EARLY BUD-BREAK 1 and EARLY BUD-BREAK 3 control resumption of poplar growth after winter dormancy

Woody perennials from seasonal climates alternate between active growth and winter dormancy. In Populus, short days induce growth cessation and dormancy establishment via photoperiodic pathways converging on FT2, LAP1, and AIL1, with downstream effects on the cell cycle. ABA signaling and deposition of callose at plasmodesmata reinforce dormancy by isolating the shoot apical meristem from growth-promoting signals. Reactivation of growth (bud-break) requires chilling followed by warm temperatures and long days, with temperature being the dominant cue. Although MADS-box genes such as DAM and SVP-like (SVL) have been implicated in dormancy and bud-break, and EBB1 was identified as a positive regulator, the regulatory relationships and temperature integration remained unclear. This study investigates how EBB1, SVL, and a newly identified AP2/ERF factor, EBB3, form a regulatory module linking low-temperature cues to cell-cycle activation and bud-break.

Prior work established that photoperiodic components (LHYs, GI, CO1/2) regulate FT2 to control seasonal growth, with LAP1 and AIL1 promoting growth and D-type cyclins regulating a major cell-cycle checkpoint. ABA promotes dormancy by enhancing plasmodesmatal callose and signaling, rendering buds insensitive to growth cues. DAM genes in fruit trees and SVP-like genes are associated with dormancy; overexpression delays bud-break and their expression can be influenced by cold and histone modifications, drawing parallels to vernalization and FLC regulation. In poplar, SVL functions as a negative regulator of bud-break and a hub in ABA-mediated dormancy control, integrating short-day and low-temperature signals. EBB1, an AP2/ERF transcription factor related to Arabidopsis DRN, was previously identified as a positive regulator of bud-break, but its connection to SVL and the temperature-to-cell cycle link was unresolved.

• Mutant discovery and genotyping: An activation tagging population in Populus tremula × alba (WT-717) was field-screened. A dominant early bud-break mutant (ebb3D) was identified. TAIL-PCR recovered sequences flanking the 4× CaMV35S enhancer insertion. BLAST positioned the tag on chromosome XII near Potri.012G108400 (RPL34e-like) and Potri.012G108500 (AP2/ERF, PtERF113).
• Transgenics: Overexpression constructs for Potri.012G108400 and Potri.012G108500 (Gateway, pK2GW7; CaMV35S) were generated and transformed via Agrobacterium. RNAi for EBB3 used a 200 bp fragment cloned into pK7GWIWG2(I); transformed into WT-T89. Multiple independent lines were produced; expression verified by qRT-PCR.
• Plant material and growth regimes: Hybrid clones WT-717 (P. tremula × alba) and WT-T89 (P. tremula × tremuloides) used. Plants propagated in vitro, acclimated, then grown in greenhouse (16 h light, 20 °C). Dormancy induction: 10 weeks short days (8 h light, 20 °C/16 h dark, 18 °C). Chilling: 4 °C for up to 5 weeks. Bud-break induction: long days (16 h) and warm temperatures (20 °C). Bud-break scored daily. Tissue sampling included apices, leaves, stems, and axillary buds; monthly field sampling of Populus tremuloides buds (Sep–Apr).
• Gene expression: RNA extracted (Qiagen/Bio-Rad kits), DNase-treated. cDNA synthesized (iScript or SuperScript II). Reference genes: ACT7 (WT-717) and UBQ (WT-T89) validated by geNorm. qRT-PCR performed (StepOnePlus, LightCycler 480) using SYBR Green; ΔΔCt for relative expression.
• Hormone treatments: ABA (50 µM, 2 h) applied to excised apices; GA3 (3 mM, 24 h) applied to shoot apices before expression analysis.
• Protein-DNA interaction assays: EMSAs used biotin-labeled promoter fragments containing ERF GCC-box motifs. HA-EBB1/HA-EBB3 expressed in protoplast extracts or purified His-tagged EBB3 (E. coli) tested for binding to SVL and CYCD3.1 promoter GCC-box sequences; mutated GCC boxes used as controls.
• ChIP-qPCR: Populus mesophyll protoplasts transfected with EBB1-GFP or EBB3-GFP (pMDC83). Chromatin crosslinked and immunoprecipitated with anti-GFP (IgG control). Enrichment at SVL and CYCD3.1 promoter regions (GCC-box-containing vs control regions) quantified as % input.
• ChIP-seq for histone marks: Apical buds collected at 0W, 6WSD, 10WSD, 4WC. ChIP with anti-H3K27me3 and anti-H3. Reads processed (FastQC, SortMeRNA, Trimmomatic), aligned (STAR, BWA-MEM), peaks called (MACS2). H3K27me3 abundance across EBB3 locus normalized to H3, log2-transformed, compared across time points.
• RNA-seq and differential expression: Apices from WT and EBB3-RNAi at 10WSD, 2WC, 5WC, 2WLD sequenced (HiSeq 2500, PE125). Reads QC-filtered, pseudo-aligned to P. tremula transcripts (kallisto), and analyzed with DESeq2 (FDR 1%). DEG counts summarized; focus on consistent changes across stages.
• Statistics: Two-tailed paired t-tests and multiple t-tests used as noted in figure legends to assess significance of phenotypes and ChIP enrichments. Biological replicates generally n=3 for expression/ChIP; multiple plants per genotype for phenotyping.

• Identification of EBB3: Activation-tagged mutant ebb3D flushed buds ~6 days earlier than WT-717 in field and controlled conditions. Two nearby genes were upregulated in ebb3D, but only overexpression of Potri.012G108500 (PtERF113) recapitulated early bud-break; overexpression of Potri.012G108400 (RPL34e-like) had no effect. PtERF113 is designated EBB3.
• Genetic validation: EBB3 overexpression (multiple independent lines) significantly accelerated bud-break versus WT-717 (P<0.05 to P<0.0001). EBB3-RNAi lines in WT-T89 significantly delayed bud-break compared with WT (P<0.05 to P<0.0001).
• Expression dynamics and temperature response: EBB3 is primarily expressed in shoot apices. It is low during growth cessation and dormancy establishment, rises during winter/spring in field samples, and is strongly induced by cold exposure (2–5 weeks at 4 °C) and further increased during warm LD-induced bud-break. EBB3 expression drops after bud-break.
• Epigenetic regulation: ChIP-seq profiling showed a significant reduction in repressive H3K27me3 marks across the EBB3 locus after chilling (4WC), correlating with EBB3 induction, indicating low-temperature-associated epigenetic activation.
• EBB1-SVL interaction: EBB1 expression increases upon chilling and bud-break; SVL shows the opposite pattern. SVL is repressed in EBB1 overexpressors and upregulated in EBB1 knockdown (amiRNA) lines. EMSA and ChIP-qPCR demonstrate EBB1 directly binds a GCC-box ~150 bp upstream in the SVL promoter, indicating direct repression.
• Position of EBB3 in the pathway: EBB3 expression increases in EBB1 overexpressors and decreases in EBB1 knockdowns. EBB3 does not bind the SVL promoter and SVL expression is unchanged in EBB3-OE or EBB3-RNAi, placing EBB3 downstream of SVL. In SVL transgenics, EBB3 is downregulated in SVL-OE and upregulated in SVL-RNAi, confirming EBB3 is negatively regulated by SVL.
• Hormonal control: ABA treatment downregulates EBB3 in apices; EBB3 expression is elevated in an ABA-insensitive abil-1 background. GA treatment did not alter EBB3 expression, indicating ABA specifically represses EBB3 downstream of the SVL/ABA feedforward loop.
• Downstream target of EBB3: RNA-seq across dormancy stages in EBB3-RNAi plants identified CYCD3.1 as the only gene consistently downregulated at all stages (FDR<0.01). CYCD3.1 is upregulated in EBB3-OE lines. EMSA and ChIP-qPCR show EBB3 directly binds a GCC-box motif in the CYCD3.1 promoter, indicating direct positive regulation of a key G1/S cell-cycle promoter.
• Proposed module: Low temperature upregulates EBB1 and reduces SVL and ABA signaling, relieving repression of EBB3. EBB3 then activates CYCD3.1 to trigger cell proliferation and bud-break.

The study clarifies how temperature cues are transduced into molecular events that enable bud-break. EBB1 is induced by chilling and directly represses SVL, disrupting the SVL/ABA feedforward loop that maintains dormancy. As SVL and ABA decline, EBB3 expression rises; its induction is accompanied by a decrease in H3K27me3 across its locus, suggesting epigenetic de-repression by low temperature. EBB3, an AP2/ERF transcription factor, directly activates CYCD3.1, providing a mechanistic link between environmental temperature sensing and cell-cycle reactivation in the shoot apex. Together, EBB1, SVL (and ABA), and EBB3 form a regulatory cascade integrating hormonal and chromatin-level regulation to control the transition from dormancy to growth. This framework aligns with, yet is distinct from, vernalization-like processes in herbaceous plants and emphasizes tree-specific adaptations for seasonal growth control. The model suggests that manipulating elements of this module can modulate bud-break timing, with implications for forestry and horticulture under changing climates.

This work identifies EBB3 as a temperature-responsive, epigenetically regulated positive regulator of bud-break in poplar, and elucidates a regulatory module in which chilling-induced EBB1 directly represses SVL, reducing ABA signaling and permitting EBB3 induction. EBB3 directly activates CYCD3.1, linking environmental cues to cell-cycle reentry at bud-break. The study integrates transcriptional, hormonal, and epigenetic mechanisms into a coherent model of dormancy release. Future research should include in planta spatiotemporal analyses of EBB1/SVL/EBB3 interactions, creation and phenotyping of double/triple mutants to test pathway hierarchy and redundancy, and exploration of natural allelic variation for breeding strategies to optimize bud phenology under climate change.

• The regulatory hierarchy, while supported by expression and binding assays, would benefit from genetic validation using double and triple mutant combinations to confirm epistasis and pathway order.
• Spatiotemporal specificity of interactions within the apex during dormancy transitions was not resolved; tissue/cell-type–specific analyses are needed.
• The correlation between H3K27me3 changes and EBB3 expression suggests epigenetic regulation, but causal mechanisms and the upstream chromatin modifiers were not identified.
• Most binding data were obtained using protoplast-based ChIP and in vitro EMSA; additional in planta evidence under native chromatin and developmental contexts would strengthen conclusions.
• Hormonal regulation was primarily assessed for ABA and GA; contributions of other hormones or signaling pathways were not comprehensively tested.