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Sulfuric Acid and Gibberellic Acid (GA3) Treatment Combined with Exposure to Cold Temperature Modulates Seed Proteins during Breaking of Dormancy to Germination in Tilia miqueliana


Tilia miqueliana produces woody seeds that exhibit deep dormancy. In this study, we used cell biology methods, including Paraffin section determination and Coomassie brilliant blue staining, as well as proteomics-based methods, including two-dimensional electrophoresis with matrix-assisted laser desorption/ionisation tandem time-of-flight mass spectrometry (2DE–MALDI–TOF/TOF), to examine the effects of H2SO4–GA3 and cold stratification (3 °C) treatment on proteins during dormancy release and germination in T. miqueliana seeds. The results revealed that during cold stratification, the area and density of proteins in the endosperm cells of H2SO4–GA3-treated seeds were significantly altered. Total protein content was continuously consumed and utilised. Storage proteins (albumin, globulin, prolamin, and glutelin) were degraded to varying degrees. Sixteen differential proteins were identified using mass spectrometry. Kyoto encyclopedia of genes (KEGG) pathway analysis revealed that the glycolysis/gluconeogenesis, secondary metabolite biosynthesis, glyoxylate and dicarboxylate metabolism, amino acid biosynthesis, and metabolic pathways were critical during dormancy release and germination. Gene ontology analysis and KEGG pathway annotation of differential proteins in the co-expression network indicated that the differential proteins are implicated in photosynthesis, glucose metabolism, biosynthesis of plant hormones, and glycolysis/gluconeogenesis. Synergistic interactions among these proteins accelerated dormancy release and germination. Therefore, H2SO4–GA3 cold stratification treatment is the best method for achieving rapid dormancy release and increasing the germination rate of T. miqueliana seeds.

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Data Availability

All data generated or analysed during this study are included in this published article.

Code Availability

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD021999. Submission details: (1) Project Name: Key protein variations during seeds germination in Tilia miqueliana, (2) Project accession: PXD021999, (3) Project DOI: Not applicable. Reviewer account details: (1) Username:, (2) Password: DIyJPxwv.


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We would like to thank the College of Forestry, Nanjing Forestry University, and Co-innovation Center for Sustainable Forestry in Southern China, Southern Tree Inspection Center National Forestry Administration.


This work was financed by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

Author information




YBS conceived the original screening and research plans; YW performed the experiments; YW designed the experiments and analyzed the data; YW conceived the project and wrote the article with contributions of all the authors. YW agrees to serve as the author responsible for contact and ensures communication. All authors have read and approved the manuscript.

Corresponding author

Correspondence to Yong Bao Shen.

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The authors declare that they have no competing interests.

Ethical Approval

We strictly comply with the Convention on the Trade in Endangered Species of Wild Fauna and Flora, and also abide by Law of the People's Republic of China on Wildlife Protection. Research permission on Tilia miqueliana tree has been obtained from Jiangsu Wildlife Protection Station. The collection of experimental intact seeds was approved by the Management Office of Huang Zangyu National Forest Park in Anhui Province, China.

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All the authors supported the publication of the article.

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Wu, Y., Shen, Y.B. Sulfuric Acid and Gibberellic Acid (GA3) Treatment Combined with Exposure to Cold Temperature Modulates Seed Proteins during Breaking of Dormancy to Germination in Tilia miqueliana. Protein J (2021).

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  • Dormancy release
  • Germination
  • H2SO4–GA3 cold stratification treatment
  • Proteome
  • Storage protein
  • Tilia miqueliana