AN INVESTIGATION OF PHOSPHORYLATION OF A SPLICING FACTOR, SR45, IN ARABIDOPSIS THALIANA

Sinead Coleman, Jason Chien, Xiao-Ning Zhang

Department of Biology, St. Bonaventure University, St. Bonaventure, NY 14778

Abstract

SR45 is an important splicing factor that is involved in multiple developmental processes in Arabidopsis thaliana. SR45 has two splicing isoforms, SR45.1 and SR45.2, that play distinct roles in root growth and flower development. SR45.1 has two hypothesized phosphorylation sites, threonine 218 (T218) and serine 219 (S219), that are missing in SR45.2. To further investigate the function of these two sites after phosphorylation, we substituted these amino acids in the existing SR45.1-GFP construct with aspartic acid (D) and glutamic acid (E) by site-direct mutagenesis. The resulting mutant genes were transformed into sr45-1 mutant plants to generate stable transgenic plants.The transgenic plant lines were screened by examining their resistance to herbicide and selected by the presence of the GFP signal. Our results show that the expression level of GFP and its sustainability are directly related to the effectiveness of the transgene. For the petal development, substitutions at either/both sites recovered the normal development to various degrees. For root growth, the double substitution resulted in overgrowth, while the substitution on T218 gave a better recovery than the substitution on S219 alone. Therefore these data suggest that the phosphorylation of both T218 and S219 may not be necessary for the normal function of SR45.1 in both root and petal development. However, a phosphorylated T218, but not a phosphorylated S219, may be required for a fully functional SR45.1 in roots and petals

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Figure 1 Experimental procedure

CATCTCCTCAACGGAAAACAGT S P Q R K T G

Alternative Acceptor Site (21 nt.)

SR45.1 (AS1)

SR45.2 (AS2)

Conclusion

In order to study the functional difference between SR45.1 and SR45.2, we identified two residues (T218 and S219) present in SR45.1 but absent in SR45.2 as potential targets for phosphorylation modification. Due to the structural similarity between the phosphorylated residues and phosphorylation mimics, namely D and E, we substituted T218 and S219 with D and E to generate a set of SR45.1 mutant genes and introduced them into sr45-1 mutant plants, separately. By analyzing the degree of recovery from mutant phenotypes in these transgenic plants, we found that both T218D and T218E had a greater effect on recovering both root growth and petal development to wild type than S219D and S219E. When both T218 and S219 were forced to be negatively charged by phosphorylation mimics, the root exhibited exaggerated growth, while the observations on petal development were not conclusive. This indicates that SR45.1 may be phosphorylated at T218 rather than S219 in its active form in roots. However, since these two residues are missing in SR45.2, SR45.2 is regulated differently from SR45.1. This study provided further evidence on the difference between two isoforms of SR45 and how it may contribute to the distinct functions of the two isoforms in regulating plant development and growth.

ReferenceX.-N. Zhang & S. M. Mount. Two alternatively spliced isoforms of the Arabidopsis thaliana SR45 protein have distinct roles during normal plant development. Plant Physiol. 2009, 150(3): 1-9.

This Project is supported by the National Science Foundation and St Bonaventure University.

Figure 4 Phosphorylation Mimicking on threonine (T218) and serine (S219) in SR45.1. A. Two SR45 isoforms: SR45.1 and SR45.2. Exons are shown as green boxes; introns are shown as straight lines; untranslated regions (UTRs) are shown as white boxes. The alternative 3’ splice site sequence that is present in SR45.1 but missing in SR45.2 is shown with its deduced amino acid sequence.B. Structure similarity of phosphoserine, phosphothreonine, aspartic acid (D) and glutamic acid (E).

Figure 3 Amino acid substitutions and their effect on root growth and flower petal development. Plants of Col wild type, sr45-1 and two independent transgenic lines for each amino acid substitution were used for all the analysis. A and B: Root growth. Root length was measured from 4-day-old seedlings. Error bars show the standard deviation (n=15). C and D: Petal development. Petal width-to-length ratio was measured from individual flowers randomly picked from plants at 10 days after flowering. Error bars show the standard deviation (n=19). * presents statistical difference compared to Col; † presents statistical difference compared to the sr45-1 mutant (p<0.05).

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Figure 2 Transgenic plants overexpressing different SR45-GFP constructs.A. Domain arrangement of SR45.1, SR45.2, and mutant constructs with different amino acid substitutions.B and C: An example of the expression of SR45.1 mutant proteins visualized by GFP. GFP signalwas detected in nucleus of root cells in the SR45.1S219E construct. B: Root tip. Scale bar represents 100µm. C: Nucleus. Scale bar represents 10µm.

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