Revealing the transitory and local effect of zebularine on development and on proteome dynamics of Salix purpurea

Abstract

IntroductionDNA methylation plays major roles in the epigenetic regulation of gene expression, transposon and transcriptional silencing, and DNA repair, with implications in developmental processes and phenotypic plasticity. Relevantly for woody species, DNA methylation constitutes a regulative layer in cell wall dynamics associated with xylogenesis. The use of methyltransferase and/or demethylase inhibitors has been proven informative to shed light on the methylome dynamics behind the regulation of these processes.MethodsThe present work employs the cytidine analog zebularine to inhibit DNA methyltransferases and induce DNA hypomethylation in Salix purpurea plantlets grown in vitro and in soil. An integrative approach was adopted to highlight the effects of zebularine on proteomic dynamics, revealing age-specific (3 weeks of in vitro culture and 1 month of growth in soil) and tissue-specific (stem and root) effects.Results and discussionAfter 3 weeks of recovery from zebularine treatment, a decrease of 5-mC levels was observed in different genomic contexts in the roots of explants that were exposed to zebularine, whereas a functionally heterogeneous subset of protein entries was differentially accumulated in stem samples, including entries related to cell wall biosynthesis, tissue morphogenesis, and hormonal regulation. Significant proteomic remodeling was revealed in the development from in vitro to in-soil culture, but no significant changes in 5-mC levels were observed. The identification of tissue-specific proteomic hallmarks in combination with hypomethylating agents provides new insights into the role of DNA methylation and proteome in early plant development in willow species. Proteomic data are available via ProteomeXchange with identifier PXD045653. WGBS data are available under BioProject accession PRJNA889596.

Impacts of in‐vitro zebularine treatment on genome‐wide DNA methylation and transcriptomic profiles in Salix purpurea L.

“…At high concentrations (100 μM), the plantlets did not survive, while no significant visual differences were observed at lower concentrations (15 μM) between the control and treated plantlets. Consistent with the findings of Pagano et al (2024), treatment with 50 μM zebularine resulted in significant developmental differences compared to the control samples, such as the absence of root development in the treated samples (Figure 1A). However, during the recovery period, in the absence of zebularine, the plantlets began to develop roots.…”

“…In the current study, we collected buds from a single hybrid tree (genotype PSP33) from the experimental station of IGR Poznan to minimize somaclonal variation and cultured the buds in vitro using ½ MS medium supplement (Sigma-Aldrich) with 30 gÁL À1 sucrose (Duchefa) that was solidified using 8 gÁL À1 agar with vitamins (Micro-agar, Duchefa). Previously, we observed that 25 and 50 μM of zebularine modify DNA methylation (Pagano et al, 2024). In the current research, we re-optimized the zebularine concentration by applying 15, 25, 50, or 100 μM of zebularine as hypomethylation (demethylating) agent.…”

“…Despite the importance of willow trees as renewable bioenergy sources, epigenetic regulation of SCW formation in this species remains unclear. Our recent research (Pagano et al, 2024) demonstrated that 50 μM of in vitro zebularine treatment for two weeks decreased total genome-wide DNA methylation after three weeks of recovery and one-month soil culture in purple willow plantlets. Compared to the control plants, functionally different proteins were accumulated in treated samples, including entries related to cell wall biosynthesis.…”