deer and goats.
For a subset of the holly trees, the team also profiled methylation patterns at 177 cytosine nucleotides across the genome using a methylation-sensitive amplified polymorphism approach that relied on four primer combinations.
On average, researchers reported seeing a slight dip in methylation at these marks in the prickly leaf samples, with half a dozen methylation marks exhibiting pronounced ties to leaf shape.
"This result demonstrates that DNA methylation differences between leaf types, rather than being randomly spread across the genome, affected predominantly certain specific markers," they noted.
"By documenting here, for the first time, a correlation between herbivory-induced heterophylly and leaf DNA methylation profiles," Herrera and Bazanga wrote, "our results provide additional support for the emerging three-way relationship between herbivory, phenotypic plasticity, and epigenetic changes in plants."