Phosphothreonine 218 is required for the function of SR45.1 in regulating flower petal development in Arabidopsis

Authors: ZHANG, XIAO-NING - St Bonaventure University; MO, CECILIA - St Bonaventure University; Garrett, Wesley; Cooper, Bret

Submitted to: Plant Signaling and Behavior
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/6/2014
Publication Date: 5/15/2014
Publication URL: http://handle.nal.usda.gov/10113/61519
Citation: Zhang, X., Mo, C., Garrett, W.M., Cooper, B. 2014. Phosphothreonine 218 is required for the function of SR45.1 in regulating flower petal development in Arabidopsis. Plant Signaling and Behavior. 9(7):e29134.

Interpretive Summary: In plant cells, RNA message is made from DNA, the heritable genetic material. In turn, proteins, which have structural and enzymatic functions, are made from messenger RNA. In a cellular process called splicing, small parts of messenger RNA are cutaway from larger RNA molecules such that the smaller RNA encodes a protein that is different (smaller) than the protein from the larger RNA. It has been demonstrated in plants and animals that splicing helps make different proteins that then have different functions. How splicing is governed and regulated is not fully resolved. It is known that a protein called SR45 is important for splicing in the model plant Arabidopsis. Interestingly, two different forms of the splicing regulator SR45 are made by differential splicing of its messenger RNA. SR45.1 is important for flower development and SR45.2 is important for root growth. This research shows that the SR45.1 protein, which is larger than SR45.2, is phosphorylated in the part of the protein encoded by the RNA not spliced away. Phosphorylation is a protein modification that controls that activity of proteins. When the SR45.1 was mutated such that it could no longer be phosphorylated, SR45.1 misbehaved and caused test plants to have incorrectly formed flowers. Thus, the research shows that not only is the splicing regulator SR45 regulated by RNA splicing, but that it is also regulated at the protein level by phosphorylation. These data are most likely to influence scientists at universities, government agencies and companies who are studying flowers or who are trying to find ways to improve root growth on drought stricken soybeans.

Technical Abstract: RNA splicing is crucial to the production of mature messenger RNAs (mRNA). The protein Arginine/Serine-rich 45 (SR45) acts as an RNA splicing activator and initiates the spliceosome assembly. It is also a peripheral component of the exon-exon junction complex, which assures the quality and availability of mRNAs. SR45 is alternatively spliced into two isoforms; isoform 1 (SR45.1) plays an important role in the flower petal development whereas isoform 2 is important for root growth. In this study, we used immunoprecipitation to isolate an SR45.1-GFP fusion protein, then analyzed it for the presence of phosphate moieties by mass spectrometry. The mass spectrometry data suggested a single phosphorylation event in a peptide region that is not in isoform 2. Substituting alanine for threonine 218, a candidate site for phosphorylation, led to significantly narrower flower petals in transgenic plants than those in wild type plants. Substituting aspartic acid or glutamic acid for threonine 218 in transgenic plants led to petal development comparable to the wild type. The results suggest that the phosphorylation of threonine 218 may be required to activate SR45.1 during petal development. Genetic evidence suggests that the Clk/Sty LAMMER kinase, Fus3-Complementing Gene 2 (AFC2), may regulate the phosphorylation of SR45.1 at threonine 218. These results show that the splicing regulator SR45.1, itself regulated by alternative splicing at the mRNA level, is also regulated post-translationally by phosphorylation.