Rice is a food crop widely cultivated around the world, and it often faces a nutrient deficiency environment during its growth, phosphorus starvation is one of the most common stresses faced by rice, and the large use of phosphate fertilizer poses a serious threat to the environment. Rice responds to low phosphorus stress by initiating phosphate starvation responses (PSR), and rice OSPHR2 is the core transcription factor on this pathway, and elucidating the regulatory mechanism of OSPHR2 is of great significance for understanding the PSR process. Brassinosteroid (BR) is widely used as a plant growth regulator in agricultural production, as a plant endogenous hormone, how BR regulates the absorption and utilization of phosphorus is still very unclear, and it is of great significance to analyze the regulatory relationship between BR and phosphorus to reduce the use of phosphorus fertilizer by BR.
Recently, South China Agricultural UniversityChu Chengcaiteam and Institute of Crops, Chinese Academy of Agricultural SciencesTong HongningTeamwork inthe plant cellPublished a post entitled :brassinosteroid-dependent phosphorylation of phosphate starvation response2 reduces its dna-binding ability in ricerevealed the mechanism of post-translational modification of brassinolide in regulating phosphorus starvation response in rice.
The study found that OSPHR2 could be phosphorylated by GSK2, the core repressor of the BR signaling pathway in rice, thereby weakening its DNA-binding activity, resulting in PSR inhibition, while low phosphorus induced GSK2 degradation. This study revealed a novel mechanism by which plants respond to low phosphorus stress, namely the degradation of GSK2 and the removal of its phosphorylation inhibition of OSPHR2. Phylogenetic analysis found that the key serine sites on OSPHR2 phosphorylated by GSK2 were present in most land plants, suggesting that this post-translational modification mechanism may be an important mechanism developed by plants to adapt to fluctuating trophic environments during evolution. Through the analysis of BR-related rice mutants, the researchers found that BR could promote the expression of phosphate starvation inducing (PSI) genes, suggesting that BR has a regulatory role in PSR. A variety of experimental methods have demonstrated the interaction between GSK2 and OSPHR2, and mass spectrometry analysis has shown that GSK2 can phosphorylate OSPHR2 serine at position 269 (OSPHR2-S269) in both in vitro and in vivo. This site exists in the Myb domain of OSPHR2, and protein structure analysis showed that the phosphorylation modification of this site did not affect the formation of OSPHR2 dimers, but affected its binding activity to DNA. Both EMSA and LUC reporter system analyses confirmed that the binding capacity of OSPHR2S269D to the downstream PSI gene promoter was significantly reduced after mutation of S269 to a mimic phosphorylated form of aspartic acid D.
The researchers further constructed an overexpression line of the wild-type OSPHR2 gene (Flag-OSPHR2), as well as an overexpression line of the S269 mimic phosphorylated form (FLAG-OSPHR2S269D) and a phosphorylated inactivated form (FLAG-OSPHR2S269A) in the background of OSPHR2, and confirmed the reduced binding ability of OSPHR2S269D to the downstream PSI gene promoter in vivo by chip-qPCR. Phenotypic analysis of transgenic plants showed that when the expression levels of different forms of OSPHR2 were similar, the phosphorus toxicity phenotype of FLAG-OSPHR2S269D leaves disappeared, the phosphorus content was significantly reduced, and the expression of PSI gene was also significantly reduced. In addition, there was no significant difference in the abundance of OSPHR2 protein in different forms of transgenic plants, and there was no significant difference in the subcellular localization of OSPHR2, OSPHR2S269D and OSPHR2S269A in rice protoplasts, indicating that the phosphorylation modification of S269 did not affect the protein expression and subcellular localization of OSPHR2. These results further indicate that the phosphorylation modification of GSK2 to S269 specifically inhibits the DNA binding activity of OSPHR2.
To confirm whether this mechanism is conserved in dicots, the researchers analyzed it in Arabidopsis thaliana and found that mutating the serine site corresponding to OSPHR2-S269 in Atphr1 to aspartic acid also showed a significant decrease in its DNA-binding activity. A comprehensive analysis of PHR family members in different species showed that the serine site identified by GSK2 was conserved in different species, but there were polymorphisms among PHR family members. For example, serine (S) is found in rice OSPHR1 2 and Arabidopsis ATPHR1 and ATPHL1 4, while proline (P) is found in rice OSPHR3 4 and Arabidopsis ATPHL2 3. Notably, previous studies have found that unlike OSPHR3 4, which is induced by low phosphorus at the transcriptional level, OSPHR1 2 is not regulated by low phosphorus at the transcriptional level. ThereforeThe post-translational modification regulation of OSPHR2 revealed in this study may represent a key new mechanism of PSR regulation. Interestingly, this serine residue is not found in bryophytes and is only found in vascular plants, suggesting that the phosphorylation modification may be an evolutionary mechanism of plant adaptation to terrestrial environments.
The results showed that phosphorus starvation induced the degradation of GSK2 protein, suggesting that plants could improve their rapid response to unfavorable nutritional conditions by removing the inhibitory effect of GSK2 on OSPHR2 in the face of low phosphorus stress, and this deinhibition working model provided a new perspective for understanding the low phosphorus response of plants. In addition, this study also showed that BR can improve the uptake and utilization of phosphorus by promoting PSR, which provides theoretical guidance for the use of BR to reduce the use of phosphorus fertilizer, and the functional phosphorylation site can also be used as an effective target for gene editing to improve the phosphorus uptake and utilization efficiency of plants.
Postdoctoral fellow, South China Agricultural UniversityZhang GuoxiaThe first author is South China Agricultural UniversityChu ChengcaiProfessor and Institute of Crop Science, Chinese Academy of Agricultural SciencesTong HongningThe researcher is the co-corresponding author. Shenzhen Institute of Agricultural Genomics, Chinese Academy of Agricultural SciencesWang HongruResearcher, Postdoctoral Fellow, Howard Hughes Medical InstituteRen XianglePh.D., graduated from the Institute of Genetics and Developmental Biology, Chinese Academy of SciencesXiao YunhuaPh.D.,Qiu YahongPh.D., Institute of Crop Science, Chinese Academy of Agricultural SciencesMeng WenjingPh.D.,Liu DapuPh.D., South China Agricultural UniversityXie QingjunProfessorsHu BinProfessors and others were involved in the work. The research was supported by the Guangdong Provincial Basic and Applied Research Major Project, the National Natural Science of China, and the Innovation Project of the Chinese Academy of Agricultural Sciences.
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