Ecological memory is a fundamental component of plant adaptation to fluctuating environments, particularly in long-lived species such as trees that experience repeated stress events over multiple years. However, the molecular mechanisms underlying short-term and long-term drought memory, and the contribution of epigenetic regulation to these processes, remain poorly understood.

In the context of climate change and increasing drought frequency, this study investigates how perennial trees perceive, integrate, and memorize water deficit. We explored drought-induced ecological memory in cambium-derived tissues of poplars (Populus spp.) by combining physiological, hormonal, transcriptomic, and epigenomic analyses.

A controlled 5-week drought stress followed by one week of re-watering was applied to two contrasting genotypes of black poplar (P. nigra), PG31 and DRA38, as well as four genetically modified P. tremula × P. alba epitypes with altered DNA methylation machinery (RNAi-dml, RNAi-ddm1, and OX-dml). To assess trans-annual memory, a subset of trees previously exposed to drought in Year 1 was subjected to a second drought episode in Year 2.

The generated datasets include genome-wide DNA methylation profiles (WGBS), transcriptomic data (RNA-seq), and alternative splicing analyses. Differentially expressed genes and differentially methylated regions were identified using the P. trichocarpa v4.1 reference genome, and functional enrichment analyses were performed using Metascape to uncover key biological processes and pathways.

Our results reveal persistent changes in hormone levels, gene expression, and DNA methylation one week after stress relief, consistent with a multilayered molecular short-term drought memory. These molecular signatures differed markedly between genotypes and epitypes, highlighting the combined influence of genetic background and epigenetic regulation on stress responses. Upon drought re-exposure in Year 2, trees previously stressed displayed distinct physiological and molecular behaviors, indicative of long-term stress imprinting.

Notably, the intrinsically more sensitive genotype exhibited higher molecular plasticity, whereas the more tolerant genotype showed greater stability. A limited set of candidate genes was reactivated during the second drought, in association with persistent drought-induced DNA methylation changes, particularly in the CG context, supporting a role for DNA methylation in long-term stress memory and potential priming.

Overall, this study identifies the cambium as a central reservoir of stress memory in trees and demonstrates that DNA methylation dynamics, shaped by genetic predisposition and acting through both cis- and trans-regulatory mechanisms, contribute to balancing growth and survival strategies over extended time scales. These findings provide new perspectives for exploiting epigenetic variation in tree breeding and forest management under increasingly drought-prone conditions.

R, 4.1.1

Metascape, 3.5

DESeq2, 2.11.40.7

TrimGalore, 0.6.3

Salmon quasi-mapping, 1.5.1

STAR, 2.5.3

rMATS, 4.0.2

SUPPA, 2.0