Professor Hongbo Zhao’s research team at Zhejiang A&F University uncovered novel DNA methylation regulation mechanisms in the rapid response of Osmanthus fragrans to low temperature for flower opening

Osmanthus fragrans, celebrated for its rich fragrance and symbolism, ranks among China’s ten traditional famous flowers. The flower bud opening after differentiation requires undergoing continuous temperatures below 22°C. Should the temperatures rise above this critical threshold, the flower buds will not open. Unlike many woody plants (such as Prunus mume, Chimonanthus praecox, and Prunus persica) requiring extended cold, Osmanthus needs just 3-4 days at an optimal 19°C to flower opening. Despite the crucial role of DNA methylation in temperature-dependent biological processes in many plants, research on how plants rapidly respond to ambient temperature through DNA methylation mechanisms to initiate flower opening is currently limited.

This study, employing WGBS technology, discovered a significant reduction in DNA methylation levels across all three contexts, especially CHH, within mature Osmanthus floral buds during rapid low-temperature response. Transcriptomic analysis revealed an enrichment of differentially expressed genes with hypo-methylated regions, in biological processes associated with auxin signaling, temperature response, and RNA processing. Paraffin section analysis showed pedicel elongation as a key marker for flowering initiation, significantly inhibited by the external auxin transport inhibitor TIBA. During the process, a notable elevation in transcription levels was observed for three DNA demethylase genes (OfROS1a, OfDML3, OfDME), while intriguingly, upregulation of DNA methyltransferase genes, with the exception of OfCMT2b, was also noted. Analysis of promoter cis-acting elements identified OfROS1a as a candidate gene harboring both low-temperature and auxin response elements. Transient transformation in Nicotiana benthamiana demonstrated  promoter activation of OfROS1a at low temperatures and IAA levels, with reduced activity at higher IAA concentrations compared to low IAA treatment, indicating temperature and IAA consentration jointly regulate OfROS1a’s activity. Overexpression of OfROS1a in petunia hybrida lowered endogenous auxin levels, highlighting its feedback loop with auxin signaling. Further mRNA sequencing revealed enhanced transcription of auxin response and spliceosome-related genes in petunia with OfROS1a overexpression.

This study unveils a novel mechanism wherein active DNA demethylation critically facilitates the rapid temperature response of Osmanthus, initiating flower opening by modulating auxin signal transduction. Additionally, it posits a novel perspective, suggesting that this process might activate the RNA-directed DNA methylation (RdDM) to adapt to rapid shifts in DNA methylation dynamics, thereby facilitating transition to the next stage—flower opening. This marks the inaugural discovery of a DNA hypomethylation regulatory mechanism in plants that promptly reacts to ambient temperature changes to induce flower opening. These findings offer fresh insights into temperature-dependent flower opening from an epigenetic standpoint and introduce innovative strategies for enhancing environmental adaptability in Osmanthus and other plant species.

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References

Authors

Shiwei Zhong, Huijun Zhu, Wenle Li, Dan Wu, Yunfeng Miao, Bin Dong, Yiguang Wang, Zhen Xiao, Qiu Fang, Jinping Deng, Hongbo Zhao

Affiliations

Zhejiang Provincial Key Laboratory of Germplasm Innovation and Utilization for Garden Plants, Key Laboratory of National Forestry and Grassland Administration on Germplasm Innovation and Utilization for Southern Garden Plants, School of Landscape and Architecture, Zhejiang A&F University, Hangzhou, Zhejiang 311300, China

About Hongbo Zhao

Professor Zhao serves as the Dean of the College of Landscape Architecture and Architecture, Professor, PhD Supervisor, Director of the Key Laboratory of Landscape Plant Innovation and Utilization in Zhejiang Province, and Director of the Key Laboratory of Southern Landscape Plant Innovation and Utilization of the National Forestry and Grassland Administration. Moreover, they hold the position of Chief Expert in the major scientific and technological project of Zhejiang Province focusing on floriculture.