Gene Regulatory Changes Associated With Phenological Transitions in an Ecologically Significant Tree Species

Abstract

Climate change is driving earlier spring leaf-out across temperate regions, but the genetic mechanisms and environmental interactions underlying this variability are poorly understood. We conducted a controlled growth chamber experiment using excised northern red oak (Quercus rubra ) branches, testing the influence of temperature and photoperiod on leaf development. Two genotypes of red oak were exposed to four different warming and daylength treatments, and gene expression was analyzed across stages of bud development. Results revealed significant phenotypic differences between genotypes and across treatments, confirming that leaf-out timing is both genetically determined and environmentally responsive. Our analysis identified several key genes involved in dormancy break and photoperiod sensitivity, including orthologs to genes identified in Populus species, suggesting conserved pathways across tree species. These genes were differentially expressed in response to environmental factors, highlighting the polygenic nature of phenological timing. Notably, modules associated with temperature and photoperiod showed overlap with dormancy break pathways, indicating shared regulatory networks. This study provides a foundational dataset for understanding phenology in red oak and offers insights into how genetic and environmental factors shape leaf development in temperate trees, setting the stage for further functional genomic research.

Keywords: Quercus rubra; climate change; genotype; growth chamber; phenology; photoperiod; red oak; temperature.

FIGURE 1

A graphic representing our experimental design. We collected branch cuttings from (A) two red oak individuals with differing leaf‐out timing, then placed them in one of four (B) experimental treatments, and (C) observed each cutting as it advanced through early leaf‐development phenophases, collecting RNA at each new phase.

FIGURE 2

Warmer temperatures and longer days lead to earlier phenological advancement. Data shown for each genotype in the (A) cold (14°C day/4°C night) and (B) hot (26°C day/14°C night) treatments, for both the short day (solid line, 10 h) and long day (dashed line, 15 h) treatments. Error bars represent standard error across samples. As we destructively harvested, the N drops by stages 4 and 5, such that it is not always possible to calculate standard error.

FIGURE 3

A summary of our phenotypic and RNA results, demonstrating the influence of genotype, photoperiod, and temperature on leaf out on phenotypes and subsequent transcription. Leaf development phenotypes at (A) stage 1 and (B) stage 3. MDS clustering plots of transcriptomic data, showing (C) clustering by genotypes and temperature treatment across dimension 1 & 2 and (D) clustering across stages of phenophase development in dimensions 3 & 4.

FIGURE 4

A heatmap summarizing the transcript clustering results and how they correlate with our experimental factors. Only significant interactions (p > = 0.05) are colored, with the correlation coefficient (range −1 to 1) indicated in text. Dormancy here is defined as transcriptional differences between stages 0 and the combined stages 1–5. Each of the 18 modules defined varied in the number of genes included, as given by the bolded section of table on the far right, as well as what percentage of total genes in the red oak genome that value represents.

FIGURE 5

Graphical representations of select transcript modules identified in our study (A–E). Hot pink highlighted genes are leaf‐out related orthologs from other species (Table S1).

FIGURE 6

Expression changes in orthologs to genes central to early leaf‐out mutants. The proposed pathway (A) of bud break in a Populus tremula × Populus alba hybrid, modified from (Azeez et al. 2021). (B) How expression has changed across the first four stages of leaf‐out (0–3) in our study for those orthologous genes, given as both raw counts and percent differences. Error bars represent standard deviation.