Proteomes and Ubiquitylomes Reveals the Regulation Mechanism of Cold Tolerance Mediated by OsGRF4 in Rice

Li Zhang ¹ Renyan Huang ² Donghai Mao ³ Jia Zeng ⁴ Pengpeng Fang ⁴ Qiang He ⁵ Fu Shu ⁵ Huafeng Deng ⁵ Wuhan Zhang ⁵ Ping Yong Sun ^5*

• ¹ State Key Laboratory of Hybrid Rice, Hunan Hybrid Rice Research Center, Changsha, China
• ² Hunan Plant Protection Institute, Hunan Academy of Agricultural Sciences, Changsha, Hunan Province, China
• ³ Key Laboratory of Agro-ecological Processes in Subtropical Region, Institute of Subtropical Agriculture, Chinese Academy of Sciences (CAS), Changsha, Hunan Province, China
• ⁴ Department of Biomedical Engineering, College of Biology, Hunan University,, Changsha, Anhui Province, China
• ⁵ State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Changsha, Hunan Province, China

Low temperature is one of the major abiotic stresses which severely restricts the development of rice. It has been demonstrated previously that OsGRF4 enhances the cold tolerance in rice, the molecular mechanism of which remain unknown. In this study, the proteome results showed that 6157 proteins were identified and 5045 proteins were quantified after 24 h cold treatment. A total of 59 proteins were up-regulated and 63 proteins were down-regulated in OX24 (overexpression OsGRF4 plants with 24 h cold stress) vs OX0 group; 27 proteins were up-regulated and 34 proteins were down-regulated in OX24 vs CK24. Finally, 3789 ubiquitination modification sites located on 1846 proteins, of which 2695 sites of 1376 proteins contained quantitative information. While, 178 sites in 131 proteins were quantified as up-regulated and 92 sites in 72 proteins were quantified as down-regulated differently ubiquitination modification proteins (DUMPs) in OX24 vs OX0. To the contrary, 82 sites in 71 proteins were identified as up-regulated and 13 sites in 12 proteins were identified as down-regulated DUMPs in CK24 vs OX24. The results suggested that global ubiquitination levels increase during cold tolerance in rice. In total, 76 differently abundant proteins and 101 DUMPs were co-localized within 50 cold or stress tolerance QTLs. The combined analysis of proteomics and ubiquitination omics found that 5 proteins demonstrated opposing changes in protein and ubiquitination, the protein Q6ZH84 (Os02g0593700) was an up-regulated DAPs but was a down-regulated DUMPs in OX24 vs OX0, which is a homologous gene of NBR1 regulated cold tolerance. Os02g0593700 should up-regulated protein expression through reducing ubiquitination modification, thus affecting cold tolerance.The enrichment pathway shows that OsGRF4 plays an important role in rice cold tolerance by ubiquitination through the glutathione metabolism and arachidonic acid metabolism. The research provides a new perspective on the molecular mechanism of cold tolerance regulated by OsGRF4.